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Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-EM. The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg 2+ and define how the Mg 2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg 2+ in the SgrAI active site as a direct result of filamentation induced conformational changes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fatty acid in a reaction catalyzed by a diiron center. The diiron center is well-coordinated by conserved histidine residues and is thought to remain with the enzyme. However, we find here that SCD1 progressively loses its activity during catalysis and becomes fully inactive after about nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center and that the addition of free ferrous ions (Fe 2+ ) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe 2+ is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe 2+ in cells could regulate SCD1 activity and hence lipid metabolism., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacterial actin MreB forms filaments composed of antiparallel double-stranded units. The wall-less helical bacterium Spiroplasma has five MreB homologs (MreB1-5), some of which are involved in an intracellular ribbon for driving the bacterium's swimming motility. Although the interaction between MreB units is important for understanding Spiroplasma swimming, the interaction modes of each ribbon component are unclear. Here, we examined the assembly properties of Spiroplasma eriocheiris MreB5 (SpeMreB5), one of the ribbon component proteins that forms sheets. Electron microscopy revealed that sheet formation was inhibited under acidic conditions and bundle structures were formed under acidic and neutral conditions with low ionic strength. We also used solution assays and identified four properties of SpeMreB5 bundles as follows: (I) bundle formation followed sheet formation; (II) electrostatic interactions were required for bundle formation; (III) the positively charged and unstructured C-terminal region contributed to promoting lateral interactions for bundle formation; and (IV) bundle formation required Mg 2+ at neutral pH but was inhibited by divalent cations under acidic pH conditions. During these studies, we also characterized two aggregation modes of SpeMreB5 with distinct responses to ATP. These properties will shed light on SpeMreB5 assembly dynamics at the molecular level., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca 2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca 2+ -sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca 2+ and Mg 2+ and a regulatory site (II) that has been thought to bind only Ca 2+ . Binding of Ca 2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca 2+ /Mg 2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca 2+ /Mg 2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg 2+ caused a 1.4-fold lower affinity for Ca 2+ . These experiments strongly suggest that cytosolic-free Mg 2+ occupies a significant population of the available site II. Interaction of Mg 2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg 2+ , such as secondary hyperparathyroidism or ischemia, respectively., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg 2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg 2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase. We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg 2+ concentration. Subsequently, the second Mg 2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (K d  = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg 2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg 2+ concentrations. An increase of the Mg 2+ concentration from 0.25 to 10 mM increases nucleotide specificity (k cat /K m ) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg 2+ binds very weakly (K d ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV reverse transcriptase binds only two metal ions prior to incorporation of a correct base pair. Molecular dynamics simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg 2+ . Molecular dynamics simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions in the specificity of DNA polymerases., Competing Interests: Conflict of interest K. A. J. is the President of KinTek Corp, which provided the AutoSF-120 stopped-flow, RQF-3 rapid-quench-flow, and KinTek Explorer software used in this study., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Ribonuclease HI, an endoribonuclease, catalyzes the hydrolysis of the RNA strand of an RNA/DNA hybrid and requires divalent metal ions for its enzymatic activity. However, the mechanistic details of the activity of ribonuclease HI and its interaction with divalent metal ions remain unclear. In this study, we performed real-time monitoring of the enzyme-substrate complex in the presence of divalent metal ions (Mn 2+ or Zn 2+ ) using electrospray ionization-mass spectrometry (ESI-MS). The findings provide clear evidence that the enzymatic activity of the ternary complex requires the binding of two divalent metal ions. The Zn 2+ ions bind to both the enzyme itself and the enzyme:substrate complex more strongly than Mn 2+ ions, and gives, in part, the ternary complex, [RNase HI:nicked RNA/DNA hybrid:2Zn 2+ ], suggesting that the ternary complex is retained, even after the hydrolysis of the substrate. The collective results presented herein shed new light on the essential role of divalent metal ions in the activity of ribonuclease HI and demonstrate how Zn 2+ ions confer inhibitory properties on the activity of this enzyme by forming a highly stable complex with the substrate., Competing Interests: Conflict of interest The authors declare no conflicts of interest in regard to this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress-inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca 2+ oscillations. As ER stress is known to reduce ER Ca 2+ levels, we tested the hypothesis that the observed increase in Ca 2+ oscillations occurred because of reduced ER Ca 2+ levels and, in turn, increased store-operated Ca 2+ entry. TM-induced cytosolic Ca 2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca 2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca 2+ depletion-activated Ca 2+ current in mediating Ca 2+ -induced insulin secretion in response to ER stress., (© 2020 Zhang et al.)

Manganese (Mn 2+ ) is extruded from the cell by the zinc transporter 10 (ZnT10). Loss of ZnT10 expression caused by autosomal mutations in the ZnT10 gene leads to hypermanganesemia in multiple organs. Here, combining fluorescent monitoring of cation influx in HEK293-T cells expressing human ZnT10 with molecular modeling of ZnT10 cation selectivity, we show that ZnT10 is exploiting the transmembrane Ca 2+ inward gradient for active cellular exchange of Mn 2+ In analyzing ZnT10 activity we used the ability of Fura-2 to spectrally distinguish between Mn 2+ and Ca 2+ fluxes. We found that ( a ) application of Mn 2+ -containing Ca 2+ -free solution to ZnT10-expressing cells triggers an influx of Mn 2+ , ( b ) reintroduction of Ca 2+ leads to cellular Mn 2+ extrusion against an inward Mn 2+ gradient, and ( c ) the cellular transport of Mn 2+ by ZnT10 is coupled to a reciprocal movement of Ca 2+ Remarkably, replacing a single asparagine residue in ZnT10 (Asp-43) with threonine (ZnT10 N43T) converted the Mn 2+ /Ca 2+ exchange to an uncoupled channel mode, permeable to both Ca 2+ and Mn 2+ The findings in our study identify the first ion transporter that uses the Ca 2+ gradient for active counter-ion exchange. They highlight a remarkable versatility in metal selectivity and mode of transport controlled by the tetrahedral metal transport site of ZnT proteins., (© 2019 Levy et al.)

