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Retinal guanylate cyclases (RetGCs) are regulated by a family of guanylate cyclase-activating proteins (called GCAP1-7). GCAPs form dimers that bind to Ca2+ and confer Ca2+ sensitive activation of RetGC during visual phototransduction. The GCAP5 homologue from zebrafish contains two nonconserved cysteine residues (Cys15 and Cys17) that bind to ferrous ion, which stabilizes GCAP5 dimerization and diminishes its ability to activate RetGC. Here, we present NMR and EPR-DEER structural analysis of a GCAP5 dimer in the Mg2+-bound, Ca2+-free, Fe2+-free activator state. The NMR-derived structure of GCAP5 is similar to the crystal structure of Ca2+-bound GCAP1 (root-mean-square deviation of 2.4 Å), except that the N-terminal helix of GCAP5 is extended by two residues, which allows the sulfhydryl groups of Cys15 and Cys17 to become more solvent exposed in GCAP5 to facilitate Fe2+ binding. Nitroxide spin-label probes were covalently attached to particular cysteine residues engineered in GCAP5: C15, C17, T26C, C28, N56C, C69, C105, N139C, E152C, and S159C. The intermolecular distance of each spin-label probe in dimeric GCAP5 (measured by EPR-DEER) defined restraints for calculating the dimer structure by molecular docking. The GCAP5 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H18, Y21, M25, F72, V76, and W93, as well as an intermolecular salt bridge between R22 and D71. The structural model of the GCAP5 dimer was validated by mutations (H18E/Y21E, H18A/Y21A, R22D, R22A, M25E, D71R, F72E, and V76E) at the dimer interface that disrupt dimerization of GCAP5 and affect the activation of RetGC. We propose that GCAP5 dimerization may play a role in the Fe2+-dependent regulation of cyclase activity in zebrafish photoreceptors.

Isocitrate dehydrogenase 1 (IDH1) is a key metabolic enzyme for maintaining cytosolic levels of α-ketoglutarate (AKG) and preserving the redox environment of the cytosol. Wild-type (WT) IDH1 converts isocitrate to AKG; however, mutant IDH1-R132H that is recurrent in human cancers catalyzes the neomorphic production of the oncometabolite d-2-hydroxyglutrate (D-2HG) from AKG. Recent work suggests that production of l-2-hydroxyglutarte in cancer cells can be regulated by environmental changes, including hypoxia and intracellular pH (pHi). However, it is unknown whether and how pHi affects the activity of IDH1-R132H. Here, we show that in cells IDH1-R132H can produce D-2HG in a pH-dependent manner with increased production at lower pHi. We also identify a molecular mechanism by which this pH sensitivity is achieved. We show that pH-dependent production of D-2HG is mediated by pH-dependent heterodimer formation between IDH1-WT and IDH1-R132H. In contrast, neither IDH1-WT nor IDH1-R132H homodimer formation is affected by pH. Our results demonstrate that robust production of D-2HG by IDH1-R132H relies on the coincidence of (1) the ability to form heterodimers with IDH1-WT and (2) low pHi or highly abundant AKG substrate. These data suggest cancer-associated IDH1-R132H may be sensitive to physiological or microenvironmental cues that lower pH, such as hypoxia or metabolic reprogramming. This work reveals new molecular considerations for targeted therapeutics and suggests potential synergistic effects of using catalytic IDH1 inhibitors targeting D-2HG production in combination with drugs targeting the tumor microenvironment.

It was previously shown that human platelet 12S-lipoxygenase (h12-LOX) exists as a dimer; however, the specific structure is unknown. In this study, we create a model of the dimer through a combination of computational methods, experimental mutagenesis, and hydrogen-deuterium exchange (HDX) investigations. Initially, Leu183 and Leu187 were replaced by negatively charged glutamate residues and neighboring aromatic residues were replaced with alanine residues (F174A/W176A/L183E/L187E/Y191A). This quintuple mutant disrupted both the hydrophobic and π-π interactions, generating an h12-LOX monomer. To refine the determinants for dimer formation further, the L183E/L187E mutant was generated and the equilibrium shifted mostly toward the monomer. We then submitted the predicted monomeric structure to protein-protein docking to create a model of the dimeric complex. A total of nine of the top 10 most energetically favorable docking conformations predict a TOP-to-TOP dimeric arrangement of h12-LOX, with the α-helices containing a Leu-rich region (L172, L183, L187, and L194), corroborating our experimental results showing the importance of these hydrophobic interactions for dimerization. This model was supported by HDX investigations that demonstrated the stabilization of four, non-overlapping peptides within helix α2 of the TOP subdomain for wt-h12-LOX, consistent with the dimer interface. Most importantly, our data reveal that the dimer and monomer of h12-LOX have distinct biochemical properties, suggesting that the structural changes due to dimerization have allosteric effects on active site catalysis and inhibitor binding.

