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ATP and GTP are exceptionally important molecules in biology with multiple, and often discrete, functions. Therefore, enzymes that bind to either of them must develop robust mechanisms to selectively utilize one or the other. Here, this specific problem is addressed by molecular studies of the human NMP kinase AK3 which uses GTP to phosphorylate AMP. AK3 plays an important role in the citric acid cycle where it is responsible for GTP/GDP recycling. By combining a structural biology approach with functional experiments, we present a comprehensive structural and mechanistic understanding of the enzyme. We discovered that AK3 functions by recruitment of GTP to the active site, while ATP is rejected and non-productively bound to the AMP binding site. Consequently, ATP acts as an inhibitor with respect to GTP and AMP. The overall features with specific recognition of the correct substrate and non-productive binding by the incorrect substrate bears strong similarity to previous findings for the ATP specific NMP kinase adenylate kinase. Taken together we are now able to provide the fundamental principles for GTP and ATP selectivity in the large NMP kinase family. As a side-result originating from non-linearity of chemical shifts in GTP and ATP titrations, we find that protein surfaces offer a general and weak binding affinity for both GTP and ATP. These non-specific interactions likely act to lower the available intra-cellular GTP and ATP concentrations and may have driven evolution to adapt the Michaelis constants of NMP kinases accordingly.

DNA polymerase epsilon (Pol epsilon), the major leading-strand DNA polymerase in eukaryotes, has a catalytic subunit (Pol2) and three non-catalytic subunits. The N-terminal half of Pol2 (Pol2(CORE)) exhibits both polymerase and exonuclease activity. It has been suggested that both the non-catalytic C-terminal domain of Pol2 (with the two cysteine motifs CysA and CysB) and Pol2(CORE) (with the CysX cysteine motif) are likely to coordinate an Fe-S cluster. Here, we present two new crystal structures of Pol2(CORE) with an Fe-S cluster bound to the CysX motif, supported by an anomalous signal at that position. Furthermore we show that purified four-subunit Pol epsilon, Pol epsilon CysA(MUT) (C2111S/C2133S), and Pol epsilon CysB(MUT) (C2167S/C2181S) all have an Fe-S cluster that is not present in Pol epsilon CysX(MUT) (C665S/C668S). Pol epsilon CysA(MUT) and Pol epsilon CysB(MUT) behave similarly to wildtype Pol epsilon in in vitro assays, but Pol epsilon CysX(MUT) has severely compromised DNA polymerase activity that is not the result of an excessive exonuclease activity. Tetrad analyses show that haploid yeast strains carrying CysX(MUT) are inviable. In conclusion, Pol epsilon has a single Fe-S cluster bound at the base of the P-domain, and this Fe-S cluster is essential for cell viability and polymerase activity.

Listeria monocytogenes is a bacterial pathogen that controls much of its virulence through the transcriptional regulator PrfA. In this study, we describe structure-guided design and synthesis of a set of PrfA inhibitors based on ring-fused 2-pyridone heterocycles. Our most effective compound decreased virulence factor expression, reduced bacterial uptake into eukaryotic cells, and improved survival of chicken embryos infected with L. monocytogenes compared to previously identified compounds. Crystal structures identified an intraprotein “tunnel” as the main inhibitor binding site (AI), where the compounds participate in an extensive hydrophobic network that restricts the protein’s ability to form functional DNA-binding helix–turn–helix (HTH) motifs. Our studies also revealed a hitherto unsuspected structural plasticity of the HTH motif. In conclusion, we have designed 2-pyridone analogues that function as site-AI selective PrfA inhibitors with potent antivirulence properties.

Summary The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes cellular uptake by reducing the expression of virulence genes. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA-binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds at two separate sites on the protein: one within a hydrophobic pocket or tunnel, located between the C- and N-terminal domains of PrfA, and the second in the vicinity of the DNA-binding helix-turn-helix motif. At both sites the compound interacts with residues important for PrfA activation and helix-turn-helix formation. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes., Graphical Abstract, Highlights • Inhibitors of L. monocytogenes infectivity reduce virulence gene expression • Binding of inhibitor to the PrfA regulator reduces affinity for its DNA motif • First crystal structure of a Crp family regulator with an inhibitor • Provides rationale for screening with Crp family transcriptional regulators, In this study, Good et al. identify ring-fused 2-pyridones which attenuate Listeria monocytogenes infectivity and reduce the expression of key virulence genes by binding to the central virulence regulator PrfA.