RcnR, a transcriptional regulator in Escherichia coli , derepresses the expression of the export proteins RcnAB upon binding Ni(II) or Co(II). Lack of structural information has precluded elucidation of the allosteric basis for the decreased DNA affinity in RcnR's metal-bound states. Here, using hydrogen-deuterium exchange coupled with MS (HDX-MS), we probed the RcnR structure in the presence of DNA, the cognate metal ions Ni(II) and Co(II), or the noncognate metal ion Zn(II). We found that cognate metal binding altered flexibility from the N terminus through helix 1 and modulated the RcnR-DNA interaction. Apo-RcnR and RcnR-DNA complexes and the Zn(II)-RcnR complex exhibited similar 2 H uptake kinetics, with fast-exchanging segments located in the N terminus, in helix 1 (residues 14-24), and at the C terminus. The largest difference in 2 H incorporation between apo- and Ni(II)- and Co(II)-bound RcnR was observed in helix 1, which contains the N terminus and His-3, and has been associated with cognate metal binding. 2 H uptake in helix 1 was suppressed in the Ni(II)- and Co(II)-bound RcnR complexes, in particular in the peptide corresponding to residues 14-24, containing Arg-14 and Lys-17. Substitution of these two residues drastically affected DNA-binding affinity, resulting in rcnA expression in the absence of metal. Our results suggest that cognate metal binding to RcnR orders its N terminus, decreases helix 1 flexibility, and induces conformational changes that restrict DNA interactions with the positively charged residues Arg-14 and Lys-17. These metal-induced alterations decrease RcnR-DNA binding affinity, leading to rcnAB expression., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Divalent metal ions are essential components of DNA polymerases both for catalysis of the nucleotidyl transfer reaction and for base excision. They occupy two sites, A and B, for DNA synthesis. Recently, a third metal ion was shown to be essential for phosphoryl transfer reaction. The metal ion in the A site is coordinated by the carboxylate of two highly conserved acidic residues, water molecules, and the 3'-hydroxyl group of the primer so that the A metal is in an octahedral complex. Its catalytic function is to lower the pK a of the hydroxyl group, making it a highly effective nucleophile that can attack the α phosphorous atom of the incoming dNTP. The metal ion in the B site is coordinated by the same two carboxylates that are affixed to the A metal ion as well as the non-bridging oxygen atoms of the incoming dNTP. The carboxyl oxygen of an adjacent peptide bond serves as the sixth ligand that completes the octahedral coordination geometry of the B metal ion. Similarly, two metal ions are required for proofreading; one helps to lower the pK a of the attacking water molecule, and the other helps to stabilize the transition state for nucleotide excision. The role of different divalent cations are discussed in relation to these two activities as well as their influence on base selectivity and misincorporation by DNA polymerases. Some, but not all, of the effects of these different metal ions can be rationalized based on their intrinsic properties, which are tabulated in this review., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The human-restricted pathogen Neisseria gonorrhoeae encodes a single N-acetylmuramyl-l-alanine amidase involved in cell separation (AmiC), as compared with three largely redundant cell separation amidases found in Escherichia coli (AmiA, AmiB, and AmiC). Deletion of amiC from N. gonorrhoeae results in severely impaired cell separation and altered peptidoglycan (PG) fragment release, but little else is known about how AmiC functions in gonococci. Here, we demonstrated that gonococcal AmiC can act on macromolecular PG to liberate cross-linked and non-cross-linked peptides indicative of amidase activity, and we provided the first evidence that a cell separation amidase can utilize a small synthetic PG fragment as substrate (GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc(pentapeptide)). An investigation of two residues in the active site of AmiC revealed that Glu-229 is critical for both normal cell separation and the release of PG fragments by gonococci during growth. In contrast, Gln-316 has an autoinhibitory role, and its mutation to lysine resulted in an AmiC with increased enzymatic activity on macromolecular PG and on the synthetic PG derivative. Curiously, the same Q316K mutation that increased AmiC activity also resulted in cell separation and PG fragment release defects, indicating that activation state is not the only factor determining normal AmiC activity. In addition to displaying high basal activity on PG, gonococcal AmiC can utilize metal ions other than the zinc cofactor typically used by cell separation amidases, potentially protecting its ability to function in zinc-limiting environments. Thus gonococcal AmiC has distinct differences from related enzymes, and these studies revealed parameters for how AmiC functions in cell separation and PG fragment release., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Members of the Zrt and Irt protein (ZIP) family are a central participant in transition metal homeostasis as they function to increase the cytosolic concentration of zinc and/or iron. However, the lack of a crystal structure hinders elucidation of the molecular mechanism of ZIP proteins. Here, we employed GREMLIN, a co-evolution-based contact prediction approach in conjunction with the Rosetta structure prediction program to construct a structural model of the human (h) ZIP4 transporter. The predicted contact data are best fit by modeling hZIP4 as a dimer. Mutagenesis of residues that comprise a central putative hZIP4 transmembrane transition metal coordination site in the structural model alter the kinetics and specificity of hZIP4. Comparison of the hZIP4 dimer model to all known membrane protein structures identifies the 12-transmembrane monomeric Piriformospora indica phosphate transporter (PiPT), a member of the major facilitator superfamily (MFS), as a likely structural homolog., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Transient receptor potential (TRP) channel melastatin subfamily member 4 (TRPM4) is a broadly expressed nonselective monovalent cation channel. TRPM4 is activated by membrane depolarization and intracellular Ca(2+), which is essential for the activation. The Ca(2+) sensitivity is known to be regulated by calmodulin and membrane phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Although these regulators must play important roles in controlling TRPM4 activity, mutation analyses of the calmodulin-binding sites have suggested that Ca(2+) binds to TRPM4 directly. However, the intrinsic binding sites in TRPM4 remain to be elucidated. Here, by using patch clamp and molecular biological techniques, we show that there are at least two functionally different divalent cation-binding sites, and the negatively charged amino acids near and in the TRP domain in the C-terminal tail of TRPM4 (Asp-1049 and Glu-1062 of rat TRPM4) are required for maintaining the normal Ca(2+) sensitivity of one of the binding sites. Applications of Co(2+), Mn(2+), or Ni(2+) to the cytosolic side potentiated TRPM4 currents, increased the Ca(2+) sensitivity, but were unable to evoke TRPM4 currents without Ca(2+). Mutations of the acidic amino acids near and in the TRP domain, which are conserved in TRPM2, TRPM5, and TRPM8, deteriorated the Ca(2+) sensitivity in the presence of Co(2+) or PI(4,5)P2 but hardly affected the sensitivity to Co(2+) and PI(4,5)P2. These results suggest a novel role of the TRP domain in TRPM4 as a site responsible for maintaining the normal Ca(2+) sensitivity. These findings provide more insights into the molecular mechanisms of the regulation of TRPM4 by Ca(2+)., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The pathogenic protozoan parasite Leishmania donovani is capable of both de novo pyrimidine biosynthesis and salvage of pyrimidines from the host milieu. Genetic analysis has authenticated L. donovani uracil phosphoribosyltransferase (LdUPRT), an enzyme not found in mammalian cells, as the focal enzyme of pyrimidine salvage because all exogenous pyrimidines that can satisfy the requirement of the parasite for pyrimidine nucleotides are funneled to uracil and then phosphoribosylated to UMP in the parasite by LdUPRT. To characterize this unique parasite enzyme, LdUPRT was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Kinetic analysis revealed apparent Km values of 20 and 99 μM for the natural substrates uracil and phosphoribosylpyrophosphate, respectively, as well as apparent Km values 6 and 7 μM for the pyrimidine analogs 5-fluorouracil and 4-thiouracil, respectively. Size exclusion chromatography revealed the native LdUPRT to be tetrameric and retained partial structure and activity in high concentrations of urea. L. donovani mutants deficient in de novo pyrimidine biosynthesis, which require functional LdUPRT for growth, are hypersensitive to high concentrations of uracil, 5-fluorouracil, and 4-thiouracil in the growth medium. This hypersensitivity can be explained by the observation that LdUPRT is substrate-inhibited by uracil and 4-thiouracil, but 5-fluorouracil toxicity transpires via an alternative mechanism. This substrate inhibition of LdUPRT provides a protective mechanism for the parasite by facilitating purine and pyrimidine nucleotide pool balance and by sparing phosphoribosylpyrophosphate for consumption by the nutritionally indispensable purine salvage process.