Enzymatic dehalogenation is an important and well-studied biological process in both the detoxification and catabolism of small molecules, many of which are anthropogenic in origin. However, dedicated dehalogenation reactions that replace a halogen atom with a hydrogen are rare in the biosynthesis of natural products. In fact, the debrominase Bmp8 is the only known example. It catalyzes the reductive debromination of the coral settlement cue and the potential human toxin 2,3,4,5-tetrabromopyrrole as part of the biosynthesis of the antibiotic pentabromopseudilin. Using a combination of protein crystallography, mutagenesis, and computational modeling, we propose a catalytic mechanism for Bmp8 that is reminiscent of that catalyzed by human deiodinases in the maintenance of thyroid hormones. The identification of the key catalytic residues enabled us to recognize divergent functional homologues of Bmp8. Characterization of one of these homologues demonstrated its debromination activity even though it is found in a completely distinct genomic context. This observation suggests that additional enzymes outside those associated with the tetrabromopyrrole biosynthetic pathway may be able to alter the lifetime of this compound in the environment.

The "guardian of the genome", p53, functions as a tumor suppressor that responds to cell stressors such as DNA damage, hypoxia, and tumor formation by inducing cell-cycle arrest, senescence, or apoptosis. Mutation of p53 disrupts its tumor suppressor function, leading to various types of human cancers. One particular mutant, R175H, is a structural mutant that inactivates the DNA damage response pathway and acquires oncogenic functions that promotes both cancer and drug resistance. Our current work aims to understand how p53 wild-type function is disrupted due to the R175H mutation. We use a series of atomistic integrative models built previously from crystal structures of the full-length p53 tetramer bound to DNA and model the R175H mutant using in silico site-directed mutagenesis. Explicitly solvated all-atom molecular dynamics (MD) simulations on wild-type and the R175H mutant p53 reveal insights into how wild-type p53 searches and recognizes DNA, and how this mechanism is disrupted as a result of the R175H mutation. Specifically, our work reveals the optimal quaternary DNA binding mode of the DNA binding domain and shows how this binding mode is altered via symmetry loss as a result of the R175H mutation, indicating a recognition mechanism that is reminiscent of the asymmetry seen in wild type p53 binding to nonspecific genomic elements. Altogether our work sheds new light into the hitherto unseen molecular mechanisms governing transcription factor, DNA recognition.

Sensory guanylate cyclases (zGCs) in zebrafish photoreceptors are regulated by a family of guanylate cyclase activator proteins (called GCAP1-7). GCAP5 contains two nonconserved cysteine residues (Cys15 and Cys17) that could in principle bind to biologically active transition state metal ions (Zn2+ and Fe2+). Here, we present nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) binding analyses that demonstrate the binding of one Fe2+ ion to two GCAP5 molecules (in a 1:2 complex) with a dissociation constant in the nanomolar range. At least one other Fe2+ binds to GCAP5 with micromolar affinity that likely represents electrostatic Fe2+ binding to the EF-hand loops. The GCAP5 double mutant (C15A/C17A) lacks nanomolar binding to Fe2+, suggesting that Fe2+ at this site is ligated directly by thiolate groups of Cys15 and Cys17. Size exclusion chromatography analysis indicates that GCAP5 forms a dimer in the Fe2+-free and Fe2+-bound states. NMR structural analysis and molecular docking studies suggest that a single Fe2+ ion is chelated by thiol side chains from Cys15 and Cys17 in the GCAP5 dimer, forming an [Fe(SCys)4] complex like that observed previously in two-iron superoxide reductases. Binding of Fe2+ to GCAP5 weakens its ability to activate photoreceptor human GC-E by decreasing GC activity >10-fold. Our results indicate a strong Fe2+-induced inhibition of GC by GCAP5 and suggest that GCAP5 may serve as a redox sensor in visual phototransduction.

Despite extensive research on the sequence-determined self-assembly of both pathogenic and nonpathogenic proteins, the question of how the sequence identity would influence the coassembly or cross-seeding of diverse proteins without distinct sequence similarity remains largely unanswered. Here, we demonstrate that the rapid coaggregation of proteins with negligible sequence similarity is fundamentally governed by preferred heteromeric interactions between their partially unfolded states via the gain of additional charge complementarity and hydrophobic interactions. The partial loss of intramolecular interactions and concurrent gain of non-native intrinsically disordered regions with sticky groups become crucial for both aggressive heteromeric primary nucleation and secondary nucleation events. The results signify the direct relevance of sequence-independent conformational cross-talk between diverse proteins to the foundational events required for the growth of biological multiprotein amyloid deposits.