The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N-terminal domain (residues 23-103) that contains two short α-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.

Thyroid-disrupting chemicals (TDCs) are xenobiotics that can interfere with the endocrine system and cause adverse effects in organisms and their offspring. TDCs affect both the thyroid gland and regulatory enzymes associated with thyroid hormone homeostasis. Transthyretin (TTR) is found in the serum and cerebrospinal fluid of vertebrates, where it transports thyroid hormones. Here, we explored the interspecies variation in TDC binding to human and fish TTR (exemplified by Gilthead seabream ( Sparus aurata)). The in vitro binding experiments showed that TDCs bind with equal or weaker affinity to seabream TTR than to the human TTR, in particular, the polar TDCs (500-fold lower affinity). Crystal structures of the seabream TTR-TDC complexes revealed that all TDCs bound at the thyroid binding sites. However, amino acid substitution of Ser117 in human TTR to Thr117 in seabream prevented polar TDCs from binding deep in the hormone binding cavity, which explains their low affinity to seabream TTR. Molecular dynamics and in silico alanine scanning simulation also suggested that the protein backbone of seabream TTR is more rigid than the human one and that Thr117 provides fewer electrostatic contributions than Ser117 to ligand binding. This provides an explanation for the weaker affinities of the ligands that rely on electrostatic interactions with Thr117. The lower affinities of TDCs to fish TTR, in particular the polar ones, could potentially lead to milder thyroid-related effects in fish.

Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the rate-limiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-Å X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic sidechains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change. published

The metalloprotease PrtV from Vibrio cholerae serves an important function for the bacteria’s ability to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102kDa) multidomain pre-pro-protein that so far has only been expressed in V. cholerae. Structural studies require high amounts of soluble protein with high purity. Previous attempts for recombinant expression have been hampered by low expression and solubility of protein fragments. Here, we describe results from parallel cloning experiments in Escherichia coli where fusion tagged constructs of PrtV fragments were designed, and protein products tested for expression and solubility. Of more than 100 designed constructs, three produced protein products that expressed well. These include the N-terminal domain (residues 23–103), the PKD1 domain (residues 755–839), and a 25kDa fragment (residues 581–839). The soluble fusion proteins were captured with Ni2+ affinity chromatography, and subsequently cleaved with tobacco etch virus protease. Purification protocols yielded ∼10–15mg of pure protein from 1L of culture. Proper folding of the shorter domains was confirmed by heteronuclear NMR spectra recorded on 15N-labeled samples. A modified protocol for the native purification of the secreted 81kDa pro-protein of PrtV is provided. Proteolytic activity measurements suggest that the 37kDa catalytic metalloprotease domain alone is sufficient for activity.

Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfAWT in complex only with DNA confirms that PrfAWT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.

Vibrio cholerae, the causative agent of cholera, releases several virulence factors including secreted proteases when it infects its host. These factors attack host cell proteins and break down tissue barriers and cellular matrix components such as collagen, laminin, fibronectin, keratin, elastin, and they induce necrotic tissue damage. The secreted protease PrtV constitutes one virulence factors of V. cholerae. It is a metalloprotease belonging to the M6 peptidase family. The protein is expressed as an inactive, multidomain, 102 kDa pre-pro-protein that undergoes several N- and C-terminal modifications after which it is secreted as an intermediate variant of 81 kDa. After secretion from the bacteria, additional proteolytic steps occur to produce the 55 kDa active M6 metalloprotease. The domain arrangement of PrtV is likely to play an important role in these maturation steps, which are known to be regulated by calcium. However, the molecular mechanism by which calcium controls proteolysis is unknown. In this study, we report the atomic resolution crystal structure of the PKD1 domain from V. cholera PrtV (residues 755–838) determined at 1.1 Å. The structure reveals a previously uncharacterized Ca2+-binding site located near linker regions between domains. Conformational changes in the Ca2+-free and Ca2+-bound forms suggest that Ca2+-binding at the PKD1 domain controls domain linker flexibility, and plays an important structural role, providing stability to the PrtV protein., Highlights • The PKD1 domain was expressed in E. coli and purified to homogeneity. • Purified PKD1 domains are not toxic for human HTC8 cells. • The atomic 1.1 Å crystal structure of the PKD1 domain revealed a Ca2+-binding site. • Ca2+ binding causes large conformational changes in the N-terminal half of the PKD1 domain. • Ca2+ stabilizes the 81 kDa pro-protein outside the bacterial cell, preventing its degradation.

