bluuebunny/crossref_metadata_2025 huggingface dataset:
The active site histidines of creatine kinase. A critical role of His 61 situated on a flexible loop
Authors: Forstner Michael , Müller Alexandre , Stolz Martin , Wallimann Theo
Published: July 2010

A histidine residue with a PKα of 7 has been inferred to act as a general acid‐base catalyst for the reaction of creatine kinase (CK), catalyzing the reversible phosphorylation of creatine by ATP. The chicken sarcomeric muscle mitochon‐drial isoenzyme Mib‐CK contains several histidine residues that are conserved throughout the family of creatine kinases. By X‐ray crystal structure analysis, three of them (His 61, His 92, and His 186) were recently shown to be located close to the active site of the enzyme. These residues were exchanged against alanine or aspartate by in vitro mutagenesis, and the six mutant proteins were expressed in E. coli and purified. Structural integrity of the mutant proteins was checked by small‐angle X‐ray scattering. Kinetic analysis showed the mutant His 61 Asp to be completely inactive in the direction of ATP consumption while exhibiting a residual activity of 1.7% of the wild‐type (wt) activity in the reverse direction. The respective His to Ala mutant of residue 61 showed approximately 1% wt activity in the forward and 10% wt activity in the reverse reaction. All other mutants showed near wt activities. Changes in the kinetic parameters Km or Kmax, as well as a significant loss of synergism in substrate binding, could be observed with all active mutants. These effects were most pronounced for the binding of creatine and phosphocreatine, whereas ATP or ADP binding were less severely affected. Based on our results, we assume that His 92 and His 186 are involved in the binding of creatine and ATP in the active site, whereas His 61 is of importance for the catalytic reaction but does not serve as an acid‐base catalyst in the transphosphorylation of creatine and ATP. In addition, our data support the idea that the flexible loop bearing His 61 is able to move towards the active site and to participate in catalysis.
Intrachain disulfide bond in the core hinge region of human IgG4
Authors: Bloom James W. , Madanat Melanie S. , Marriott David , Wong Teresa , Chan Sham‐Yuen
Published: July 2010

IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four‐chain structure is maintained by strong noncovalent interactions between the amino‐terminal half of pairs of heavy‐light chains and between the carboxyl‐terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy‐light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor‐a (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half‐IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP571(S229P), or with an IgGl rather than IgG4 hinge region, CDP571(γ1), only trace amounts of nondisulfide bonded half‐IgG tetramers were observed. Trypsin digest reverse‐phase HPLC peptide mapping studies of CDP571 and CDP571(γ1) with on‐line electrospray ionization mass spectros‐copy supplemented with Edman sequencing identified the chemical factor preventing inter‐heavy chain disulfide bond formation between half‐IgG molecules: the two cysteines in the IgG4 and IgGl core hinge region (CPSCP and CPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low‐energy conformations of 31‐33 kcal/mol and 40‐42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP pep‐tide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgGl molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgGl and thus may permit formation of a stable intrachain disulfide bond more readily.
P100, a transcriptional coactivator, is a human homologue of staphylococcal nuclease
Authors: Ponting Christopher P.
Published: July 2010

Staphylococcus aureus nuclease (SNase) homologues, previously thought to be restricted to bacteria and archaea, are demonstrated by sequence analysis to be present also in eukary‐otes. The human cellular coactivator pi00 is shown to contain four repeats, each of which is a SNase homologue. Surprisingly, these repeats are unlikely to possess SNase‐like activities as each lacks equivalent SNase catalytic residues, yet they may mediate plOO's single‐stranded DNA‐binding function. Products of Corydalis sem‐pervirens and Saccharomyces cerevisiae open reading frames are predicted to adopt the same fold and possess similar functions as SNase. Five additional hypothetical proteins of bacterial origin are also predicted to be active SNase‐like nucleases, including one that appears to be C‐terminally truncated in a manner analogous to an engineered active SNase variant. Conservation of Asp‐19 and Asp‐83 among these homologues suggests a re‐evaluation of the roles of these residues in Ca2+‐binding and/or catalysis.
Taxonomy and function of C1 protein kinase C homology domains
Authors: Hurley James H. , Newton Alexandra C. , Parker Peter J. , Blumberg Peter M. , Nishizuka Yasutomi
Published: July 2010

