RESUMO
Novel strategies are proposed to quantitatively analyze and relate biological pathways to drug responses using gene expression and small-molecule growth inhibition data (GI(50)) derived from the National Cancer Institute's 60 cancer cells (NCI(60)). We have annotated groups of drug GI(50) responses with pathways defined by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and BioCarta, and functional categories defined by Gene Ontology (GO), through correlations between pathway gene expression patterns and drug GI(50) profiles. Drug-gene-pathway relationships may then be utilized to find drug targets or target-specific drugs. Significantly correlated pathways and the gene products involved represent interesting targets for further exploration, whereas drugs that are significantly correlated with only certain pathways are more likely to be target specific. Separate pathway clustering finds that pathways engaged in the same biological process tend to have similar drug correlation patterns. The biological and statistical significances of our method are established by comparison to known small-molecule inhibitor-gene target relationships reported in the literature and by standard randomization procedures. The results of our pathway, gene expression and drug-induced growth inhibition associations, can serve as a basis for proposing testable hypotheses about potential anticancer drugs, their targets, and mechanisms of action.
Assuntos
Antineoplásicos/farmacologia , Bases de Dados Factuais , Bases de Dados Genéticas , Ensaios de Seleção de Medicamentos Antitumorais , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Antineoplásicos/classificação , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Armazenamento e Recuperação da Informação/métodos , Dose Letal Mediana , National Institutes of Health (U.S.) , Neoplasias/genética , Valor Preditivo dos Testes , Células Tumorais Cultivadas/efeitos dos fármacos , Estados UnidosRESUMO
The Protein Data Bank (PDB; http://www.rcsb.org/pdb/) is the single worldwide archive of structural data of biological macromolecules. This paper describes the data uniformity project that is underway to address the inconsistency in PDB data.
Assuntos
Bases de Dados Factuais , Proteínas/química , Armazenamento e Recuperação da Informação , Internet , Espectroscopia de Ressonância Magnética , Conformação ProteicaRESUMO
The molecular mechanisms that evolution has been employing to adapt to environmental temperatures are poorly understood. To gain some further insight into this subject we solved the crystal structure of triosephosphate isomerase (TIM) from the hyperthermophilic bacterium Thermotoga maritima (TmTIM). The enzyme is a tetramer, assembled as a dimer of dimers, suggesting that the tetrameric wild-type phosphoglycerate kinase PGK-TIM fusion protein consists of a core of two TIM dimers covalently linked to 4 PGK units. The crystal structure of TmTIM represents the most thermostable TIM presently known in its 3D-structure. It adds to a series of nine known TIM structures from a wide variety of organisms, spanning the range from psychrophiles to hyperthermophiles. Several properties believed to be involved in the adaptation to different temperatures were calculated and compared for all ten structures. No sequence preferences, correlated with thermal stability, were apparent from the amino acid composition or from the analysis of the loops and secondary structure elements of the ten TIMs. A common feature for both psychrophilic and T. maritima TIM is the large number of salt bridges compared with the number found in mesophilic TIMs. In the two thermophilic TIMs, the highest amount of accessible hydrophobic surface is buried during the folding and assembly process.
