Ion channel activity of transmembrane segment 6 of Escherichia coli proton‐dependent manganese transporter

Synthetic peptides corresponding to the sixth transmembrane segment (TMS6) of secondary‐active transporter MntH (Proton‐dependent Manganese Transporter) from Escherichia coli and its two mutations in the functionally important conserved histidine residue were used as a model for structure–function study of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformations in lipid membranes. Electrophysiological experiments demonstrated that TMS6 was able to form multi‐state ion channels in model biological membranes. Electrophysiological properties of these weakly cation‐selective ion channels were strongly dependent on the surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS6 channels, influenced the transition between closed and open states, and affected the peptide conformations. Moreover, functional properties of peptides carrying two different mutations of His²¹¹ were analogous to in vivo functional characteristics of Nramp/MntH proteins mutated at homologous residues. Hence, a single functionally important TMS can retain some of the functional properties of the full‐length protein. These findings could contribute to understanding the structure–function relationship at the molecular level. However it remains unclear to what extent the peptide‐specific channel activity represents a functional aspect of the full‐length membrane carrier protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 718–726, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The effect of the side chain length of Asp and Glu on coordination structure of Cu2+ in a de novo designed protein

Metal ions in proteins are important not only for the formation of the proper structures but also for various biological activities. For biological functions such as hydrolysis and oxidation, metal ions often adopt unusual coordination structures. We constructed a stable scaffold for metal binding to create distorted metal coordination structures. A stable four stranded α‐helical coiled‐coil structure was used as the scaffold, and the metal binding site was in the cavity created at the center of the structure. Two His residues and one Asp or Glu residue were used to coordinate the metal ions, AM2D and AM2E, respectively. Cu²⁺ bound to AM2D with an equatorial planar coordination structure with two His, one Asp, and H₂O as detected by electron spin resonance and UV spectral analyzes. On the other hand, Cu²⁺ had a slightly distorted square planar structure when it bound two His and Glu in AM2E, due to the longer side‐chain of the Glu residue as compared to the Asp residue. Computational analysis also supported the distorted coordination structure of Cu²⁺ in AM2E. This construct should be useful to create various coordinations of metal ions for catalytic functions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 907–916, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Three-dimensional structure of porcine pancreatic procarboxypeptidase A. A comparison of the A and B zymogens and their determinants for inhibition and activation.

The three-dimensional structure of procarboxypeptidase A (PCPA) from porcine pancreas has been determined at 2 A resolution and refined to a crystallographic R-factor of 0.198, with a root-mean-square deviation from ideal values for bond lengths of 0.015 A and for angles of 2.1 degrees. It is compared with procarboxypeptidase B (PCPB) from the same tissue. The 94/95 residue activation segments of PCPA/PCPB have equivalent folds: an N-terminal globular region with an open sandwich antiparallel alpha/antiparallel beta topology, followed by an extended alpha-helical segment, the connection to the enzyme. Alignment of the secondary structures of the activation segments of PCPA and PCPB (residues A1 to A99) indicates a two residue insertion between residues A34 and A35 and a C-terminal helix that is two turns longer in PCPA compared to PCPB. A deletion is observed between residues A43 to A45, the region containing the short 3(10) helix that covers the active site in PCPB. The globular region (A4 to A80) shields the preformed active center of carboxypeptidase A (CPA), but none of the residues involved in catalysis makes direct contacts with the activation segment. In contrast, subsites S2, S3 and S4 of the enzyme, involved in the binding of peptidic substrates, are blocked by specific contacts with residues AspA36, TrpA38, ArgA47, AspA53 and GluA86 of the activation segment. It has been described that several residues of CPA exhibit different conformations in the free enzyme compared to when substrate is bound: Arg127, Arg145, Glu270 and Tyr248. In PCPA all of these residues are found in the "active" conformation, as if substrate were actually bound. The presence of a ligand, tentatively interpreted as a free amino acid (Val) in the active center could explain this fact. The connecting region (A80 to A99), the target for proteolytic activation, establishes fewer contacts with the enzyme in PCPA than in PCPB. The activation segment of PCPA (A4 to A99) remains bound to the enzyme after the first trypsin cleavage between ArgA99-Ala1 probably due to the stability conferred on it by the alpha-helix (alpha 3) of the connecting segment. These and other structural features may explain the differences in intrinsic activity and different rates or proteolytic activation of each zymogen.     

