bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel, multilayer structure of a helical peptide
Authors: Taylor K. S. , Lou M.‐Z. , Chin T.‐M. , Yang N. C. , Garavito R. M.
Published: July 2010

X‐ray diffraction analysis at 1.5 A resolution has confirmed the helical conformation of a de novo designed 18‐residue peptide. However, the crystal structure reveals the formation of continuous molecular layers of parallel‐packed amphiphilic helices as a result of much more extensive helix‐helix interactions than predicted. The crystal packing arrangement, by virtue of distinct antiparallel packing interactions, segregates the polar and apolar surfaces of the helices into discrete and well‐defined interfacial regions. An extensive “ridges‐into‐grooves” interdigitation characterizes the hydrophobic interface, whereas an extensive network of salt bridges and hydrogen bonds dominates the corresponding hydrophilic interface.
The structure of bovine mitochondrial adenylate kinase: Comparison with isoenzymes in other compartments
Authors: Schlauderer G. J. , Schulz G. E.
Published: July 2010

In vertebrates, there are different adenylate kinases in the compartments cytosol, mitochondrial intermembrane space, and mitochondrial matrix. Here, we report the spatial structure of the intermembrane species established in two crystal forms by X‐ray diffraction analyses at 1.92 and 2.1 Å resolution. In both structures, the enzyme is unligated, and thus in an “open” conformation. The enzyme was prepared from bovine liver, containing at least five variants arisen from posttranscriptional and posttranslational modifications. It could only be crystallized after removing some of these variants. A comparison with the known structures of the adenylate kinases from cytosol and mitochondrial matrix reveals structural differences that should play a role in protein targeting because none of these enzymes contains a cleavable signal peptide. A further comparison with adenylate kinases from Gram‐positive bacteria showed that the structural Zn2+ ion of these species is replaced by a strictly conserved assembly of hydrogen bonded residues.
Electrospray mass spectrometric investigation of the chaperone SecB
Authors: Smith Virginia F. , Schwartz Brenda L. , Randall Linda L. , Smith Richard D.
Published: July 2010

Electrospray ionization mass spectrometry was used to investigate the structure of the Escherichia coli chaperone protein SecB. It was determined that the N‐terminal methionine of SecB has been removed and that more than half of all SecB monomers are additionally modified, most likely by acetylation of the N‐terminus or a lysine. The use of gentle mass spectrometer interface conditions showed that the predominant, oligomeric form of SecB is a tetramer that is stable over a range of solution pH conditions and mass spectrometer interface heating (i.e., inlet capillary temperatures). At very high pH, SecB dimers are observed. SecB contains a region that is hypersensitive to cleavage by proteinase K and is thought to be involved in conformational changes that are crucial to the function of SecB. We identified the primary site of cleavage to be between Leu 141 and Gln 142. Fourteen amino acids are removed, but the truncated form remains a tetramer with stability similar to that of the intact form.
Three‐dimensional solution structure of the HIV‐1 protease complexed with DMP323, a novel cyclic urea‐type inhibitor, determined by nuclear magnetic resonance spectroscopy
Authors: Yamazaki Toshimasa , Hinck Andrew P. , Wang Yun‐Xing , Nicholson Linda K. , Torchia Dennis A. , Wingfield Paul , Stahl Stephen J. , Kaufman Joshua D. , Chang Chong‐Hwan , Domaille Peter J. , Lam Patrick Y.S.
Published: July 2010

The three‐dimensional solution structure of the HIV‐1 protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea‐based inhibitor, DMP323, is reported. This is the first solution structure of an HIV protease/inhibitor complex that has been elucidated. Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints. Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 Å or 5°, respectively. Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 Å relative to the average structure. The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure. The latter structure is similar to the 1.8‐Å X‐ray structure of the protease/DMP323 complex (Chang CH et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 Å. As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible. The flexibility of residues 37–42 and 50–51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps. Flexibility of residues 4–8 may play a role in protease regulation by facilitating autolysis.
On the entropy of protein folding
Authors: Makhatadze George I. , Privalov Peter L.
Published: July 2010

The failure to appreciate that the hydration of polar groups is a major contribution to the entropy of protein unfolding has led to considerable underestimates for the loss of configurational freedom when a protein chain folds.
Control of aggregation in protein refolding: The temperature‐leap tactic
Authors: Xie Yansheng , Wetlaufer Donald B.
Published: July 2010

