bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural and functional consequences of the presence of a fourth disulfide bridge in the scorpion short toxins: Solution structure of the potassium channel inhibitor HsTX1
Authors: Savarin Philippe , Romi‐Lebrun Régine , Zinn‐Justin Sophie , Gilquin Bernard , Ménez André , Lebrun Bruno , Nakajima Terumi 
Published: February 2009

AbstractWe have determined the three‐dimensional structure of the potassium channel inhibitor HsTX1, using nuclear magnetic resonance and molecular modeling. This protein belongs to the scorpion short toxin family, which essentially contains potassium channel blockers of 29 to 39 amino acids and three disulfide bridges. It is highly active on voltage‐gated Kv1.3 potassium channels. Furthermore, it has the particularity to possess a fourth disulfide bridge. We show that HsTX1 has a fold similar to that of the three‐disulfide‐bridged toxins and conserves the hydrophobic core found in the scorpion short toxins. Thus, the fourth bridge has no influence on the global conformation of HsTX1. Most residues spatially analogous to those interacting with voltage‐gated potassium channels in the three‐disulfide‐bridged toxins are conserved in HsTX1. Thus, we propose that Tyr21, Lys23, Met25, and Asn26 are involved in the biological activity of HsTX1. As an additional positively charged residue is always spatially close to the aromatic residue in toxins blocking the voltage‐gated potassium channels, and as previous mutagenesis experiments have shown the critical role played by the C‐terminus in HsTX1, we suggest that Arg33 is also important for the activity of the four disulfide‐bridged toxin. Docking calculations confirm that, if Lys23 and Met25 interact with the GYGDMH motif of Kv1.3, Arg33 can contact Asp386 and, thus, play the role of the additional positively charged residue of the toxin functional site. This original configuration of the binding site of HsTX1 for Kv1.3, if confirmed experimentally, offers new structural possibilities for the construction of a molecule blocking the voltage‐gated potassium channels.
Inhibition of the HIV‐1 and HIV‐2 proteases by a monoclonal antibody
Authors: Lescar Julien , Brynda Jiri , Bentley Graham A. , Riottot Marie‐Madeleine , Chitarra Véronique , Rezacova Pavlina , Stouracova Renata , Fabry Milan , Horejsi Magda , Sedlacek Juraj 
Published: February 2009

AbstractThe monoclonal antibody 1696, directed against the HIV‐1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV‐1 and HIV‐2 isolates. This antibody cross‐reacts with peptides that include the N‐terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigenbinding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV‐1 and HIV‐2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 Å resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease‐inhibiting molecules of broad specificity.
Robustness of protein folding kinetics to surface hydrophobic substitutions
Authors: Gu H. , Doshi N. , Kim D.E. , Simons K.T. , Santiago J.V. , Nauli S. , Baker David 
Published: February 2009

AbstractWe use both combinatorial and site‐directed mutagenesis to explore the consequences of surface hydrophobic substitutions for the folding of two small single domain proteins, the src SH3 domain, and the IgG binding domain of Peptostreptococcal protein L. We find that in almost every case, destabilizing surface hydrophobic substitutions have much larger effects on the rate of unfolding than on the rate of folding, suggesting that nonnative hydrophobic interactions do not significantly interfere with the rate of core assembly.
Structural determinants in domain II of human glutathione transferase M2–2 govern the characteristic activities with aminochrome, 2‐cyano‐1, 3‐dimethyl‐1‐nitrosoguanidine, and 1, 2‐dichloro‐4‐nitrobenzene
Authors: Hansson Lars O. , Widersten Mikael , Mannervik Bengt , Bolton‐Grob Robyn 
Published: February 2009