Model peptide‐based system used for the investigation of metal ions binding to histidine‐containing polypeptides

The reaction of histidine‐containing polypeptides with toxic and essential metals and the molecular mechanism of complexation has yet to be determined, particularly with respect to the conformational changes of the interacting macromolecules. Therefore, a system of oligopeptides containing histidine residues in various positions of Ala or Gly sequences has been designed and used in heavy metal comparatively binding experiments. The role of spacing residues (Gly and Ala repeats) in selecting the various conformations was investigated. The newly synthesized peptides and metal ion adducts have been characterized by Fourier transform infrared spectroscopy (FTIR) as well as electrospray ion trap mass spectrometry (ESI–MS) and circular dichroism (CD). The analysis of CD‐spectra of the four peptides in water revealed that the secondary structure depends much on the position of each amino acid in the peptide backbone. Our peptides system reveals various binding mechanisms of metal ions to peptides depending on the position of histidine residue and the corresponding conformations of Ala or Gly sequences. Biological and medical consequences of conformational changes of metal‐bound peptides are further discussed. Thus, the binding of heavy metals to four peptides may serve as a model system with respect to the conformational consequences of the metal addition on the amino acid repeats situated in prion protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93:497–508, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Crystal structure of the complex between carboxypeptidase A and the biproduct analog inhibitor L-benzylsuccinate at 2.0 A resolution.

The X-ray crystal structure of the carboxypeptidase A-L-benzylsuccinate complex has been refined at 2.0 A resolution to a final R-factor of 0.166. One molecule of the inhibitor binds to the enzyme active site. The terminal carboxylate forms a salt link with the guanidinium group of Arg145 and hydrogen bonds with Tyr248 and Asn144. The second carboxylate group binds to the zinc ion in an asymmetric bidentate fashion replacing the water molecule of the native structure. The zinc ion moves 0.5 A from its position in the native structure to accommodate the inhibitor binding. The overall stereochemistry around the zinc can be considered a distorted tetrahedron, although six atoms of the co-ordinated groups lie within 3.0 A from the zinc ion. The key for the strong inhibitory properties of L-benzylsuccinate can be found in its ability both to co-ordinate the zinc and to form a short carboxyl-carboxylate-type hydrogen bond (2.5 A) with Glu270.     

Chaperone‐like activities of different molecular forms of β‐casein. Importance of polarity of N‐terminal hydrophilic domain

As a member of intrinsically unstructured protein family, β‐casein (β‐CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone‐like activity in vitro. Like many chaperones, native β‐CN does not contain cysteinyl residues and exhibits strong tendencies for self‐association. The chaperone‐like activities of three recombinant β‐CNs wild type (WT) β‐CN, C4 β‐CN (with cysteinyl residue in position 4) and C208 β‐CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native β‐CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (β‐CND) of C4‐β‐CN and C208 β‐CN were also studied and their chaperone‐like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT β‐CN, C208 β‐CN, C4 β‐CN and C4 β‐CND exhibited significantly lower chaperone‐like activities than native β‐CN. Dimerization of C208 β‐CN with two distal hydrophilic domains considerably improved its chaperone‐like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N‐terminal hydrophilic domain as important functional elements in enhancing the chaperone‐like activity of native β‐CN. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 623–632, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Analysis of functional divergence within two structurally related glycoside hydrolase families

Two glycoside hydrolase (GH) families were analyzed to detect the presence of functional divergence using the program DIVERGE. These two families, GH7 and GH16, each contain members related by amino acid sequence similarity, retaining hydrolytic mechanisms, and catalytic residue identity. GH7 and GH16 comprise GH Clan B, with a shared β‐jelly roll topology and mechanism. GH7 contains fungal cellobiohydrolases and endoglucanases and is divided into five main subfamilies, four of the former and one of the latter. Cluster comparisons between three of the cellobiohydrolase subfamilies and the endoglucanase subfamily identified specific amino acid residues that play a role in the functional divergence between the two enzyme types. GH16 contains subfamilies of bacterial agarases, xyloglucosyl transferases, 1,3‐β‐D ‐glucanases, lichenases, and other enzymes with various substrate specificities and product profiles. Four cluster comparisons between these four main subfamilies again have identified amino acid residues involved in functional divergence between the subfamilies. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 478–495, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The crystal structure of the H116Q mutant of NDM-1: An enzyme devoid of zinc ions