The Parkinson disease-associated kinase Pink1 is targeted to mitochondria where it is thought to regulate mitochondrial quality control by promoting the selective autophagic removal of dysfunctional mitochondria. Nevertheless, the targeting mode of Pink1 and its submitochondrial localization are still not conclusively resolved. The aim of this study was to dissect the mitochondrial import pathway of Pink1 by use of a highly sensitive in vitro assay. Mutational analysis of the Pink1 sequence revealed that its N terminus acts as a genuine matrix localization sequence that mediates the initial membrane potential (Δψ)-dependent targeting of the Pink1 precursor to the inner mitochondrial membrane, but it is dispensable for Pink1 import or processing. A hydrophobic segment downstream of the signal sequence impeded complete translocation of Pink1 across the mitochondrial inner membrane. Additionally, the C-terminal end of the protein promoted the retention of Pink1 at the outer membrane. Thus, multiple targeting signals featured by the Pink1 sequence result in the final localization of both the full-length protein and its major Δψ-dependent cleavage product to the cytosolic face of the outer mitochondrial membrane. Full-length Pink1 and deletion constructs resembling the natural Pink1 processing product were found to assemble into membrane potential-sensitive high molecular weight protein complexes at the mitochondrial surface and displayed similar cytoprotective effects when expressed in vivo, indicating that both species are functionally relevant.

Calcium-independent phospholipase A(2)γ (iPLA(2)γ) (PNPLA8) is the predominant phospholipase activity in mammalian mitochondria. However, the chemical mechanisms that regulate its activity are unknown. Here, we utilize iPLA(2)γ gain of function and loss of function genetic models to demonstrate the robust activation of iPLA(2)γ in murine myocardial mitochondria by Ca(2+) or Mg(2+) ions. Calcium ion stimulated the production of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) from 1-palmitoyl-2-[(14)C]arachidonoyl-sn-glycero-3-phosphocholine during incubations with wild-type heart mitochondrial homogenates. Furthermore, incubation of mitochondrial homogenates from transgenic myocardium expressing iPLA(2)γ resulted in 13- and 25-fold increases in the initial rate of radiolabeled 2-AA-LPC and arachidonic acid (AA) production, respectively, in the presence of calcium ion. Mass spectrometric analysis of the products of calcium-activated hydrolysis of endogenous mitochondrial phospholipids in transgenic iPLA(2)γ mitochondria revealed the robust production of AA, 2-AA-LPC, and 2-docosahexaenoyl-LPC that was over 10-fold greater than wild-type mitochondria. The mechanism-based inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (iPLA(2)γ selective), but not its enantiomer, (S)-BEL (iPLA(2)β selective) or pyrrolidine (cytosolic PLA(2)α selective), markedly attenuated Ca(2+)-dependent fatty acid release and polyunsaturated LPC production. Moreover, Ca(2+)-induced iPLA(2)γ activation was accompanied by the production of downstream eicosanoid metabolites that were nearly completely ablated by (R)-BEL or by genetic ablation of iPLA(2)γ. Intriguingly, Ca(2+)-induced iPLA(2)γ activation was completely inhibited by long-chain acyl-CoA (IC(50) ∼20 μm) as well as by a nonhydrolyzable acyl-CoA thioether analog. Collectively, these results demonstrate that mitochondrial iPLA(2)γ is activated by divalent cations and inhibited by acyl-CoA modulating the generation of biologically active metabolites that regulate mitochondrial bioenergetic and signaling functions.

The human small heat-shock protein αB-crystallin (αB) rescues misfolded proteins from irreversible aggregation during cellular stress. Binding of Cu(II) was shown to modulate the oligomeric architecture and the chaperone activity of αB. However, the mechanistic basis of this stimulation is so far not understood. We provide here first structural insights into this Cu(II)-mediated modulation of chaperone function using NMR spectroscopy and other biophysical approaches. We show that the α-crystallin domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion with picomolar binding affinity. Putative Cu(II) ligands are His(83), His(104), His(111), and Asp(109) at the dimer interface. These loop residues are conserved among different metazoans, but also for human αA-crystallin, HSP20, and HSP27. The involvement of Asp(109) has direct implications for dimer stability, because this residue forms a salt bridge with the disease-related Arg(120) of the neighboring monomer. Furthermore, we observe structural reorganization of strands β2-β3 triggered by Cu(II) binding. This N-terminal region is known to mediate both the intermolecular arrangement in αB oligomers and the binding of client proteins. In the presence of Cu(II), the size and the heterogeneity of αB multimers are increased. At the same time, Cu(II) increases the chaperone activity of αB toward the lens-specific protein β(L)-crystallin. We therefore suggest that Cu(II) binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher order assemblies of αB.

The vesicular nucleotide transporter (VNUT) is a secretory vesicle protein that is responsible for the vesicular storage and subsequent exocytosis of ATP (Sawada, K., Echigo, N., Juge, N., Miyaji, T., Otsuka, M., Omote, H., and Moriyama, Y. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 5683-5686). Because VNUT actively transports ATP in a membrane potential (Δψ)-dependent manner irrespective of divalent cations such as Mg(2+) and Ca(2+), VNUT recognizes free ATP as a transport substrate. However, whether or not VNUT transports chelating complexes with divalent cations remains unknown. Here, we show that proteoliposomes containing purified VNUT actively took up Mg(2+) when ATP was present, as detected by atomic absorption spectroscopy. The VNUT-containing proteoliposomes also took up radioactive Ca(2+) upon imposing Δψ (positive-inside) but not ΔpH. The Δψ-driven Ca(2+) uptake required ATP and a millimolar concentration of Cl(-), which was inhibited by Evans blue, a specific inhibitor of SLC17-type transporters. VNUT in which Arg-119 was specifically mutated to alanine, the counterpart of the essential amino acid residue of the SLC17 family, lost the ability to take up both ATP and Ca(2+). Ca(2+) uptake was also inhibited in the presence of various divalent cations such as Mg(2+). Kinetic analysis indicated that Ca(2+) or Mg(2+) did not affect the apparent affinity for ATP. RNAi of the VNUT gene in PC12 cells decreased the vesicular Mg(2+) concentration to 67.7%. These results indicate that VNUT transports both nucleotides and divalent cations probably as chelating complexes and suggest that VNUT functions as a divalent cation importer in secretory vesicles under physiological conditions.