Amyloid formation of the plasma protein transthyretin (TTR) has been linked to familial amyloid polyneuropathy and senile systemic amyloidosis. Binding of ligands within its natural hormone binding site can stabilize the tetrameric structure and impair amyloid formation. We have recently shown that the flavonoid luteolin stabilizes TTR in human plasma with a very high selectivity. Luteolin, however, is inactivated in vivo via glucuronidation for which the preferred site is the hydroxy group at position 7 on its aromatic A-ring. We have evaluated the properties of two luteolin variants in which the 7-hydroxy group has been exchanged for a chlorine (7-Cl-Lut) or a methoxy group (7-MeO-Lut). Using an in vitro model, based on human liver microsomes, we verified that these modifications increase the persistence of the drug. Crystal structure determinations show that 7-Cl-Lut binds similarly to luteolin. The larger MeO substituent cannot be accommodated within the same space as the chlorine or hydroxy group and as a result 7-MeO-Lut binds in the opposite direction with the methoxy group in position 7 facing the solvent. Both 7-Cl-Lut and 7-MeO-Lut qualify as high-affinity binders, but in contrast to luteolin, they display a highly non-specific binding to other plasma components. The binding of the two conformations and the key-interactions to TTR are discussed in detail. Taken together, these results show a proof-of-concept that the persistence of luteolin towards enzymatic modification can be increased. We reveal two alternative high-affinity binding modes of luteolin to TTR and that modification in position 7 is restricted only to small substituents if the original orientation of luteolin should be preserved. In addition, the present work provides a general and convenient method to evaluate the efficacy of TTR-stabilizing drugs under conditions similar to an in vivo environment.

Thyroid disruption by xenobiotics is associated with a broad spectrum of severe adverse outcomes. One possible molecular target of thyroid hormone disrupting chemicals (THDCs) is transthyretin (TTR), a thyroid hormone transporter in vertebrates. To better understand the interactions between TTR and THDCs, we determined the crystallographic structures of human TTR in complex with perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and 2,2',4,4'-tetrahydroxybenzophenone (BP2). The molecular interactions between the ligands and TTR were further characterized using molecular dynamics simulations. A structure-based virtual screening (VS) protocol was developed with the intention of providing an efficient tool for the discovery of novel TTR-binders from the Tox21 inventory. Among the 192 predicted binders, 12 representatives were selected, and their TTR binding affinities were studied with isothermal titration calorimetry, of which seven compounds had binding affinities between 0.26 and 100 mu M. To elucidate structural details in their binding to TTR, crystal structures were determined of TTR in complex with four of the identified compounds including 2,6-dinitro-p-cresol, bisphenol S, clonixin, and triclopyr. The compounds were found to bind in the TTR hormone binding sites as predicted. Our results show that the developed VS protocol is able to successfully identify potential THDCs, and we suggest that it can be used to propose THDCs for future toxicological evaluations.

The biological role of human DNA polymerase θ (POLQ) is not yet clearly defined, but it has been proposed to participate in several cellular processes based on its translesion synthesis capabilities. POLQ is a low-fidelity polymerase capable of efficient bypass of blocking lesions such as abasic sites and thymine glycols as well as extension of mismatched primer termini. Here, we show that POLQ possesses a DNA polymerase activity that appears to be template independent and allows efficient extension of single-stranded DNA as well as duplex DNA with either protruding or multiply mismatched 3'-OH termini. We hypothesize that this DNA synthesis activity is related to the proposed role for POLQ in the repair or tolerance of double-strand breaks.

In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed in order to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involv ...