C1 domains are compact α/β structural units of about 50 amino acids which tightly bind two zinc ions. These domains were first discovered as the loci of phorbol ester and diacylglycerol binding to conventional protein kinase C isozymes, which contain two C1 domains (CIA and C1B) in their N‐terminal regulatory regions. We present a comprehensive list of 54 C1 domains occurring singly or doubly in 34 different proteins. Many C1 domains and C1 domain‐containing proteins bind phorbol esters, but many others do not. By combining analysis of 54 C1 domain sequences with information from previously reported solution and crystal structure determinations and site‐directed mutagenesis, profiles are derived and used to classify C1 domains. Twenty‐six C1 domains fit the profile for phorbol‐ester binding and are termed “typical.” Twenty‐eight other domains fit the profile for the overall C1 domain fold but do not fit the profile for phorbol ester binding, and are termed “atypical.” Proteins containing typical C1 domains are predicted to be regulated by diacylglycerol, whereas those containing only atypical domains are not.
Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals
Authors: Mozzarelli Andrea , Rivetti Claudio , Rossi Gian Luigi , Eaton William A. , Henry Eric R.
Published: July 2010

In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of G‐state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry 52:2888–2906) suggested that there are high‐ and low‐affinity subunit conformations of G, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of G‐state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the “strong” allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10‐30‐fold. We find no change in affinity (<10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two‐state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol 12:88–118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the α and β subunits. This was shown by calculating the separate α and β subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X‐ray structures of deoxygenated and fully oxygenated T‐state molecules determined by Paoli M et al. (1996, J Mol Biol 256:775–792).
Structural comparison of cupredoxin domains: Domain recycling to construct proteins with novel functions
Authors: Murphy MICHAEL E. P. , Lindley Peter F. , Adman Elinor T.
Published: July 2010

The three‐dimensional structures of the copper‐containing enzymes ascorbate oxidase, ceruloplasmin, and nitrite reductase, comprised of multiple domains with a cupredoxin fold, are consistent with having evolved from a common ancestor. The presence or absence of copper sites has complicated ascertaining the structural and evolutionary relationship among these and related proteins. Simultaneous structural superposition of the enzyme domains and their known cupredoxin relatives shows clearly that there are at least six cupredoxin classes, and that the evolution of the conserved core of these domains is independent of the presence or absence of copper sites. Relationships among the variable loops in these structures show that the two‐domain ancestor of the blue oxidases contained a trinuclear‐copper interface but could not have functioned in a monomeric state. Comparison of the sequence of the copper‐containing, iron‐regulating protein. Ferrous transport (Fet3) from yeast to the structurally defined core and loop residues of the cupredoxins suggests specific residues that could be involved in the ferroxidase activity of Fet3.
Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X‐ray crystallography
Authors: Su Shaoyu , Gao Yi‐Gui , Zhang Hong , Terwilliger Thomas C. , Wang ANDREW H.‐J.
Published: July 2010

The high‐resolution crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, f1, fd) has been solved recently for the wild‐type and two surface mutant (Y41F and Y41H) proteins, leading to a plausible model for the polymeric GVP‐ssDNA complex (Guan Y, Zhang H, Wang AHJ, 1995, Protein Sci 4:187–197). The model of the complex shows extensive contacts between neighboring dimer GVPs involving electrostatic interactions between the K69 from one and the D79 and R82 from the next dimer. In addition, hydrophobic interactions between the amino acids L32 and L44 from one and G23 from the next dimer also contribute to the dimer‐dimer interactions. Mutations at the L32, K69, and R82 amino acid sites generally destabilize the protein and many of these affect the function of the phage. We have studied the structural effects of three mutant proteins involving those sites, i.e., L32R, K69H, and R82C, by X‐ray crystallographic analysis at 2.0 Å resolution. In L32R GVP, the structural perturbation is localized, whereas in K69H and R82C GVPs, some long‐range effects are also detected in addition to the local perturbation. We have interpreted the protein stability and the functional properties associated with those mutations in terms of the observed structural perturbations.
Intermolecular tuning of calmodulin by target peptides and proteins: Differential effects on Ca2+ binding and implications for kinase activation
Authors: Peersen Olve B. , Madsen Travis S. , Falke Joseph J.
Published: July 2010

Ca2+‐activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM‐regulated proteins: phosphorylase kinase, CaM‐dependent protein kinase 11, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca2+‐ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N‐terminal regulatory domain from 14‐ to 350‐fold and slow its Ca2+ dissociation kinetics from 60‐ to 140‐fold. Smaller effects are observed for the C‐terminal domain, where peptides increase the apparent Ca2+ affinity 8‐ to 100‐fold and slow dissociation kinetics 13‐ to 32‐fold. In full‐length skeletal myosin light chain kinase the inter‐molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM‐protein complex that has a 10‐fold lower Ca2+ affinity than the analogous CaM‐peptide complex. Unlike the CaM‐peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N‐terminal domain are substantially occluded in the CaM‐protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free CaZ2+‐CaM or Cq2+‐CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate‐limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C‐terminal domain or the dissociation of Ca2+ loaded C‐terminal domain from skMLCK. The observed target‐induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.
Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol
Authors: De Laureto Patrizia Polverino , Scaramella Elena , De Filippis Vincenzo , Bruix Marta , Rico Manuel , Fontana Angelo
Published: July 2010