Ion channels gate at membrane-embedded domains by changing their conformation along the ion conduction pathway. Inward rectifier K(+) (Kir) channels possess a unique extramembrane cytoplasmic domain that extends this pathway. However, the relevance and contribution of this domain to ion permeation remain unclear. By qualitative x-ray crystallographic analysis, we found that the pore in the cytoplasmic domain of Kir3.2 binds cations in a valency-dependent manner and does not allow the displacement of Mg(2+) by monovalent cations or spermine. Electrophysiological analyses revealed that the cytoplasmic pore of Kir3.2 selectively binds positively charged molecules and has a higher affinity for Mg(2+) when it has a low probability of being open. The selective blocking of chemical modification of the side chain of pore-facing residues by Mg(2+) indicates that the mode of binding of Mg(2+) is likely to be similar to that observed in the crystal structure. These results indicate that the Kir3.2 crystal structure has a closed conformation with a negative electrostatic field potential at the cytoplasmic pore, the potential of which may be controlled by conformational changes in the cytoplasmic domain to regulate ion diffusion along the pore.

Chondroitin sulfate is a linear polysaccharide of alternating D-glucuronic acid and N-acetyl-D-galactosamine residues with sulfate groups at various positions of the sugars. It interacts with and regulates cytokine and growth factor signal transduction, thus influencing development, organ morphogenesis, inflammation, and infection. We found chondroitinase activity in medium conditioned by baculovirus-infected insect cells and identified a novel chondroitinase. Sequence analysis revealed that the enzyme was a truncated form of occlusion-derived virus envelope protein 66 (ODV-E66) of Autographa californica nucleopolyhedrovirus. The enzyme was a novel chondroitin lyase with distinct substrate specificity. The enzyme was active over a wide range of pH (pH 4-9) and temperature (30-60 °C) and was unaffected by divalent metal ions. The ODV-E66 truncated protein digested chondroitin most efficiently followed by chondroitin 6-sulfate. It degraded hyaluronan to a minimal extent but did not degrade dermatan sulfate, heparin, and N-acetylheparosan. Further analysis using chemo-enzymatically synthesized substrates revealed that the enzyme specifically acted on glucuronate residues in non-sulfated and chondroitin 6-sulfate structures but not in chondroitin 4-sulfate structures. These results suggest that this chondroitinase is useful for detailed structural and compositional analysis of chondroitin sulfate, preparation of specific chondroitin oligosaccharides, and study of baculovirus infection mechanism.

Cu(II) ions are implicated in the pathogenesis of Alzheimer disease by influencing the aggregation of the amyloid-β (Aβ) peptide. Elucidating the underlying Cu(II)-induced Aβ aggregation is paramount for understanding the role of Cu(II) in the pathology of Alzheimer disease. The aim of this study was to characterize the qualitative and quantitative influence of Cu(II) on the extracellular aggregation mechanism and aggregate morphology of Aβ(1-40) using spectroscopic, microelectrophoretic, mass spectrometric, and ultrastructural techniques. We found that the Cu(II):Aβ ratio in solution has a major influence on (i) the aggregation kinetics/mechanism of Aβ, because three different kinetic scenarios were observed depending on the Cu(II):Aβ ratio, (ii) the metal:peptide stoichiometry in the aggregates, which increased to 1.4 at supra-equimolar Cu(II):Aβ ratio; and (iii) the morphology of the aggregates, which shifted from fibrillar to non-fibrillar at increasing Cu(II):Aβ ratios. We observed dynamic morphological changes of the aggregates, and that the formation of spherical aggregates appeared to be a common morphological end point independent on the Cu(II) concentration. Experiments with Aβ(1-42) were compatible with the conclusions for Aβ(1-40) even though the low solubility of Aβ(1-42) precluded examination under the same conditions as for the Aβ(1-40). Experiments with Aβ(1-16) and Aβ(1-28) showed that other parts than the Cu(II)-binding His residues were important for Cu(II)-induced Aβ aggregation. Based on this study we propose three mechanistic models for the Cu(II)-induced aggregation of Aβ(1-40) depending on the Cu(II):Aβ ratio, and identify key reaction steps that may be feasible targets for preventing Cu(II)-associated aggregation or toxicity in Alzheimer disease.

Editing of adenosine (A) to inosine (I) at the first anticodon position in tRNA is catalyzed by adenosine deaminases acting on tRNA (ADATs). This essential reaction in bacteria and eukarya permits a single tRNA to decode multiple codons. Bacterial ADATa is a homodimer with two bound essential Zn(2+). The ADATa crystal structure revealed residues important for substrate binding and catalysis; however, such high resolution structural information is not available for eukaryotic tRNA deaminases. Despite significant sequence similarity among deaminases, we continue to uncover unexpected functional differences between Trypanosoma brucei ADAT2/3 (TbADAT2/3) and its bacterial counterpart. Previously, we demonstrated that TbADAT2/3 is unique in catalyzing two different deamination reactions. Here we show by kinetic analyses and inductively coupled plasma emission spectrometry that wild type TbADAT2/3 coordinates two Zn(2+) per heterodimer, but unlike any other tRNA deaminase, mutation of one of the key Zn(2+)-coordinating cysteines in TbADAT2 yields a functional enzyme with a single-bound zinc. These data suggest that, at least, TbADAT3 may play a role in catalysis via direct coordination of the catalytic Zn(2+). These observations raise the possibility of an unusual Zn(2+) coordination interface with important implications for the function and evolution of editing deaminases.

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α(L) subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K(D)) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn(2+) was replaced sequentially by Mg(2+) and Ca(2+). Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4(+) and CD8(+) T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.

Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia, hepatocerebral syndrome with mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg(2+) ions, and elevated ionic strength was required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis, or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.

Dyshomeostasis of transition metals iron and copper as well as accumulation of oxidative DNA damage have been implicated in multitude of human neurodegenerative diseases, including Alzheimer disease and Parkinson disease. These metals oxidize DNA bases by generating reactive oxygen species. Most oxidized bases in mammalian genomes are repaired via the base excision repair pathway, initiated with one of four major DNA glycosylases: NTH1 or OGG1 (of the Nth family) or NEIL1 or NEIL2 (of the Nei family). Here we show that Fe(II/III) and Cu(II) at physiological levels bind to NEIL1 and NEIL2 to alter their secondary structure and strongly inhibit repair of mutagenic 5-hydroxyuracil, a common cytosine oxidation product, both in vitro and in neuroblastoma (SH-SY5Y) cell extract by affecting the base excision and AP lyase activities of NEILs. The specificity of iron/copper inhibition of NEILs is indicated by a lack of similar inhibition of OGG1, which also indicated that the inhibition is due to metal binding to the enzymes and not DNA. Fluorescence and surface plasmon resonance studies show submicromolar binding of copper/iron to NEILs but not OGG1. Furthermore, Fe(II) inhibits the interaction of NEIL1 with downstream base excision repair proteins DNA polymerase beta and flap endonuclease-1 by 4-6-fold. These results indicate that iron/copper overload in the neurodegenerative diseases could act as a double-edged sword by both increasing oxidative genome damage and preventing their repair. Interestingly, specific chelators, including the natural chemopreventive compound curcumin, reverse the inhibition of NEILs both in vitro and in cells, suggesting their therapeutic potential.

