bluuebunny/crossref_metadata_2025 huggingface dataset:
A genetic screen to identify variants of bovine pancreatic trypsin inhibitor with altered folding energetics
Authors: Coplen Lonnie J. , Frieden Richard W. , Goldenberg David P.
Published: May 2005

A genetic screening procedure has been developed to identify mutant forms of bovine pancreatic trypsin inhibitor (BPTI) that can fold to an active conformation but are inactivated more rapidly than the wild type protein. Small cultures of Escherichia coli containing plasmids with mutagenized BPTI genes were grown in microtiter plates, lysed, and treated with dithiothreitol (DTT). Under these conditions, unfolding and inactivation of wild‐type protein has a half‐time of about 10 hours. Variants of BPTI that are inactivated within 1 hour were identified by adding trypsin and a chromogenic substrate. Approximately 11,000 mutagenized clones were screened in this way and 75 clones that produce proteins that can fold but are inactivated by DTT were isolated. The genes coding for 68 “DTT‐sensitive” mutant proteins were sequenced, and 25 different single amino acid substitutions at 15 of the 58 residues of the protein were identified. Most of the altered residues are largely buried in the core of the naive wild‐type structure and are highly conserved among proteins homologous to BPTI. These results indicate that a large fraction of the sequence of the protein contributes to the kinetic stability of the active conformation, but it also appears that substitutions can be tolerated a most sites without completely preventing folding Because this genetics, further studies of the isolated mutants are expected to provide information about the roles of the altered residues in folding and unfolding.
An expectation maximization (EM) algorithm for the identification and characterization of common sites in unaligned biopolymer sequences
Authors: Lawrence Charles E. , Reilly Andrew A.
Published: May 2005

Statistical methodology for the identification and characterization of protein binding sites in a set of unaligned DNA fragments is presented. Each sequence must contain at least one common site. No alignment of the sites is required. Instead, the uncertainty in the location of the sites is handled by employing the missing information principle to develop an “expectation maximization” (EM) algorithm. This approach allows for the simultaneous identification of the sites and characterization of the binding motifs. The reliability of the algorithm increases with the number of fragments, but the computations increase only linearly. The method is illustrated with an example, using known cyclic adenosine monophophate receptor protein (CRP) binding sites. The final motif is utilized in a search for undiscovered CRP binding sites.
Polyproline, β‐turn helices. Novel secondary structures proposed for the tandem repeats within rhodopsin, synaptophysin, synexin, gliadin, RNA polymerase II, hordein, and gluten
Authors: Matsushima Norio , Creutz Carl. E. , Kretsinger Robert H.
Published: May 2005

Seven proteins each contain 8 to 52 tandem repeats of a unique class of oligopeptide. The consensus peptide for each is

rhodopsin Tyr Pro Pro Gln Gly

synapto‐physin Tyr Gly Pro Gln Gly

synexin Tyr Pro Pro Pro Pro Gly

gliadin Tyr Pro Pro Pro Gln Pro

RNA polymerase II Tyr Ser Pro Thr Ser Pro Ser

hordein Phe Pro Gln Gln Pro Gln Gln Pro

gluten Tyr Pro Thr Ser Pro Gln Gn Gly Tyr

Although there is obvious variations of sequence and of length, the penta‐to nonapeptides share an initial Tyr(or Phe) and have high Pro contents and abundant Gly, Gln, and Ser. We have evaluated helical models that both recognize the uniqueness of these sequence repeats and accommodate variations on the basic theme.

We have developed a group of related heical model for these proteins with about three oligopeptide repeats per turn of 10–20 Å. These models share several common features: Most of the ϕ dihedral angels are −54°, to accommodate Pro at all positions expect the first (Tyr). Except for the β‐turns, most ψ dihedral angles are near +140° as found in polyproline. Each oligopeptide has at least one β‐turn; several have two. Some contains a cis‐Tyr, Pro peptide bond; a few have a cis‐bond plus one β‐turn. Tyr side chains vary from totally exposed to buried within the helices and could mode to accommodate either external hydrophobic interactions or phosphorylation. The several related structures seem to be readily interconverted without major change in the overall helical parameters, and therein may lie the key to their functions.
Substrate recognition by the EcoRI endonuclease
Authors: Heitman Joseph , Model Peter
Published: May 2005

The EcoRI restriction endonuclease is one of the most widely used tools for recombinant DNA manipulations. Because the EcoRI enzyme has been extremely well characterized biochemically and its structure is known at 3 Å resolution as an enzyme–DNA complex, EcoRI also serves as an important model of DNA–protein interactions. To facilitate a genetic analysis of the EcoRI enzyme, we devised an in vivo DNA scission assay based on our finding that DNA double‐strand breaks induce the Escherichia coli SOS response and thereby increase β‐galactosidase expression from SOS::lacZ gene fusions. By site‐directed mutagenesis, 50 of 60 possible point mutations were generated at three amino acids (E144, R145, and R200) implicated in substrate recognition by the crystal structure. Although several of these mutant enzymes retain partial endonuclease activity, none are altered in substrate specificity in vivo or in vitro. These findings argue that, in addition to the hydrogen bond interactions revealed by the crystal structure, the EcoRI enzyme must make additional contacts to recognize its substrate.
Modelling of the human intercellular adhesion molecule‐1, the human rhinovirus major group receptor
Authors: Giranda Vincent L. , Chapman Michael S. , Rossmann Michael G.
Published: May 2005