New Delhi metallo-β-lactamase 1 (NDM-1) is an important causative factor of antimicrobial resistance due to its efficient hydrolysis of a broad range of β-lactam compounds. The two zinc ions at the active site play essential roles in the NDM-1 catalytic activities. In a previous work, H116, one of the three ligands at the Zn1 site, was mutated in order to investigate the nature of zinc ion chelation. We report here the crystal structure of the NDM-1 H116Q mutant, that was designed to convert a B1 di-zinc enzyme into a B3 type, which either still binds two zinc ions or binds only one at the Zn2 site. The effect of mutation on the overall structure is minimal. Unexpectedly, no zinc ion was observed in the crystal structure. The Zn2-site ligating residue C221 forms a covalent bond with the nearby K121, a residue important in maintaining the active-site structure. The largest conformational changes were found at main-chain and side-chain atoms at residues 232–236 (loop 10), the proper configuration of which is known to be essential for substrate binding. The catalytic-site mutation caused little local changes, yet the effects were amplified and propagated to the substrate binding residues. There were big changes in the ψ angles of residues G232 and L234, which resulted in the side chain of N233 being displaced away from the substrate-binding site. In summary, we failed in turning a B1 enzyme into a B3 enzyme, yet we produced a zinc-less NDM-1 with residual activities.     

Human procarboxypeptidase B: three-dimensional structure and implications for thrombin-activatable fibrinolysis inhibitor (TAFI)

Besides their classical role in alimentary protein degradation, zinc-dependant carboxypeptidases also participate in more selective regulatory processes like prohormone and neuropeptide processing or fibrinolysis inhibition in blood plasma. Human pancreatic procarboxypeptidase B (PCPB) is the prototype for those human exopeptidases that cleave off basic C-terminal residues and are secreted as inactive zymogens. One such protein is thrombin-activatable fibrinolysis inhibitor (TAFI), also known as plasma PCPB, which circulates in human plasma as a zymogen bound to plasminogen. The structure of human pancreatic PCPB displays a 95-residue pro-segment consisting of a globular region with an open-sandwich antiparallel-alpha antiparallel-beta topology and a C-terminal alpha-helix, which connects to the enzyme moiety. The latter is a 309-amino acid residue catalytic domain with alpha/beta hydrolase topology and a preformed active site, which is shielded by the globular domain of the pro-segment. The fold of the proenzyme is similar to previously reported procarboxypeptidase structures, also in that the most variable region is the connecting segment that links both globular moieties. However, the empty active site of human procarboxypeptidase B has two alternate conformations in one of the zinc-binding residues, which account for subtle differences in some of the key residues for substrate binding. The reported crystal structure, refined with data to 1.6A resolution, permits in the absence of an experimental structure, accurate homology modelling of TAFI, which may help to explain its properties.     

Crystal structure of the stromelysin catalytic domain at 2.0 A resolution: inhibitor-induced conformational changes.

Matrix metalloproteinases are believed to play an important role in pathological conditions such as osteoarthritis, rheumatoid arthritis and tumor invasion. Stromelysin is a zinc-dependent proteinase and a member of the matrix metalloproteinase family. We have solved the crystal structure of an active uninhibited form of truncated stromelysin and a complex with a hydroxamate-based inhibitor. The catalytic domain of the enzyme of residues 83-255 is an active fragment. Two crystallographically independent molecules, A and B, associate as a dimer in the crystals. There are three alpha-helices and one twisted, five-strand beta-sheet in each molecule, as well as one catalytic Zn, one structural Zn and three structural Ca ions. The active site of stromelysin is located in a large, hydrophobic cleft. In particular, the S1' specificity site is a deep and highly hydrophobic cavity. The structure of a hydroxamate-phosphinamide-type inhibitor-bound stromelysin complex, formed by diffusion soaking, has been solved as part of our structure-based design strategy. The most important feature we observed is an inhibitor-induced conformational change in the S1' cavity which is triggered by Tyr223. In the uninhibited enzyme structure, Tyr223 completely covers the S1' cavity, while in the complex, the P1' group of the inhibitor displaces the Tyr223 in order to fit into the S1' cavity. Furthermore, the displacement of Tyr223 induces a major conformational change of the entire loop from residue 222 to residue 231. This finding provides direct evidence that Tyr223 plays the role of gatekeeper of the S1' cavity. Another important intermolecular interaction occurs at the active sit of molecule A, in which the C-terminal tail (residues 251-255) from molecule B inserts. The C-terminal tail interacts extensively with the active site of molecule A, and the last residue (Thr255) coordinated to the catalytic zinc as the fourth ligand, much like a product inhibitor would. The inhibitor-induced conformational change and the intermolecular C-terminal-zinc coordination are significant in understanding the structure-activity relationships of the enzyme.     