Human guanylate binding proteins (hGBPs), which are large GTPases, are crucial for cell-autonomous immunity, including antiviral activity. hGBPs contain two domains: an N-terminal catalytic domain and a C-terminal helical domain. hGBP3 and its splice variant hGBP3ΔC have been shown to possess anti-influenza activity in lung epithelial cells. These two proteins have identical catalytic domains but different helical domains. It is unclear whether this difference affects GTPase activity or protein oligomerization. Using combined approaches, we show that both proteins hydrolyze GTP to GDP and further to GMP. However, they form different oligomers. hGBP3 exists as a hexamer in the free form, whereas hGBP3ΔC forms large oligomers, indicating that helical domain modifications of the splice variant result in distinct oligomers. Furthermore, unlike other homologues, neither protein changes its oligomeric state upon substrate binding or hydrolysis. Deleting the helical domain of hGBP3 (hGBP3 1-309 ) yields a monomer, suggesting that the helical domain promotes the hexamerization of hGBP3. We overexpressed hGBP3 and hGBP3ΔC to test their efficacy against HCV growth and found that hGBP3 inhibits HCV multiplication, while the splice variant has little effect. Our mutational studies on hGBP3 show that substrate hydrolysis, rather than substrate binding, is required for inhibiting HCV growth. This suggests that substrate hydrolysis generates a protein conformation essential for anti-HCV activity. Additionally, truncated hGBP3 1-309 does not exhibit anti-HCV activity. Altogether, these findings suggest that the helical domain of hGBP3 is crucial for reducing HCV growth through hexamer formation and that its variations result in different oligomers and antiviral activities.

The protein periostin is a matricellular protein that is expressed in connective tissue. It is composed of five globular domains arranged in an elongated structure with an extensive disordered C-terminal tail. Periostin contains 11 cysteine residues, of which one is unpaired and the rest form five intramolecular disulfide bonds. Periostin plays an important role during wound healing and is also involved in driving the inflammatory state in atopic diseases. This study provides a comprehensive biochemical characterization of periostin in human skin and in dermal and pulmonary fibroblasts in vitro . Through the application of Western blotting, co-immunoprecipitation, and LC-MS/MS, we show for the first time that periostin is a disulfide-bonded homodimer and engages in a novel disulfide-bonded complex with fibronectin both in vivo and in vitro . This inherent characteristic of periostin holds the potential to redefine our approach to exploring and understanding its functional role in future research endeavors.

The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF1R) are homodimeric transmembrane glycoproteins that transduce signals across the membrane on binding of extracellular peptide ligands. The structures of IR/IGF1R fragments in apo and liganded states have revealed that the extracellular subunits of these receptors adopt Λ-shaped configurations to which are connected the intracellular tyrosine kinase (TK) domains. The binding of peptide ligands induces structural transitions in the extracellular subunits leading to potential dimerization of transmembrane domains (TMDs) and autophosphorylation in TKs. However, the activation mechanisms of IR/IGF1R, especially the role of TMDs in coordinating signal-inducing structural transitions, remain poorly understood, in part due to the lack of structures of full-length receptors in apo or liganded states. While atomistic simulations of IR/IGF1R TMDs showed that these domains can dimerize in single component membranes, spontaneous unbiased dimerization in a plasma membrane having a physiologically representative lipid composition has not been observed. We address this limitation by employing coarse-grained (CG) molecular dynamics simulations to probe the dimerization propensity of IR/IGF1R TMDs. We observed that TMDs in both receptors spontaneously dimerized independent of their initial orientations in their dissociated states, signifying their natural propensity for dimerization. In the dimeric state, IR TMDs predominantly adopted X-shaped configurations with asymmetric helical packing and significant tilt relative to the membrane normal, while IGF1R TMDs adopted symmetric V-shaped or parallel configurations with either no tilt or a small tilt relative to the membrane normal. Our results suggest that IR/IGF1R TMDs spontaneously dimerize and adopt distinct dimerized configurations.

Phototransduction in vertebrate photoreceptor cells is controlled by Ca 2+ -dependent feedback loops involving the membrane-bound guanylate cyclase GC-E that synthesizes the second messenger guanosine-3',5'-cyclic monophosphate. Intracellular Ca 2+ -sensor proteins named guanylate cyclase-activating proteins (GCAPs) regulate the activity of GC-E by switching from a Ca 2+ -bound inhibiting state to a Ca 2+ -free/Mg 2+ -bound activating state. The gene GUCY2D encodes for human GC-E, and mutations in GUCY2D are often associated with an imbalance of Ca 2+ and cGMP homeostasis causing retinal disorders. Here, we investigate the Ca 2+ -dependent inhibition of the constitutively active GC-E mutant V902L. The inhibition is not mediated by GCAP variants but by Ca 2+ replacing Mg 2+ in the catalytic center. Distant from the cyclase catalytic domain is an α-helical domain containing a highly conserved helix-turn-helix motif. Mutating the critical amino acid position 804 from leucine to proline left the principal activation mechanism intact but resulted in a lower level of catalytic efficiency. Our experimental analysis of amino acid positions in two distant GC-E domains implied an allosteric communication pathway connecting the α-helical and the cyclase catalytic domains. A computational connectivity analysis unveiled critical differences between wildtype GC-E and the mutant V902L in the allosteric network of critical amino acid positions.