Alzheimer's disease, a neurodegenerative disorder causing synaptic impairment and neuronal cell death, is strongly correlated with aggregation of the amyloid-beta peptide (Abeta). Divalent metal ions such as Cu(2+) and Zn(2+) are known to significantly affect the rate of aggregation and morphology of Abeta assemblies in vitro and are also found at elevated levels within cerebral plaques in vivo. The present investigation characterized the architecture of the aggregated forms of Abeta(1-40) and Abeta(1-42) in the presence or absence of either Cu(2+) or Zn(2+) using quenched hydrogen/deuterium exchange combined with solution NMR spectroscopy. The NMR analyses provide a quantitative and residue-specific structural characterization of metal-induced Abeta aggregates, showing that both the peptide sequence and the type of metal ion exert an impact on the final architecture. Common features among the metal-complexed peptide aggregates are two solvent-protected regions with an intervening minimum centered at Asn27, and a solvent-accessible N-terminal region, Asp1-Lys16. Our results suggest that Abeta in complex with either Cu(2+) or Zn(2+) can attain an aggregation-prone beta-strand-turn-beta-strand motif, similar to the motif found in fibrils, but where the metal binding to the N-terminal region guides the peptide into an assembly distinctly different from the fibril form.

Human transthyretin (hTTR) is one of several proteins known to cause amyloid disease. Conformational changes in its native structure result in aggregation of the protein, leading to insoluble amylo ...

Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd=10.6+/-1.7nM) and T4 (Kd=9.8+/-0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3+/-15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.

The signal-recognition particle (SRP) is a ubiquitous protein–RNA complex that targets proteins to cellular membranes for insertion or secretion. A key player in SRP-mediated protein targeting is the evolutionarily conserved core consisting of the SRP RNA and the multidomain protein SRP54. Communication between the SRP54 domains is critical for SRP function, where signal sequence binding at the M domain directs receptor binding at the GTPase domain (NG domain). These SRP activities are linked to domain rearrangements, for which the role of SRP RNA is not clear. In free SRP, a direct interaction of the GTPase domain with SRP RNA has been proposed but has never been structurally verified. In this study, we present the crystal structure at 2.5-Å resolution of the SRP54–SRP19–SRP RNA complex of Methanococcus jannaschii SRP. The structure reveals an RNA-bound conformation of the SRP54 GTPase domain, in which the domain is spatially well separated from the signal peptide binding site. The association of both the N and G domains with SRP RNA in free SRP provides further structural evidence for the pivotal role of SRP RNA in the regulation of the SRP54 activity.

The use of high temperatures in the purification procedures of heat-stable proteins is a well established technique. Recently, rapid pre-heat treatment of protein samples prior to crystallization trials was described as a final polishing step to improve the diffraction properties of crystals [Pusey et al. (2005), Prog. Biophys. Mol. Biol. 88, 359-386]. The present study demonstrates that extended high-temperature incubation (328 K for 48 h) of the highly amyloidogenic transthyretin mutant TTR G53S/E54D/L55S successfully removes heterogeneities and allows the reproducible growth of well diffracting crystals. Heat treatment might be applied as an optimization method to other cases in which the protein/biomolecule fails to form diffracting crystals.

The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre‐pro‐protein that undergoes several N‐ and C‐terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N‐terminal domain (residues 23–103) that contains two short α‐helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C‐terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important., Interactive Figure 1 | PDB Code(s): 5abk

An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions., Adenylate kinase (AdK) plays a key role in cellular energy homeostasis by catalysing the reversible magnesium-dependent formation of ADP from AMP and ATP. Here the authors present a detailed analysis of adenylate kinase's conformational dynamics and characterize a high-energy state of AdK indispensable for catalysis.

In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO2 and water into carbohydrates. The light-driven oxidation of water that provides the electrons and protons for the subsequent CO2 fixation takes place in photosystem II (PSII). Recent studies show that in higher plants, HCO3 – increases PSII activity by acting as a mobile acceptor of the protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested to improve proton removal from PSII, possibly by rapid reformation of HCO3 – from CO2. In this study, we investigated the interplay between PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometry measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and 2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in α-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 function with dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO2/HCO3 – on PSII activity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSII preparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at low pH and CO2 concentration., This work was supported by Solar Fuels at Umeå University, the Artificial Leaf Project Umeå (Knut and Alice Wallenberg’s Foundation), the Kempe Foundation, the Swedish Research Council (to J.M., G.S., and A.E.S.-E.), Energimyndigheten (to J.M.), and Stiftelsen Olle Engkvist Byggmästare (to A.E.S.-E.).

