bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kringle‐kringle interactions in multimer kringle structures
Authors: Padmanabhan K. , Wu T.‐P. , Ravichandran K. G. , Tulinsky A. 
Published: July 2010

AbstractThe crystal structure of a monoclinic form of human plasminogen kringle 4 (PGK4) has been solved by molecular replacement using the orthorhombic structure as a model and it has been refined by restrained least‐squares methods to an R factor of 16.4% at 2.25 Å resolution. The X‐PLOR structure of kringle 2 of tissue plasminogen activator (t‐PAK2) has been refined further using PROFFT (R = 14.5% at 2.38 Å resolution). The PGK4 structure has 2 and t‐PAK2 has 3 independent molecules in the asymmetric unit. There are 5 different noncrystallographic symmetry “dimers” in PGK4. Three make extensive kringle‐kringle interactions related by noncrystallographic 2, screw axes without blocking the lysine binding site. Such associations may occur in multikringle structures such as prothrombin, hepatocyte growth factor, plasminogen (PG), and apolipoprotein [a]. The t‐PAK2 structure also has noncrystallographic screw symmetry (31) and mimics fibrin binding mode by having lysine of one molecule interacting electrostatically with the lysine binding site of another kringle. This ligand‐like binding interaction may be important in kringle‐kringle interactions involving non‐lysine binding kringles with lysine or pseudo‐lysine binding sites. Electrostatic intermolecular interactions involving the lysine binding site are also found in the crystal structures of PGK1 and orthorhombic PGK4. Anions associate with the cationic centers of these and t‐PAK2 that appear to be more than occasional components of lysine binding site regions.
Receptor binding properties of four‐helix‐bundle growth factors deduced from electrostatic analysis
Authors: Demchuk Eugene , Mueller Thomas , Oschkinat Hartmut , Sebald Walter , Wade Rebecca C. 
Published: July 2010

AbstractHormones of the hematopoietin class mediate signal transduction by binding to specific transmembrane receptors. Structural data show that the human growth hormone (hGH) forms a complex with a homodimeric receptor and that hGH is a member of a class of hematopoietins possessing an antiparallel 4‐α‐helix bundle fold. Mutagenesis experiments suggest that electrostatic interactions may have an important influence on hormone‐receptor recognition. In order to examine the specificity of hormone‐receptor complexation, an analysis was made of the electrostatic potentials of hGH, interleukin‐2 (IL‐2), interleukin‐4 (IL‐4), granulocyte colony‐stimulating factor (G‐CSF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and the hGH and IL‐4 receptors.The binding surfaces of hGH and its receptor, and of IL‐4 and its receptor, show complementary electrostatic potentials. The potentials of the hGH and its receptor display approximately 2‐fold rotational symmetry because the receptor subunits are identical. In contrast, the potentials of GM‐CSF and IL‐2 lack such symmetry, consistent with their known high affinity for hetero‐oligomeric receptors. Analysis of the electrostatic potentials supports a recently proposed hetero‐oligomeric model for a high‐affinity IL‐4 receptor and suggests a possible new receptor binding mode for G‐CSF; it also provides valuable information for guiding structural and mutagenesis studies of signal‐transducing proteins and their receptors.
A comparison of X‐ray and NMR structures for human endothelin‐1
Authors: Wallace B.A. , Janes Robert W. , Bassolino Donna A. , Krystek Stanley R. 
Published: July 2010

AbstractDirect comparisons between the recently solved X‐ray and NMR structures of human endothelin‐1 with respect to secondary structure, RMS deviations, surface accessibilities, and side‐chain conformers indicate important differences in conformation, especially in the C‐terminus, but also in the central loop region, that are important for defining the specificity of binding. These differences are larger than seen for other X‐ray and NMR structures that have been compared. Comparisons between the X‐ray structure and the NMR NOE constraints highlight the regions of flexibility and environment‐induced diversity in the endothelin structures.
A procedure for detecting structural domains in proteins
Authors: Swindells Mark B. 
Published: July 2010