A model has been built of the amino‐terminal domain of the intercellular adhesion molecule‐1 (ICAM‐1), the receptor for most human rhinovirus serotypes. The model was based on sequence and presumed structural homology to immunoglobulin constant domains. It fits well into the putative receptors attachment site, the canyon, on the human rhinovirus‐14 (HRV14) surface in a manner consistent with most of the mutational data for ICMA‐1 (Staunton, D. E., Dustin, M. L., Erickson, H. P., Springer, T. A. Cell, in press, 1989) and HRV14 (Colonno, R. J., Condra, J. H., Mizutani, S., Callahan, P. L., Davies, M. E., Murcko, M. A. Proc. Natl. Acad. Sci. U.S.A. 85: 5449‐5453, 1988).
Automated docking of substrates to proteins by simulated annealing
Authors: Goodsell David S. , Olson Arthur J.
Published: May 2005

The Metropolis technique of conformation searching is combined with rapid energy evaluation using molecular affinity potentials to give an efficient procedure for docking substrates to macromolecules of known structure. The procedure works well on a number of crystallographic test systems, functionally reproducing the observed binding modes of several substrates.
Models of δ‐hemolysin membrane channels and crystal structures
Authors: Raghunathan G. , Seetharamulu P. , Brooks B. R. , Guy H. R.
Published: May 2005

Molecular modeling and energy calculations have been used to study how δ‐hemolysin and melittin helices may aggregate on membrane surfaces and insert through membranes to form channels. In these models adjacent antiparallel amphipathic helices form planar “raft” structures, in which one surface is hydrophobic and the other hydrophilic. Models of δ‐hemolysin crystal structure were developed using these “rafts.” These models are based on the unit cell constants and the crystal symmetry obtained from the preliminary crystal data. Energy calculations favor channel models of δ‐hemolysin with six or eight monomers per channel.
Changes in secondary structure follow the dissociation of human insulin hexamers: A circular dichroism study
Authors: Melberg Steen G. , Johnson W. Curtis
Published: May 2005

Vacuum UV circular dichroism spectra measured down to 178 nm for hexameric 2‐zinc human insulin, zinc‐free human insulin, and the two engineered and biologically active monomeric mutants, [B/S9D] and [BS9D, T27E] human insulin, show significant differences. The secondary structure analysis of the 2‐zinc human insulin (T6) in neutral solution was determined: 57% helix, 1% β‐strand, 18% turn, and 24% random coil. This is very close to the corresponding crystal structure showing that the solution and solid structures are similar. The secondary structure of the monomer shows a 10–15% increase in antiparallel β‐structure and a corresponding reduction in random coil structure. These structural changes are consistent with an independent analysis of the corresponding difference spectra. The advantage of secondary structure analyses of difference spectra is that the contribution of odd spectral features stemming mainly from side chain chromophores is minimized and the sensitivity of the analyses improved. Analysis of the CD spectra of T6 2‐zinc, zinc‐free human insulin and monomeric mutant insulin by singular value decomposition indicates that the secondary structure changes following the dissociation of hexamers into dimers and monomers are two‐state processes.
The effect of solvent viscosity on the rate‐determining step of fatty acid synthetase
Authors: Kyushiki Hiroyuki , Ikai Atsushi
Published: May 2005

The overall activity of animal fatty acid synthetase at the saturation level of substrate concentration decreased when the solvent viscosity, η, of the reaction mixture was increased with viscogens such as glycerol, sucrose, and polyethylene glycol. The activity of the enzyme changed roughly proportional to η−p, where p = 1.0 for glycerol, p = 0.66 for sucrose, and p < 0.6 for polyethylene glycol with different molecular sizes. The thioesterase activity, which catalyzes the final partial reaction in the multifunctional enzyme, was not affected by 5‐fold increase of solvent viscosity with sucrose. These results suggested that the rate‐determining step of the enzyme other than the thioesterase reaction involves a microscopic transport step, the rate of which is influenced by the solvent viscosity. The microscopic transport step may be related to the transfer of the reaction intermediate from one active site to another or to the motion of a larger part of the enzyme requisite for the catalytic reaction. In the solution containing glycerol, the rate‐determining motion was primarily diffusion limited since the inverse of the initial rate was proportional to η, i.e., p = 1. Since the substrate concentration was at a saturation level in this experiment, the viscosity‐dependent step cannot be the encounter between the enzyme and substrates, but must be intramolecular in origin, most probably the reaction catalyzed by β‐ketoacyl synthetase. In solutions containing other viscogens, however, p was less than 1.0, indicating a significant involvement of chemical steps in the rate‐determining step as well. Bovine serum albumin, when used as a proteinic viscogen, also decreased the initial rate. In the living cells therefore, the rate‐determining intramolecular motion of fatty acid synthetase could be affected by the presence of high concentrations of cytoplasmic proteins.
Hormone phage: An enrichment method for variant proteins with altered binding properties
Authors: Bass Steven , Greene Ronald , Wells James A.
Published: May 2005