We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE‐resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124‐residue chain of RNase in its TFE state (20–42 °C, 6–24 h) at peptide bond Asn 34‐Leu 35, followed by a slower cleavage at peptide bond Thr 45‐Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1–34 and 35–124) (RNase Th1) or three (1–34, 35–45 and 46–124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Th1 maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (‐ΔTm 16 °C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Th1 is closely similar to that of the intact protein was confirmed unambiguously by two‐dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30–41, i.e., at residues nearby the cleaved Asn 34‐Leu 35 peptide bond. RNase Th1 retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 31–39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41–48 region forms a β‐strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Th1 and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE‐state that likely dictate the selective proteolysis phenomenon by thermolysin.
Characterization and crystallization of a minimal catalytic core domain from mammalian type II adenylyl cyclase
Authors: Zhang Gongyi , Liu Yu , Qin Jun , Vo Binh , Tang Wei‐Jen , Ruoho Arnold E. , Hurley James H.
Published: July 2010

Adenylyl cyclases play a pivotal role in signal transduction by carrying out the regulated synthesis of cyclic AMP. The nine cloned mammalian adenylyl cyclases all share two conserved regions of sequence, C1and C2, which are homologous to each other and are together responsible for catalytic activity. Recombinant C1and C2domains catalyze the synthesis of cyclic AMP when they are mixed and activated by forskolin, and C2domains alone also manifest reduced levels of forskolin‐stimulated enzyme activity. Using limited proteolysis and mass spectrometry, we have mapped the boundaries of a minimal stable and active C2catalytic domain to residues 871–1090 of type II adenylyl cyclase. We report the properties and crystallization of this trimmed domain, termed IIC2‐Δ4. Crystals belong to space group P4n212, wheren= 1 or 3;a=b= 81.3, andc= 180.5 Å; and there are two molecules per asymmetric unit related by an approximate body centering operation. Flash‐frozen crystals diffract anisotropically to 2.2 Å along thec*direction and to 2.8 Å along thea*andb*directions using synchrotron radiation.
How do potentials derived from structural databases relate to “true” potentials?
Authors: Zhang Li , Skolnick Jeffrey
Published: July 2010

Knowledge‐based potentials are used widely in protein folding and inverse folding algorithms. Two kinds of derivation methods are used. (1) The interactions in a database of known protein structures are assumed to obey a Boltzmann distribution. (2) The stability of the native folds relative to a manifold of misfolded structures is optimized. Here, a set of previously derived contact and secondary structure propensity potentials, taken as the “true” potentials, are employed to construct an artificial protein structural database from protein fragments. Then, new sets of potentials are derived to see how they are related to the true potentials. Using the Boltzmann distribution method, when the stability of the structures in the database lies within a certain range, both contact potentials and secondary structure propensities can be derived separately with remarkable accuracy. In general, the optimization method was found to be less accurate due to errors in the “excess energy” contribution. When the excess energy terms are kept as a constraint, the true potentials are recovered exactly.
Contaminant inclusion into protein crystals analyzed by electrospray mass spectrometry and X‐ray crystallography
Authors: Fontecilla‐Camps Juan Carlos , Halgand Frédéric , Forest Eric , Hirschler Joachim
Published: July 2010

The inclusion of protein contaminants into crystals of turkey egg white lysozyme (TEWL) was investigated by electrospray mass spectrometry of the dissolved crystals. The results show that significant amounts of the structurally related contaminant hen egg white lysozyme (HEWL) are included in the crystals of TEWL. The structurally unrelated contaminant RNAse A, on the other hand, is not included. The X‐ray diffraction data statistics of a hybrid TEWL/HEWL crystal and an uncontaminated TEWL crystal were of similar quality. This indicates that, even though the crystals contain much higher levels of the contaminant than one would have expected after a recrystallization experiment, they are still suitable for X‐ray diffraction experiments. However, attempts to detect the presence of the contaminant in the crystal by crystallographic structure refinement did not yield conclusive results.
Cloning, overexpression, purification, and spectroscopic characterization of human S100P
Authors: Gribenko Alexey , Lopez Maria M. , Richardson John M. , Makhatadze George I.
Published: July 2010