Lipoprotein lipase (LPL) is responsible for the hydrolysis of triglycerides from circulating lipoproteins. Whereas most identified mutations in the LPL gene are deleterious, one mutation, LPLS447X, causes a gain of function. This mutation truncates two amino acids from LPL's C-terminus. Carriers of LPLS447X have decreased VLDL levels and increased HDL levels, a cardioprotective phenotype. LPLS447X is used in Alipogene tiparvovec, the gene therapy product for individuals with familial LPL deficiency. It is unclear why LPLS447X results in a serum lipid profile more favorable than that of LPL. In vitro reports vary as to whether LPLS447X is more active than LPL. We report a comprehensive, biochemical comparison of purified LPLS447X and LPL dimers. We found no difference in specific activity on synthetic and natural substrates. We also did not observe a difference in the Ki for ANGPTL4 inhibition of LPLS447X relative to that of LPL. Finally, we analyzed LPL-mediated uptake of fluorescently labeled lipoprotein particles and found that LPLS447X enhanced lipoprotein uptake to a greater degree than LPL did. An LPL structural model suggests that the LPLS447X truncation exposes residues implicated in LPL binding to uptake receptors.

G protein-coupled receptors (GPCRs) detect a wide variety of physical and chemical signals and transmit that information across the cellular plasma membrane. Dimerization is a proposed modulator of GPCR signaling, but the structure and stability of class A GPCR dimerization have been difficult to establish. Here we investigated the dimerization affinity and binding interface of human cone opsins, which initiate and sustain daytime color vision. Using a time-resolved fluorescence approach, we found that human red cone opsin exhibits a strong propensity for dimerization, whereas the green and blue cone opsins do not. Through mutagenesis experiments, we identified a dimerization interface in the fifth transmembrane helix of human red cone opsin involving amino acids I230, A233, and M236. Insights into this dimerization interface of red cone opsin should aid ongoing investigations of the structure and function of GPCR quaternary interactions in cell signaling. Finally, we demonstrated that the same residues needed for dimerization are also partially responsible for the spectral tuning of red cone opsin. This last observation has the potential to open up new lines of inquiry regarding the functional role of dimerization for red cone opsin.

The 11-cis-retinylidene chromophore of visual pigments isomerizes upon interaction with a photon, initiating a downstream cascade of signaling events that ultimately lead to visual perception. 11-cis-Retinylidene is regenerated through enzymatic transformations collectively called the visual cycle. The first and rate-limiting enzymatic reaction within this cycle, i.e., the reduction of all-trans-retinal to all-trans-retinol, is catalyzed by retinol dehydrogenases. Here, we determined the structure of Drosophila melanogaster photoreceptor retinol dehydrogenase (PDH) isoform C that belongs to the short-chain dehydrogenase/reductase (SDR) family. This is the first reported structure of a SDR that possesses this biologically important activity. Two crystal structures of the same enzyme grown under different conditions revealed a novel conformational change of the NAD+ cofactor, likely representing a change during catalysis. Amide hydrogen-deuterium exchange of PDH demonstrated changes in the structure of the enzyme upon dinucleotide binding. In D. melanogaster, loss of PDH activity leads to photoreceptor degeneration that can be partially rescued by transgenic expression of human RDH12. Based on the structure of PDH, we analyzed mutations causing Leber congenital amaurosis 13 in a homology model of human RDH12 to obtain insights into the molecular basis of RDH12 disease-causing mutations.

Known for its distinct metamorphic behavior, XCL1 interconverts between a canonical chemokine folded monomer (XCL1mon) that interacts with the receptor, XCR1, and a unique dimer (XCL1dim) that interacts with glycosaminoglycans and inhibits HIV-1 activity. This study presents the first detailed analysis of the GAG binding properties of XCL1dim. Basic residues within a conformationally selective dimeric variant of XCL1 (W55D) were mutated and analyzed for their effects on heparin binding. Mutation of Arg23 and Arg43 greatly diminished the level of heparin binding in both heparin Sepharose chromatography and surface plasmon resonance assays. To assess the contributions of different GAG structures to XCL1 binding, we developed a solution fluorescence polarization assay and correlated affinity with the length and level of sulfation of heparan sulfate oligosaccharides. It was recently demonstrated that the XCL1 GAG binding form, XCL1dim, is responsible for preventing HIV-1 infection through interactions with gp120. This study defines a GAG binding surface on XCL1dim that includes residues that are important for HIV-1 inhibition.