Human growth hormone (hGH), a 191 residue protein containing two disulfide bonds, was fused to the carboxyl‐terminal domain of the gene III protein, a minor coat protein exposed at one end of the filamentous phage M13. The gene fusion was cloned into a plasmid containing origins of replication for Escherichia coli and filamentous phage and was packaged into phagemid particles upon infection by an M13KO7 helper phage. Transcription of the hGH‐gene III fusion was controlled so that usually no more than one copy of the fusion protein was displayed along with the four copies of the wild‐type gene III protein. The hGH‐gene III fusion protein was properly folded, as judged by reactivity with six hGH monoclonal antibodies whose epitopes are sensitive to the folded conformation of hGH. Moreover, the hGH–gene III phagemid particles were enriched over 5000‐fold from non‐hGH phage, and 8‐fold from a mutant hGH phagemid following a single hGH‐specific elution step from hGH receptor‐coated beads. The hGH phagemid should be useful for isolating new receptor binding mutants of hGH. More generally, this expression system may allow other large proteins with discontinuous binding epitopes to be displayed, and binding selections applied to their mutated gene III fusions on filamentous phage.
Fragment peptide library for classification and functional prediction of proteins
Authors: Seto Yasuhiko , Ikeuchi Yoshinori , Kanehisa Minoru
Published: May 2005

From protein sequence comparison data found in the literature, a library was organized using peptide fragment sequences which are common to related proteins. Each of the fragments was then examined for its occurrence in all the protein superfamilies defined by the NBRF‐PIR data base. We have selected those fragment peptides that appear exclusively in one or a few superfamilies, and thus made a library of fragment peptides that characterize specific superfamilies. Such characteristic peptides are, in general, five to seven residues long and contain unusually high proportions of glycine and cysteine. This collection is a useful resource for the classification and functional prediction of protein molecules.
Refinement of the NMR structures for acyl carrier protein with scalar coupling data
Authors: Kim Yangmee , Prestegard James H.
Published: May 2005

Structure determination of small proteins using NMR data is most commonly pursued by combining NOE derived distance constraints with inherent constraints based on chemical bonding. Ideally, one would make use of a variety of experimental observation, not just distance constraints. Here, coupling constant constraints have been added to molecular mechanics and molecular dynamics protocols for structure determination in the form of a psuedoenergy function that is minimized in a search for an optimum molecular conformation. Application is made to refinement of a structure for a 77 amino acid protein involved in fatty acid synthesis, Escherichia coli acyl carrier protein (ACP). 54 3JHNα coupling constants, 12 coupling constants for stereospecifically assigned side chain protons, and 450 NOE distance contraints were used to calculate the 3‐D structure of ACP. A three‐step protocol for a molecular dynamics calculation is described, in analogy to the protocol previously used in molecular mechanics calculations. The structures calculated with the molecular mechanics approach and the molecular dynamics apporach using a rigid model for the protein show similar molecular energies and similar agreement with experimental distance and coupling constant constraints. The molecular dynamics approach shows some advantage in overcoming local minimum problems, but only when a two‐state averaging model for the protein was used, did molecular energies drop significantly.
Modeling of agonist binding to the ligand‐gated ion channel superfamily of receptors
Authors: Cockcroft Victor B. , Osguthorpe David J. , Barnard Eric A. , Lunt George G.
Published: May 2005

A generalized model is presented of agonist binding to ligand‐gated ion channels (LGICs). Broad similarity in the structure of agonists suggests that the binding sites of LGICs may have evolved from a protobinding site. Aligned sequence data identified as a candidate for such a site a highly conserved 15 residue stretch of primary structure in the N‐terminal extracellular region of all known LGIC subunits. We modeled this subregion, termed the cys‐loop, as a rigid, amphiphilic β‐hairpin and propose that it may form a major determinant of a conserved structural binding cleft.

In the model of the binding complex (1) an invariant aspartate residue at position 11 of the cys‐loop is the anionic site interacting with the positively charged amine group of agonists, (2) a local dipole within the π‐electron system of agonists is favorably oriented in the electrostatic field of the invariant aspartate, (3) the ε ring‐proton of a conserved aromatic residue at the turn of the cys‐loop interacts orthogonally with the agonist α‐electron density at its electronegative center, and (4) selective recognition is partly a result of the type of amino acid residue at position 6 of the cys‐loop. Additionally, the formation of a hydrogen bond between the electronegative atom of the π‐electron system of agonist and a complementary group in the receptor may be important in the high‐affinity binding of agonists.
Predicted three‐dimensional structure of the protease inhibitor domain of the Alzheimer's disease β‐amyloid precursor
Authors: Struthers R. Scott , Kitson David H. , Hagler Arnold T.
Published: May 2005