The calcium‐binding protein S100P has been found to be associated with human prostate cancer. We have overexpressed S100P in Escherichia coli using a T7 expression system. A rapid two‐step procedure for the isolation of overexpressed S100P leads to a preparation of >95% pure protein with a yield of ∼ 150 mg per liter of culture. The structural integrity of recombinant S100P was analyzed using CD and fluorescence spectroscopic techniques. The far‐UV CD shows that secondary structure of recombinant S100P consists predominantly of α‐helical structure. Both near‐UV CD and tyrosine fluorescence spectra show that aromatic residues are involved in the formation of a specific, well packed structure, indicating that the recombinant S100P protein adopts a compact folded conformation. Ca2+ has a profound effect on S100P structure. Near‐UV CD and fluorescence intensity of both internal (tyrosine) and external (ANS) probes suggest significant structural rearrangements in the tertiary structure of the molecule. The similarity of far‐UV CD spectrum of S100P in the presence and in the absence of Ca2+ suggests that Ca2+ binding has only minor effects on secondary structure.
Hirunorms are true hirudin mimetics. The crystal structure of human α‐thrombin‐hirunorm V complex
Authors: De Simone Giuseppina , Lombardi Angela , Galdiero Stefania , Nastri Flavia , Morte Rossella Della , Staiano Norma , Pedone Carlo , Bolognesi Martino , Pavone Vincenzo
Published: July 2010

A novel class of synthetic, multisite‐directed thrombin inhibitors, known as hirunorms, has been described recently. These compounds were designed to mimic the binding mode of hirudin, and they have been proven to be very strong and selective thrombin inhibitors. Here we report the crystal structure of the complex formed by human α‐thrombin and hirunorm V, a 26‐residue polypeptide containing non‐natural amino acids, determined at 2.1 Å resolution and refined to anR‐factor of 0.176. The structure reveals that the inhibitor binding mode is distinctive of a true hirudin mimetic, and it highlights the molecular basis of the high inhibitory potency (Ki, is in the picomolar range) and the strong selectivity of hirunorm V.

Hirunorm V interacts through the N‐terminal tetrapeptide with the thrombin active site in a nonsubstrate mode; at the same time, this inhibitor specifically binds through the C‐terminal segment to the fibrinogen recognition exosite. The backbone of the N‐terminal tetrapeptide Chg1″‐Val2″‐2‐Nal3″‐Thr4″(Chg, cyclohexyl‐glycine; 2‐Nal, β‐(2‐naphthyl)‐alanine) forms a short β‐strand parallel to thrombin main‐chain residues Ser2l4‐Gly219. The Chg1″side chain fills the S2 subsite, Val2″is located at the entrance of S1, whereas 2‐Nal3″side chain occupies the aryl‐binding site. Such backbone orientation is very close to that observed for the N‐terminal residues of hirudin, and it is similar to that of the synthetic retro‐binding peptide BMS‐183507, but it is opposite to the proposed binding mode of fibrinogen and of small synthetic substrates. Hirunorm V C‐terminal segment binds to the fibrinogen recognition exosite, similarly to what observed for hirudin C‐terminal tail and related compounds. The linker polypeptide segment connecting hirunorm V N‐and C‐terminal regions is not observable in the electron density maps. The crystallographic analysis proves the correctness of the design and it provides a compelling proof on the interaction mechanism for this novel class of high potency multisite‐directed synthetic thrombin inhibitors.
Exploring sequence constraints on an interhelical turn using in vivo selection for catalytic activity
Authors: Macbeath Gavin , Kast Peter , Hilvert Donald
Published: July 2010

The role of interhelical turns in determining protein structure has been investigated previously in relatively simple four‐helix‐bundle proteins using combinatorial mutagenesis coupled with screening for functional variants. To assess the tolerance to sequence substitution of a short, interhelical turn in a larger, more complicated protein, we have exploited a more sensitive in vivo selection for catalytic activity. Randomization of three solvent‐exposed turn residues in Escherichia coli chorismate mutase (Ala65, His66, and His67), followed by selection, indicated that > 63% of tripeptides, including some with significantly altered backbone conformations, can functionally replace the native sequence. The increased sensitivity of the catalytic assay allowed optimal sequences to be distinguished from less appropriate ones, revealing a statistically significant preference for hydrophilic residues in solvent‐exposed positions. It also enabled investigation of the extent to which either secondary structure or tertiary interactions influence substitution patterns. Randomization of an α‐helical residue (Lys64), together with the adjacent solvent‐exposed tripeptide, Ala65‐His66‐His67, showed that the secondary structure at position 64 does not limit the range of side chains allowed at this site. In contrast, randomization of a buried turn residue (Leu68), together with the same tripeptide, revealed an extremely strict requirement for hydrophobic aliphatic amino acids at this position. The strong constraint imposed by the tertiary interaction, in contrast to the weak influence of secondary structure, has important implications for protein design.
Reactivities of the S2 and S3 subsite residues of thrombin with the native and heparin‐induced conformers of antithrombin
Authors: Rezaie Alireza R.
Published: July 2010