The transthyretin-related protein (TRP) family comprises proteins predicted to be structurally related to the homotetrameric transport protein transthyretin (TTR). The function of TRPs is not yet fully established, but recent data suggest that they are involved in purine catabolism. We have determined the three-dimensional structure of the Escherichia coli TRP in two crystal forms; one at 1.65 A resolution in the presence of zinc, and the other at 2.1 A resolution in the presence of zinc and bromide. The structures revealed five zinc-ion-binding sites per monomer. Of these, the zinc ions bound at sites I and II are coordinated in tetrahedral geometries to the side chains of residues His9, His96, His98, Ser114, and three water molecules at the putative ligand-binding site. Of these four residues, His9, His98, and Ser114 are conserved. His9 and His98 bind the central zinc (site I) together with two water molecules. The side chain of His98 also binds to the zinc ion at site II. Bromide ions bind at site I only, replacing one of the water molecules coordinated to the zinc ion. The C-terminal four amino acid sequence motif Y-[RK]-G-[ST] constitutes the signature sequence of the TRP family. Two Tyr111 residues form direct hydrogen bonds to each other over the tetramer interface at the area, which in TTR constitutes the rear part of its thyroxine-binding channel. The putative substrate/ligand-binding channel of TRP is consequently shallower and broader than its counterpart in TTR.

Alzheimer disease is a neurodegenerative disorder that is tightly linked to the self-assembly and amyloid formation of the 39-43-residue-long amyloid-beta (Abeta) peptide. Considerable evidence suggests a correlation between Alzheimer disease development and the longer variants of the peptide, Abeta-(1-42/43). Currently, a molecular understanding for this behavior is lacking. In the present study, we have investigated the hydrogen/deuterium exchange of Abeta-(1-42) fibrils under physiological conditions, using solution NMR spectroscopy. The obtained residue-specific and quantitative map of the solvent protection within the Abeta-(1-42) fibril shows that there are two protected core regions, Glu11-Gly25 and Lys28-Ala42, and that the residues in between, Ser26 and Asn27, as well as those in the N terminus, Asp1-Tyr10, are solvent-accessible. This result reveals considerable discrepancies when compared with a previous investigation on Abeta-(1-40) fibrils and suggests that the additional residues in Abeta-(1-42), Ile41 and Ala42, significantly increase the solvent protection and stability of the C-terminal region Lys28-Ala42. Consequently, our findings provide a molecular explanation for the increased amyloidogenicity and toxicity of Abeta-(1-42) compared with shorter Abeta variants found in vivo.

The Tyr114Cys substitution in the human plasma protein transthyretin leads to a particularly aggressive form of familial amyloidotic polyneuropathy. In a previous study we demonstrated that ATTR Tyr114Cys forms intermolecular disulfide bonds, which partly impair fibril formation and result in a more amorphous morphology. Apart from the introduced cysteinyl group in position 114, the native sequence contains one cysteine located at position 10. To deduce the role of intermolecular disulfide bridging in fibril formation we generated and characterized the TTR Cys10Ala/Tyr114Cys double mutant. Our results suggest that an intermolecular cysteine bridge at position 114 enhances the exposure of cysteine 10, thereby facilitating additional intermolecular cysteine assemblies. We also purified a disulfide-linked dimeric form of TTR Cys10Ala/Tyr114Cys, which was recognized by the anti-TTR amyloid-specific monoclonal antibody MAb (39-44). Moreover, this dimeric molecule can form protofibrils indistinguishable from the fibrils formed under reducing conditions, as judged by atomic force microscopy. Assuming that both molecules of the dimer are part of the core of the fibril, the assembly is incompatible with a preserved native or near-native dimeric interphase. Our findings raise the question of whether TTR-amyloid architecture is indeed the result of one highly stringent assembly of structures or if different fibrils may be built from different underlying structures.

Transthyretin amyloid formation occurs through a process of tetramer destabilization and partial unfolding. Small molecules, including the natural ligand thyroxine, stabilize the tetrameric form of the protein, and serve as inhibitors of amyloid formation. Crucial for TTR's ligand-binding properties are its three halogen-binding sites situated at the hormone-binding channel. In this study, we have performed a structural characterization of the binding of two halides, iodide and chloride, to TTR. Chlorides are known to shield charge repulsions at the tetrameric interface of TTR, which improve tetramer stability of the protein. Our study shows that iodides, like chlorides, provide tetramer stabilization in a concentration-dependent manner and at concentrations approximately 15-fold below that of chlorides. To elucidate binding sites of the halides, we took advantage of the anomalous scattering of iodide and used the single-wavelength anomalous dispersion (SAD) method to solve the iodide-bound TTR structure at 1.8 A resolution. The structure of chloride-bound TTR was determined at 1.9 A resolution using difference Fourier techniques. The refined structures showed iodides and chlorides bound at two of the three halogen-binding sites located at the hydrophobic channel. These sites therefore also function as halide-binding sites.