The kinetics of renaturation of bovine carbonic anhydrase II (CAII) were studied from 4° to 36°, at the relatively high [CAII] of 4 mg/mL. Following dilution to 1 M guanidinium chloride, aggregate formation is very rapid and reduces the formation of active enzyme. The CAII activity yield at 150 min, 20° (∼60%), is greater than that at either 4° or 36°. However, if refolding is conducted at 4°, aggregation is reduced dramatically and 37% yield is obtained at 120 min. If the solution is then rapidly warmed to 36°, the yield rises rapidly to 95% at 150 min. This is an example of the “temperature leap” tactic. These results can be understood on the basis of two slow‐folding intermediates whose kinetics have been studied. Only the first of these forms aggregates. Kinetic simulations show that, at 4°, the first intermediate is depleted after 120 min, and the second intermediate rapidly isomerizes to active enzyme on warming. A series of experiments was conducted where the initial (120 min) folding temperature was systematically varied, followed by a “leap” to 36° for 30 additional minutes. With initial incubations from 4° to 12°, the final yield is >90%, drops rapidly from 12° to 20°, and decreases more gradually to ∼45% at 36°. The overall results qualitatively fit the simple idea of ordinary temperature‐accelerated reactions in competition with hydrophobic aggregation, which is strongly suppressed in the cold. Qualifications are discussed for the temperature‐leap approach to find application in refolding other proteins.
Mass spectrometric‐based revision of the structure of a cysteine‐rich peptide toxin with γ‐carboxyglutamic acid, TxVIIA, from the sea snail, Conus textile
Authors: Nakamura Takemichi , Yu Zhonghua , Fainzilber Michael , Burlingame Alma L.
Published: July 2010

A mollusk‐specific toxin, TxVIIA, having potent paralytic activity was isolated from the venom of sea snail Conus textile (Fainzilber M et al., 1991, Eur J Biochem 202:589–595). The structure reported above was based upon amino acid analysis and the Edman degradation. We have recently reinvestigated this toxin employing some of the most novel techniques in mass spectrometry. We now report a revised structure based primarily on high‐energy collision‐induced dissociation analysis of the two Asp17‐N peptides of the reduced, pyridinylethyl derivative representing the entire sequence using matrix‐assisted laser desorption ionization (MALDI) as CGGYSTYCγVDSγ CCSDNCVRSYCTLF‐NH2 (γ, γ‐carboxyglutamic acid or Gla).

The N‐terminus of the previous sequence was incorrect, apparently due to a side reaction of reduction and alkylation, which led to the erroneous assignment of Trp for the N‐terminal residue. In addition, the last two C‐terminal amino acids and the C‐terminal amidation had not been detected. Also, a combination of electrospray ionization mass spectrometry and positive and negative ion MALDI mass spectrometry provided information on the molecular weights of the native and derivatized toxin and presence of two Gla residues. Thus, TxVIIA does not have an “unusual” sequence as previously reported, but in fact belongs to the conserved Cys framework for ω‐ and δ‐conotoxins. However, the four net negative charges with the cysteine‐rich structure of this revised sequence is highly unusual for conopeptides.
Classification of mutations in the human CD40 ligand, gp39, that are associated with X‐linked hyper IgM syndrome
Authors: Bajorath Jürgen , Seyama Kuniaki , Nonoyama Shigeaki , Ochs Hans D. , Aruffo Alejandro
Published: July 2010

The interaction between the T cell activation antigen gp39 and CD40, its receptor CD40 on B cells, plays a critical role in the regulation of humoral immune responses. Using a detailed three‐dimensional model of the gp39 extracellular region, we have analyzed 20 mutations in gp39 that were, with one exception, isolated from patients with X‐linked hyper IgM (XHIM) syndrome. On the basis of this analysis, the mutations were classified according to their predicted locations and effects. Twelve mutations are thought to compromise the gp39 structure by affecting interactions in hydrophobic core regions or at monomer interfaces, whereas seven others map closely to gp39 residues important for interaction with CD40. The latter mutations may thus, directly or indirectly, interfere with CD40 binding. One naturally occurring mutant whose carrier displays normal immune responses maps to a solvent‐exposed position in a loop region of the molecule.
Simulated annealing with restrained molecular dynamics using CONGEN: Energy refinement of the NMR solution structures of epidermal and type‐α transforming growth factors
Authors: Tejero Roberto , Bassolino‐Klimas Donna , Bruccoleri Robert E. , Montelione Gaetano T.
Published: July 2010

The new functionality of the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137–168; Bassolino‐Klimas D et al., 1996, Protein Sci 5:593–603) has been applied for energy refinement of two previously determined solution NMR structures, murine epidermal growth factor (mEGF) and human type‐α transforming growth factor (hTGFα). A summary of considerations used in converting experimental NMR data into distance constraints for CONGEN is presented. A general protocol for simulated annealing with restrained molecular dynamics is applied to generate NMR solution structures using CONGEN together with real experimental NMR data. A total of 730 NMR‐derived constraints for mEGF and 424 NMR‐derived constraints for hTGFα were used in these energy‐refinement calculations. Different weighting schemes and starting conformations were studied to check and/or improve the sampling of the low‐energy conformational space that is consistent with all constraints. The results demonstrate that loosened (i.e., “relaxed”) sets of the EGF and hTGFα internuclear distance constraints allow molecules to overcome local minima in the search for a global minimum with respect to both distance restraints and conformational energy. The resulting energy‐refined structures of mEGF and hTGFα are compared with structures determined previously and with structures of homologous proteins determined by NMR and X‐ray crystallography.
Redox‐dependent dynamics of putidaredoxin characterized by amide proton exchange
Authors: Lyons Teresa A. , Ratnaswamy Gayathri , Pochapsky Thomas C.
Published: July 2010