A pentasaccharide (PS) fragment of heparin capable of activating antithrombin (AT) markedly accelerates the inhibition of factor Xa by AT, but has insignificant effect on inhibition of thrombin. For inhibition of thrombin, the bridging function of a longer polysaccharide chain is required to accelerate the reaction. To study the basis for the similar reactivity of thrombin with the native or heparin‐activated conformers of AT, several residues surrounding the active site pocket of thrombin were targeted for mutagenesis study. Leu99 and Glu192, the variant residues influencing the S2 and S3 subsite specificity of thrombin were replaced with Tyr and Gin. The Tyr60a, Pro60b, Pro60c, and Trp60d residues forming part of the S2 specificity pocket were deleted from the B‐insertion loop of the wild‐type and Leu99/Glu192 Tyr/Gln thrombins. Kinetic studies indicated that the reactivities of all mutants with AT were moderately or severely impaired. Although heparin largely corrected the defect in reactivities, it also markedly elevated the stoichiometrics of inhibition with the mutants. Interestingly, PS also accelerated AT inhibition of the mutants 5‐68‐fold, suggesting that the mutants are able to discriminate between the native and activated conformers of AT. Based on these results and the recent crystal structure determination of AT in complex with PS, a model for thrombin‐AT interaction is proposed in which the S2 and S3 subsite residues of thrombin are critical for recognition of the P2 and P3 residues of AT in the native conformation. In the activated conformation, other residues are made accessible for interaction with the protease, and the similar reactivity of thrombin with the native and heparin‐activated conformers of AT may be coincidental. The results further suggest that the S2 and S3 subsite residues are crucial in controlling the partitioning of the thrombin‐AT intermediate into the alternative inhibitory or substrate pathways of the reaction.
Thioredoxin reductase from Escherichia coli: Evidence of restriction to a single conformation upon formation of a crosslink between engineered cysteines
Authors: Veine Donna M. , Ohnishi Kenji , Williams Charles H.
Published: July 2010

Thioredoxin reductase is a flavoprotein which catalyzes the reduction of the small protein thioredoxin by NADPH. It contains a redox active disulfide and an FAD in each subunit of its dimeric structure. Each subunit is further divided into two domains, the FAD and the pyridine nucleotide binding domains. The orientation of the two domains determined from the crystal structure and the flow of electrons determined from mechanistic studies suggest that thioredoxin reductase requires a large conformational change to carry out catalysis (Williams CH Jr, 1995, FASEB J 9:1267‐1276). The constituent amino acids of an ion pair, E48/R130, between the FAD and pyridine nucleotide binding domains, were mutagenized to cysteines to form E48C, R130C (CC mutant). Formation of a stable bridge between these cysteines was expected to restrict the enzyme largely in the conformation observed in the crystal structure. Crosslinking with the bifunctional reagent N, N,1,2 phenylenedimaleimide, spanning 4‐9 Å, resulted in a <95 % decrease in thioredoxin reductase and transhydrogenase activity. SDS‐PAGE confirmed that the crosslink in the CC‐mutant was intramolecular. Dithionite titration showed an uptake of electrons as in wild‐type enzyme, but anaerobic reduction of the flavin with NADPH was found to be impaired. This indicates that the crosslinked enzyme is in the conformation where the flavin and the active site disulfide are in close proximity but the flavin and pyridinium rings are too far apart for effective electron transfer. The evidence is consistent with the hypothesis that thioredoxin reductase requires a conformational change to complete catalysis.
Engineering protein for X‐ray crystallography: The murine major histocompatibility complex class II molecule I‐Ad
Authors: Scott Christoper A. , Christoper Garcia K. , Stura Enrico A. , Peterson Per A. , Wilson Ian A. , Tetton Luc
Published: July 2010

Class II Major Histocompatibility (MHC) molecules are cell surface heterodimeric glycoproteins that play a central role in the immune response by presenting peptide antigens for surveillance by T cells. Due to the inherent instability of the class II MHC heterodimer, and its dependence on bound peptide for proper assembly, the production of electrophoretically pure samples of class II MHC proteins in complex with specific peptides has been problematic. A soluble form of the murine class II MHC molecule, I‐Ad, with a leucine zipper tail added to each chain to enhance dimer assembly and secretion, has been produced inDrosophila melanogasterSC2 cells. To facilitate peptide loading, a high affinity ovalbumin peptide was covalently engineered to be attached by a six‐residue linker to the amino terminus of the I‐Adβ chain. This modified I‐Admolecule was purified using preparative IEF and one fraction, after removal of the leucine zipper tails, produced crystals suitable for X‐ray crystallographic analysis. The protein engineering and purification methods described here should be of general value for the expression of I‐A and other class II MHC‐peptide complexes.
Expansion of the genetic code: Site‐directed p‐fluoro‐phenylalanine incorporation in Escherichia coli
Authors: Furter Rolf
Published: July 2010