Proper assembly of large protein–RNA complexes requires sequential binding of the proteins to the RNA. The signal recognition particle (SRP) is a multiprotein–RNA complex responsible for the cotranslational targeting of proteins to biological membranes. Here we describe the crystal structure at 2.6-Å resolution of the S-domain of SRP RNA from the archeon Methanococcus jannaschii. Comparison of this structure with the SRP19-bound form reveals the nature of the SRP19-induced conformational changes, which promote subsequent SRP54 attachment. These structural changes are initiated at the SRP19 binding site and transmitted through helix 6 to looped-out adenosines, which form tertiary RNA interaction with helix 8. Displacement of these adenosines enforces a conformational change of the asymmetric loop structure in helix 8. In free RNA, the three unpaired bases A195, C196, and C197 are directed toward the helical axis, whereas upon SRP19 binding the loop backbone inverts and the bases are splayed out in a conformation that resembles the SRP54-bound form. Nucleotides adjacent to the bulged nucleotides seem to be particularly important in the regulation of this loop transition. Binding of SRP19 to 7S RNA reveals an elegant mechanism of how protein-induced changes are directed through an RNA molecule and may relate to those regulating the assembly of other RNPs.

Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3 -triiodo- L-thyronine (T3) and 3,5,3 ,5 -tetraiodo-L-thyronine (thyroxine, T4). Human TTR has higher affinity for T4 than T3, whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 Å resolution and bound to ligands T3 and T4, both at 1.9 Å resolution. The apo structure is similar to human TTR with structural changes only at -strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR T4 complex shows the T4-binding site to be similar but not identical to human TTR, whereas the TTR T3 complex shows the I3 halogen situated at the site normally occupied by the hydroxyl group of T4. The significantly wider entrance of the hormone- binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T3 and T4. We thank Anders Olofsson, Uwe H. Sauer, Andreas Ho¨rnberg, and Terese Bergfors for valuable discussions and critical reading of the manuscript.

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that associates with ribosomes to mediate the targeting of membrane and secretory proteins to biological membranes. In higher eukaryotes, SRP biogenesis involves the sequential binding of SRP19 and SRP54 proteins to the S domain of 7S RNA. The recently determined crystal structures of SRP19 in complex with the S domain, and that of the ternary complex of SRP19, the S domain and the M domain of SRP54, provide insight into the molecular basis of S-domain assembly and SRP function.

A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretin-related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand-binding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used double-stranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity.

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein. It associates with ribosomes to mediate co-translational targeting of membrane and secretory proteins to biological membranes. In mammalian cells, the SRP consists of a 7S RNA and six protein components. The S domain of SRP comprises the 7S.S part of RNA bound to SRP19, SRP54 and the SRP68/72 heterodimer; SRP54 has the main role in recognizing signal sequences of nascent polypeptide chains and docking SRP to its receptor. During assembly of the SRP, binding of SRP19 precedes and promotes the association of SRP54 (refs 4, 5). Here we report the crystal structure at 2.3 A resolution of the complex formed between 7S.S RNA and SRP19 in the archaeon Methanococcus jannaschii. SRP19 bridges the tips of helices 6 and 8 of 7S.S RNA by forming an extensive network of direct protein RNA interactions. Helices 6 and 8 pack side by side; tertiary RNA interactions, which also involve the strictly conserved tetraloop bases, stabilize helix 8 in a conformation competent for SRP54 binding. The structure explains the role of SRP19 and provides a molecular framework for SRP54 binding and SRP assembly in Eukarya and Archaea.