Assuntos
Thermotoga maritima/química , Triose-Fosfato Isomerase/química , Sequência de Aminoácidos , Cristalografia por Raios X , Calefação , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Estrutura Secundária de Proteína , Alinhamento de SequênciaRESUMO
Triosephosphate isomerase (TIM) is a very stable dimer. In order to understand better the importance of dimerization for stability and catalytic activity, we have constructed a monomeric double-mutation variant. The dimer interface residues Thr75 and Gly76, which are at the tip of loop 3, have been substituted by an arginine and a glutamate, respectively. In wild type TIM, these two residues are at a distance of 27 A from the active site (as measured within the same subunit). This new variant, RE-TIM, was expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Sedimentation equilibrium ultracentrifugation runs showed that RE-TIM is a monomer in solution. Far-UV CD spectra indicate that this new variant is folded properly and that the secondary structure contents of RE-TIM are similar to those of wild type TIM. The monomeric RE-TIM has residual TIM activity. The thermal stability of RE-TIM is lower than that for wild type TIM. CD melting curves for RE-TIM and wild type TIM show Tm values of 52 and 57 degrees C, respectively, in the presence of the active site ligand 2-phosphoglycolate at 1 mM. Previously, we have characterized two other monomeric forms of TIM: monoTIM and H47N-TIM. The properties of RE-TIM, H47N-TIM, and monoTIM are compared, and it is argued that the properties of RE-TIM will be very similar to those of wild type monomeric subunits. This implies that wild type monomeric subunits have some stability and are catalytically active. It is also inferred that these monomeric subunits have flexible loops which rigidify at the dimer interface on dimerization, causing a 1000-fold increase of kcat and a 10-fold decrease of Km.
Assuntos
Mutagênese Sítio-Dirigida , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/genética , Catálise , Dimerização , Ativação Enzimática/genética , Estabilidade Enzimática/genética , Variação Genética , Cinética , Termodinâmica , Triose-Fosfato Isomerase/isolamento & purificaçãoRESUMO
Protein engineering experiments have been carried out with loop-1 of monomeric triosephosphate isomerase (monoTIM). Loop-1 of monoTIM is disordered in every crystal structure of liganded monoTIM, but in the wild-type TIM it is a very rigid dimer interface loop. This loop connects the first beta-strand with the first alpha-helix of the TIM-barrel scaffold. The first residue of this loop, Lys13, is a conserved catalytic residue. The protein design studies with loop-1 were aimed at rigidifying this loop such that the Lys13 side chain points in the same direction as seen in wild type. The modelling suggested that the loop should be made one residue shorter. With the modelling package ICM the optimal sequence of a new seven-residue loop-1 was determined and its structure was predicted. The new variant could be expressed and purified and has been characterized. The catalytic activity and stability are very similar to those of monoTIM. The crystal structure (at 2.6 A resolution) shows that the experimental loop-1 structure agrees well with the modelled loop-1 structure. The direct superposition of the seven loop residues of the modelled and experimental structures results in an r.m.s. difference of 0.5 A for the 28 main chain atoms. The good agreement between the predicted structure and the crystal structure shows that the described modelling protocol can be used successfully for the reliable prediction of loop structures.
Assuntos
Modelos Moleculares , Triose-Fosfato Isomerase/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Primers do DNA/genética , Estabilidade Enzimática , Cinética , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Engenharia de Proteínas , Soluções , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismoRESUMO
The structure of orthorhombic crystals of monellin, a sweet protein extracted from African serendipity berries, has been solved by molecular replacement and refined to 2.3 A resolution. The final R factor was 0.150 for a model with excellent geometry. A monellin molecule consists of two peptides that are non-covalently bound, with chain A composed of three beta-strands interconnected by loop regions and chain B composed of two beta-strands interconnected by an alpha-helix. The N terminus of chain A is in close proximity to the C terminus of chain B. The two molecules in the asymmetric unit are related by a non-crystallographic twofold axis and form a dimer, similar to those previously observed in other crystal forms of both natural and single-chain monellin. The r.m.s, deviation between the Calpha atoms in the two independent molecules is 0.60 A, while the deviations from the individual molecules in the previously reported monoclinic crystals are 0.50-0.57 A. This result proves that the structure of monellin is not significantly influenced by crystal packing forces.