Site‐directed incorporation of the amino acid analogue p‐fluoro‐phenylalanine (p‐F‐Phe) was achieved in Escherichia coli. A yeast suppressor tRNAPheamber/phenylalanyl‐tRNA synthetase pair was expressed in an analogue‐resistant E. coli strain to direct analogue incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64‐75% as p‐F‐Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p‐F‐Phe incorporation was 11‐21‐fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analogue incorporation. Protein expression yields of 8‐12 mg/L of culture, corresponding to about two thirds of the expression level of the wild‐type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19F‐NMR spectroscopy and other sample‐intensive methods. The use of a nonessential “21 st” tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.
A conserved deamidation site at asn 2 in the catalytic subunit of mammalian cAMP‐dependent protein kinase detected by capillary LC‐MS and tandem mass spectrometry
Authors: Jedrzejewski Paul T. , Girod Andreas , Tholey Andreas , König Norbert , Thullner Sandra , Kinzel Volker , Bossemeyer Dirk
Published: July 2010

The N‐terminal sequence myr‐Gly‐Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes C, Cβ, and O. By capillary LC‐MS and tandem MS, we show that, in approximately one third of the C and Cβ enzyme populations from cattle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to Asp 2. This deamidation accounts for the major isoelectric variants of the cAPK C‐subunits formerly called CA and CB. Deamidation also includes characteristic isoaspartate isomeric peptides from C and Cβ. Asn 2 deamidation does not occur during C‐subunit preparation and is absent in recombinant myristoylated Cα (rCα) from Escherichia coli. Deamidation appears to be the exclusive pathway for introduction of an acidic residue adjacent to the myristoylated N‐terminal glycine, verified by the myristoylation negative phenotype of an rCa(Asn 2 Asp) mutant. This is the first report thus far of a naturally occurring myr‐Gly‐Asp sequence. Asp 2 seems to be required for the well‐characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a conserved site for deamidation in vivo. Comparable myr‐Gly‐Asn sequences are found in several signaling proteins. This may be especially significant in view of the recent knowledge that negative charges close to myristic acid in some proteins contribute to regulating their cellular localization.
Genfold: A genetic algorithm for folding protein structures using NMR restraints
Authors: Bayley Martin J. , Jones Gareth , Willett Peter , Williamson Michael P.
Published: July 2010

We report the development and validation of the program GENFOLD, a genetic algorithm that calculates protein structures using restraints obtained from NMR, such as distances derived from nuclear Overhauser effects, and dihedral angles derived from coupling constants. The program has been tested on three proteins: the POU domain (a small three‐helix DNA‐binding protein), bovine pancreatic trypsin inhibitor (BPTI), and the starch‐binding domain from Aspergillus niger glucoamylase I, a 108‐residue β‐sheet protein. Structures were calculated for each protein using published NMR restraints. In addition, structures were calculated for BPTI using artificial restraints generated from a high‐resolution crystal structure. In all cases the fittest calculated structures were close to the target structure, and could be refined to structures indistinguishable from the target structures by means of a low‐temperature simulated annealing refinement. The effectiveness of the program is similar to that of distance geometry and simulated annealing methods, and it is capable of using a very wide range of restraints as input. It can thus be readily extended to the calculation of structures of large proteins, for which few NOE restraints may be available.
Subunit asymmetry in the three‐dimensional structure of a human CuZnSOD mutant found in familial amyotrophic lateral sclerosis
Authors: Eisenberg David , Hart P. John , Liu Hongbin , Pellegrini Matteo , Nersissian Aram M. , Gralla Edith B. , Valentine Joan S.
Published: July 2010

The X‐ray crystal structure of a human copper/zinc superoxide dismutase mutant (G37R CuZnSOD) found in some patients with the inherited form of Lou Gehrig's disease (FALS) has been determined to 1.9 Å resolution. The two SOD subunits have distinct environments in the crystal and are different in structure at their copper binding sites. One subunit (subunitintact) shows a four‐coordinate ligand geometry of the copper ion, whereas the other subunit (subunitbroken) shows a three‐coordinate geometry of the copper ion. Also, subunitintact, displays higher atomic displacement parameters for backbone atoms ( = 30 ± 10 Å2) than subunitbroken ( = 24 ± 11 Å2). This structure is the first CuZnSOD to show large differences between the two subunits. Factors that may contribute to these differences are discussed and a possible link of a looser structure to FALS is suggested.
Structures of murine carbonic anhydrase IV and human carbonic anhydrase II complexed with brinzolamide: Molecular basis of isozyme‐drug discrimination
Authors: Stams Travis , Chen Yun , Christianson David W. , Boriack‐Sjodin P. Ann , Hurt Jonathan D. , Laipis Philip , Silverman David N. , Liao John , May Jesse A. , Dean Tom
Published: July 2010