AbstractA procedure is described for detecting domains in proteins of known structure. The method is based on the intuitively simple idea that each domain should contain an identifiable hydrophobic core. By applying the algorithm described in the companion paper (Swindells MB, 1995, Protein Sci 4:93–102) to identify distinct cores in multi‐domain proteins, one can use this information to determine both the number and the location of the constituent domains. Tests have shown the procedure to be effective on a number of examples, even when the domains are discontinuous along the sequence. However, deficiencies also occur when hydrophobic cores from different domains continue through the interface region and join one another.
Identification of a eukaryotic‐like protein kinase gene in Archaebacteria
Authors: Smith Randall F. , King Katherine Y. 
Published: July 2010

AbstractPrimary sequence patterns based on known conserved sites in eukaryotic protein kinases were used to search for eukaryotic‐like protein kinase sequences in a six‐frame translation of the bacterial subsection of GenBank. This search identified a previously unrecognized eukaryotic‐like protein kinase gene in three related methanogenic archaebacteria, Methanococcus vannielii, M. voltae, and M. thermolithotrophicus. The proposed coding sequences are located in orthologous open reading frames (ORFs): ORF547, ORF294, and ORF114, respectively. The C‐terminus of the ORFs contains 9 of the 11 subdomains characteristically conserved within the eukaryotic protein kinase catalytic domain. The N‐terminus of the ORFs is similar to a putative glycoprotease in Pasteurella haemolytica and its homologue in Escherichia coli, the orfX gene. This is the first report of a eukaryotic‐like protein kinase sequence observed in Archaebacteria.
Identification of the Serratia endonuclease dimer: Structural basis and implications for catalysis
Authors: Miller Mitchell D. , Krause Kurt L. 
Published: July 2010

AbstractThe Serratia endonuclease is an extracellularly secreted enzyme capable of cleaving both single‐ and double‐stranded forms of DNA and RNA. It is the first member of a large class of related and usually dimeric endonucleases for which a structure is known. Using X‐ray crystallography, the structure of monomer of this enzyme was reported by us previously (Miller MD et al., 1994, Nature Struct Biol 1:461–468). We now confirm the dimeric nature of this enzyme through light‐scattering experiments and identify the physiologic dimer interface through crystal packing analysis. This dimerization occurs through an isologous twofold interaction localized to the carboxy‐terminal subdomain of the enzyme. The dimer is a prolate ellipsoid with dimensions 30 Å× ± 35 Å × 90 Å. The dimer interface is flat and contains four salt links, several hydrogen bonds, and nonpolar interactions. Buried water is prominent in this interface and it includes an unusual “cubic” water cluster. The position of the two active sites in the dimer suggests that they can act independently in their cleavage of DNA, but have a geometrical advantage in attacking substrate relative to the monomer.
A model for the regulation of D‐3‐phosphoglycerate dehydrogenase, a Vmax‐type allosteric enzyme
Authors: Grant Gregory A. , Schuller David J. , Banaszak Leonard J. 
Published: July 2010

AbstractEscherichia coli D‐3‐phosphoglycerate dehydrogenase (ePGDH) is a tetramer of identical subunits that is alloste‐rically inhibited by L‐serine, the end product of its metabolic pathway. Because serine binding affects the velocity of the reaction and not the binding of substrate or cofactor, the enzyme is classified as of the Vmax type. Inhibition by a variety of amino acids and analogues of L‐serine indicate that all three functional groups of serine are required for optimal interaction. Removing or altering any one functional group results in an increase in inhibitory concentration from micromolar to millimolar, and removal or alteration of any two functional groups removes all inhibitory ability. Kinetic studies indicate at least two serine‐binding sites, but the crystal structure solved in the presence of bound serine and direct serine‐binding studies show that there are a total of four serine‐binding sites on the enzyme. However, approximately 85% inhibition is attained when only two sites are occupied. The three‐dimensional structure of ePGDH shows that the serine‐binding sites reside at the interface between regulatory domains of adjacent subunits. Two serine molecules bind at each of the two regulatory domain interfaces in the enzyme. When all four of the serines are bound, 100% inhibition of activity is seen. However, because the domain contacts are symmetrical, the binding of only one serine at each interface is sufficient to produce approximately 85% inhibition. The tethering of the regulatory domains to each other through multiple hydrogen bonds from serine to each subunit apparently prevents the body of these domains from undergoing the reorientation that must accompany a catalytic cycle. It is suggested that part of the conformational change may involve a hinge formed in the vicinity of the union of two antiparallel β‐sheets in the regulatory domains. The tethering function of serine, in turn, appears to prevent the substrate‐binding domain from closing the cleft between it and the nucleotide‐binding domain, which may be necessary to form a productive hydrophobic environment for hydride transfer. Thus, the structure provides a plausible model that is consistent with the binding and inhibition data and that suggests that catalysis and inhibition in this rare Vmax‐type allosteric enzyme is based on the movement of rigid domains about flexible hinges.
The crystal structure of trypanothione reductase from the human pathogen Trypanosoma cruzi at 2.3 Å resolution
Authors: Zhang Yihong , Bond Charles S. , Bailey Susan , Cunningham Mark L. , Fairlamb Alan H. , Hunter William N. 
Published: July 2010