Alzheimer's disease is characterized by the deposition of amyloid β‐protein as plaques and tangles in the brains of its victims. The amyloid precursor can be expressed with or without the inclusion of a protease inhibitor domain, the potential role of which in amyloidogenesis has prompted the generation of a model of its three‐dimensional structure based on the known structure of a related inhibitor. The model structure predicts that the mutated residues are almost entirely on the surface of the inhibitor domain, while conserved residues constitute the hydrophobic core. In addition, several pairs of structurally complementary, or concerted, mutations are seen. These structural features provide strong evidence for the validity of the modeled structure, and it is suggested that the presence of complementary mutations may be used as a criterion for evaluating protein structures built by homology, in addition to the (spatial) location of the mutations. The terminal residues delimiting the domain are among those furthest from the protease binding site and are in close proximity to one another, thus suggesting the ability of the domain to function as a structural cassette within the context of a larger protein. The electrostatic potentials of the inhibitor and of the related bovine pancreatic trypsin inhibitor reveal how two inhibitors with very different net charges can bind with approximately the same binding constant to trypsin and suggest a mutation of trypsin that might selectively enhance the binding of the amyloid inhibitor domain. The model provides a structural basis for understanding the functional roles of residues in the domain and for designing simpler molecules to test as pharmacologic agents for intervention in Alzheimer's disease.
Assignment of the nucleotide binding sites and the mechanism of substrate inhibition of Escherichia coli adenylate kinase
Authors: Liang Peng , Phillips George N. , Glaser Michael
Published: May 2005

Site‐directed mutagenesis of key amino acids of adenylate kinase has been used to suggest a new model for the location of the AMP and ATP binding sites. Phe‐86 and Tyr‐133, which are in close contact with the inhibitor Ap5A according to previous crystallographic results, have been independently changed to tryptophan and other amino acids. The Phe‐86→Trp mutant had a 3‐ to 6‐fold change in the Km for ATP and a 44‐fold increase in the Km for AMP with a simultaneous loss of AMP substrate inhibition. Thus Phe‐86 is probably in close contact with bound AMP. The Tyr‐133→Trp mutant showed no large effects on enzyme kinetics and suggests that the previous assignment of Ap5A occupying natural adenosine binding sites is probably incorrect. A temperature‐sensitive Leu‐107→Gln mutant showed a 6‐fold decrease in the Km for ATP and no effect on AMP binding, suggesting that this amino acid is near the ATP binding site.

Changes in the fluorescence of single tryptophan‐containing mutant enzymes provided specific information about AMP and ATP binding. The fluorescence results are consistent with the kinetic studies, and also suggest that AMP substrate inhibition is caused by the formation of an abortive complex that prevents the release of product.
Distribution and complementarity of hydropathy in mutisunit proteins
Authors: Korn Alex P. , Burnett Roger M.
Published: May 2005

A survey of 40 multisubunit proteins and 2 protein–protein complexes was performed to assay quantitatively the distribution of hydropathy among the exterior surface, interior, contact surface, and noncontact exterior surface of the isolated subunits. We suggest a useful way to present this distribution by using a “hydropathy level diagram.” Additionally, we have devised a function called “hydropathy complementarity” to quantitate the degree to which interacting surfaces have matching hydropathy distributions. Our survey revealed the following patters: (1) The difference in hydropathy between the interior and exterior of subunits is a fairly invariant quantity. (2) On average, the hydropathy of the contact surface is higher than that of the exterior surface, but is not greater than that of the protein as a whole. There was variation, however, among the proteins. In some instances, the contact surface was more hydrophilic than the noncontact exterior, and in a few cases the contact surface was as hydrophobic as the protein interior. (3) The average interface manifests significant hydropathy complementarity, signifying that proteins interact by placing hydrophobic centers of one surface against hydrophobic centers of the other surface, and by similarly matching hydrophilic centers. As a measure of recognition and specificity, hydropathy complementarity could be a useful tool for predicting correct docking of interacting proteins. We suggest that high hydropathy complementarity is associated with static inflexible interactions. (4) We have found that some subunits that bind predominantly through hydrophilic forces, such as hydrogen bonds, ionic pairs, and water and metal bridges, are involved in dynamic quaternary organization and allostery.
Refined structure of melittin bound to perdeuterated dodeclylphoscholine micelles as studied by 2D‐NMR and distance geometry calculation
Authors: Ikura Teikichi , Gō Nobuhiro , Inagaki Fuyuhiko
Published: May 2005

In our previous paper we reported the conformation of melittin bound to perdeuterated dodecylphosphocholine micelles as studied by 1H NMR experiment and distance geometry calculation. No hydrogen bonds were taken into consideration explicitly in the calculation. However, mostly α‐helical conformations were obtained as results of the calculation even with no explicitly assumed hydrogen bonds. In the present paper we refined the distance geometry calculation by incorporating hydrogen bonds suggested by the previous calculation. As a result, we obtained the conformation of melittin, which was consistent with both NMR data and the additional hydrogen bonding data. The α‐helical rod in the refined conformation also has a kink at Thr‐11 and Gly‐12, but its bent angle is now a bit narrowly distributed in 135° × 15°. In the present study another distortion at Trp‐19 and IIe‐20 becomes conspicuous. The average root‐mean‐square displacement of atoms is now much smaller and is 1.5 Å for all backbone atoms. In the present paper side chain conformations are also analyzed.
The three‐dimensional structure of glutathione reductase from Escherichia coli at 3.0 Å resolution
Authors: Ermler Ulrich , Schulz Georg E.
Published: May 2005

The structure of glutathione reductase from Escherichia coli has been solved at 3 Å resolution using multiple isomorphous replacement, solvent flattening, and molecular replacement on the basis of the homologous (53% identical residues) and structurally well‐established human enzyme. The structures of both enzyme species agree with each other in a global way; there is no domain rearrangement. In detail, clear structural differences can be observed. The structure analysis of the E. coli enzyme was tackled in order to understand sitedirected mutants, the most spectacular of which changed the cofactor specificity of this enzyme from NADP to NAD (Scrutton et al., 1990, Nature 343:38–43).
Conformational perturbation of interleukin‐2: A strategy for the design of cytokine analogs
Authors: Landgraf Bryan E. , Williams Diane P. , Murphy John R. , Smith Kendall A. , Ciardelli Thomas L.
Published: May 2005