GMP catalyzes the formation of GDP-Man, a fundamental precursor for protein glycosylation and bacterial cell wall and capsular polysaccharide biosynthesis. Crystal structures of GMP from the thermophilic bacterium Thermotoga maritima in the apo form, in complex with the substrates mannose-1-phosphate or GTP and bound with the end product GDP-Man in the presence of the essential divalent cation Mg(2+), were solved in the 2.1-2.8 A resolution range. The T. maritima GMP molecule is organized in two separate domains: a N-terminal Rossman fold-like domain and a C-terminal left-handed beta-helix domain. Two molecules associate into a dimer through a tail-to-tail arrangement of the C-terminal domains. Comparative analysis of the structures along with characterization of enzymatic parameters reveals the bases of substrate specificity of this class of sugar nucleotidyltransferases. In particular, substrate and product binding are associated with significant changes in the conformation of loop regions lining the active center and in the relative orientation of the two domains. Involvement of both the N- and C-terminal domains, coupled to the catalytic role of a bivalent metal ion, highlights the catalytic features of bacterial GMPs compared with other members of the pyrophosphorylase superfamily.

Tight junctions are dynamic components of epithelial and endothelial cells that regulate the paracellular transport of ions, solutes, and immune cells. The assembly and permeability of these junctions is dependent on the zonula occludens (ZO) proteins, members of the membrane-associated guanylate kinase homolog (MAGUK) protein family, which are characterized by a core Src homology 3 (SH3)-GUK module that coordinates multiple protein-protein interactions. The structure of the ZO-1 SH3-GUK domain confirms that the interdependent folding of the SH3 and GUK domains is a conserved feature of MAGUKs, but differences in the orientation of the GUK domains in three different MAGUKs reveal interdomain flexibility of the core unit. Using pull-down assays, we show that an effector loop, the U6 region in ZO-1, forms a novel intramolecular interaction with the core module. This interaction is divalent cation-dependent and overlaps with the binding site for the regulatory molecule calmodulin on the GUK domain. These findings provide insight into the previously observed ability of the U6 region to regulate TJ assembly in vivo and the structural basis for the complex protein interactions of the MAGUK family.

Her4 (ErbB-4) and Her2/neu (ErbB-2) are receptor-tyrosine kinases belonging to the epidermal growth factor receptor (EGFR) family. Crystal structures of EGFR and Her4 kinase domains demonstrate kinase dimerization and activation through an allosteric mechanism. The kinase domains form an asymmetric dimer, where the C-lobe surface of one monomer contacts the N-lobe of the other monomer. EGFR kinase dimerization and activation in vitro was previously reported using a nickel-chelating lipid-liposome system, and we now apply this system to all other members of the EGFR family. Polyhistidine-tagged Her4, Her2/neu, and Her3 kinase domains are bound to these nickel-liposomes and are brought to high local concentration, mimicking what happens to full-length receptors in vivo following ligand binding. Addition of nickel-liposomes to Her4 kinase domain results in 40-fold activation in kinase activity and marked enhancement of C-terminal tail autophosphorylation. Activation of Her4 shows a sigmoidal dependence on kinase concentration, consistent with a cooperative process requiring kinase dimerization. Her2/neu kinase activity is also activated by nickel-liposomes, and is increased further by heterodimerization with Her3 or Her4. The ability of Her3 and Her4 to heterodimerize and activate other family members is studied in vitro. Her3 kinase domain readily activates Her2/neu but is a poor activator of Her4, which differs from the prediction made by the asymmetric dimer model. Mutation of Her3 residues (952)ENI(954) to the corresponding sequence in Her4 enhanced the ability of Her3 to activate Her4, demonstrating that sequence differences on the C-lobe surface influence the heterodimerization and activation of ErbB kinase domains.

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity.

Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca(2+) and Mn(2+). Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors.

We have determined the structure of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in an E2.P(i)-like form stabilized as a complex with MgF(4)(2-), an ATP analog, adenosine 5'-(beta,gamma-methylene)triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5A resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca(2+)-ATPases, e.g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing key residues at or near the ATP binding site. A structural comparison to the Na(+),K(+)-ATPase reveals that the Phe(93) side chain occupies the equivalent binding pocket of the CPA site in SERCA, suggesting an important role of this residue in stabilization of the potassium-occluded E2 state of Na(+),K(+)-ATPase.

The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca(2+) and Mg(2+) permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg(2+) permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca(2+) permeability along with concentration-dependent inhibition of Na(+)-carrying currents by Ca(2+). Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca(2+)-permeable TRPM channels, substantially increased Ca(2+) permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca(2+) permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.

Dictyostelium myosin-5b is the gene product of myoJ and one of two closely related myosin-5 isoenzymes produced in Dictyostelium discoideum. Here we report a detailed investigation of the kinetic and functional properties of the protein. In standard assay buffer conditions, Dictyostelium myosin-5b displays high actin affinity in the presence of ADP, fast ATP hydrolysis, and a high steady-state ATPase activity in the presence of actin that is rate limited by ADP release. These properties are typical for a processive motor that can move over long distances along actin filaments without dissociating. Our results show that a physiological decrease in the concentration of free Mg(2+)-ions leads to an increased rate of ADP release and shortening of the fraction of time the motor spends in the strong actin binding states. Consistently, the ability of the motor to efficiently translocate actin filaments at very low surface densities decreases with decreasing concentrations of free Mg(2+)-ions. In addition, we provide evidence that the observed changes in Dd myosin-5b motor activity are of physiological relevance and propose a mechanism by which this molecular motor can switch between processive and non-processive movement.

Divalent metal ions play a critical role in the removal of N-terminal methionine from nascent proteins by methionine aminopeptidase (MetAP). Being an essential enzyme for bacteria, MetAP is an appealing target for the development of novel antibacterial drugs. Although purified enzyme can be activated by several divalent metal ions, the exact metal ion used by MetAP in cells is unknown. Many MetAP inhibitors are highly potent on purified enzyme, but they fail to show significant inhibition of bacterial growth. One possibility for the failure is a disparity of the metal used in activation of purified MetAP and the metal actually used by MetAP inside bacterial cells. Therefore, the challenge is to elucidate the physiologically relevant metal for MetAP and discover MetAP inhibitors that can effectively inhibit cellular MetAP. We have recently discovered MetAP inhibitors with selectivity toward different metalloforms of Escherichia coli MetAP, and with these unique inhibitors, we characterized their inhibition of MetAP enzyme activity in a cellular environment. We observed that only inhibitors that are selective for the Fe(II)-form of MetAP were potent in this assay. Further, we found that only these Fe(II)-form selective inhibitors showed significant inhibition of growth of five E. coli strains and two Bacillus strains. We confirmed their cellular target as MetAP by analysis of N-terminal processed and unprocessed recombinant glutathione S-transferase proteins. Therefore, we conclude that Fe(II) is the likely metal used by MetAP in E. coli and other bacterial cells.