Substitution of Pro for Thr199 in the active site of human carbonic anhydrase II (HCA II)(1) reduces its catalytic efficiency about 3000-fold. X-ray crystallographic structures of the T199P/C206S variant have been determined in complex with the substrate bicarbonate and with the inhibitors thiocyanate and beta-mercaptoethanol. The latter molecule is normally not an inhibitor of wild-type HCA II. All three ligands display novel binding interactions to the T199P/C206S mutant. The beta-mercaptoethanol molecule binds in the active site area with its sulfur atom tetrahedrally coordinated to the zinc ion. Thiocyanate binds tetrahedrally coordinated to the zinc ion in T199P/C206S, in contrast to its pentacoordinated binding to the zinc ion in wild-type HCA II. Bicarbonate binds to the mutant with two of its oxygens at the positions of the zinc water (Wat263) and Wat318 in wild-type HCA II. The environment of this area is more hydrophilic than the normal bicarbonate-binding site of HCA II situated in the hydrophobic part of the cavity normally occupied by the so-called deep water (Wat338). The observation of a new binding site for bicarbonate has implications for understanding the mechanism by which the main-chain amino group of Thr199 acquired an important role for orientation of the substrate during the evolution of the enzyme.

The signal recognition particle (SRP) recognizes and binds the signal sequence of nascent proteins as they emerge from the ribosome. We present here the 3.0-Å structure of a signal sequence bound to the Methanococcus jannaschii SRP core. Structural comparison with the free SRP core shows that signal-sequence binding induces formation of the GM-linker helix and a 180° flip of the NG domain-structural changes that ensure a hierarchical succession of events during protein targeting.

Transthyretin (TTR) is a homotetrameric plasma protein with amyloidogenic properties that has been linked to the development of familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The in vivo role of TTR is associated with transport of thyroxine hormone T4 and retinol-binding protein. Loss of the tetrameric integrity of TTR is a rate-limiting step in the process of TTR amyloid formation, and ligands with the ability to bind within the thyroxin binding site (TBS) can stabilize the tetramer, a feature that is currently used as a therapeutic approach for FAP. Several different flavonoids have recently been identified that impair amyloid formation. The flavonoid luteolin shows therapeutic potential with low incidence of unwanted side effects. In this work, we show that luteolin effectively attenuates the cytotoxic response to TTR in cultured neuronal cells and rescues the phenotype of a Drosophila melanogaster model of FAP. The plant-derived luteolin analogue cynaroside has a glucoside group in position 7 of the flavone A-ring and as opposed to luteolin is unable to stabilize TTR tetramers and thus prevents a cytotoxic effect. We generated high-resolution crystal-structures of both TTR wild type and the amyloidogenic mutant V30M in complex with luteolin. The results show that the A-ring of luteolin, in contrast to what was previously suggested, is buried within the TBS, consequently explaining the lack of activity from cynaroside. The flavonoids represent an interesting group of drug candidates for TTR amyloidosis. The present investigation shows the potential of luteolin as a stabilizer of TTR in vivo. We also show an alternative orientation of luteolin within the TBS which could represent a general mode of binding of flavonoids to TTR and is of importance concerning the future design of tetramer stabilizing drugs.

Transthyretin is a tetrameric plasma protein associated with two forms of amyloid disease. The structure of the highly amyloidogenic transthyretin triple mutant TTRG53S/E54D/L55S determined at 2.3 A resolution reveals a novel conformation: the beta-slip. A three-residue shift in beta strand D places Leu-58 at the position normally occupied by Leu-55 now mutated to serine. The beta-slip is best defined in two of the four monomers, where it makes new protein-protein interactions to an area normally involved in complex formation with retinol-binding protein. This interaction creates unique packing arrangements, where two protein helices combine to form a double helix in agreement with fiber diffraction and electron microscopy data. Based on these findings, a novel model for transthyretin amyloid formation is presented.