RESUMO
MonoTIM is a stable monomeric variant of the dimeric trypanosomal enzyme triose phosphate isomerase (TIM) with less, but significant, catalytic activity. It is known that in TIM, three residues, Lys 13 (loop 1), His 95 (loop 4), and Glu 167 (loop 6) are the crucial catalytic residues. In the wild-type TIM dimer, loop 1 and loop 4 are very rigid because of tight interactions with residues of the other subunit. Previous structural studies indicate that Lys 13 and His 95 have much increased conformational flexibility in monoTIM. Using site-directed mutagenesis, it is shown here that Lys 13 and His 95 are nevertheless essential for optimal catalysis by monoTIM: monoTIM-K13A is completely inactive, although it can still bind substrate analogues, and monoTIM-H95A is 50 times less active. The best inhibitors of wild-type TIM are phosphoglycolohydroxamate (PGH) and 2-phosphoglycolate (2PG), with KI values of 8 microM and 26 microM, respectively. The affinity of the monoTIM active site for PGH has been reduced approximately 60-fold, whereas for 2PG, only a twofold weakening of affinity is observed. The mode of binding, as determined by protein crystallographic analysis of these substrate analogues, shows that, in particular, 2PG interacts with Lys 13 and His 95 in a way similar but not identical to that observed for the wild-type enzyme. This crystallographic analysis also shows that Glu 167 has the same interactions with the substrate analogues as in the wild type. The data presented suggest that, despite the absence of the second subunit, monoTIM catalyzes the interconversion of D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate via the same mechanism as in the wild type.
Assuntos
Modelos Moleculares , Conformação Proteica , Proteínas de Protozoários/química , Triose-Fosfato Isomerase/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Catálise , Galinhas/metabolismo , Dicroísmo Circular , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ácido Glutâmico/química , Glicolatos/metabolismo , Glicolatos/farmacologia , Histidina/química , Ácidos Hidroxâmicos/metabolismo , Ácidos Hidroxâmicos/farmacologia , Cinética , Lisina/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/enzimologia , Especificidade da Espécie , Relação Estrutura-Atividade , Especificidade por Substrato , Triose-Fosfato Isomerase/antagonistas & inibidores , Triose-Fosfato Isomerase/genética , Trypanosoma/enzimologia , Trypanosoma/genéticaRESUMO
The purpose of this study was to bacterially express, purify, and refold combinations of the extracellular immunoglobulin (Ig)-like domains (2-3, 1-3, and 1-5) of the human alpha-platelet-derived growth factor receptor (alpha PDGFR) to characterize molecular interactions with its ligand, platelet-derived growth factor (PDGF). The far UV circular dichroism spectroscopy of the alpha-PDGFR extracellular domains (ECDs) revealed a predominantly beta-sheet protein, with a structure consistent with folded Ig-like domains. The addition of PDGF-BB to these ECD types changed the conformation of all three types with a decrease in mean residue ellipticity in the following rank order: 1-5 = 1-3 > 2-3. In striking contrast, addition of PDGF-AA to these ECD types markedly changed the conformation of ECD 2-3, by an increased mean residue ellipticity but no changes were observed for ECDs 1-3 and 1-5. PDGF-AA bound to the immobilized ECD types 2-3, 1-3, and 1-5 at concentrations of 20, 11, and 7.5 nM, respectively. In contrast, PDGF-BB bound the ECD types 2-3, 1-3, and 1-5 at concentrations of 3, 3, and 2.2 nM, respectively. Scatchard analysis of binding studies using labeled ECDs indicated that PDGF-BB bound ECD 1-3 and ECD 2-3 with KD values of 74 and 72 nM, respectively. While, PDGF-AA bound ECD 1-3 and ECD 2-3 with KD values of 33 and 87 nM, respectively. Therefore, our results indicated that the loss of ECD 1 impaired the binding affinity of alpha PDGFR ECD 1-3 toward PDGF-AA without having a similar effect on PDGF-BB binding. Together all of our data suggest that ECD 1 is differentially required for proper orientation of PDGF-AA but not PDGF-BB binding determinant within ECDs 2 and 3.