Multidimensional NMR methods were used to obtain 1H‐15N correlations and 15N resonance assignments for amide and side‐chain nitrogens of oxidized and reduced putidaredoxin (Pdx), the Fe2S2 ferredoxin, which acts as the physiological reductant of cytochrome P‐450cam (CYP101). A model for the solution structure of oxidized Pdx has been determined recently using NMR methods (Pochapsky TC, Ye XM, Ratnaswamy G, Lyons TA, 1994, Biochemistry 53:6424–6432) and redox‐dependent 1H NMR spectral features have been described (Pochapsky TC, Ratnaswamy G, Patera A, 1994, Biochemistry 55:6433–6441). 15N assignments were made with NOESY‐(1H/15N) HMQC and TOCSY‐(1H/15N) HSQC spectra obtained using samples of Pdx uniformly labeled with 15N. Local dynamics in both oxidation states of Pdx were then characterized by comparison of residue‐specific amide proton exchange rates, which were measured by a combination of saturation transfer and H2O/D2O exchange methods at pH 6.4 and 7.4 (uncorrected for isotope effects). In general, where exchange rates for a given site exhibit significant oxidation‐state dependence, the oxidized protein exchanges more rapidly than the reduced protein. The largest dependence of exchange rate upon oxidation state is found for residues near the metal center and in a region of compact structure that includes the loop‐turn Val 74‐Ser 82 and the C‐terminal residues (Pro 102‐Trp 106). The significance of these findings is discussed in light of the considerable dependence of the binding interaction between Pdx and CYP101 upon the oxidation state of Pdx.
Structure of a secreted aspartic protease from C. albicans complexed with a potent inhibitor: Implications for the design of antifungal agents
Authors: Abad‐Zapatero Cele , Goldman Robert , Muchmore Steven W. , Hutchins Charles , Stewart Kent , Navaza Jorge , Payne Candia D. , Ray Thomas L.
Published: July 2010

The three‐dimensional structure of a secreted aspartic protease from Candida albicans complexed with a potent inhibitor reveals variations on the classical aspartic protease theme that dramatically alter the specificity of this class of enzymes. The structure presents: (1) an 8‐residue insertion near the first disulfide (Cys 45‐Cys 50, pepsin numbering) that results in a broad flap extending toward the active site; (2) a 7‐residue deletion replacing helix hN2 (Ser 110‐Tyr 114), which enlarges the S3 pocket; (3) a short polar connection between the two rigid body domains that alters their relative orientation and provides certain specificity; and (4) an ordered 11‐residue addition at the carboxy terminus. The inhibitor binds in an extended conformation and presents a branched structure at the P3 position. The implications of these findings for the design of potent antifungal agents are discussed.
The pH dependence of hydrogen‐deuterium exchange in trp repressor: The exchange rate of amide protons in proteins reflects tertiary interactions, not only secondary structure
Authors: Finucane Michael D. , Jardetzky Oleg
Published: July 2010

The pH dependence of amide proton exchange rates have been measured for trp‐repressor. One class of protons exchanges too fast to be measured in these experiments. Among the protons that have measurable hydrogen‐deuterium exchange rates, two additional classes may be distinguished. The second class of protons are in elements of secondary structure that are mostly on the surface of the protein, and exchange linearly with increasing base concentration (log kex versus pH). The third class of amide protons is characterized by much higher protection against exchange at higher pH. These protons are located in the core of the protein, in helices B and C. The exchange rate in the core region does not increase linearly with pH, but rather goes through a minimum around pH 6.

The mechanism of exchange for the slowly exchanging core protons is interpreted in terms of the two‐process model of Hilton and Woodward (1979, Biochemistry 75:5834–5841), i.e., exchange through both a local mechanism that does not require unfolding of the protein, and a mechanism involving global unfolding of the protein. The increase in exchange rates at low pH is attributed to a partial unfolding of the repressor. It is concluded that the formation of secondary structure alone is insufficient to account for the high protection factors seen in the core of native proteins at higher pH, and that tertiary interactions are essential to stabilize the structure.
Crystal structure of cod liver class I alcohol dehydrogenase: Substrate pocket and structurally variable segments
Authors: Ramaswamy S. , Ahmad Mustapha El , Danielsson Olle , Jörnvall Hans , Eklund Hans
Published: July 2010

The structural framework of cod liver alcohol dehydrogenase is similar to that of horse and human alcohol dehydrogenases. In contrast, the substrate pocket differs significantly, and main differences are located in three loops. Nevertheless, the substrate pocket is hydrophobic like that of the mammalian class I enzymes and has a similar topography in spite of many main‐chain and side‐chain differences. The structural framework of alcohol dehydrogenase is also present in a number of related enzymes like glucose dehydrogenase and quinone oxidoreductase. These enzymes have completely different substrate specificity, but also for these enzymes, the corresponding loops of the substrate pocket have significantly different structures. The domains of the two subunits in the crystals of the cod enzyme further differ by a rotation of the catalytic domains by about 6°. In one subunit, they close around the coenzyme similarly as in coenzyme complexes of the horse enzyme, but form a more open cleft in the other subunit, similar to the situation in coenzyme‐free structures of the horse enzyme. The proton relay system differs from the mammalian class I alcohol dehydrogenases. His 51, which has been implicated in mammalian enzymes to be important for proton transfer from the buried active site to the surface is not present in the cod enzyme. A tyrosine in the corresponding position is turned into the substrate pocket and a water molecule occupies the same position in space as the His side chain, forming a shorter proton relay system.
Roles of Glu 349 and Asp 352 in membrane insertion and translocation by diphtheria toxin
Authors: Kaul Poonam , Silverman Jared , Shen Wei Hai , Blanke Steven R. , Huynh Paul D. , Finkelstein Alan , John Collier R.
Published: July 2010