TDP-43 is a ubiquitously expressed, multidomain functional protein that is distinctively known to form aggregates in many fatal neurodegenerative disorders. However, the information for arresting TDP-43 aggregation is missing due to a lack of understanding of the molecular mechanism of the aggregation and structural properties of TDP-43. TDP-43 is inherently prone to aggregation and has minimal protein solubility. Multiple studies have been performed on the smaller parts of TDP-43 or the full-length protein attached to a large solubilization tag. However, the presence of co-solutes or solubilization tags is observed to interfere with the molecular properties and aggregation mechanism of full-length TDP-43. Notably, this study populated and characterized the native, dimeric state of TDP-43 without the interference of co-solutes or protein modifications. We observed that the electrostatics of the local environment is capable of the partial unfolding and monomerization of the native dimeric state of TDP-43 into an amyloidogenic molten globule. By employing the tools of thermodynamics and kinetics, we reveal the structural characteristics and temporal order of the early intermediates and transition states during the transition of the molten globule to β-rich, amyloid-like aggregates of TDP-43, which is governed by the electrostatics of the environment. The current advanced understanding of the nature of native and early aggregation-prone intermediates, early steps, and the influence of electrostatics in TDP-43 aggregation is essential for drug design.

Hepatitis B virus (HBV) displays remarkable self-assembly capabilities that interest the scientific community and biotechnological industries as HBV is leading to an annual mortality of up to 1 million people worldwide (especially in Africa and Southeast Asia). When the ionic strength is increased, hepatitis B virus-like particles (VLPs) can assemble from dimers of the first 149 residues of the HBV capsid protein core assembly domain (Cp149). Using solution small-angle X-ray scattering, we investigated the disassembly of the VLPs by titrating guanidine hydrochloride (GuHCl). Measurements were performed with and without 1 M NaCl, added either before or after titrating GuHCl. Fitting the scattering curves to a linear combination of atomic models of Cp149 dimer (the subunit) and T = 3 and T = 4 icosahedral capsids revealed the mass fraction of the dimer in each structure in all the titration points. Based on the mass fractions, the variation in the dimer-dimer association standard free energy was calculated as a function of added GuHCl, showing a linear relation between the interaction strength and GuHCl concentration. Using the data, we estimated the energy barriers for assembly and disassembly and the critical nucleus size for all of the assembly reactions. Extrapolating the standard free energy to [GuHCl] = 0 showed an evident hysteresis in the assembly process, manifested by differences in the dimer-dimer association standard free energy obtained for the disassembly reactions compared with the equivalent assembly reactions. Similar hysteresis was observed in the energy barriers for assembly and disassembly and the critical nucleus size. The results suggest that above 1.5 M, GuHCl disassembled the capsids by attaching to the protein and adding steric repulsion, thereby weakening the hydrophobic attraction.

The intrinsically disordered protein (IDP) stathmin plays an important regulatory role in cytoskeletal maintenance through its helical binding to tubulin and microtubules. However, it lacks a stable fold in the absence of its binding partner. Although stathmin has been a focus of research over the past two decades, the solution-phase conformational dynamics of this IDP are poorly understood. It has been reported that stathmin is purely monomeric in solution and that it bears a short helical region of persistent foldedness, which may act to nucleate helical folding in the C-terminal direction. Here we report a comprehensive study of the structural equilibria local to this region in stathmin that contradicts these two claims. Using the technique of electron paramagnetic resonance (EPR) spectroscopy on spin-labeled stathmin mutants in the solution-phase and when immobilized on Sepharose solid support, we show that all sites in the helical nucleation region of stathmin exhibit multiple spectral components that correspond to dynamic states of differing mobilities and stabilities. Importantly, a state with relatively low mobility dominates each spectrum with an average population greater than 50%, which we suggest corresponds to an oligomerized state of the protein. This is in contrast to a less populated, more mobile state, which likely represents a helically folded monomeric state of stathmin, and a highly mobile state, which we propose is the random coil conformer of the protein. Our interpretation of the EPR data is confirmed by further characterization of the protein using the techniques of native and SDS PAGE, gel filtration chromatography, and multiangle and dynamic light scattering, all of which show the presence of oligomeric stathmin in solution. Collectively, these data suggest that stathmin exists in a diverse equilibrium of states throughout the purported helical nucleation region and that this IDP exhibits a propensity toward oligomerization.