Carbonic anhydrase IV (CAIV) is a membrane‐associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8‐Å resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azopt™). The overall structure of murine CAIV is generally similar to that of human CAIV; however, some local structural differences are found in the active site resulting from amino acid sequence differences in the “130′s segment” and the residue‐63 loop (these may affect the nearby catalytic proton shuttle, His‐64). Similar to human CAIV, the C‐terminus of murine CAIV is surrounded by a substantial electropositive surface potential that may stabilize the interaction with the phospholipid membrane. Binding interactions observed for brinzolamide rationalize the generally weaker affinity of inhibitors used in glaucoma therapy toward CAIV compared with CAII.
Cadmium‐induced crystallization of proteins: II. Crystallization of the Salmonella typhimuri histidine‐binding protein in complex with L‐histidine, L‐arginine, or L‐lysine
Authors: Ames Giovanna Ferro‐Luzzi , Kreimer David I. , Trakhanov Sergei , Parkin Sean , Rupp Bernhard
Published: July 2010

To further investigate favorable effects of divalent cations on the formation of protein crystals, three complexes of Salmonella typhimurium histidine‐binding protein were crystallized with varying concentrations of cadmium salts. For each of the three histidine‐binding protein complexes, cadmium cations were found to promote or improve crystallization. The optimal cadmium concentration is ligand specific and falls within a narrow concentration range. In each case, crystals grown in the presence of cadmium diffract to better than 2.0 Å resolution and belong to the orthorhombic space group P212121. From our results and from the analysis of cadmium sites in well‐refined protein structures, we propose that cadmium addition provides a generally useful technique to modify crystal morphology and to improve diffraction quality.
Anion binding to the ubiquitin molecule
Authors: Makhatadze George I. , Lopez Marin M. , Richardson John M. , Thmos Susan T.
Published: July 2010

Effects of different salts (NaCl, MgCl2, CaCl2, GdmCl, NaBr, NaC1O4, NaH2PO4, Na2SO4) on the stability of the ubiquitin molecule at pH 2.0 have been studied by differential scanning calorimetry, circular dichroism, and Tyr fluorescence spectroscopies. It is shown that all of the salts studied significantly increase the thermostability of the ubiquitin molecule, and that this stabilization can be interpreted in terms of anion binding. Estimated thermodynamic parameters of binding for Cl− show that this binding is relatively weak (Kd = 0.15 M) and is characterized by a negative enthalpy of ‐15 kJ/mol per site. Particularly surprising was the observed stabilizing effect of GdmCl through the entire concentration range studied (0.01‐2 M), however, to a lesser extent than stabilization by NaCl. This stabilizing effect of GdmCl appears to arise from the binding of Cl− ions. Analysis of the observed changes in the stability of the ubiquitin molecule in the presence of GdmCl can be adequately described by combining the thermodynamic model of denaturant binding with Cl− binding effects.
Tautomeric state and pKa of the phosphorylated active site histidine in the N‐terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: Sugar phosphotransferase system
Authors: Garrett Daniel S. , Clore G.Marius , Gronenborn Angela M. , Seok Yeong‐Jae , Peterkfsky Alan
Published: July 2010

The phosphorylated form of the N‐terminal domain of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one‐bond and long‐range 1H ‐ 1.5N correlation spectroscopy. The active site His 189 is phosphorylated at the Nϵ2 position and has a pKa of 7.3, which is one pH unit higher than that of unphosphorylated His 189. Because the neutral form of unphosphorylated His 189 is in the Nδ1‐H tautomer, and its Nϵ2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side‐chain conformation of His 189, specifically from a X2 angle in the g+ conformer in the unphosphorylated state to the g− conformer in the phosphorylated state.
S100B(ββ) inhibits the protein kinase C‐dependent phosphorylation of a peptide derived from p53 in a Ca2+‐dependent manner
Authors: Wilder Paul T. , Rustandi Richard R. , Drohat Alexander C. , Weber David J.
Published: July 2010

S100B(ββ) is a dimeric Ca2+‐binding protein that is known to inhibit the protein kinase C (PKC)‐dependent phosphorylation of several proteins. To further characterize this inhibition, we synthesized peptides based on the PKC phosphorylation domains of p53 (residues 367‐388), neuromodulin (residues 37‐53), and the regulatory domain of PKC (residues 19‐31), and tested them as substrates for PKC. All three peptides were shown to be good substrates for the catalytic domain of PKC. As for full‐length p53 (Baudier J, Delphin C, Grunwald D, Khochbin S, Lawrence JJ. 1992. Proc Natl Acad Sci USA 89:11627‐11631), S100B(ββ) binds the p53 peptide and inhibits its PKC‐dependent phosphorylation (IC50 = 10 ± 7μM) in a Ca2+‐dependent manner. Similarly, phosphorylation of the neuromodulin peptide and the PKC regulatory domain peptide were inhibited by S100B(ββ) in the presence of Ca2+ (IC50 = 17 ± 5 μM; IC50 = 1 ± 0.5 μM, respectively). At a minimum, the C‐terminal EF‐hand Ca2+‐binding domain (residues 61‐72) of each S100β subunit must be saturated to inhibit phosphorylation of the p53 peptide as determined by comparing the Ca2+ dependence of inhibition (CaIC50 = 29.3 ± 17.6 μM) to the dissociation of Ca2+ from the C‐terminal EF‐hand Ca2+‐ binding domain of S100B(ββ).
Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin‐biotin system
Authors: Chu Vano , Stayton Partrick S. , Freitag Stefanie , Le Trong Isolde , Stenkamp Ronald E.
Published: July 2010