Interleukin‐2 (IL‐2) is a representative of a growing family of small proteins termed lymphokines which are responsible for mediating cell differentiation, growth and function in the immune system. Many of these proteins are being evaluated for their clinical potential. From the perspective of drug development, structure–function analysis of these molecules and their receptors require the use methodologies different than those traditionally employed for small peptides and other natural products. However, similar pharmacologic principles apply and an understanding of ligand‐receptor interactions and the asssociated responses is required in order to efficiently pursue agonist and antagonist design.

Although IL‐2 is a protein of only 133 amino acid residues for which a low resolution X‐ray structure does exist, the complexity of its receptor system has provided an added challenge to structure–function studies. Consequently, little is known concerning the receptor contact residues for this protein. We have attempted to utilize established principles of protein and peptide structure to manipulate the conformation of IL‐2 in a manner which has provided analogs helpful for receptor interactions studies. These proteins have not only providing useful information on the nature of the IL‐2 receptor but have also revealed potential strategies for the design of IL‐2 agonists and antagonists.
Phospholipase A2 at the bilayer interface
Authors: Ramirez Fausto , Jain Mahendra Kumar
Published: May 2005

Interfacial catalysis is a necessary consequence for all enzymes that act on amphipathic substrates with a strong tendency to form aggregates in aqueous dispersions. In such cases the catalytic event occurs at the interface of the aggregated substrate, the overall turnover at the interface is processive, and it is influenced the molecular organization and dynamics of the interface. Such enzymes can access the substrate only at the interface because the concentration of solitary monomers of the substrate in the aqueous phase is very low. Moreover, the microinterface between the bound enzyme and the organized substrate not only facilitates formation of the enzyme–substrate complex, but a longer residence time of the enzyme at the substrate interface also promotes high catalytic processivity.

Binding of the enzyme to the substrate interface as an additional step in the overall catalytic turnover permits adaptation of the Michaelis–Menten formalism as a basis to account for the kinetics of interfacial catalysis. As shown for the action of phospholipase A2 on bilayer vesicles, binding equilibrium has two extreme kinetic consequences. During catalysis in the scooting mode the enzyme does not leave the surface of the vesicle to which it is bound. On the other hand, in the hopping mode the absorption and desorption steps are a part of the catalytic turnover.

In this minireview we elaborate on the factors that control binding of pig pancreatic phospholipase A2 to the bilayer interface. Binding of PLA2 to the interface occurs through ionic interactions and is further promoted by hydrophobic interactions which probably occur along a face of the enzyme, with a hydrophobic collar and a ring of cationic residues, through which the catalytic site is accessible to substrate molecules in the bilayer. An enzyme molecule binds to the surface occupied by about 35 lipid molecules with an apparent dissociation constant of less than 0.1 pM for the enzyme on anionic vesicles compared to 10 mM on zwitterionic vesicles. Results at hand also show that aggregation or acylation of the protein is not required for the high affinity binding or catalytic interaction at the interface.
Hexamers of subunit II from Limulus hemocyanin (a 48‐mer) have the same quaternary structure as whole Panulirus hemocyanin molecules
Authors: Magnus Karen A. , Lattman Eaton E. , Volbeda Anne , Hol Wim G. J.
Published: May 2005

Hemocyanins are copper‐containing proteins that transport oxygen in a variety of invertebrates. Considerable evidence has accumulated that arthropodan hemocyanins are multimers of a fundamental hexameric unit. X‐Ray crystallographic structure determination has revealed that the hemocyanin molecule from the spiny lobster Panulirus interruptus is a single hexamer having 32 point group symmetry. Using crystals of subunit II, one of 8 polypeptide types comprising the octahexameric hemocyanin of the horseshoe crab Limulus polyphemus, and the molecular replacement method for crystallographic phase determination we show that subunit II forms assemblies with the same hexameric quaternary structure as the whole Panulirus hemocyanin molecule. Observation of the same hexameric motif in two widely separated species provides strong additional evidence that this quaternary structural unit is a universal building block of arthropodan hemocyanins.
Thiol protease‐like active site found in the enzyme dienelactone hydrolase: Localization using biochemical, genetic, and structural tools
Authors: Pathak Dushyant , Ashley Gary , Ollis David
Published: May 2005