The nitric oxide synthase (NOS) dimer is stabilized by a Zn(2+) ion coordinated to four symmetry-related Cys residues exactly along the dimer 2-fold axis. Each of the two essential tetrahydrobiopterin (H4B) molecules in the dimer interacts directly with the heme, and each H4B molecule is ~15 Å from the Zn(2+). We have determined the crystal structures of the bovine endothelial NOS dimer oxygenase domain bound to three different pterin analogues, which reveal an intimate structural communication between the H4B and Zn(2+) sites. The binding of one of these compounds, 6-acetyl-2-amino-7,7-dimethyl-7,8-dihydro-4(3H)-pteridinone (1), to the pterin site and Zn(2+) binding are mutually exclusive. Compound 1 both directly and indirectly disrupts hydrogen bonding between key residues in the Zn(2+) binding motif, resulting in destabilization of the dimer and a complete disruption of the Zn(2+) site. Addition of excess Zn(2+) stabilizes the Zn(2+) site at the expense of weakened binding of 1. The unique structural features of 1 that disrupt the dimer interface are extra methyl groups that extend into the dimer interface and force a slight opening of the dimer, thus resulting in disruption of the Zn(2+) site. These results illustrate a very delicate balance of forces and structure at the dimer interface that must be maintained to properly form the Zn(2+), pterin, and substrate binding sites.

Catalytic subunits of the IκB kinase (IKK), IKK1/IKKα, and IKK2/IKKβ function in vivo as dimers in association with the necessary scaffolding subunit NEMO/IKKγ. Recent X-ray crystal structures of IKK2 suggested that dimerization might be mediated by a smaller protein-protein interaction than previously thought. Here, we report that removal of a portion of the scaffold dimerization domain (SDD) of human IKK2 yields a kinase subunit that remains monomeric in solution. Expression in baculovirus-infected Sf9 insect cells and purification of this engineered monomeric human IKK2 enzyme allows for in vitro analysis of its substrate specificity and mechanism of activation. We find that the monomeric enzyme, which contains all of the amino-terminal kinase and ubiquitin-like domains as well as the more proximal portions of the SDD, functions in vitro to direct phosphorylation exclusively to residues S32 and S36 of its IκBα substrate. Thus, the NF-κB-inducing potential of IKK2 is preserved in the engineered monomer. Furthermore, we observe that our engineered IKK2 monomer readily autophosphorylates activation loop serines 177 and 181 in trans. However, when residues that were previously observed to interfere with IKK2 trans autophosphorylation in transfected cells are mutated within the context of the monomer, the resulting Sf9 cell expressed and purified proteins were significantly impaired in their trans autophosphorylation activity in vitro. This study further defines the determinants of substrate specificity and provides additional evidence in support of a model in which activation via trans autophosphorylation of activation loop serines in IKK2 requires transient assembly of higher-order oligomers.

The histidine kinase, CheA, couples environmental stimuli to changes in bacterial swimming behavior, converting a sensory signal to a chemical signal in the cytosol via autophosphorylation. The kinase activity is regulated in the platform of chemotaxis signaling complexes formed by CheW, chemoreceptors, and the regulatory domain of CheA. Our previous computational and mutational studies have revealed that two interdomain linkers play important roles in CheA's enzymatic activity. Of the two linkers, one that connects the dimerization and ATP binding domains is essential for both basal autophosphorylation and activation of the kinase. However, the mechanistic role of this linker remains unclear, given that it is far from the autophosphorylation reaction center (the ATP binding site). Here we investigate how this interdomain linker is coupled to CheA's enzymatic activity. Using modern nuclear magnetic resonance (NMR) techniques, we find that by interacting with the catalytic domain, the interdomain linker initiates long-range structural and dynamic changes directed toward the catalytic center of the autophosphorylation reaction. Subsequent biochemical assays define the functional relevance of these NMR-based observations. These findings extend our understanding of the chemotaxis signal transduction pathway.

N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac, the most common form of sialic acid) to form pyruvate and N-acetyl-d-mannosamine. Although equilibrium favors sialic acid cleavage, these enzymes can be used for high-yield chemoenzymatic synthesis of structurally diverse sialic acids in the presence of excess pyruvate. Engineering these enzymes to synthesize structurally modified natural sialic acids and their non-natural derivatives holds promise in creating novel therapeutic agents. Atomic-resolution structures of these enzymes will greatly assist in guiding mutagenic and modeling studies to engineer enzymes with altered substrate specificity. We report here the crystal structures of wild-type Pasteurella multocida N-acetylneuraminate lyase and its K164A mutant. Like other bacterial lyases, it assembles into a homotetramer with each monomer folding into a classic (β/α)₈ TIM barrel. Two wild-type structures were determined, in the absence of substrates, and trapped in a Schiff base intermediate between Lys164 and pyruvate, respectively. Three structures of the K164A variant were determined: one in the absence of substrates and two binary complexes with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Both sialic acids bind to the active site in the open-chain ketone form of the monosaccharide. The structures reveal that every hydroxyl group of the linear sugars makes hydrogen bond interactions with the enzyme, and the residues that determine specificity were identified. Additionally, the structures provide some clues for explaining the natural discrimination of sialic acid substrates between the P. multocida and Escherichia coli NALs.