Acidic conditions within the endosomal lumen induce the T domain of receptor‐bound diphtheria toxin (DT) to insert into the endosomal membrane and mediate translocation of the toxin's catalytic domain to the cytosol. A conformational rearrangement in the toxin occurring near pH 5 allows a buried apolar helical hairpin of the native T domain (helices TH8 and TH9) to undergo membrane insertion. If the inserted hairpin spans the bilayer, as hypothesized, then the two acidic residues within the TL5 interhelical loop, Glu 349 and Asp 352, should become exposed at the neutral cytosolic face of the membrane and reionize. To investigate the roles of these residues in toxin action, we characterized mutant toxins in which one or both acidic residues had been replaced with nonionizable ones. Each of two double mutants examined showed a several‐fold reduction in cytotoxicity in 24‐h Vero cell assays (sixfold for E349A+D352A and fourfold for E349Q+D352N), whereas the individual E349Q and D352N mutations caused smaller reductions in toxicity. The single and double mutations also attenuated the toxin's ability to permeabilize Vero cells to Rb+ at low pH and decreased channel formation by the toxin in artificial planar bilayers. Neither of the double mutations affected the pH‐dependence profile of the toxin's conformational rearrangement in solution, as measured by binding of the hydrophobic fluorophore, 2‐p‐toluidinyl‐naphthalene 6‐sulfonate. The results demonstrate that, although there is no absolute requirement for an acidic residue within the TL5 loop for toxicity, Glu 349 and Asp 352 do significantly enhance the biological activity of the protein. The data are consistent with a model in which ionization of these residues at the cytosolic face of the endosomal membrane stabilizes the TH8/TH9 hairpin in a transmembrane configuration, thereby facilitating channel formation and translocation of the toxin's catalytic chain.
Xylanase homology modeling using the inverse protein folding approach
Authors: Chen Xiannong , Whitmire David , Bowen J. Phillip
Published: July 2010

Xylanase has been used in wood pulp bleaching in an effort to reduce chlorine release into the environment and pollution associated with paper production. The three‐dimensional structure of xylanase is important to enable better understanding of the enzyme mechanism and to help design a more thermostable xylanase mutant. At the time this work was begun, there was no sequence homologous protein available for traditional sequence‐based homology modeling. In order to circumvent this problem, the inverse protein folding approach was undertaken to find a suitable template structure. Model structures of Bacillus circulans xylanase were built based on the database search results of related proteins. The model structures were refined and compared to the recently solved xylanase X‐ray crystal structure. The overall structural similarity between the theoretical model and experimental structure demonstrate the usefulness of this approach. Disagreement in folding topology, however, warrants further research into the inverse protein folding approach.
Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: Characterization of the active site and evidence for an interdomain carboxy‐terminal helix in ornithine transcarbamoylase
Authors: Murata Lauren B. , Schachman H.K.
Published: July 2010

Predictions of tertiary structures of proteins from their amino acid sequences are facilitated greatly when the structures of homologous proteins are known. On this basis, structural features of Escherichia coli ornithine transcarbamoylase (OTCase) were investigated by site‐directed mutagenesis experiments based on the known tertiary structure of the catalytic (c) chain of E. coli aspartate transcarbamoylase (ATCase). In ATCase, each c chain is composed of two globular domains connected by two interdomain helices, one of which is near the C‐terminus and is critical for the in vivo folding of the chains and their assembly into trimers. Each active site is located at the interface between two chains and requires the participation of residues from each of the adjacent chains. OTCase, a trimeric enzyme, has been proposed to be similar in structure to the ATCase trimer on the basis of sequence identity (32%), the nature of the reaction catalyzed by the enzyme, and secondary structure predictions. As shown here, analysis of OTCase and ATCase sequences revealed extensive evolutionary conservation in portions corresponding to the ATCase active site and the C‐terminal helix. Truncations and substitutions within the predicted C‐terminal helix of OTCase had effects on activity and thermal stability strikingly similar to those caused by analogous alterations in ATCase. Similarly, substitutions at either of two conserved residues, Ser 55 and Lys 86, in the proposed active site of OTCase had deleterious effects parallel to those caused by the analogous ATCase substitutions. Hybrid trimers comprised of chains from both these relatively inactive OTCase mutants exhibited dramatically increased activity, as predicted for shared active sites located at the chain interfaces. These results strongly support the hypothesis that the tertiary and quaternary structures of the two enzymes are similar.
Subunit dissociations in natural and recombinant hemoglobins
Authors: Manning Lois R. , Jenkins W. Terry , Hess John R. , Vandegriff Kim , Winslow Robert M. , Manning James M.
Published: July 2010