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Robust kinetic analyses of the reaction mechanism are complicated by the instability of ferrous iron in aqueous solution, particularly at alkaline pH values. At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant ferrochelatase from both murine and yeast sources inserts not only ferrous iron but also divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability. Ferrous iron is the preferred metal ion substrate in terms of apparent k(cat) and is also the only metal ion substrate not subject to severe substrate inhibition. Substrate inhibition occurs in the order Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) and can be alleviated by the addition of metal complexing agents such as beta-mercaptoethanol or imidazole to the reaction buffer. These data indicate the presence of two catalytically significant metal ion binding sites that may coordinately regulate a selective processivity for the various potential metal ion substrates.

TRPM7 is a divalent cation-permeable channel that is ubiquitously expressed. Recently, mouse TRPM7 has been shown to be sensitive to, and even permeable to, protons when heterologously expressed. Here we have demonstrated that human TRPM7 expressed either heterologously or endogenously also exhibits proton conductivity. The gene silencing of TRPM7 by small interfering RNA suppressed H+ currents in human cervical epithelial HeLa cells. In HEK293T cells transfected with human TRPM7, the inward proton conductance was suppressed by extracellular Mg2+ or Ca2+ with IC(50) values of 0.5 and 1.9 mm, respectively. Anomalous mole fraction behavior of H+ currents in the presence of Mg2+ or Ca2+ indicated that these divalent cations compete with protons for binding sites. Systematic mutation of negatively charged amino acid residues within the putative pore-forming region of human TRPM7 into the neutral amino acid alanine was tested. E1047A resulted in non-functional channels, and D1054A abolished proton conductance, whereas E1052A and D1059A only partially reduced proton conductivity. Thus, it is concluded that Asp-1054 is an essential determinant of the proton conductivity, whereas Glu-1047 might be required for channel formation, and the remaining negatively charged amino acids in the pore region (Glu-1052 and Asp-1059) may play a facilitating role in the proton conductivity of human TRPM7. It is suggested that proton conductivity of endogenous human TRPM7 plays a role in physiologically/pathologically acidic situations.

Retinoic acid-inducible gene I (RIG-I) is a cytosolic receptor that recognizes viral RNA and activates the interferon-mediated innate antiviral response. To understand the mechanism of signal activation at the receptor level, we cloned, expressed, and purified human RIG-I containing the two caspase activation and recruitment domains (CARDs) followed by the C-terminal helicase domain. We found that recombinant RIG-I is a functional protein that interacts with double-stranded RNA with substantially higher affinity as compared with single-stranded RNA structures unless they contain a 5'-triphosphate group. Viral RNA binding to RIG-I stimulates the velocity of ATP hydrolysis by 33-fold, which at the cellular level translates into a 43-fold increase of interferon-beta expression. In contrast, the isolated ATPase/helicase domain is constitutively activated while also retaining its RNA ligand binding properties. These results support the recent model by which RIG-I signaling is autoinhibited in the absence of RNA by intra-molecular interactions between the CARDs and the C terminus. Based on pH profile and metal ion dependence experiments, we propose that the active site of RIG-I cannot efficiently accommodate divalent cations under the RNA-free repressed conformation. Overall, these results show a direct correlation between RNA binding and ATPase enzymatic function leading to signal transduction and suggest that a tight control of ATPase activity by the CARDs prevents RIG-I signaling in the absence of viral RNA.

The prion protein (PrP) is a cell-surface Cu(2+)-binding glycoprotein that when misfolded is responsible for a number of transmissible spongiform encephalopathies. Full-length PrP-(23-231) and constructs in which the octarepeat region has been removed, or His(95) and His(110) is replaced by alanine residues, have been used to elucidate the order and mode of Cu(2+) coordination to PrP-(23-231). We have built on our understanding of the appearance of visible CD spectra and EPR for various PrP fragments to characterize Cu(2+) coordination to full-length PrP. At physiological pH, Cu(2+) initially binds to full-length PrP in the amyloidogenic region between the octarepeats and the structured domain at His(95) and His(110). Only subsequent Cu(2+) ions bind to single histidine residues within the octarepeat region. Ni(2+) ions are used to further probe metal binding and, like Cu(2+), Ni(2+) will bind individually to His(95) and His(110), involving preceding main chain amides. Competitive chelators are used to determine the affinity of the first mole equivalent of Cu(2+) bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu(2+) binding sites support the suggestion that PrP could act as a sacrificial quencher of free radicals generated by copper redox cycling.

NRAMP-1 (natural resistance-associated macrophage protein-1) has been associated with innate resistance to unrelated intracellular pathogen infections, up-regulation of proinflammatory phagocyte functions, and susceptibility to autoimmune diseases. It is still unclear how the divalent cation transport function of NRAMP-1 accounts for the associated pleiotropic effects. In this study, we evaluated the impact of murine macrophage NRAMP-1 expression on the activity of protein-tyrosine phosphatases (PTPs) as an upstream event contributing to the NRAMP-1 regulation of signal transduction and control of effector macrophage functions. Functional expression of NRAMP-1 results in lower macrophage PTP activity and increased protein phosphorylation. Decreased PTP activity is not a result of changes in protein expression but rather a reversible regulatory mechanism involving the interaction with NRAMP-1 metal substrates. In the context of intracellular infections, NRAMP-1 expression prevents full macrophage PTP induction by Leishmania infection, correlating with higher nitric oxide production and lower parasite survival. We suggest that NRAMP-1 divalent cation transport leads to transient inhibition of PTPs via direct PTP-metal interaction and/or by reactive oxygen species-dependent PTP oxidation, consequently promoting positive signal transduction, as a backbone for the induction of proinflammatory phagocyte functions.

Slc11a1 (formerly Nramp1) is a proton/divalent cation transporter that regulates cation homeostasis in macrophages. Slc11a2 mediates divalent cation uptake via the gut and delivery into cells. The mode of action of the two transporters remains controversial. Heterologous expression in frog oocytes shows Slc11a2 is a symporter, whereas Slc11a1 is an antiporter fluxing divalent cations against the proton gradient. This explains why Slc11a2, but not Slc11a1, can complement EGTA sensitivity in smf1Delta/smf2Delta/smf3Delta yeast. However, some studies of transport in mammalian cells suggest Slc11a1 is a symporter. We now demonstrate that Slc11a1, but not Slc11a2, complements a divalent cation stress phenotype in bsd2Delta/rer1Delta yeast. This is the first description of a yeast complementation assay for Slc11a1 function. Given the prior demonstration in frog oocytes that Slc11a1 acts as an antiporter, the most plausible interpretation of the data is that Slc11a1 is rescuing bsd2Delta/rer1Delta yeast by exporting divalent cations. Chimaeras define the N terminus, and a segment of the protein core preceding transmembrane domain 9 through transmembrane domain 12, as important in rescuing the divalent cation stress phenotype. EGTA sensitivity and divalent cation stress phenotypes in yeast expressing Slc11a orthologues show that symport activity is ancestral. Molecular changes that mediate rescue of the divalent cation stress phenotype post-date frogs and co-evolved with Slc11a1 orthologues that regulate divalent cation homeostasis in macrophages and resistance to infection in chickens and mammals.