The crystal structure of carbonic anhydrase from Neisseria gonorrhoeae has been solved to a resolution of 1.78 A by molecular replacement using human carbonic anhydrase II as a template. After refinement the R factor was 17.8% (Rfree=23.2%). There are two molecules per asymmetric unit (space group P21), but they have essentially identical structures. The fold of the N. gonorrhoeae enzyme is very similar to that of human isozyme II; 192 residues, 74 of which are identical in the two enzymes, have equivalent positions in the three-dimensional structures. This corresponds to 85% of the entire polypeptide chain of the bacterial enzyme. The only two cysteine residues in the bacterial enzyme, which has a periplasmic location in the cell, are connected by a disulfide bond. Most of the secondary structure elements present in human isozyme II are retained in N. gonorrhoeae carbonic anhydrase, but there are also differences, particularly in the few helical regions. Long deletions in the bacterial enzyme relative to human isozyme II have resulted in a considerable shortening of three surface loops. One of these deletions, corresponding to residues 128 to 139 in the human enzyme, leads to a widening of the entrance to the hydrophobic part of the active site cavity. Practically all the amino acid residues in the active site of human isozyme II are conserved in the N. gonorrhoeae enzyme and have similar structural positions. However, the imidazole ring of a histidine residue, which has been shown to function as a proton shuttle in the catalytic mechanism of the human enzyme, interacts with an extraneous entity, which has tentatively been identified as a 2-mercaptoethanol molecule from the crystallization medium. When this entity is removed by soaking the crystal in a different medium, the side-chain of His66 becomes quite mobile. The structure of a complex with the sulfonamide inhibitor, acetazolamide, has also been determined. Its position in the active site is very similar to that observed in human carbonic anhydrase II.

DNA polymerase ɛ (Pol ɛ) is a high-fidelity polymerase that has been shown to participate in leading-strand synthesis during DNA replication in eukaryotic cells. We present here a ternary structure of the catalytic core of Pol ɛ (142 kDa) from Saccharomyces cerevisiae in complex with DNA and an incoming nucleotide. This structure provides information about the selection of the correct nucleotide and the positions of amino acids that might be critical for proofreading activity. Pol ɛ has the highest fidelity among B-family polymerases despite the absence of an extended β-hairpin loop that is required for high-fidelity replication by other B-family polymerases. Moreover, the catalytic core has a new domain that allows Pol ɛ to encircle the nascent double-stranded DNA. Altogether, the structure provides an explanation for the high processivity and high fidelity of leading-strand DNA synthesis in eukaryotes.

Bacterial virulence systems are attractive targets for development of new antibacterial agents. Yersinia spp. utilize the type III secretion (T3S) system to secrete and translocate Yersinia outer proteins (Yop effectors) into the cytosol of the target cell and thereby overcome host defenses to successfully establish an infection. Thus, the Yop effectors constitute attractive targets for drug development. In the present study we apply small molecule screening to identify inhibitors of one of the secreted proteins YopH, a tyrosine phosphatase required for virulence. Characterization of seven inhibitors indicated that both competitive and noncompetitive inhibitors were identified with IC50 values of 6–20 μM.

The signal recognition particle (SRP) is a protein-RNA complex that associates with ribosomes to mediate co-translational targeting of membrane and secretory proteins to biological membranes. The universally conserved core of SRP consists of SRP RNA and the SRP54 protein, and plays the key role in signal-sequence recognition and binding to the SRP receptor. Critical for SRP function is communication between the two conserved SRP54 domains, the GTPase- and the M-domain, so that signal-sequence binding at the M domain directs receptor binding at the GTPase domain. The structural basis for signal-sequence binding by SRP and subsequent signaling is still poorly understood. By studying the structures of the SRP RNA in its free form as well as in complex with its different protein partners, we have made steady progress towards the elucidation of structural states of the SRP, using the archaeon Methanococcus jannaschii as model system. Together with other structures of SRP proteins and RNA-protein complexes, these structures provide new insights into the mechanisms of SRP-mediated protein targeting.

The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion - a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO(2)-HCO(3) (-) interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.

In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involved in the regulation of fimbriae gene clusters. Recent findings suggest that members from this family of proteins may form homomeric or heteromeric complexes and exert both positive and negative effects on the transcription of fimbriae genes. To elucidate the detailed function of FocB, we have determined its crystal structure at 1.4 A resolution. FocB is an all alpha-helical protein with a helix-turn-helix motif. Interestingly, conserved residues important for DNA-binding are located not in the postulated recognition helix of the motif, but in the preceding helix. Results from protein-DNA-binding studies suggest that FocB interacts with the minor groove of its cognate DNA target, which is indicative of a DNA interaction that is unusual for this motif. FocB crystallizes in the form of dimers. Packing interactions in the crystals give two plausible dimerization interfaces. Conserved residues, known to be important for protein oligomerization, are present at both interfaces, suggesting that both sites could play a role in a functional FocB protein.