A precise and rapid procedure employing gel filtration on Superose‐12 to measure the tetramer‐dimer dissociation constants of some natural and recombinant hemoglobins in the oxy conformation is described. Natural sickle hemoglobin was chosen to verify the validity of the results by comparing the values with those reported using an independent method not based on gel filtration. Recombinant sickle hemoglobin, as well as a sickle double mutant with a substitution at the Val‐6(β) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in the α1β2 subunit interface, one was found to be extensively dissociated and the other completely dissociated. In addition, the absence of an effect of the allosteric regulators DPG and IHP on the dissociation constant was demonstrated. Thus, a tetramer dissociation constant can now be determined readily and used together with other criteria for characterization of hemoglobins and their interaction with small regulatory molecules.
Comparison of the structures of the cyclotheonamide a complexes of human α‐thrombin and bovine β‐trypsin
Authors: Ganesh V. , Tulinsky A. , Lee A.Y. , Clardy J.
Published: July 2010

Thrombin, a trypsin‐like serine protease present in blood, plays a central role in the regulation of thrombosis and hemostasis. A cyclic pentapeptide, cyclotheonamide A (CtA), isolated from sponges of the genus Theonella, inhibits thrombin, trypsin, and certain other serine proteases. Enzyme inhibition data for CtA indicate that it is a moderate inhibitor of α‐thrombin (Ki = 1.0 nM), but substantially more potent toward trypsin (Ki = 0.2 nM). The comparative study of the crystal structures of the CtA complexes of α‐thrombin and β‐trypsin reported here focuses on structure‐function relationships in general and the enhanced specificity of trypsin, in particular.

The crystal structures of the CtA complexes of thrombin and trypsin were solved and refined at 1.7 and 2.0 Å resolution, respectively. The structures show that CtA occupies the active site with the Pro‐Arg motif positioned in the S2 and S1 binding sites. The α‐keto group of CtA is involved in a tetrahedral intermediate hemiketal structure with Ser 195 OG of the catalytic triad and is positioned within bonding distance from, and orthogonal to, the re‐face of the carbonyl of the arginine of CtA. As in other productive binding modes of serine proteases, the Ser 214‐Gly 216 segment runs in a twisted antiparallel β‐strand manner with respect to the diaminopropionic acid (Dpr)‐Arg segment of CtA. The Tyr 60A‐Thr 601 insertion loop of thrombin makes a weak aromatic stacking interaction with the v‐Tyr of CtA through Trp 60D. The Glu 39 Tyr and Leu 41 Phe substitutions in trypsin produce an enhanced aromatic interaction with D‐Phe of CtA, which also leads to different orientations of the side chains of D‐Phe and the V‐Tyr.

The comparison of the CtA complexes of thrombin and trypsin shows that the gross structural features of both in the active site region are the same, whereas the differences observed are mainly due to minor insertions and substitutions. In trypsin, the substitution of Ile 174‐Arg 175 by Gly 174‐Gln 175 makes the S3 aryl site more polar because the Arg 175 side chain is directed away from thrombin and into the solvent, whereas Gln 175 is not. Because the site is occupied by the Dpr group of CtA, the occupancy of the S3 site is better in trypsin than in thrombin. In trypsin, the D‐Phe side chain of CtA fits between Tyr 39 and Phe 41 in a favorable manner, whereas in thrombin, these residues are Glu 39 and Leu 41. The higher degree of specificity for trypsin is most likely the result of these substitutions and the absence of the fairly rigid Tyr 60A‐Thr 601 insertion loop of thrombin, which narrows access to the active site and forces less favorable orientations for the D‐Phe and v‐Tyr residues.
Structural investigation of the alpha‐1‐antichymotrypsin: Prostate‐specific antigen complex by comparative model building
Authors: Villoutreix Bruno O. , Teleman Olle , Lilja Hans , Pettersson Kim , Lövgren Timo
Published: July 2010

Prostate‐specific antigen (PSA), produced by prostate cells, provides an excellent serum marker for prostate cancer. It belongs to the human kallikrein family of enzymes, a second prostate‐derived member of which is human glandular kallikrein‐1 (hK2). Active PSA and hK2 are both 237‐residue kallikrein‐like proteases, based on sequence homology. An hK2 model structure based on the serine protease fold is presented and compared to PSA and six other serine proteases in order to analyze in depth the role of the surface‐accessible loops surrounding the active site. The results show that PSA and hK2 share extensive structural similarity and that most amino acid replacements are centered on the loops surrounding the active site. Furthermore, the electrostatic potential surfaces are very similar for PSA and hK2.

PSA interacts with at least two serine protease inhibitors (serpins): alpha‐1‐antichymotrypsin (ACT) and protein C inhibitor (PCI). Three‐dimensional model structures of the uncleaved ACT molecule were developed based upon the recent X‐ray structure of uncleaved antithrombin. The serpin was docked both to PSA and hK2. Amino acid replacements and electrostatic complementarities indicate that the overall orientation of the proteins in these complexes is reasonable. In order to investigate PSA's heparin interaction sites, electrostatic computations were carried out on PSA, hK2, protein C, ACT, and PCI. Two heparin binding sites are suggested on the PSA surface and could explain the enhanced complex formation between PSA and PCI, while inhibiting the formation of the ACT‐PSA complex. PSA, hK2, and their preliminary complexes with ACT should facilitate the understanding and prediction of structural and functional properties for these important proteins also with respect to prostate diseases.
Purification and characterization of dihydroorotate dehydrogenase a from lactococcus lactis, crystallization and preliminary X‐ray diffraction studies of the enzyme
Authors: Nielsen Finn S. , Jensen Kaj Frank , Rowland Paul , Larsen Sine
Published: July 2010