Assuntos
Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/química , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Becaplermina , Sítios de Ligação , Cromatografia de Afinidade , Dicroísmo Circular , Espaço Extracelular/fisiologia , Humanos , Radioisótopos do Iodo , Cinética , Modelos Estruturais , Reação em Cadeia da Polimerase , Conformação Proteica , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-sis , Ensaio Radioligante , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Receptores do Fator de Crescimento Derivado de Plaquetas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por RestriçãoRESUMO
BACKGROUND: Wild-type triosephosphate isomerase (TIM) is a very stable dimeric enzyme. This dimer can be converted into a stable monomeric protein (monoTIM) by replacing the 15-residue interface loop (loop-3) by a shorter, 8-residue, loop. The crystal structure of monoTIM shows that two active-site loops (loop-1 and loop-4), which are at the dimer interface in wild-type TIM, have acquired rather different structural properties. Nevertheless, monoTIM has residual catalytic activity. RESULTS: Three new structures of variants of monoTIM are presented, a double-point mutant crystallized in the presence and absence of bound inhibitor, and a single-point mutant in the presence of a different inhibitor. These new structures show large structural variability for the active-site loops, loop-1, loop-4 and loop-8. In the structures with inhibitor bound, the catalytic lysine (Lys13 in loop-1) and the catalytic histidine (His95 in loop-4) adopt conformations similar to those observed in wild-type TIM, but very different from the monoTIM structure. CONCLUSIONS: The residual catalytic activity of monoTIM can now be rationalized. In the presence of substrate analogues the active-site loops, loop-1, loop-4 and loop-8, as well as the catalytic residues, adopt conformations similar to those seen in the wild-type protein. These loops lack conformational flexibility in wild-type TIM. The data suggest that the rigidity of these loops in wild-type TIM, resulting from subunit-subunit contacts at the dimer interface, is important for optimal catalysis.
Assuntos
Variação Genética , Mutação Puntual , Estrutura Secundária de Proteína , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/genética , Sequência de Aminoácidos , Clonagem Molecular , Cristalografia por Raios X/métodos , Escherichia coli , Histidina , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Triose-Fosfato Isomerase/biossínteseRESUMO
Pleckstrin homology (PH) domains are approximately 110 amino acid residues in length and are structurally conserved in a number of intracellular signaling proteins. A role for these domains has been postulated for beta ARK, which binds to G beta gamma subunits. We have quantified the binding of individual (His)6-tag PH domains of human Db1, human Sos1, rat IRS-1, human beta ARK, and human beta ARK with an extra 33-residue C-terminal extension (beta ARK + C) to G beta gamma subunits. Our in vitro binding studies show that all of the PH domains (apart from Sos1), bind G beta gamma subunits in a dose-dependent manner, but beta ARK + C binds 4 times as much G beta gamma at saturation as the others. The IRS-1 PH domain has a similar half-maximal concentration of G beta gamma binding (18 nM) to beta ARK + C (30 nM), suggesting that the IRS-1 PH domain has sufficient determinants for G beta gamma binding. The beta ARK PH domain alone has a half-maximal value of 45 nM but a drastically reduced extent of G beta gamma binding, suggesting that both the PH domain and the C-terminal 33 residues are necessary for maximal binding. Db1 has a half-maximum concentration of G beta gamma binding of 45 nM and a maximal extent of binding similar to that of beta ARK, but it is difficult to demonstrate saturable binding of G beta gamma to Sos1. Since it was previously predicted that the C-terminal PH domain of Pleckstrin [Tyers, M., et al. (1988) Nature 333, 470-473] contains a potential calcium binding site, we have tested the different PH domains for calcium binding. Only the PH domain of Db1 bound 45Ca2+ with a Kd of 10 microM. CD spectroscopy of the purified recombinant PH domains indicated that they are predominantly beta-sheet structures.