AbstractTwo human Mu class glutathione transferases, hGST M1–1 and hGST M2–2, with high sequence identity (84%) exhibit a 100‐fold difference in activities with the substrates aminochrome, 2‐cyano‐1, 3‐dimethyl‐1‐nitrosoguanidine (cyanoDMNG), and 1, 2‐dichloro‐4‐nitrobenzene (DCNB), hGST M2–2 being more efficient. A sequence alignment with the rat Mu class GST M3–3, an enzyme also showing high activities with aminochrome and DCNB, demonstrated an identical structural cluster of residues 164–168 in the α6‐helices of rGST M3–3 and hGST M2–2, a motif unique among known sequences of human, rat, and mouse Mu class GSTs. A putative electrostatic network Arg107‐Asp161‐Arg165‐Glu164(‐Gln167) was identified based on the published three‐dimensional structure of hGST M2–2. Corresponding variant residues of hGSTM1–1 (Leu165, Asp164, and Arg167) as well as the active site residue Ser209 were targeted for point mutations, introducing hGST M2–2 residues to the framework of hGST M1–1, to improve the activities with substrates characteristic of hGST M2–2. In addition, chimeric enzymes composed of hGST M1–1 and hGST M2–2 sequences were analyzed. The activity with 1‐chloro‐2, 4‐dinitrobenzene (CDNB) was retained in all mutant enzymes, proving that they were catalytically competent, but none of the point mutations improved the activities with hGST M2–2 characteristic substrates. The chimeric enzymes showed that the structural determinants of these activities reside in domain II and that residue Arg 165 in hGST M2–2 appears to be important for the reactions with cyanoDMNG and DCNB. A mutant, which contained all the hGST M2–2 residues of the putative electrostatic network, was still lacking one order of magnitude of the activities with the characteristic substrates of wild‐type hGST M2–2. It was concluded that a limited set of point mutations is not sufficient, but that indirect secondary structural affects also contribute to the hGST M2–2 characteristic activities with aminochrome, cyanoDMNG, and DCNB.
Human liver mitochondrial aldehyde dehydrogenase: Three‐dimensional structure and the restoration of solubility and activity of chimeric forms
Authors: Ni Li , Zhou Jianzhong , Weiner Henry , Hurley Thomas D. 
Published: February 2009

AbstractHuman liver cytosolic and mitochondrial isozymes of aldehyde dehydrogenase share 70% sequence identity. However, the first 21 residues are not conserved between the human isozymes (15% identity). The three‐dimensional structures of the beef mitochondrial and sheep cytosolic forms have virtually identical three‐dimensional structures. Here, we solved the structure of the human mitochondrial enzyme and found it to be identical to the beef enzyme. The first 21 residues are found on the surface of the enzyme and make no contact with other subunits in the tetramer. A pair of chimeric enzymes between the human isozymes was made. Each chimera had the first 21 residues from one isozyme and the remaining 479 from the other. When the first 21 residues were from the mitochondrial isozyme, an enzyme with cytosolic‐like properties was produced. The other was expressed but was insoluble. It was possible to restore solubility and activity to the chimera that had the first 21 cytosolic residues fused to the mitochondrial ones by making point mutations to residues at the N‐terminal end. When residue 19 was changed from tyrosine to a cysteine, the residue found in the mitochondrial form, an active enzyme could be made though the Km for NAD+ was 35 times higher than the native mitochondrial isozyme and the specific activity was reduced by 75%. This residue interacts with residue 203, a nonconserved, nonactive site residue. A mutation of residue 18, which also interacts with 203, restored solubility, but not activity. Mutation to residue 15, which interacts with 104, also restored solubility but not activity. It appears that to have a soluble or active enzyme a favorable interaction must occur between a residue in a surface loop and a residue elsewhere in the molecule even though neither make contact with the active site region of the enzyme.
Feasibility in the inverse protein folding protocol
Authors: Ota Motonori , Nishikawa Ken 
Published: February 2009

AbstractMethods for protein structure (3D)–sequence (1D) compatibility evaluation (threading) have been developed during the past decade. The protocol in which a sequence can recognize its compatible structure in the structural library (i.e., the fold recognition or the forward‐folding search) is available for the structure prediction of new proteins. However, the reverse protocol, in which a structure recognizes its homologous sequences among a sequence database, named the inverse‐folding search, is a more difficult application. In this study, we have investigated the feasibility of the latter approach. A structural library, composed of about 400 well‐resolved structures with mutually dissimilar sequences, was prepared, and 163 of them had remote homologs in the library. We examined whether they could correctly seek their homologs by both forward‐and inverse‐folding searches. The results showed that the inverse‐folding protocol is more effective than the forward‐folding protocol, once the reference states of the compatibility functions are appropriately adjusted. This adjustment only slightly affects the ability of the forward‐folding search. We noticed that the scoring, in which a given sequence is re‐mounted onto a structure according to the 3D–1D alignment determined by the dynamic programming method, is only effective in the forward‐folding protocol and not in the inverse‐folding protocol. Namely, the inverse‐folding search works significantly better with the score given by the 3D–1D alignment per se, rather than that obtained by the re‐mounting. The implications of these results are discussed.
Macromolecular docking of a three‐body system: The recognition of human growth hormone by its receptor
Authors: Hendrix Donna K. , Klein Teri E. , Kuntz Irwin D. 
Published: February 2009