Lactococcus lactis is the only organism known to contain two dihydroorotate dehydrogenases, i.e., the A‐ and B‐forms. In this paper, we report the overproduction, purification, and crystallization of dihydroorotate dehydrogenase A. In solution, the enzyme is bright yellow. It is a dimer of subunits (34 kDa) that contain one molecule of flavin mononucleotide each. The enzyme shows optimal function in the pH range 7.5–9.0. It is specific for L‐dihydroorotate as substrate and can use dichlorophenolindophenol, potassium hexacyanoferrate(III), and, to a lower extent, also molecular oxygen as acceptors of the reducing equivalents, whereas the pyridine nucleotide coenzymes (NAD+, NADP+) and the respiratory quinones (i.e., vitamins Q6, Q10 and K2) were inactive. The enzyme has been crystallized from solutions of 30% polyethylene glycol, 0.2 M sodium acetate, and 0.1 M Tris‐HC1, pH 8.5. The resulting yellow crystals diffracted well and showed little sign of radiation damage during diffraction experiments. The crystals are monoclinic, space group P2, with unit cell dimensions a = 54.19 Å, b = 109.23 Å, c = 67.17 Å, and β = 104.5°. A native data set has been collected with a completeness of 99.3% to 2.0 Å and an Rsym value of 5.2%. Analysis of the solvent content and the self‐rotation function indicates that the two subunits in the asymmetric unit are related by a noncrystallographic twofold axis perpendicular to the crystallographic b and c axes.
Protein design automation
Authors: Dahiyat Bassil I. , Mayo Stephen L.
Published: July 2010

We have conceived and implemented a cyclical protein design strategy that couples theory, computation, and experimental testing. The combinatorially large number of possible sequences and the incomplete understanding of the factors that control protein structure are the primary obstacles in protein design. Our protein design automation algorithm objectively predicts protein sequences likely to achieve a desired fold. Using a rotamer description of the side chains, we implemented a fast discrete search algorithm based on the Dead‐End Elimination Theorem to rapidly find the globally optimal sequence in its optimal geometry from the vast number of possible solutions. Rotamer sequences were scored for steric complementarity using a van der Waals potential. A Monte Carlo search was then executed, starting at the optimal sequence, in order to find other high‐scoring sequences. As a test of the design methodology, high‐scoring sequences were found for the buried hydrophobic residues of a homodimeric coiled coil based on GCN4‐p1. The corresponding peptides were synthesized and characterized by CD spectroscopy and size‐exclusion chromatography. All peptides were dimeric and nearly 100% helical at 1 °C, with melting temperatures ranging from 24 °C to 57 °C. A quantitative structure activity relation analysis was performed on the designed peptides, and a significant correlation was found with surface area burial. Incorporation of a buried surface area potential in the scoring of sequences greatly improved the correlation between predicted and measured stabilities and demonstrated experimental feedback in a complete design cycle.
A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: Identification of duplicated repeats and potential active site residues
Authors: Ponting C.P. , Kerr I.D.
Published: July 2010

Phosphatidylcholine‐specific phospholipase D (PLD) enzymes catalyze hydrolysis of phospholipid phosphodiester bonds, and also transphosphatidylation of phospholipids to acceptor alcohols. Bacterial and plant PLD enzymes have not been shown previously to be homologues or to be homologous to any other protein. Here we show, using sequence analysis methods, that bacterial and plant PLDs show significant sequence similarities both to each other, and to two other classes of phospholipid‐specific enzymes, bacterial cardiolipin synthases, and eukaryotic and bacterial phosphatidylserine synthases, indicating that these enzymes form an homologous family. This family is suggested also to include two Poxviridae proteins of unknown function (p37K and protein K4), a bacterial endonuclease (nuc), an Escherichia coli putative protein (o338) containing an N‐terminal domain showing similarities with helicase motifs V and VI, and a Synechocystis sp. putative protein with a C‐terminal domain likely to possess a DNA‐binding function. Surprisingly, four regions of sequence similarity that occur once in nuc and o338, appear twice in all other homologues, indicating that the latter molecules are bi‐lobed, having evolved from an ancestor or ancestors that underwent a gene duplication and fusion event. It is suggested that, for each of these enzymes, conserved histidine, lysine, aspartic acid, and/or asparagine residues may be involved in a two‐step ping‐pong mechanism involving an enzyme‐substrate intermediate.
Preliminary crystallographic studies of an anti‐HIV‐1 protease antibody that inhibits enzyme activity
Authors: Lescar Julien , Stouracova Renata , Riottot Marie‐Madeleine , Chitarra Véronique , Brynda Jiri , Fabry Milan , Horejsi Magda , Sedlacek Juraj , Bentley Graham A.
Published: July 2010