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas Sanguíneas/química , Proteínas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Cálcio/metabolismo , Dicroísmo Circular , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/química , Fatores de Troca do Nucleotídeo Guanina , Humanos , Técnicas In Vitro , Proteínas Substratos do Receptor de Insulina , Cinética , Dados de Sequência Molecular , Estrutura Molecular , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas , Ratos , Proteínas Oncogênicas de Retroviridae/química , Proteínas Oncogênicas de Retroviridae/genética , Proteínas Oncogênicas de Retroviridae/metabolismo , Homologia de Sequência de Aminoácidos , Quinases de Receptores Adrenérgicos betaRESUMO
To investigate the effects of metal ion binding to the alpha-PDGFR kinase insert domain, a PCR product representing amino acid residues 691-795 (104 amino acids) was bacterially expressed and purified. Secondary structure prediction and circular dichroism spectroscopy indicated this domain to be a mixed alpha + beta protein with a large coil/turn contribution. This 16 kDa, soluble, nonphosphorylated domain bound to 45Ca2+ and 65Zn2+ through a common shared site. Of the unlabeled divalent and trivalent metal ions tested, Ho3+ = Zn2+ > Ni2+ > Ca2+ = Mn2+ > Mg2+, Ba2+ in competing for 45Ca2+ binding to this domain. In the presence of Ca2+ ions, the conformation of the KI domain changed significantly, and this changed conformation was resistant to subtilisin proteolysis. However, in the presence of Zn2+ ions, the conformation of the KI domain changed only slightly. Nevertheless, Zn2+ ions were more effective in rendering the KI domain resistant to proteolysis as compared to that shown by Ca2+ ions. In vitro binding studies using purified baculovirus-expressed alpha-PDGFR showed a marked increase in binding the p85 N-SH2 domain in the presence of Ca2+ or Zn2+ ions (KD = 0.5 microM), suggesting that metal ion binding enhances association of the p85 N-SH2 domain with the receptor. To confirm this, association of the alpha-PDGFR with the p85 N-SH2 domain was tested in the presence of the KI domain. The nonphosphorylated KI domain was effective in competing with the alpha-PDGFR for the binding of the p85 N-SH2 domain. This effect was more pronounced in the presence of Ca2+ ions. Microinjection of this domain into Xenopus oocytes delayed maturation in the presence of insulin but not progesterone. This suggests that the KI domain has a correctly folded three-dimensional structure compatible with biological activity. Together these findings indicate that the recombinant alpha-PDGFR KI domain binds the p85 N-SH2 domain and this binding is modulated by the presence of a novel divalent metal ion binding site within its structure.
Assuntos
Cálcio/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Cátions Bivalentes , Dicroísmo Circular , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/química , Oócitos , Fosfatidilinositol 3-Quinases , Fosfatidilinositóis/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Subtilisinas/farmacologia , Xenopus laevisRESUMO
Src-homology region 2 (SH2) domains are stretches of about 100 amino acids which are found to be structurally conserved in a number of signaling molecules. These regions have been shown to bind with high affinity to phosphotyrosine residues within activated receptor tyrosine kinases. Here we report the bacterial expression and purification of individual N-terminal SH2 (NSH2) domains of phosphatidylinositol 3-kinase (PI-3K) binding subunit (p85) and Ras GTPase activating protein (GAP) in amounts suitable for structure-function studies. The p85NSH2 domain stains dark purple and absorbs around 620-640 nm with Stains-all, a dye known to bind to calcium binding proteins. This effect was not observed for the GAPNSH2 domain. Circular dichroism analysis of the N-terminal SH2 domain of these proteins shows that p85NSH2, but not GAPNSH2, undergoes a significant dose-dependent change in conformation in the presence of increasing calcium concentrations. Moreover, the conformational change of p85NSH2 induced by calcium could be replicated by addition of a phosphorylated hexapeptide (DYpMDMK) representing the alpha-PDGFR binding site for p85. Limited proteolysis studies showed a significant calcium-dependent increase in protection of p85NSH2 but not GAPNSH2 from degradation by subtilisin. Our results further indicate that holmium, a trivalent lanthanide ion, which has been previously shown to substitute for calcium, could also protect the p85NSH2 domain from proteolysis even at 10-fold lower concentrations. In vitro binding studies using purified preparations of activated alpha-PDGFR show that calcium did not affect the binding of GAPNSH2 domains to activated alpha-PDGFR.