The active site of dienelactone hydrolase (DLH), a microbial enzyme of the β‐ketoadipate pathway, has been conclusively located using a combination of crystallographic, biochemical, and genetic techniques. DLH hydrolyzes a dienelactone to maleylacetate and has esterase activity on p‐nitrophenyl acetate and trans‐cinnamoyl imidazole. The identification of Cys‐123 as containing the essential thiol confirms the localization of the active site as suggested by the crystal structure of DLH, and disproves an earlier hypothesis regarding its location. Two mutant proteins have been engineered in which Cys‐123 has been converted to a serine (C123S DLH) and an alanine (C123A DLH), respectively. C123S DLH (Km = 9900±2300 μM; Vmax = 4.4±0.8 μmol/min‐mg) displays burst kinetics with p‐nitrophenyl acetate and is 10% as active as DLH (Km = 170±7 μM; Vmax = 21.1±0.4 μmol/min‐mg). C123A DLH is inactive. The structures of DLH, C123S DLH, and C123A DLH have been refined at 1.8, 2.2, and 2.0 Å, respectively. Comparison of the structures of these proteins demonstrates that the only differences between them are centered at residue 123. The structures of the active sites of DLH, papain, and subtilisin are similar and are suggestive of the three enzymes having evolved convergently to similar active sites with similar enzymic mechanisms.
Energetic approach to the folding of α/β barrels
Authors: Chou Kuo‐Chen , Carlacci Louis
Published: May 2005

The folding of a polypeptide into a parallel (α/β)8 barrel (which is also called a circularly permuted β8α8 barrel) has been investigated in terms of energy minimization. According to the arrangement of hydrogen bonds between two neighboring β‐strands of the central barrel therein, such an α/β barrel structure can be folded into six different types: (1) left‐tilted, left‐handed crossover; (2) left‐tilted, right‐handed crossover; (3) nontilted, left‐handed crossover; (4) nontilted, right‐handed crossover; (5) right‐tilted, left‐handed crossover; and (6) right‐tilted, right‐handed crossover. Here “tilt” refers to the orientational relation of the β‐strands to the axis of the central β‐barrel, and “crossover” to the βαβ folding connection feature of the parallel β‐barrel. It has been found that the right‐tilted, right‐handed crossover α/β barrel possesses much lower energy than the other five types of α/β barrels, elucidating why the observed α/β barrels in proteins always assume the form of right tilt and right‐handed crossover connection. As observed, the β‐strands in the energy‐minimized right‐tilted, right‐handed crossover (α/β)8‐barrel are of strong right‐handed twist. The value of root‐mean‐square fits also indicates that the central barrel contained in the lowest energy (α/β)8 structure thus found coincides very well with the observed 8‐stranded parallel β‐barrel in triose phosphate isomerase (TIM). Furthermore, an energetic analysis has been made demonstrating why the right‐tilt, right‐handed crossover barrel is the most stable structure. Our calculations and analysis support the principle that it is possible to account for the main features of frequently occurring folding patterns in proteins by means of conformational energy calculations even for very complicated structures such as (α/β)8 barrels.
Calcium‐free calmodulin is a substrate of proteases from human immunodeficiency viruses 1 and 2
Authors: Tomasselli Alfredo G. , Howe W. Jeffrey , Hui John O. , Sawyer Tomi K. , Reardon Ilene M. , DeCamp Dianne L. , Craik Charles S. , Heinrikson Robert L.
Published: May 2005

Calcium‐free calmodulin‐(CaM) is rapidly hydrolyzed by proteases from both human immunodeficiency viruses (HIV) 1 and 2. Kinetic analysis reveals a sequential order of cleavage by both proteases which initiates in regions of the molecule known from X‐ray crystallographic analysis of Ca2+/CaM to be associated with calcium binding. Although HIV‐1 and HIV‐2 proteases hydrolyze two bonds in common, the initial site of cleavage required for subsequent events differs in each case. The first bond hydrolyzed by the HIV‐1 protease in the Asn‐Tyr linkage in the sequence,‐N‐I‐D‐G‐D‐G‐Q‐V‐N‐Y‐E‐E, found in the fourth calcium binding loop. In contrast, it is an Ala‐Ala bond in the third calcium loop, ‐D‐K‐D‐G‐N‐G‐Y‐I‐S‐A‐A‐E‐, that is first hydrolyzed by the HIV‐2 enzyme, followed in short order by cleavage of the same Asn‐Tyr linkage described above. Thereafter, both enzymes proceed to hydrolyze additional peptide bonds, some in common, some not. Considerable evidence exists that inhibitors are bound to the protease in an extended conformation and yet all of the cleavages we observed occur within, or at the beginning of helices in Ca2+/CaM, regions that also appear to be insufficiently exposed for protease binding. Molecular modeling studies indicate that CaM in solution must adopt a conformation in which the first cleavage site observed for each enzyme is unshielded and extended, and that subsequent cleavages involve further unwinding of helices. The conclusion that the conformation of CaM is different from that of Ca2+/CaM is supported by the observation that Ca2+/CaM is resistant to hydrolysis by either enzyme. As well as demonstrating conformational differences between CaM and Ca2+/CaM, these studies provide further evidence that the two highly homologous human retroviral proteases may be distinguished enzymologically in terms of differential substrate specificities. In addition, some new and unpredicted sequences have been identified that undergo cleavage by these enzymes. Finally, the fact that an abundant, ubiquitous, and biologically important cellular protein is broken down by the HIV proteases could be physiologically relevant to HIV infection if the viral enzyme ever displays activity within the host cell.
The mutation β99 Asp‐Tyr stabilizes Y—A new, composite quaternary state of human hemoglobin
Authors: Smith Francine R. , Lattman Eaton E. , Carter Charles W.
Published: May 2005