AbstractHuman growth hormone (hGH) binds to its receptor (hGHr) in a three‐body interaction: one molecule of the hormone and two identical monomers of the receptor form a trimer. Curiously, the hormone‐receptor interactions in the trimer are not equivalent and the formation of the complex occurs in a specific kinetic order (Cunningham BC, Ultsch M, De Vos AM, Mulkerrin MG, Clauser KR, Wells JA, 1991, Science 254:821–825). In this paper, we model the recognition of hGH to the hGHr using shape complementarity of the three‐dimensional structures and macromolecular docking to explore possible binding modes between the receptor and hormone. The method, reported previously (Hendrix DK, Kuntz ID, 1998, Pacific symposium on biocomputing 1998, pp 1234–1244), is based upon matching complementary‐shaped strategic sites on the molecular surface. We modify the procedure to examine three‐body systems. We find that the order of binding seen experimentally is also essential to our model. We explore the use of mutational data available for hGH to guide our model. In addition to docking hGH to the hGHr, we further test our methodology by successfully reproducing 16 macromolecular complexes from X‐ray crystal structures, including enzyme‐inhibitor, antibody‐antigen, protein dimer, and protein‐DNA complexes.
Ternary complex structure of human hgprtase, prpp, mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding
Authors: Balendiran Ganesaratnam K. , Molina JOSÉ A. , Xu Yiming , Torres‐Martinez Jan , Stevens Robert , Focia Pamela J. , Eakin Ann E. , Sacchettini James C. , Craig Sydney P. 
Published: February 2009

AbstractSite‐directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one‐to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (kcat/Km) of the forward and reverse reactions were more severely reduced (6‐ to 30‐fold), and the mutant enzyme showed positive cooperativity in binding of α‐D‐5‐phosphoribosyl‐1‐pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7‐hydroxy [4, 3‐d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo – Fc)ϕcalc] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.
Prediction of the location and type of β‐turns in proteins using neural networks
Authors: Shepherd Adrian J. , Gorse Denise , Thornton Janet M. 
Published: February 2009

AbstractA neural network has been used to predict both the location and the type of β‐turns in a set of 300 nonhomologous protein domains. A substantial improvement in prediction accuracy compared with previous methods has been achieved by incorporating secondary structure information in the input data. The total percentage of residues correctly classified as β‐turn or not‐β‐turn is around 75% with predicted secondary structure information. More significantly, the method gives a Matthews correlation coefficient (MCC) of around 0.35, compared with a typical MCC of around 0.20 using other β‐turn prediction methods. Our method also distinguishes the two most numerous and well‐defined types of β‐turn, types I and II, with a significant level of accuracy (MCCs 0.22 and 0.26, respectively).
Expression, purification, and crystallography of the conserved methionine‐rich domain of human signal recognition particle 54 kda protein
Authors: Gowda Krishne , Clemons William M. , Zwieb Christian , Black Shaun D. 
Published: February 2009

AbstractProtein SRP54 is an essential component of eukaryotic signal recognition particle (SRP). The methionine‐rich M‐domain (SRP54M or 54M) interacts with the SRP RNA and is also involved in the binding to signal peptides of secretory proteins during their targeting to cellular membranes. To gain insight into the molecular details of SRP‐mediated protein targeting, we studied the human 54M polypeptide. The recombinant human protein was expressed successfully in Escherichia coli and was purified to homogeneity. Our studies determined the sites that were susceptible to limited proteolysis, with the goal to design smaller functional mutant derivatives that lacked nonessential amino acid residues from both termini. Of the four polypeptides produced by V8 protease or chymotrypsin, 54MM‐2 was the shortest (120 residues; Mr = 13, 584.8), but still contained the conserved amino acids suggested to associate with the signal peptide or the SRP RNA. 54MM‐2 was cloned, expressed, purified to homogeneity, and was shown to bind human SRP RNA in the presence of protein SRP19, indicating that it was functional. Highly reproducible conditions for the crystallization of 54MM‐2 were established. Examination of the crystals by X‐ray diffraction showed an orthorhombic unit cell of dimensions a = 29.127 Å, b = 63.693 Å, and c = 129.601 Å, in space group P212121, with reflections extending to at least 2.0 Å.
Energetic analysis of an antigen/antibody interface: Alanine scanning mutagenesis and double mutant cycles on the hyhel‐10/lysozyme interaction
Authors: Pons Jaume , Rajpal Arvind , Kirsch Jack F. 
Published: February 2009