AbstractTrypanothione reductase (TR) is an NADPH‐dependent flavoprotein unique to protozoan parasites from the genera Trypanosoma and Leishmania and is an important target for the design of improved trypanocidal drugs. We present details of the structure of TR from the human pathogen Trypanosoma cruzi, the agent responsible for Chagas' disease or South American trypanosomiasis. The structure has been solved by molecular replacement, using as the starting model the structure of the enzyme from the nonpathogenic Crithidia fasciculata, and refined to an R‐factor of 18.9% for 53,868 reflections with F κ σF between 8.0 and 2.3 Å resolution. The model comprises two subunits (968 residues), two FAD prosthetic groups, two maleate ions, and 419 water molecules. The accuracy and geometry of the enzyme model is improved with respect to the C. fasciculata enzyme model. The new structure is described and specific features of the enzyme involved in substrate interactions are compared with previous models of TR and related glutathione reductases from human and Escherichia coli. Structural differences at the edge of the active sites suggest an explanation for the differing specificities toward glutathionylspermidine disulfide.
A recombinant peptide model of the first nucleotide‐binding fold of the cystic fibrosis transmembrane conductance regulator: Comparison of wild‐type and ΔF508 mutant forms
Authors: Yike Iwona , Ye Jin , Zhang Yan , Manavalan Parthasarathy , Gerken Thomas A. , Dearborn Dorr G. 
Published: July 2010

AbstractA series of recombinant peptides, each including the sequence proposed to be the first nucleotide‐binding fold of cystic fibrosis transmembrane conductance regulator (CFTR), has been produced in an attempt to find a model peptide that would autologously fold into a soluble structure with native‐like properties. The peptide NBD1F, which contains the 267‐amino acid sequence of CFTR from 384 to 650, meets these requirements. The peptide was produced with a high expression bacterial plasmid pRSET, purified from inclusion bodies following solubilization with 6 M guanidine‐HCl and refolded from 8 M urea. Competitive displacement of trinitrophenol‐ATP by nucleotides reveals binding of ATP and related nucleotides with KDs in the low micromolar range; the KD for ATP‐γS is 1.0 ± 0.4 μM and for ADP 8.8 ± 3.1 μM. The native‐like character of the model peptide's structure is further supported by the findings that the KD for the ATP analog, 5′‐adenylimidodiphosphate, is fourfold lower than the KD for the methylene analog, 5′‐adenylmethylenediphosphonate, and that ATP binding slows the trypsin proteolysis of NBD1F. The CD spectra of NBD1F and the parallel peptide containing the most common cystic fibrosis mutation, deletion of Phe 508, are essentially indistinguishable, both spectra indicating 28% α‐helix and 23% β‐sheet, with insignificant differences in the amounts of β‐turns and random structure. Extensive investigation using multiple conditions with highly purified preparations of the model peptides demonstrates that they do not support ATP hydrolysis. These large recombinant peptides offer practical models for the investigation of the first nucleotide‐binding domain of CFTR.
Contact interactions method: A new algorithm for protein folding simulations
Authors: Toma Lucio , Toma Salvatore 
Published: July 2010