A circularly permuted streptavidin (CP51/46) has been designed to remove the flexible polypeptide loop that undergoes an open to closed conformational change when biotin is bound. The original termini have been joined by a tetrapeptide linker, and four loop residues have been removed, resulting in the creation of new N‐ and C‐termini. Isothermal titration calorimetric studies show that the association constant has been reduced approximately six orders of magnitude below that of wild‐type streptavidin to 107 M−1. The δH° of biotin association for CP51/46 is reduced by 11.1 kcal/mol. Crystal structures of CP51/46 and its biotin complex show no significant alterations in the binding site upon removal of the loop. A hydrogen bond between Ser45 and Ser52 found in the absence of biotin is broken in the closed conformation as the side‐chain hydroxyl of Ser45 moves to hydrogen bond to a ureido nitrogen of biotin. This is true in both the wild‐type and CP51/46 forms of the protein, and the hydrogen bonding interaction might thus help nucleate closure of the loop. The reduced entropic cost of binding biotin to CP51/46 is consistent with the removal of this loop and a reduction in entropic costs associated with loop closure and immobilization. The reduced enthalpic contribution to the free energy of binding is not readily explainable in terms of the molecular structure, as the binding contacts are nearly entirely conserved, and only small differences in solvent accessible surfaces are observed relative to wild‐type streptavidin.
Mutagenesis of histidine 26 demonstrates the importance of loop‐loop and loop‐protein interactions for the function of iso‐1‐cytochrome c
Authors: Fetrow Jacquelyn S. , Dreher Ulrike , Wiland Debra J. , Schaak Diane L. , Boose Terry L.
Published: July 2010

In yeast iso‐1‐cytochrome c, the side chain of histidine 26 (His26) attaches omega loop A to the main body of the protein by forming a hydrogen bond to the backbone atom carbonyl of glutamic acid 44. The His26 side chain also forms a stabilizing intra‐loop interaction through a hydrogen bond to the backbone amide of asparagine 31. To investigate the importance of loop‐protein attachment and intra‐loop interactions to the structure and function of this protein, a series of site‐directed and random‐directed mutations were produced at His26. Yeast strains expressing these variant proteins were analyzed for their ability to grow on non‐fermentable carbon sources and for their intracellular production of cytochrome c. While the data show that mutations at His26 lead toslightly decreased intracellular amounts of cytochrome c, the level of cytochrome c function is decreased more. The data suggest that cytochrome c reductase binding is affected more than cytochrome c oxidase or lactate dehydrogenase binding. We propose that mutations at this residue increase loop mobility, which, in turn, decreases the protein's ability to bind redox partners.
Characterization of the recombinant extracellular domain of the neurotrophin receptor TrkA and its interaction with nerve growth factor (NGF)
Authors: Woo Sang B. , Neet Kenneth E. , Whalen Christopher
Published: July 2010

Nerve growth factor (NGF) is the prototype of a family of neurotrophins that support important neuronal programs such as differentiation and survival of a subset of sympathetic, sensory, and brain neurons. NGF binds to two classes of cell surface receptors: p75LANR and pl40TrkA. NGF binding to pl40TrkA initiates the neuronal signaling pathway through activation of the tyrosine kinase activity, which subsequently results in a rapid signal transduction through a phosphorylation cascade. To examine this crucial signaling step in more detail, the TrkA extracellular domain polypeptide (TrkA‐RED) was overexpressed in Sf21 insect cells and purified to homogeneity. The recombinant TrkA‐RED is a 70 kDa acidic glycoprotein with a p1 of 5.1, and mimics the intact TrkA receptor for NGF binding with a dissociation constant, Kd, of 2.9 nM. Thus, the recombinant TrkA‐RED is functionally competent and can be used to elucidate the interaction of NGF and TrkA receptor. Circular dichroism difference spectra indicated that, upon association of NGF with TrkA‐RED, a minor conformational change occurred to form a complex with decreased ordered secondary structure. Interaction between NGF and TrkA‐RED was also demonstrated by size exclusion chromatography, light scattering, and chemical crosslinking with evidence for formation of a higher molecular weight complex consistent with a (TrkA‐RED)2–(NGF dimer) complex. Association and dissociation rates of 5.6 × 105 M−1 s−1 and 1.6 × lO−3 s−1 respectively, were determined by biosensor technology. Thus, initiation of signaling may stem from NGF‐induced receptor dimerization concomitant with a small conformational change.