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cálcio/farmacologia , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Conformação Proteica/efeitos dos fármacos , Proteínas/química , Sequência de Aminoácidos , Carbocianinas/farmacologia , Dicroísmo Circular , Relação Dose-Resposta a Droga , Proteínas Ativadoras de GTPase , Modelos Químicos , Dados de Sequência Molecular , Proteína Oncogênica pp60(v-src)/genética , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/efeitos dos fármacos , Fragmentos de Peptídeos/genética , Fosfatidilinositol 3-Quinases , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Biossíntese de Proteínas , Proteínas/efeitos dos fármacos , Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Espectrofotometria Ultravioleta , Proteínas Ativadoras de ras GTPaseRESUMO
Energy calculations have been used to study the hydration sites around the polar groups of serine, threonine and tyrosine side chains. These hydration sites depend not only on the hybridization of the polar group but also on the local secondary structure, the chi 1 side chain torsion angle and the position of the hydroxyl hydrogen atom. For tyrosine side chains, two solvent sites are found approximately in the plane of the ring. Even for serine and threonine side chains only two minimum energy sites are found in general of which one is in an expected position within hydrogen bonding of the hydroxyl hydrogen atom (unless this is blocked from interaction with solvent molecules by, for example, Oi-4 or Oi-3. The position of the second of these sites depends not only on the position of the hydroxyl oxygen but also on neighbouring main chain atoms to which it can also hydrogen bond. There is good agreement with the solvent distributions obtained from crystallographic data.
Assuntos
Aminoácidos/química , Estrutura Secundária de Proteína , Água , Simulação por Computador , Ligação de Hidrogênio , Computação Matemática , Modelos Químicos , Modelos Moleculares , Serina/química , Software , Treonina/química , Tirosina/químicaRESUMO
The structure of a crystal complex of recombinant human immunodeficiency virus type 1 (HIV-1) protease with a peptide-mimetic inhibitor containing a dihydroxyethylene isostere insert replacing the scissile bond has been determined. The inhibitor is Noa-His-Hch psi [CH(OH)CH(OH)]Vam-Ile-Amp (U-75875), and its Ki for inhibition of the HIV-1 protease is < 1.0 nM (Noa = 1-naphthoxyacetyl, Hch = a hydroxy-modified form of cyclohexylalanine, Vam = a hydroxy-modified form of valine, Amp = 2-pyridylmethylamine). The structure of the complex has been refined to a crystallographic R factor of 0.169 at 2.0 A resolution by using restrained least-squares procedures. Root mean square deviations from ideality are 0.02 A and 2.4 degrees, for bond lengths and angles, respectively. The bound inhibitor diastereomer has the R configurations at both of the hydroxyl chiral carbon atoms. One of the diol hydroxyl groups is positioned such that it forms hydrogen bonds with both the active site aspartates, whereas the other interacts with only one of them. Comparison of this X-ray structure with a model-built structure of the inhibitor, published earlier, reveals similar positioning of the backbone atoms and of the side-chain atoms in the P2-P2' region, where the interaction with the protein is strongest. However, the X-ray structure and the model differ considerably in the location of the P3 and P3' end groups, and also in the positioning of the second of the two central hydroxyl groups. Reconstruction of the central portion of the model revealed the source of the hydroxyl discrepancy, which, when corrected, provided a P1-P1' geometry very close to that seen in the X-ray structure.