F11.2.32, a monoclonal antibody directed against the HIV‐1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme. The antibody cross‐reacts with peptides 36–46 and 36–57 from the protease. Crystals of the Fab have been obtained both in the free state and as complexes formed with the protease peptide fragments, 36–46 and 36–57. Diffraction data have been collected for the free and complexed forms of Fab F11.2.32 and preliminary models for the crystal structures were obtained by molecular replacement.
Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S‐transferase, 1–1
Authors: Wang Jibo , Barycki Joseph J. , Colman Roberta F.
Published: July 2010

Reaction of rat liver glutathione S‐transferase, isozyme 1–1, with 4‐(fluorosulfonyl)benzoic acid (4‐FSB), a xenobiotic substrate analogue, results in a time‐dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with l‐chloro‐2,4‐dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4‐FSB from 0.25 mM to 9 mM, characterized by a KI of 0.78 mM and kmax of 0.011 min−1. S‐Hexylglutathione or the xenobiotic substrate analogue, 2,4‐dinitrophenol, protects against inactivation of the enzyme by 4‐FSB, whereas S‐methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5‐3H]‐4‐FSB into the enzyme in the absence and presence of S‐hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4‐FSB, but only Tyr 8 is protected against 4‐FSB by S‐hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4‐FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4‐FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme‐catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pKa, which has been interpreted as representing the ionization of enzyme‐bound glutathione; however, this pKa of about 7.4 for modified enzyme remains far below the pK of 9.1 for the ‐SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S‐transferase, but is not required for enzyme activity.
A natural variant of type I antifreeze protein with four ice‐binding repeats is a particularly potent antifreeze
Authors: Chao Heman , Hodges Robert S. , Kay Cyril M. , Gauthier Sherry Y. , Davies Peter L.
Published: July 2010

A 4.3‐kDa variant of Type I antifreeze protein (AFP9) was purified from winter flounder serum by size exclusion chromatography and reversed‐phase HPLC. By the criteria of mass, amino acid composition, and N‐terminal sequences of tryptic peptides, this variant is the posttranslationally modified product of the previously characterized AFP gene 21a. It has 52 amino acids and contains four 11‐amino acid repeats, one more than the major serum AFP components. The larger protein is completely α‐helical at 0 °C, with a melting temperature of 18 °C. It is considerably more active as an antifreeze than the three‐repeat winter flounder AFP and the four‐repeat yellowtail flounder AFP, both on a molar and a mg/mL basis. Several structural features of the four‐repeat winter flounder AFP, including its larger size, additional ice‐binding residues, and differences in ice‐binding motifs might contribute to its greater activity. Its abundance in flounder serum, together with its potency as an antifreeze, suggest that AFP9 makes a significant contribution to the overall freezing point depression of the host.
Microheterogeneity of human filaggrin: Analysis of a complex peptide mixture using mass spectrometry
Authors: Thulin Craig D. , Taylor J. Alex , Walsh Kenneth A.
Published: July 2010

Filaggrin is the product of posttranslational processing of the large, epidermal protein profilaggrin, which consists of 10 or more tandem filaggrin domains plus an amino and a carboxyl domain. According to fragmentary cDNA sequences, the filaggrin domains in the human protein vary at 40% of the amino acid positions; hence, mature filaggrin is a population of homologous but heterogeneous proteins, even within one individual. Available gene sequences give only a limited picture of the heterogeneity of human filaggrin protein because no complete human profilaggrin gene has been sequenced. Questions about the extent of heterogeneity of filaggrin within and between individuals have not been answered, nor have questions concerning the limited proteolytic cleavage of human profilaggrin that generates filaggrin in vivo.

In order to address these questions and to provide an analysis of the primary structure of human filaggrins, we employed various methods of mass spectrometry. The intact protein and a tryptic digest of the mixture of human filaggrins were examined by matrix‐assisted laser desorption time‐of‐flight mass spectrometry. Tryptic digests of human filaggrin from single individuals were also separated and analyzed by liquid chromatography/mass spectrometry (LC/MS) (using electrospray mass spectrometry), and specific peptides were identified by tandem mass spectrometry (MS/MS). A robust data analysis program, Sherpa, was developed to facilitate the interpretation of both LC/MS and MS/MS. These experiments show that human filaggrin includes heterogeneity not yet seen in cDNA sequences, but that much structure is highly conserved. Interestingly, we found that the heterogeneity is conserved among individuals. An approximation of the regions linking filaggrins in human profilaggrin is developed. These investigations provide a unique test of the limits of tryptic mapping of complex mixtures using mass spectrometry.
Preliminary crystallographic analysis of the oxidized form of a two mono‐nuclear iron centres protein from desulfovibrio desulfuricans ATCC 27774
Authors: Coelho Ana V. , Matias Pedro M. , Carrondo Maria A. , Tavares P. , Moura J. J. G. , Moura Isabel , Fülop V. , Hajdu J. , Le Gall Jean
Published: July 2010