AbstractComputer simulations of simple exact lattice models are an aid in the study of protein folding process; they have sometimes resulted in predictions experimentally proved. The contact interactions (CI) method is here proposed as a new algorithm for the conformational search in the low‐energy regions of protein chains modeled as copolymers of hydrophobic and polar monomers configured as self‐avoiding walks on square or cubic lattices. It may be regarded as an extension of the standard Monte Carlo method improved by the concept of cooperativity deriving from nonlocal contact interactions. A major difference with respect to other algorithms is that criteria for the acceptance of new conformations generated during the simulations are not based on the energy of the entire molecule, but cooling factors associated with each residue define regions of the model protein with higher or lower mobility. Nine sequences of length ranging from 20 to 64 residues were used on the square lattice and 15 sequences of length ranging from 46 to 136 residues were used on the cubic lattice. The CI algorithm proved very efficient both in two and three dimensions, and allowed us to localize energy minima not localized by other searching algorithms described in the literature. Use of this algorithm is not limited to the conformational search, because it allows the exploration of thermodynamic and kinetic behavior of model protein chains.
Identification of arginine 331 as an important active site residue in the Class II fructose‐1,6‐bisphosphate aldolase of Escherichia coli
Authors: Qamar Seema , Marsh Katherine , Berry Alan 
Published: July 2010

AbstractTreatment of the Class II fructose‐1,6‐bisphosphate aldolase of Escherichia coli with the arginine‐specific α‐dicarbonyl reagents, butanedione or phenylglyoxal, results in inactivation of the enzyme. The enzyme is protected from inactivation by the substrate, fructose 1,6‐bisphosphate, or by inorganic phosphate. Modification with [7‐14C] phenylglyoxal in the absence of substrate demonstrates that enzyme activity is abolished by the incorporation of approximately 2 moles of reagent per mole of enzyme. Sequence alignment of the eight known Class II FBP‐aldolases shows that only one arginine residue is conserved in all the known sequences. This residue, Arg‐331, was mutated to either alanine or glutamic acid. The mutant enzymes were much less susceptible to inactivation by phenylglyoxal. Measurement of the steady‐state kinetic parameters revealed that mutation of Arg‐331 dramatically increased the Km for fructose 1,6‐bisphosphate. Comparatively small differences in the inhibitor constant Ki for dihydroxyacetone phosphate or its analogue, 2‐phosphoglycolate, were found between the wild‐type and mutant enzymes. In contrast, the mutation caused large changes in the kinetic parameters when glyceraldehyde 3‐phosphate was used as an inhibitor. Kinetic analysis of the oxidation of the carbanionic aldolase‐substrate intermediate of the reaction by hexacyanoferrate (III) revealed that the Km for dihydroxyacetone phosphate was again unaffected, whereas that for fructose 1,6‐bisphosphate was dramatically increased. Taken together, these results show that Arg‐331 is critically involved in the binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C‐6 phosphate group of the substrate.
Extending the C2 domain family: C2s in PKCs δ, ϵ,η,θ, phospholipases, GAPs, and perforin
Authors: Pointing C.P. , Parker P.J. 
Published: July 2010

AbstractVarious membrane lipid metabolites, generated by phospholipases C and D (PLCs, PLDs), are known to regulate the activities of protein kinases C (PKCs) and GTP‐ase activating proteins (GAPs) in a range of cellular processes. Conventional Ca2+‐dependent PKCs (α, βI, βII, and γ), PLCs, and various GAPs are all known to contain copies of a phospholipid‐binding domain, termed C2 or CalB. Here we recognize that C2 domains are also present in “new” Ca2+ ‐independent PKCs (δ, η, η, and θ), other kinases, a eukaryotic PLD, the breakpoint cluster region (BCR) gene product, and two further GAPS. Twenty‐two previously unrecognized C2 domain sequences are presented, which include a single copy in the mammalian poreforming proteins, perforin.
An alternative topological model for Escherichia coli OmpA
Authors: Stathopoulos Christos 
Published: July 2010

AbstractThe current topological model for the Escherichia coli outer membrane protein OmpA predicts eight N‐terminal transmembrane segments followed by a long periplasmic tail. Several recent reports have raised serious doubts about the accuracy of this prediction. An alternative OmpA model has been constructed using (1) computer‐aided predictions developed specifically to predict topology of bacterial outer membrane porins, (2) the results of two reports that identified sequence homologies between OmpA and other peptidoglycan‐associated proteins, and (3) biochemical, immunochemical, and genetic topological data on proteins of the OmpA family provided by numerous previous studies. The new model not only agrees with the varied experimental data concerning OmpA but also provides an improved understanding of the relationship between the structure and the multifunctional role of OmpA in the bacterial outer membrane.
Rapid screening for structural integrity of expressed proteins by heteronuclear NMR spectroscopy
Authors: Gronenborn Angela M. , Clore G. Marius 
Published: July 2010