Activation of lactoperoxidase by heme‐linked protonation and heme‐independent iodide binding

Lactoperoxidase (LPO), a mammalian secretory heme peroxidase, catalyzes the oxidation of thiocyanate by hydrogen peroxide to produce hypothiocyanate, an antibacterial agent. Although LPO is known to be activated at acidic pH and in the presence of iodide, the structural basis of the activation is not well understood. We have examined the effects of pH and iodide concentration on the catalytic activity and the structure of LPO. Electrochemical and colorimetric assays have shown that the catalytic activity is maximized at pH 4.5. The heme Soret absorption band exhibits a small red‐shift at pH 5.0 upon acidification, which is ascribable to a structural transition from a neutral to an acidic form. Resonance Raman spectra suggest that the heme porphyrin core is slightly contracted and the Fe‐His bond is strengthened in the acidic form compared to the neutral form. The structural change of LPO upon activation at acidic pH is similar to that observed for myeloperoxidase, another mammalian heme peroxidase, upon activation at neutral pH. Binding of iodide enhances the catalytic activity of LPO without affecting either the optimum pH of activity or the heme structure, implying that the iodide binding occurs at a protein site away from the heme‐linked protonation site. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 113–120, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

β‐Sheet aggregation of kisspeptin‐10 is stimulated by heparin but inhibited by amphiphiles

The murine 10‐residue neurohormone kisspeptin (YNWNSFGLRY) is an important regulator of reproductive behavior and gonadotrophin secretion. It is known to form a random coil in solution, but undergoes a structural change in the presence of membranes although the nature of this change is not fully determined. The peptide's conformational versatility raises the question whether it is also able to form ordered aggregates under physiological conditions, which might be relevant as a storage mechanism. Here we show that heparin induces kisspeptin to form β‐sheet rich amyloid aggregates both at neutral (pH 7.0) and slightly acidic (pH 5.2) conditions. Addition of heparin leads to aggregation after a certain lag phase, irrespective of the time of addition of heparin, indicating that heparin is needed to facilitate the formation of fibrillation nuclei. Aggregation is completely inhibited by submicellar concentrations of zwitterionic and anionic surfactants. Unlike previous reports, our NMR data do not indicate persistent structure in the presence of zwitterionic surfactant micelles. Thus kisspeptin can aggregate under physiologically relevant conditions provided heparin is present, but the process is highly sensitive to the presence of amphiphiles, highlighting the very dynamic nature of the peptide conformation and suggesting that kisspeptin aggregation is a biologically regulatable process. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 678–689, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

X-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family 19 chitinases

The crystal structure of a chitinase from Carica papaya has been solved by the molecular replacement method and is reported to a resolution of 1.5 A. This enzyme belongs to family 19 of the glycosyl hydrolases. Crystals have been obtained in the presence of N-acetyl- d-glucosamine (GlcNAc) in the crystallization solution and two well-defined GlcNAc molecules have been identified in the catalytic cleft of the enzyme, at subsites -2 and +1. These GlcNAc moieties bind to the protein via an extensive network of interactions which also involves many hydrogen bonds mediated by water molecules, underlying their role in the catalytic mechanism. A complex of the enzyme with a tetra-GlcNAc molecule has been elaborated, using the experimental interactions observed for the bound GlcNAc saccharides. This model allows to define four major substrate interacting regions in the enzyme, comprising residues located around the catalytic Glu67 (His66 and Thr69), the short segment E89-R90 containing the second catalytic residue Glu89, the region 120-124 (residues Ser120, Trp121, Tyr123, and Asn124), and the alpha-helical segment 198-202 (residues Ile198, Asn199, Gly201, and Leu202). Water molecules from the crystal structure were introduced during the modeling procedure, allowing to pinpoint several additional residues involved in ligand binding that were not previously reported in studies of poly-GlcNAc/family 19 chitinase complexes. This work underlines the role played by water-mediated hydrogen bonding in substrate binding as well as in the catalytic mechanism of the GH family 19 chitinases. Finally, a new sequence motif for family 19 chitinases has been identified between residues Tyr111 and Tyr125.     

Importance of the spatial display of charged residues in heparin–peptide interactions

Many studies have examined consensus sequences required for protein‐glycosaminoglycan interactions. Through the synthesis of helical heparin binding peptides, this study probes the relationship between spatial arrangement of positive charge and heparin binding affinity. Peptides with a linear distribution of positive charge along one face of the α‐helix had the highest affinity for heparin. Moving the basic residues away from a single face resulted in drastic changes in heparin binding affinity of up to three orders of magnitude. These findings demonstrate that amino acid sequences, different from the known heparin binding consensus sequences, will form high affinity protein‐heparin binding interactions when the charged residues are aligned linearly. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 290–298, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformation dependence of the CαDα stretch mode in peptides: Side‐chain influence in dipeptide structures

We have shown by theoretical studies of alanine peptides that the C^αD^α stretch frequency could be particularly useful for determining peptide structure because of its sensitivity to the φ,ψ torsion angles at the C^α atom. To demonstrate that this is a robust methodology worthy of experimental exploration, we have also shown that this mode is even more determinative of conformation in aqueous solution, mainly as a result of the development of differential C^α? D^α···O(water) interactions. As further assurance, we now determine the influence of the side chain on this band, showing for aliphatic, a polar, and an aromatic side chains that the dependence is minor and explaining why this is also expected for other side chains. These results should stimulate new experimental methodologies in the field of peptide structure determination. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1065–1071, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization.

Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. The structure of a C-terminal truncated form of the human beta subunit has been determined by X-ray crystallography to 1.7 A resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely alpha-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among beta subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation.     

Role of hydrogen bonding in the active site of human manganese superoxide dismutase

The side chain of Gln143, a conserved residue in manganese superoxide dismutase (MnSOD), forms a hydrogen bond with the manganese-bound solvent and is critical in maintaining catalytic activity. The side chains of Tyr34 and Trp123 form hydrogen bonds with the carboxamide of Gln143. We have replaced Tyr34 and Trp123 with Phe in single and double mutants of human MnSOD and measured their catalytic activity by stopped-flow spectrophotometry and pulse radiolysis. The replacements of these side chains inhibited steps in the catalysis as much as 50-fold; in addition, they altered the gating between catalysis and formation of a peroxide complex to yield a more product-inhibited enzyme. The replacement of both Tyr34 and Trp123 in a double mutant showed that these two residues interact cooperatively in maintaining catalytic activity. The crystal structure of Y34F/W123F human MnSOD at 1.95 A resolution suggests that this effect is not related to a conformational change in the side chain of Gln143, which does not change orientation in Y34F/W123F, but rather to more subtle electronic effects due to the loss of hydrogen bonding to the carboxamide side chain of Gln143. Wild-type MnSOD containing Trp123 and Tyr34 has approximately the same thermal stability compared with mutants containing Phe at these positions, suggesting the hydrogen bonds formed by these residues have functional rather than structural roles.     

Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.     

Comparing four different approaches for the determination of inter‐residue interactions provides insight for the structure prediction of helical membrane proteins

Studying inter‐residue interactions provides insight into the folding and stability of both soluble and membrane proteins and is essential for developing computational tools for protein structure prediction. As the first step, various approaches for elucidating such interactions within protein structures have been proposed and proven useful. Since different approaches may grasp different aspects of protein structural folds, it is of interest to systematically compare them. In this work, we applied four approaches for determining inter‐residue interactions to the analysis of three distinct structure datasets of helical membrane proteins and compared their correlation to the three individual quality measures of structures in these datasets. These datasets included one of 35 structures of rhodopsin receptors and bacterial rhodopsins determined at various resolutions, one derived from the HOMEP benchmark dataset previously reported, and one comprising of 139 homology models. It was found that the correlation between the average number of inter‐residue interactions obtained by applying the four approaches and the available structure quality measures varied quite significantly among them. The best correlation was achieved by the approach focusing exclusively on favorable inter‐residue interactions. These results provide interesting insight for the development of objective quality measure for the structure prediction of helical membrane proteins. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 547–556, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center

Formation of N-terminal pyroglutamate (pGlu or pE) from glutaminyl or glutamyl precursors is catalyzed by glutaminyl cyclases (QC). As the formation of pGlu-amyloid has been linked with Alzheimer's disease, inhibitors of QCs are currently the subject of intense development. Here, we report three crystal structures of N-glycosylated mammalian QC from humans (hQC) and mice (mQC). Whereas the overall structures of the enzymes are similar to those reported previously, two surface loops in the neighborhood of the active center exhibit conformational variability. Furthermore, two conserved cysteine residues form a disulfide bond at the base of the active center that was not present in previous reports of hQC structure. Site-directed mutagenesis suggests a structure-stabilizing role of the disulfide bond. At the entrance to the active center, the conserved tryptophan residue, W(207), which displayed multiple orientations in previous structure, shows a single conformation in both glycosylated human and murine QCs. Although mutagenesis of W(207) into leucine or glutamine altered substrate conversion significantly, the binding constants of inhibitors such as the highly potent PQ50 (PBD150) were minimally affected. The crystal structure of PQ50 bound to the active center of murine QC reveals principal binding determinants provided by the catalytic zinc ion and a hydrophobic funnel. This study presents a first comparison of two mammalian QCs containing typical, conserved post-translational modifications.