Crystals of the fully oxidized form of desulfoferrodoxin were obtained by vapor diffusion from a solution containing 20% PEG 4000, 0.1 M HEPES buffer, pH 7.5, and 0.2 M CaCl2. Trigonal and/or rectangular prisms could be obtained, depending on the temperature used for the crystal growth. Trigonal prisms belong to the rhombohedral space group R32, with a = 112.5 Å and c = 63.2 Å; rectangular prisms belong to the monoclinic space group C2, with a = 77.7 Å, b = 80.9 Å, c = 53.9 Å, and β =98.1°. The crystallographic asymmetric unit of the rhombohedral crystal form contains one molecule. There are two molecules in the asymmetric unit of the monoclinic form, in agreement with the self‐rotation function.
Subcloning of a dna fragment encoding a single cohesin domain of the clostridium thermocellum cellulosome‐integrating protein cipA: Purification, crystallization, and preliminary diffraction analysis of the encoded polypeptide
Authors: Béguin Pierre , Raynaud Odette , Chaveroche Marie‐Kim , Dridi Abel , Alzari Pedro M.
Published: July 2010

An Escherichia coli clone encoding a single cohesin domain of the cellulosome‐integrating protein CipA from Clostridium thermocellum was constructed, and the corresponding polypeptide was purified, treated with papain, and crystallized from a PEG 8000 solution. Crystals exhibit orthorhombic symmetry, space group P212121, with cell dimensions a = 37.7 Å, b = 80.7 Å, c = 93.3 Å, and four or eight molecules in the unit cell. The crystals diffract X‐rays to beyond 2 Å resolution and are suitable for further crystallographic studies.
Friends and relations of the cystatin superfamily—new members and their evolution
Authors: Brown W.M. , Dziegielewska K.M.
Published: July 2010

The cystatin “superfamily” encompasses proteins that contain multiple cystatin‐like sequences. Some of the members are active cysteine protease inhibitors, while others have lost or perhaps never aquired this inhibitory activity. In recent years, several new members of the superfamily have been characterized, including proteins from insects and plants. Based on partial amino acid homology, new members, such as the invariant chain (Ii), and the transforming growth factor‐β receptor type II (TGF‐β receptor II) may, in fact, represent members of an emerging family within the superfamily that may have used some common building blocks to form functionally diverse proteins. Cystatin super‐family members have been found throughout evolution and members of each family of the superfamily are present in mammals today. In this review, the new and older, established members of the family are arranged into a possible evolutionary order, based on sequence homology and functional similarities.
Structure of the β2 homodimer of bacterial luciferase from vibrio harveyi: X‐ray analysis of a kinetic protein folding trap
Authors: Thoden James B. , Holden Hazel M. , Fisher Andrew J. , Sinclair James F. , Wesenberg Gary , Baldwin Thomas O. , Rayment Ivan
Published: July 2010

Luciferase, as isolated from Vibrio harveyi, is an aβ heterodimer. When allowed to fold in the absence of the α subunit, either in vitro or in vivo, the β subunit of the enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 °C. This form of the β subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol 1: 320–326). Here we describe the X‐ray crystal structure of the β2 homodimer of luciferase from V. harveyi determined and refined at 1.95 Å resolution. Crystals employed in the investigation belonged to the orthorhombic space group P212121 with unit cell dimensions of a = 58.8 Å, b = 62.0 Å, and c = 218.2 Å and contained one dimer per asymmetric unit. Like that observed in the functional luciferase αβ heterodimer, the major tertiary structural motif of each β subunit consists of an (α/β)8 barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three‐dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 Å resolution. Biochemistry 34: 6581–6586). The root‐mean‐square deviation of the α‐carbon coordinates between the β subunits of the hetero‐ and homodimers is 0.7 Å. This high resolution X‐ray analysis demonstrates that “domain” or “loop” swapping has not occurred upon formation of the β2 homodimer and thus the stability of the β2 species to denaturation cannot be explained in such simple terms. In fact, the subunit:subunit interfaces observed in both the β2 homodimer and αβ heterodimer are remarkably similar in hydrogen‐bonding patterns and buried surface areas.
Molecular modeling of the structures of human and rat pancreatic cholesterol esterases
Authors: Feaster Shawn R. , Quinn Daniel M. , Barnett Bobby L.
Published: July 2010

Structural models have been generated for rat and human cholesterol esterases by molecular modeling. For rat cholesterol esterase, three separate models were generated according to the following procedure: (1) the cholesterol esterase sequence was aligned with those of three template enzymes: Torpedo californica acetylcholinesterase, Geotrichum candidum lipase and Candida rugosa lipase; (2) the X‐ray structure coordinates of the three template enzymes were used to construct cholesterol esterase models by amino acid replacements of matched sequence positions and by making sequence insertions and deletions as required; (3) bad contacts in each of the cholesterol esterase models were relaxed by molecular dynamics and mechanics; (4) the three cholesterol esterase models were merged into one by arithmetic averaging of atomic coordinates; (5) Ramachandran analysis indicated that the model generated from the AChE template possessed the best set of ϕ/Ψ angles. Therefore, this model was subjected to molecular dynamics, with harmonic constraints imposed on the Cα coordinates to drive them toward the coordinates of the averaged model. (6) Subsequent relaxation by molecular mechanics produced the final rat cholesterol esterase model.

A model for human cholesterol esterase was produced by repeating steps 1–3 above, albeit with the rat cholesterol esterase model as the template. Hydrophobic and electrostatic analyses of the rat and human cholesterol esterase models suggest the structural origins of molecular recognition of hydrophobic substrates and interfaces, of charged interfaces, and of bile salt activators.