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca²⁺-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca²⁺-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron-electron resonance (DEER) experiments on recoverin that probe Ca²⁺-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 ± 0.1 nm for Ca²⁺-free recoverin and 3.7 ± 0.1 nm for Ca²⁺-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca²⁺-bound recoverin may represent an intermolecular distance between C39 and N120. ¹⁵N NMR relaxation analysis and CW-EPR experiments both confirm that Ca²⁺-bound recoverin forms a dimer at protein concentrations above 100 μM, whereas Ca²⁺-free recoverin is monomeric. We propose that Ca²⁺-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca²⁺-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca²⁺-induced dimerization is functionally important but not well understood.

In this study, we investigated the trimerization mechanism and structure of heat shock factor 1 (HSF1) using western blotting, tryptophan (Trp) fluorescence spectroscopy, and molecular modeling. First, we examined the DNA-binding domains of human ( Homo sapiens ), goldfish ( Carassius auratus ), and walleye pollock ( Gadus chalcogrammus ) HSF1s by mutating key residues (36 and 103) that are thought to directly affect trimer formation. Human, goldfish, and walleye pollock HSF1s contain cysteine at residue 36 but cysteine (C), tyrosine (Y), and phenylalanine (F), respectively, at residue 103. The optimal trimerization temperatures for the wild-type HSF1s of each species were found to be 42, 37, and 20 °C, respectively. Interestingly, a mutation experiment revealed that trimerization occurred at 42 °C when residue 103 was cysteine, at 37 °C when it was tyrosine, and at 20 °C when it was phenylalanine, regardless of the species. In addition, it was confirmed that when residue 103 of the three species was mutated to alanine, trimerization did not occur. This suggests that in addition to trimerization via disulfide bond formation between the cysteine residues in human HSF1, trimerization can also occur via the formation of a different type of bond between cysteine and aromatic ring residues such as tyrosine and phenylalanine. We also confirmed that at least one cysteine is required for the trimerization of HSF1s, regardless of its position (residue 36 or 103). Additionally, it was shown that the trimer formation temperature is related to growth and survival in fish.

Cyanovirin-N (CV-N) binds high-mannose oligosaccharides on enveloped viruses with two carbohydrate-binding sites, one bearing high affinity and one low affinity to Manα(1-2)Man moieties. A tandem repeat of two CV-N molecules (CVN2) was tested for antiviral activity against human immunodeficiency virus type I (HIV-1) by using a domain-swapped dimer. CV-N was shown to bind N -acetylmannosamine (ManNAc) and N -acetyl-d-glucosamine (GlcNAc) when the carbohydrate-binding sites in CV-N were free to interact with these monosaccharides independently. CVN2 recognized ManNAc at a K d of 1.4 μM and bound this sugar in solution, regardless of the lectin making amino acid side chain contacts on the targeted viral glycoproteins. An interdomain cross-contacting residue Glu41, which has been shown to be hydrogen bonding with dimannose, was substituted in the monomeric CV-N. The amide derivative of glucose, GlcNAc, achieved similar high affinity to the new variant CVN-E41T as high-mannose N -glycans, but binding to CVN2 in the nanomolar range with four binding sites involved or binding to the monomeric CVN-E41A. A stable dimer was engineered and expressed from the alanine-to-threonine-substituted monomer to confirm binding to GlcNAc. In summary, low-affinity binding was achieved by CVN2 to dimannosylated peptide or GlcNAc with two carbohydrate-binding sites of differing affinities, mimicking biological interactions with the respective N -linked glycans of interest and cross-linking of carbohydrates on human T cells for lymphocyte activation.

Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5 R22A ) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5 WT ). Here, we present ITC, NMR, and functional analysis of GCAP5 R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5 R22A binds a total of 3 Ca 2+ , including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5 WT were not detected by ITC, suggesting that R22A may affect the binding of Ca 2+ to these sites. The NMR-derived structure of GCAP5 R22A is overall similar to that of GCAP5 WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5 R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5 R22A with ∼25% higher affinity compared to GCAP5 WT , consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.

Aldehyde dehydrogenase enzymes (ALDHs) are widely studied for their roles in disease propagation and cell metabolism. Their use in biocatalysis applications, for the conversion of aldehydes to carboxylic acids, has also been recognized. Understanding the structural features and functions of both prokaryotic and eukaryotic ALDHs is key to uncovering novel applications of the enzyme and probing its role in disease propagation. The thermostable enzyme ALDH Tt originating from Thermus thermophilus , strain HB27, possesses a unique extension of its C-terminus, which has been evolutionarily excluded from mesophilic counterparts and other thermophilic enzymes in the same genus. In this work, the thermophilic adaptation is studied by the expression and optimized purification of mutant ALDH Tt- 508, with a 22-amino acid truncation of the C-terminus. The mutant shows increased activity throughout production compared to native ALDH Tt , indicating an opening of the active site upon C-terminus truncation and giving rationale into the evolutionary exclusion of the C-terminal extension from similar thermophilic and mesophilic ALDH proteins. Additionally, the C-terminus is shown to play a role in controlling substrate specificity of native ALDH, particularly in excluding catalysis of certain large and certain aromatic ortho-substituted aldehydes, as well as modulating the protein's pH tolerance by increasing surface charge. Dynamic light scattering and size-exclusion HPLC methods are used to show the role of the C-terminus in ALDH Tt oligomeric stability at the cost of catalytic efficiency. Studying the aggregation rate of ALDH Tt with and without a C-terminal extension leads to the conclusion that ALDH Tt follows a monomolecular reaction aggregation mechanism.