Carbonmonoxy hemoglobin Ypsilanti (β99 Asp‐Tyr) exhibits a quaternary form distinctly different from any structures previously observed for human hemoglobins. The relative orientation of αβ dimers in the new quaternary form lies well outside the range of values observed for normal unliganded and liganded tetramers (Baldwin, J., Chothia, C., J. Mol. Biol. 129:175–220, 1979). Despite this large quaternary structural difference between carbonmonoxy hemoglobin Ypsilanti and the two canonical structures, the new quaternary structure's hydrogen bonding interactions in the “switch” region, and packing interactions in the “flexible joint” region, show noncovalent interactions characteristic of the α1β2 contacts of both unliganded and liganded normal hemoglobins. In contrast to both canonical structures, the β97 histidine residue in carbonmonoxy hemoglobin Ypsilanti is disengaged from quaternary packing interactions that are generally believed to enforce two‐state behavior in ligand binding. These features of the new quaternary structure, denoted Y, may therefore be representative of quaternary states that occur transiently along pathways between the normal unliganded, T, and liganded, R, hemoglobin structures.
Projection of monte carlo and molecular dynamics trajectories onto the normal mode axes: Human lysozyme
Authors: Horiuchi Tokio , Gō Nobuhiro
Published: May 2005

A method is presented to describe the internal motions of proteins obtained from molecular dynamics or Monte Carlo simulations as motions of normal mode variables. This method calculates normal mode variables by projecting trajectories of these simulations onto the axes of normal modes and expresses the trajectories as a linear combination of normal mode variables. This method is applied to the result of the molecular dynamics and the Monte Carlo simulations of human lysozyme. The motion of the lowest frequency mode extracted from the simulations represents the hinge bending motion very faithfully. Analysis of the obtained motions of the normal mode variables provides an explanation of the anharmonic aspects of protein dynamics as due first to the anharmonicity of the actual potential energy surface near a minimum and second to trans‐minimum conformational changes.
Modelling the three‐dimensional structure and electrostatic potential field of the two Cu,Zn superoxide dismutase variants from xenopus laevis
Authors: Falconi Mattia , Rotilio Giuseppe , Desideri Alessandro
Published: May 2005

The crystallographic structure of bovine superoxide dismutase has been used as a template for the graphic reconstruction of the three‐dimensional structures of the two Xenopus laevis variants (Schinina', M. E. et al. Arch. Biochem. Biophys. 272:507–515, 1989). In these models the structure‐essential residues maintain their position and their structural role, and the interactions between the subunits and the close packing within the β‐barrel are maintained with conservative substitutions and even increased with “aromatic pairs.” Because of the same topological motif and surface location of charges, arising from the model building of the two variants with respect to the bovine enzyme, we have calculated the electrostatic potential fields around the models of the two Xenopus laevis variants by numerically solving the Poisson–Boltzmann equation. We show that conservation of a specific space‐relationship of charges maintains the potential field pattern already observed in the bovine enzyme, where a negative potential field surrounds the protein surface and specific positive regions wrap up the copper center active site. This electrostatic potential field distribution supports the idea that electrostatic interactions control, like in the bovine enzyme, the mechanism of enzyme–substrate recognition in the Xenopus laevis Cu,Zn superoxide dismutases, suggesting that coordinated mutation of charged residues has occurred in the evolution of this enzyme.
Crystallographic refinement of ricin to 2.5 Å
Authors: Rutenber Earl , Katzin Betsy J. , Ernst Stephen , Collins Edward J. , Mlsna Debra , Ready Michael P. , Robertus Jon D.
Published: May 2005

The plant cytotoxin ricin consists of two disulfide‐linked chains, each of about 30,000 daltons. An initial model based on a 2.8 Å MIR electron density map has been refined against 2.5 Å data using rounds of hand rebuilding coupled with either a restrained least squares algorithm or molecular dynamics (XPLOR). The last model (9) has an R factor of 21.6% and RMS deviations from standard bond lengths and angles of 0.021 Å and 4.67°, respectively. Refinement required several peptide segments in the original model to be adjusted translationally along the electron density. A wide range of lesser changes were also made. The RMS deviation of backbone atoms between the original and model 9 was 1.89 Å. Molecular dynamics proved to be a very powerful refinement tool. However, tests showed that it could not replace human intervention in making adjustments such as local translations of the peptide chain. The R factor is not a completely satisfactory indicator of refinement progress; difference Fouriers, when observed carefully, may be a better monitor.
Structure of ricin B‐chain at 2.5 Å resolution
Authors: Rutenber Earl , Robertus Jon D.
Published: May 2005

The heterodimeric plant toxin ricin has been refined to 2.5 Å resolution. The B‐chain lectin (RTB) is described in detail. The protein has two major domains, each of which has a galactose binding site. RTB has no regular secondary structure but displays several Ω loops. Each RTB domain is made of three copies of a primitive 40 residue folding unit, which pack around a pseudo threefold axis. In each domain, galactose binds in a shallow cleft formed by a three residue peptide kink on the bottom and an aromatic ring on the top. At the back of the cleft, an aspartate forms hydrogen bonds to the C3 and C4 hydroxyls of galactose, whereas a glutamine bonds to the C4 alcohol, helping to define specific epimer binding. In addition to analyzing the sugar binding mechanism, the assembly of subdomain units around the pseudo threefold axis of each domain is described. The subdomains contribute conserved Trp, Leu, and Ile residues to a compact central hydrophobic core. This tight threefold binding probably drives the peptide folding and stabilizes the protein structure.
Active site conformation in myoglobin as determined by X‐ray absorption spectroscopy
Authors: Zhang K. , Reddy K. S. , Bunker G. , Chance B.
Published: May 2005