The yeast vacuole is the storage depot for cellular iron. In this report we quantify the import-export balance in the vacuole because of the import of iron by Ccc1p and to export by the combined activity of Smf3p and the ferroxidase, permease pair of proteins, Fet5p and Fth1p. Our data indicate that the two efflux pathways are equally efficient in trafficking iron out of the vacuole. A major focus of this work was to identify the ferrireductase(s) that supplies the Fe(II) for efflux whether by Smf3p or the Fet5p-Fth1p complex. Using a combination of flameless atomic absorption spectrophotometry to quantify vacuolar and whole cell iron content and a reporter assay for cytoplasmic iron we demonstrate that Fre6p supplies Fe(II) to both efflux systems, while Fre7p plays no role in Fe-efflux from the vacuole. Enzymatic assay shows the two fusions to have similar reductase activity, however. Confocal fluorescence microscopy demonstrates that Fre6:GFP localizes to the vacuolar membrane; in contrast, Fre7:GFP fusions exhibit a variable and diffuse cellular distribution. Demonstrating a role for a vacuolar metalloreductase in Fe-efflux supports the model that iron is stored in the vacuole in the ferric state.

Annexins A5, A2, and A6 (Anx-A5, -A2, and -A6) are quantitatively major proteins of the matrix vesicle nucleational core that is responsible for mineral formation. Anx-A5 significantly activated the induction and propagation of mineral formation when incorporated into synthetic nucleation complexes made of amorphous calcium phosphate (ACP) and Anx-A5 or of phosphatidylserine (PS) plus ACP (PS-CPLX) and Anx-A5. Incorporation of Anx-A5 markedly shortened the induction time, greatly increasing the rate and overall amount of mineral formed when incubated in synthetic cartilage lymph. Constructed by the addition of Ca(2+) to PS, emulsions prepared in an intracellular phosphate buffer matched in ionic composition to the intracellular fluid of growth plate chondrocytes, these biomimetic PS-CPLX nucleators had little nucleational activity. However, incorporation of Anx-A5 transformed them into potent nucleators, with significantly greater activity than those made from ACP without PS. The ability of Anx-A5 to enhance the nucleation and growth of mineral appears to stem from its ability to form two-dimensional crystalline arrays on PS-containing monolayers. However, some stimulatory effect also may result from its ability to exclude Mg(2+) and HCO(-)(3) from nucleation sites. Comparing the various annexins for their ability to activate PS-CPLX nucleation yields the following: avian cartilage Anx-A5 > human placental Anx-A5 > avian liver Anx-A5 > or = avian cartilage Anx-A6 >> cartilage Anx-A2. The stimulatory effect of human placental Anx-A5 and avian cartilage Anx-A6 depended on the presence of PS, since in its absence they either had no effect or actually inhibited the nucleation activity of ACP. Anx-A2 did not significantly enhance mineralization.

The channel kinases TRPM6 and TRPM7 have recently been discovered to play important roles in Mg2+ and Ca2+ homeostasis, which is critical to both human health and cell viability. However, the molecular basis underlying these channels' unique Mg2+ and Ca2+ permeability and pH sensitivity remains unknown. Here we have created a series of amino acid substitutions in the putative pore of TRPM7 to evaluate the origin of the permeability of the channel and its regulation by pH. Two mutants of TRPM7, E1047Q and E1052Q, produced dramatic changes in channel properties. The I-V relations of E1052Q and E1047Q were significantly different from WT TRPM7, with the inward currents of 8- and 12-fold larger than TRPM7, respectively. The binding affinity of Ca2+ and Mg2+ was decreased by 50- to 140-fold in E1052Q and E1047Q, respectively. Ca2+ and Mg2+ currents in E1052Q were 70% smaller than those of TRPM7. Strikingly, E1047Q largely abolished Ca2+ and Mg2+ permeation, rendering TRPM7 a monovalent selective channel. In addition, the ability of protons to potentiate inward currents was lost in E1047Q, indicating that E1047 is critical to Ca2+ and Mg2+ permeability of TRPM7, and its pH sensitivity. Mutation of the corresponding residues in the pore of TRPM6, E1024Q and E1029Q, produced nearly identical changes to the channel properties of TRPM6. Our results indicate that these two glutamates are key determinants of both channels' divalent selectivity and pH sensitivity. These findings reveal the molecular mechanisms underpinning physiological/pathological functions of TRPM6 and TRPM7, and will extend our understanding of the pore structures of TRPM channels.

The Ni(2+)-dependent transcription factor NikR is widespread among microbes. The two experimentally characterized NikR orthologs, from Helicobacter pylori and Escherichia coli, display vastly different regulatory capabilities in response to increased intracellular Ni(2+). Here, we demonstrate that the nine-residue N-terminal arm present in H. pylori NikR plays a critical role in the expanded regulatory capabilities of this NikR family member. Specifically, the N-terminal arm is required to inhibit NikR binding to low affinity and nonspecific DNA sequences and is also linked to a cation requirement for NikR binding to the nixA promoter. Site-directed mutagenesis and arm-truncation variants of NikR indicate that two residues, Asp-7 and Asp-8, are linked to the cation requirement for binding. Pro-4 and Lys-6 are required for maximal DNA binding affinity of the full-length protein to both the nixA and ureA promoters. The N-terminal arm is highly variable among NikR family members, and these results suggest that it is an adaptable structural feature that can tune the regulatory capabilities of NikR to the nickel physiology of the microbe in which it is found.

The recent discoveries of Stim1 and Orai proteins have shed light on the molecular makeup of both the endoplasmic reticulum Ca(2+) sensor and the calcium release-activated calcium (CRAC) channel, respectively. In this study, we investigated the regulation of CRAC channel function by extracellular Ca(2+) for channels composed primarily of Orai1, Orai2, and Orai3, by co-expressing these proteins together with Stim1, as well as the endogenous channels in HEK293 cells. As reported previously, Orai1 or Orai2 resulted in a substantial increase in CRAC current (I(crac)), but Orai3 failed to produce any detectable Ca(2+)-selective currents. However, sodium currents measured in the Orai3-expressing HEK293 cells were significantly larger in current density than Stim1-expressing cells. Moreover, upon switching to divalent free external solutions, Orai3 currents were considerably more stable than Orai1 or Orai2, indicating that Orai3 channels undergo a lesser degree of depotentiation. Additionally, the difference between depotentiation from Ca(2+) and Ba(2+) or Mg(2+) solutions was significantly less for Orai3 than for Orai1 or -2. Nonetheless, the Na(+) currents through Orai1, Orai2, and Orai3, as well as the endogenous store-operated Na(+) currents in HEK293 cells, were all inhibited by extracellular Ca(2+) with a half-maximal concentration of approximately 20 mum. We conclude that Orai1, -2, and -3 channels are similarly inhibited by extracellular Ca(2+), indicating similar affinities for Ca(2+) within the selectivity filter. Orai3 channels appeared to differ from Orai1 and -2 in being somewhat resistant to the process of Ca(2+) depotentiation.