Assuntos
Antivirais/química , Inibidores da Protease de HIV/química , Protease de HIV/química , Oligopeptídeos/farmacologia , Conformação Proteica , Sequência de Aminoácidos , Antivirais/farmacologia , Sítios de Ligação , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/enzimologia , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Oligopeptídeos/química , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Difração de Raios XRESUMO
We have analysed the hydration of main-chain carbonyl and amide groups in 24 high-resolution well-refined protein structures as a function of the secondary structure in which these polar groups occur. We find that main-chain atoms in beta-sheets are as hydrated as those in alpha-helices, with most interactions involving "free" amide and carbonyl groups that do not participate in secondary structure hydrogen bonds. The distributions of water molecules around these non-bonded carbonyl groups reflect specific steric interactions due to the local secondary structure. Approximately 20% and 4%, respectively of bonded carbonyl and amide groups interact with solvent. These include interactions with carbonyl groups on the exposed faces of alpha-helices that have been correlated previously with bending of the helix. Water molecules interacting with alpha-helices occur mainly at the amino and carbonyl termini of the helices, in which case the solvent sites maintain the hydrogen bonding by bridging between residues i and i-3 or i-4 at the amino terminus and between i and i+3 or i+4 at the carbonyl terminus. We also see a number of solvent-mediated Ncap and Ccap interactions. The water molecules interacting with beta-sheets occur mainly at the edges, in which case they extend the sheet structure, or at the ends of strands, in which case they extend the beta-ladder. In summary, the solvent networks appear to extend the hydrogen-bonding structure of the secondary structures. In beta-turns, which usually occur at the surface of a protein, exposed amide and carbonyl groups are often hydrated, especially close to glycine residues. Occasionally water molecules form a bridge between residues i and i+3 in the turn and this may provide extra stabilization.
Assuntos
Conformação Proteica , Proteínas/química , Amidas/química , Sequência de Aminoácidos , Carbono/química , Humanos , Ligação de Hidrogênio , Dados de Sequência Molecular , Oxigênio/química , Solubilidade , Relação Estrutura-Atividade , Água/química , Difração de Raios XRESUMO
Previous analysis of experimental data on the solvation of high resolution protein structures has shown that preferred interaction sites for water molecules exist around most amino acid side chains. We have extended this analysis to look in more detail at the distributions around serine, threonine and tyrosine. We find that for serine and threonine side chains the preferred interaction sites of solvent molecules with the hydroxyl group depends on secondary structure and the chi 1 torsion angle of the side chain. For tyrosine side chains the hydroxyl group is too far from the main chain to reflect secondary structure influences. Specific patterns of hydration are observed in which water molecules 'bridge' between the hydroxyl side-chain atom and another main chain or side-chain atom.
Assuntos
Aminoácidos/análise , Proteínas/metabolismo , Serina/metabolismo , Treonina/metabolismo , Tirosina/metabolismo , Água/análise , Conformação Proteica , Estereoisomerismo , Difração de Raios XRESUMO
The atomic co-ordinates from 16 high-resolution (less than or equal to 1.7 A = 0.1 nm), non-homologous proteins have been used to study the distributions of water molecule sites around the 20 different amino acid residues. The proportion of residues whose main-chain atoms are in contact with water molecules was fairly constant (between 40% and 60%), irrespective of the nature of the side-chain. However, the proportion of residues whose side-chain atoms were in contact with water molecules showed a clear (inverse) correlation with the hydrophobicity of the residue, being as low as 14% for leucine and isoleucine but greater than 80% for asparagine and arginine. Despite the problems in determining accurate water molecule sites from X-ray diffraction data and the complexity of the protein surface, distinct non-random distributions of water molecules were found. These hydration patterns are consistent with the expected stereochemistry of the potential hydrogen-bonding sites on the polar side-chains. The water molecules around apolar side-chains lie predominantly at van der Waals' contact distances, but most of these have a primary, shorter contact with a neighbouring polar atom. Further analysis of these distributions, combined with energy minimization techniques, should lead to improved modelling of protein structures, including their primary shells of hydration.