bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractInterleukin‐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

AbstractInterfacial 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

AbstractHemocyanins 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

AbstractThe 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

AbstractThe 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

AbstractCalcium‐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

AbstractCarbonmonoxy 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

AbstractA 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

AbstractThe 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

AbstractThe 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

AbstractThe 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

AbstractX‐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

AbstractThe 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

AbstractConformational 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

AbstractEscherichia 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.