HdeA and HdeB are dimeric ATP-independent acid-stress chaperones, which protect the periplasmic proteins of enteric bacteria at pH 2.0 and 4.0, respectively, during their passage through the acidic environment of the mammalian stomach. Despite being structurally similar, they exhibit distinct functional pH optima and conformational prerequisite for their chaperone action. HdeA undergoes a dimer-to-monomer transition at pH 2.0, whereas HdeB remains dimeric at pH 4.0. The monomerization of HdeA exposes its hydrophobic motifs, which facilitates its interaction with the partially folded substrates. How HdeB functions despite maintaining its dimeric conformation has been poorly elucidated in the literature. Herein, we characterized the conformational states and stability of HdeB at its physiologically relevant pH and compared the data with those of HdeA. At pH 4.0, HdeB exhibited distinct spectroscopic signatures and higher stability against heat and guanidine-HCl-induced denaturation than at pH 7.5. We affirm that the pH 4.0 conformer of HdeB was distinct from that at pH 7.5 and that these two conformational states were hierarchically unrelated. Salt-bridge mutations that perturbed HdeB's intersubunit interactions resulted in the loss of its stability and function at pH 4.0. In contrast, mutations affecting intrasubunit interactions enhanced its function, albeit with a reduction in stability. These findings suggest that, unlike HdeA, HdeB acts as a noncanonical chaperone, where pH-dependent stability and conformational rearrangement at pH 4.0 play a core role in its chaperone function rather than its surface hydrophobicity. Such rearrangement establishes a stability-function trade-off that allows HdeB to function while maintaining its stable dimeric state.

Enzyme polymerization (also known as filamentation) has emerged as a new layer of enzyme regulation. SgrAI is a sequence-dependent DNA endonuclease that forms polymeric filaments with enhanced DNA cleavage activity as well as altered DNA sequence specificity. To better understand this unusual regulatory mechanism, full global kinetic modeling of the reaction pathway, including the enzyme filamentation steps, has been undertaken. Prior work with the primary DNA recognition sequence cleaved by SgrAI has shown how the kinetic rate constants of each reaction step are tuned to maximize activation and DNA cleavage while minimizing the extent of DNA cleavage to the host genome. In the current work, we expand on our prior study by now including DNA cleavage of a secondary recognition sequence, to understand how the sequence of the bound DNA modulates filamentation and activation of SgrAI. The work shows that an allosteric equilibrium between low and high activity states is modulated by the sequence of bound DNA, with primary sequences more prone to activation and filament formation, while SgrAI bound to secondary recognition sequences favor the low (and nonfilamenting) state by up to 40-fold. In addition, the degree of methylation of secondary sequences in the host organism, Streptomyces griseus , is now reported for the first time and shows that as predicted, these sequences are left unprotected from the SgrAI endonuclease making sequence specificity critical in this unusual filament-forming enzyme.

The most frequent ERK2 (MAPK1) mutation in cancers, E322K, lies in the common docking (CD) site, which binds short motifs made up of basic and hydrophobic residues present in the activators MEK1 (MAP2K1) and MEK2 (MAP2K2), in dual specificity phosphatases (DUSPs) that inactivate the kinases, and in many of their substrates. Also, part of the CD site, but mutated less often in cancers, is the preceding aspartate (D321N). These mutants were categorized as gain of function in a sensitized melanoma system. In Drosophila developmental assays, we found that the aspartate but not the glutamate mutant caused gain-of-function phenotypes. Here, we catalogued additional properties of these mutants to accrue greater insight into their functions. A modest increase in nuclear retention of E322K was noted. Binding of ERK2 E322K and D321N to a small group of substrates and regulatory proteins was similar, in spite of differences in CD site integrity. Interactions with a second docking site, the F site, which should be more accessible in E322K, were modestly reduced rather than increased. The crystal structure of ERK2 E322K also indicated a disturbed dimer interface, and reduced dimerization was detected by a two-hybrid test; yet, it was detected in dimers in EGF-treated cells, although to a lesser extent than D321N or wt ERK2. These findings indicate a range of small differences in behaviors that may contribute to increased function of E322K in certain cancers.