X‐ray absorption fine structure experiments were performed to study structural and dynamic aspects of the active site of various forms of myoglobin. The structures determined for deoxyMb, MbCO, and MbO2 are consistent with the structure established by X‐ray absorption fine structure experiment and X‐ray crystallography. The first shell of ferrous MbNO determined contains 5 nitrogens located at 2.02 Å and a short NO bond length of 1.76 Å. This study focuses on the change of the XAFS Debye–Waller factor with temperature, which is a measure of thermal and static disorder. It was found that the changes of Debye–Waller factor with temperature for the Mb proteins, except deoxyMb, are consistent with a simple Einstein model, in which a single frequency was assumed for the bond stretching modes. In contrast, the temperature dependence of deoxyMb cannot be fitted to the Einstein model and a large disorder was found at low temperatures, which indicates the existence of conformational substates of the active site.
Comparative analysis of the sequences and structures of HIV‐1 and HIV‐2 proteases
Authors: Gustchina Alla , Weber Irene T.
Published: May 2005

The different isolates available for HIV‐1 and HIV‐2 were compared for the region of the protease (PR) sequence, and the variations in amino acids were analyzed with respect to the crystal structure of HIV‐1 PR with inhibitor. Based on the extensive homology (39 identical out of 99 residues), models were built of the HIV‐2 PR complexed with two different aspartic protease inhibitors, acetylpepstatin and a renin inhibitor, H‐261. Comparison of the HIV‐1 PR crystal structure and the HIV‐2 PR model structure and the analysis of the changes found in different isolates showed that correlated substitutions occur in the hydrophobic interior of the molecule and at surface residues involved in ionic or hydrogen bond interactions. The substrate binding residues of HIV‐1 and HIV‐2 PRs show conservative substitutions of four residues. The difference in affinity of HIV‐1 and HIV‐2 PRs for the two inhibitors appears to be due in part to the change of Val 32 in HIV‐1 PR to Ile in HIV‐2 PR.
Exploration of disorder in protein structures by X‐ray restrained molecular dynamics
Authors: Kuriyan John , Ösapay Klara , Burley Stephen K. , Brünger Axel T. , Hendrickson Wayne A. , Karplus Martin
Published: May 2005

Conformational disorder in crystal structures of ribonuclease‐A and crambin is studied by including two independent structures in least‐squares optimizations against X‐ray data. The optimizations are carried out by X‐ray restrained molecular dynamics (simulated annealing refinement) and by conventional least‐squares optimization. Starting from two identical structures, the optimizations against X‐ray data lead to significant deviations between the two, with rms backbone displacements of 0.45 Å for refinement of ribonuclease at 1.53 Å resolution, and 0.31 Å for crambin at 0.945 Å. More than 15 independent X‐ray restrained molecular dynamics runs have been carried out for ribonuclease, and the displacements between the resulting structures are highly reproducible for most atoms. These include residues with two or more conformations with significant dihedral angle differences and alternative hydrogen bonding, as well as groups of residues that undergo displacements that are suggestive of rigid‐body librations. The crystallographic R‐values obtained are ≈ 13%, as compared to 15.3% for a comparable refinement with a single structure. Least‐squares optimization without an intervening restrained molecular dynamics stage is sufficient to reproduce most of the observed displacements. Similar results are obtained for crambin, where the higher resolution of the X‐ray data allows for refinement of unconstrained individual anisotropic temperature factors. These are shown to be correlated with the displacements in the two‐structure refinements.
The electrostatic potential of Escherichia coli dihydrofolate reductase
Authors: Bajorath Jüurgen , Kitson David H. , Hagler Arnold T. , Kraut Joseph
Published: May 2005

Escherichia coli dihydrofolate reductase (DHFR) carries a net charge of −10 electrons yet it binds ligands with net charges of −4 (NADPH) and −2 (folate or dihydrofolate). Evaluation and analysis of the electrostatic potential of the enzyme give insight as to how this is accomplished. The results show that the enzyme is covered by an overall negative potential (as expected) except for the ligand binding sites, which are located inside “pockets” of positive potential that enable the enzyme to bind the negatively charged ligands. The electrostatic potential can be related to the asymmetric distribution of charged residues in the enzyme.

The asymmetric charge distribution, along with the dielectric boundary that occurs at the solvent‐protein interface, is analogous to the situation occurring in superoxide dismutase. Thus DHFR is another case where the shape of the active site focuses electric fields out into solution.

The positive electrostatic potential at the entrance of the ligand binding site in E. coli DHFR is shown to be a direct consequence of the presence of three positively charged residues at positions 32, 52, and 57–residues which have also been shown recently to contribute significantly to electronic polarization of the ligand folate. The latter has been postulated to be involved in the catalytic process. A similar structural motif of three positively charged amino acids that gives rise to a positive potential at the entrance to the active site is also found in DHFR from chicken liver, and is suggested to be a common feature in DHFRs from many species. It is noted that, although the net charges of DHFRs from different species vary from +3 to −10, the enzymes are able to bind the same negatively charged ligands, and perform the same catalytic function.