Acyl carrier protein (ACP), a small protein essential for bacterial growth and pathogenesis, interacts with diverse enzymes during the biosynthesis of fatty acids, phospholipids, and other specialized products such as lipid A. NMR and hydrodynamic studies have previously shown that divalent cations stabilize native helical ACP conformation by binding to conserved acidic residues at two sites (A and B) at either end of the "recognition" helix II. To examine the roles of these amino acids in ACP structure and function, site-directed mutagenesis was used to replace individual site A (Asp-30, Asp-35, Asp-38) and site B (Glu-47, Glu-53, Asp-56) residues in recombinant Vibrio harveyi ACP with the corresponding amides, along with combined mutations at each site (SA, SB) or both sites (SA/SB). Like native V. harveyi ACP, all individual mutants were unfolded at neutral pH but adopted a helical conformation in the presence of millimolar Mg(2+) or upon fatty acylation. Mg(2+) binding to sites A or B independently stabilized native ACP conformation, whereas mutant SA/SB was folded in the absence of Mg(2+), suggesting that charge neutralization is largely responsible for ACP stabilization by divalent cations. Asp-35 in site A was critical for holo-ACP synthase activity, while acyl-ACP synthetase and UDP-N-acetylglucosamine acyltransferase (LpxA) activities were more affected by mutations in site B. Both sites were required for fatty acid synthase activity. Overall, our results indicate that divalent cation binding site mutations have predicted effects on ACP conformation but unpredicted and variable consequences on ACP function with different enzymes.

The binding of integrin alpha(L)beta(2) to its ligand intercellular adhesion molecule-1 is required for immune responses and leukocyte trafficking. Small molecule antagonists of alpha(L)beta(2) are under intense investigation as potential anti-inflammatory drugs. We describe for the first time a small molecule integrin agonist. A previously described alpha/beta I allosteric inhibitor, compound 4, functions as an agonist of alpha(L)beta(2) in Ca(2+) and Mg(2+)and as an antagonist in Mn(2+). We have characterized the mechanism of activation and its competitive and noncompetitive inhibition by different compounds. Although it stimulates ligand binding, compound 4 nonetheless inhibits lymphocyte transendothelial migration. Agonism by compound 4 results in accumulation of alpha(L)beta(2) in the uropod, extreme uropod elongation, and defective de-adhesion. Small molecule integrin agonists open up novel therapeutic possibilities.

HisB from Escherichia coli is a bifunctional enzyme catalyzing the sixth and eighth steps of l-histidine biosynthesis. The N-terminal domain (HisB-N) possesses histidinol phosphate phosphatase activity, and its crystal structure shows a single domain with fold similarity to the haloacid dehalogenase (HAD) enzyme family. HisB-N forms dimers in the crystal and in solution. The structure shows the presence of a structural Zn(2+) ion stabilizing the conformation of an extended loop. Two metal binding sites were also identified in the active site. Their presence was further confirmed by isothermal titration calorimetry. HisB-N is active in the presence of Mg(2+), Mn(2+), Co(2+), or Zn(2+), but Ca(2+) has an inhibitory effect. We have determined structures of several intermediate states corresponding to snapshots along the reaction pathway, including that of the phosphoaspartate intermediate. A catalytic mechanism, different from that described for other HAD enzymes, is proposed requiring the presence of the second metal ion not found in the active sites of previously characterized HAD enzymes, to complete the second half-reaction. The proposed mechanism is reminiscent of two-Mg(2+) ion catalysis utilized by DNA and RNA polymerases and many nucleases. The structure also provides an explanation for the inhibitory effect of Ca(2+).

Guanylyl cyclase-activating protein 1 (GCAP-1) is an EF-hand protein that activates retinal guanylyl cyclase (RetGC) in photoreceptors at low free Ca2+ in the light and inhibits it in the dark when Ca2+ concentrations rise. We present the first direct evidence that Mg2+-bound form of GCAP-1, not its cation-free form, is the true activator of RetGC-1 under physiological conditions. Of four EF-hand structures in GCAP-1, three bound Ca2+ ions and could exchange Ca2+ for Mg2+. At concentrations of free Ca2+ and Mg2+ typical for the light-adapted photoreceptors, all three metal-binding EF-hands were predominantly occupied by Mg2, and the presence of bound Mg2+ in GCAP-1 was essential for its ability to stimulate RetGC-1. In the Mg2+-bound form of GCAP-1 all three Trp residues became more exposed to the polar environment compared with its apo form. The replacement of Mg2+ by Ca2+ in the EF-hands 2 and 3 further exposed Trp-21 to the solution in a non-metal-binding EF-hand domain 1 that interacts with RetGC. Contrary to that, replacement of Mg2+ by Ca2+ in the EF-hand 4 moved Trp-94 in the entering alpha-helix of the EF-hand 3 back to the non-polar environment. Our results demonstrate that Mg2+ regulates GCAP-1 not only by adjusting its Ca2+ sensitivity to the physiological conditions in photoreceptors but also by creating the conformation required for RetGC stimulation.

YiiP is a representative member of the cation diffusion facilitator (CDF) family, a class of ubiquitous metal transporters that play an essential role in metal homeostasis. Recently, a pair of Zn2+/Cd2+-selective binding sites has been localized to two highly conserved aspartyl residues (Asp157), each in a 2-fold-symmetry-related transmembrane segment 5 (TM5) of a YiiP homodimer. Here we report the functional and structural interactions between Asp157 and yet another highly conserved Asp49 in the TM2. Calorimetric binding analysis indicated that Asp49 and Asp157 contribute to a common Cd2+ binding site in each subunit. Copper phenanthroline oxidation of YiiP(D49C), YiiP(D157C), and YiiP(D49C/D157C) yielded inter- and intra-subunit cross-links among Cys49 and Cys157, consistent with the spatial proximity of two (Asp49-Asp157) sites at the dimer interface. Hg2+ binding to YiiP(D49C) or YiiP(D49C/D157C) also yielded a Cys49-Hg2+-Cys49 biscysteinate complex across the dimer interface, further establishing the interfacial location of a (Asp49-Asp157)2 bimetal binding center. Two bound Cd2+ ions were found transported cooperatively with a sigmoidal dependence on the Cd2+ concentration (n = 1.4). The binding affinity, transport cooperativity, and rate were modestly reduced by either a D49C or D157C mutation, but greatly diminished when all the bidentate aspartate O-ligands in (Asp49-Asp157)2 were replaced by the monodentate cysteine S-ligands. The functional significance of these findings is discussed based on the unique coordination chemistry of aspartyl residues and a model for the translocation pathway of metal ions at the YiiP dimer interface.