Abstractsimple and rapid method based on 15N labeling and 1H‐15N heteronuclear single quantum coherence spectroscopy is presented to directly assess the structural integrity of overexpressed proteins in crude Escherichia coli extracts without the need for any purification. The method is demonstrated using two different expression systems and two different proteins, the B1 immunoglobulin‐binding domain of streptococcal protein G (56 residues) and human interleukin‐1β (153 residues). It is shown that high quality 1H‐15N correlation spectra, recorded in as little as 15 min and displaying only cross‐peaks arising from the overexpressed protein of interest, can be obtained from crude E. coli extracts.
Structural resiliency of an EGF‐like subdomain bound to its target protein, thrombin
Authors: Hrabal Richard , Ni Feng , Komives Elizabeth A. 
Published: July 2010

AbstractThe thrombin‐bound structures of native peptide fragments from the fifth EGF‐like domain of thrombomodulin were determined by use of NMR and transferred NOE spectroscopy. The bound peptides assume an EGF‐like structure of an antiparallel β‐sheet, a novel structural motif observed for a bound peptide in protein‐peptide complexes. There is a remarkable structural resiliency of this structure motif manifested in its ability to accommodate a different number of residues within the disulfide loop. Docking experiments revealed that the key contacts with thrombin are hydrophobic interactions between the side chains of residues Ile 414 and Ile 424 of thrombomodulin and a hydrophobic pocket on the thrombin surface. Residues Leu 415, Phe 419, and Ile 420, which would have been buried in intact EGF‐like domains, are unfavorably exposed in the complex of thrombin with the EGF‐like thrombomodulin fragment, thus providing a rationale for the enhancement of binding affinity upon the deletion of Ile 420. The unique β‐sheet structures of the bound peptides are specified by the presence of disulfide bridges in the peptides because a corresponding linear thrombomodulin fragment folds into a sheet structure with a different backbone topology. The different bound conformations for the linear and the cyclized peptides indicate that side‐chain interactions within a specific environment may dictate the folding of bound peptides in protein‐peptide complexes.
Are turns required for the folding of ribonuclease T1?
Authors: Garrett James B. , Mullins Leisha S. , Raushel Frank M. 
Published: July 2010

AbstractRibonuclease T1 (RNase T1) is a small, globular protein of 104 amino acids for which extensive thermodynamic and structural information is known. To assess the specific influence of variations in amino acid sequence on the mechanism for protein folding, circularly permuted variants of RNase T1 were constructed and characterized in terms of catalytic activity and thermodynamic stability. The disulfide bond connecting Cys‐2 and Cys‐10 was removed by mutation of these residues to alanine (C2, 10A) to avoid potential steric problems imposed by the circular permutations. The original amino‐terminus and carboxyl‐terminus of the mutant (C2, 10A) were subsequently joined with a tripeptide linker to accommodate a reverse turn and new termini were introduced throughout the primary sequence in regions of solvent‐exposed loops at Ser‐35 (cp35Sl), Asp‐49 (cp49D1), Gly‐70 (cp70G1), and Ser‐96 (cp96S1). These circularly permuted RNase T1 mutants retained 35–100% of the original catalytic activity for the hydrolysis of guanylyl(3′ → 5′)cytidine, suggesting that the overall tertiary fold of these mutants is very similar to that of wild‐type protein. Chemical denaturation curves indicated thermodynamic stabilities at pH 5.0 of 5.7, 2.9, 2.6, and 4.6 kcal/mol for cp35S1, cp49D1, cp70G1, and cp96S1, respectively, compared to a value of 10.1 kcal/mol for wild‐type RNase T1 and 6.4 kcal/mol for (C2, 10A) T1. A fifth set of circularly permuted variants was attempted with new termini positioned in a tight β‐turn between Glu‐82 and Gln‐85. New termini were inserted at Asn‐83 (cp83N1), Asn‐84 (cp84N1), and Gln‐85 (cp85Q1). No detectable amount of protein was ever produced for any of the mutations in this region, suggesting that this turn may be critical for the proper folding and/or thermodynamic stability of RNase T1.