AbstractAlanine scanning mutagenesis of the HyHEL‐10 paratope of the HyHEL‐10/HEWL complexdemonstrates that the energetically important side chains (hot spots) of both partners are in contact.A plot of ΔΔGHyHEL‐10_mutant vs.ΔΔGhewl_mutant for the five of six interacting side‐chain hydrogen bonds is linear (Slope = 1).Only 3 of the 13 residues in the HEWL epitope contribute >4 kcal/mol to the free energy of formation of the complexwhen replaced by alanine, but 6 of the 12 HyHEL‐10 paratope amino acids do.Double mutant cycle analysis of the single crystallographically identified salt bridge, D32H/K97, shows that there is a significant energetic penalty when either partner is replaced with a neutral side‐chain amino acid, but the D32HN/K97M complex is as stable as the WT.The role of the disproportionately high number of Tyr residues in the CDR was evaluated by comparing theΔΔG values of the Tyr Phe vs. the corresponding Tyr Ala mutations.The nonpolar contacts in the light chain contribue only about one‐half of the totalΔΔG observed for the Tyr Ala mutation, while they are significantly more important in the heavy chain.Replacement of the N31L/K96 hydrogen bond with a salt bridge, N31DL/K96, destabilizes the complex by 1.4 kcal/mol. The free energy of interaction,ΔΔGint, obtained from double mutant cycle analysis showed that ΔΔGint for any complex for which the HEWL residue probed is a major immunodeterminant is very close to the loss of free energy observed for the HyHEL‐10 single mutant.Error propagation analysis of double mutant cycles shows that data of atypically high precision are required to use this method meaningfully, except where large ΔΔG values are analyzed.
The variable and conserved interfaces of modeled olfactory receptor proteins
Authors: Pilpel Yitzhak , Lancet Doron 
Published: February 2009

AbstractThe accumulation of hundreds of olfactory receptor (OR) sequences, along with the recent availability of detailed models of other G‐protein‐coupled receptors, allows us to analyze the OR amino acid variability patterns in a structural context. A Fourier analysis of 197 multiply aligned olfactory receptor sequences showed an α‐helical periodicity in the variability profile. This was particularly pronounced in the more variable transmembranal segments 3, 4, and 5. Rhodopsin‐based homology modeling demonstrated that the inferred variable helical faces largely point to the interior of the receptor barrel. We propose that a set of 17 hypervariable residues, which point to the barrel interior and are more extracellularly disposed, constitute the odorant complementarity determining regions. While 12 of these residues coincide with established ligand‐binding contact postions in other G‐protein‐coupled receptors, the rest are suggested to form an olfactory‐unique aspect of the binding pocket. Highly conserved olfactory receptor‐specific sequence motifs, found in the second and third intracellular loops, may comprise the G‐protein recognition epitope. The prediction of olfactory receptor functional sites provides concrete suggestions of site‐directed mutagenesis experiments for altering ligand and G‐protein specificity.
ChloroP, a neural network‐based method for predicting chloroplast transit peptides and their cleavage sites
Authors: Emanuelsson Olof , Nielsen Henrik , Heijne Gunnar Von 
Published: February 2009

AbstractWe present a neural network based method (ChloroP) for identifying chloroplast transit peptides and their cleavage sites. Using cross‐validation, 88% of the sequences in our homology reduced training set were correctly classified as transit peptides or nontransit peptides. This performance level is well above that of the publicly available chloroplast localization predictor PSORT. Cleavage sites are predicted using a scoring matrix derived by an automatic motif‐finding algorithm. Approximately 60% of the known cleavage sites in our sequence collection were predicted to within 62 residues from the cleavage sites given in SWISS‐PROT. An analysis of 715 Arabidopsis thaliana sequences from SWISS‐PROT suggests that the ChloroP method should be useful for the identification of putative transit peptides in genome‐wide sequence data. The ChloroP predictor is available as a web‐server at http://www.cbs.dtu.dk/services/ChloroP/.
Polymer principles and protein folding
Authors: Dill Ken A. 
Published: February 2009

AbstractThis paper surveys the emerging role of statistical mechanics and polymer theory in protein folding. In the polymer perspective, the folding code is more a solvation code than a code of local ØΨ propensities. The polymer perspective resolves two classic puzzles: (1) the Blind Watchmaker' Paradox that biological proteins could not have originated from random sequences, and (2) Levinthal' Paradox that the folded state of a protein cannot be found by random search. Both paradoxes are traditionally framed in terms of random unguided searches through vast spaces, and vastness is equated with impossibility. But both processes are partly guided. The searches are more akin to balls rolling down funnels than balls rolling aimlessly on flat surfaces. In both cases, the vastness of the search is largely irrelevant to the search time and success. These ideas are captured by energy and fitness landscapes. Energy landscapes give a language for bridging between microscopics and macroscopics, for relating folding kinetics to equilibrium fluctuations, and for developing new and faster computational search strategies.