RESUMO
Collagenase is a zinc-dependent endoproteinase and is a member of the matrix metalloproteinase (MMP) family of enzymes. The MMPs participate in connective tissue remodeling events and aberrant regulation has been associated with several pathologies. The 2.4 angstrom resolution structure of the inhibited enzyme revealed that, in addition to the catalytic zinc, there is a second zinc ion and a calcium ion which play a major role in stabilizing the tertiary structure of collagenase. Despite scant sequence homology, collagenase shares structural homology with two other endoproteinases, bacterial thermolysin and crayfish astacin. The detailed description of protein-inhibitor interactions present in the structure will aid in the design of compounds that selectively inhibit individual members of the MMP family. Such inhibitors will be useful in examining the function of MMPs in pathological processes.
Assuntos
Colagenases/química , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Colagenases/metabolismo , Gráficos por Computador , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Metaloproteinase 8 da Matriz , Inibidores de Metaloproteinases de Matriz , Metaloendopeptidases/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Termolisina/química , Zinco/metabolismoRESUMO
Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/química , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , AMP Cíclico/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalização , Cristalografia por Raios X , AMP Cíclico/química , GMP Cíclico/química , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Ligação de Hidrogênio , Hidrólise , Metais/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
Most traditional cytotoxic anticancer agents ablate the rapidly dividing epithelium of the hair follicle and induce alopecia (hair loss). Inhibition of cyclin-dependent kinase 2 (CDK2), a positive regulator of eukaryotic cell cycle progression, may represent a therapeutic strategy for prevention of chemotherapy-induced alopecia (CIA) by arresting the cell cycle and reducing the sensitivity of the epithelium to many cell cycle-active antitumor agents. Potent small-molecule inhibitors of CDK2 were developed using structure-based methods. Topical application of these compounds in a neonatal rat model of CIA reduced hair loss at the site of application in 33 to 50% of the animals. Thus, inhibition of CDK2 represents a potentially useful approach for the prevention of CIA in cancer patients.
Assuntos
Alopecia/induzido quimicamente , Alopecia/prevenção & controle , Antineoplásicos/toxicidade , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Folículo Piloso/efeitos dos fármacos , Indóis/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Animais Recém-Nascidos , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Ciclofosfamida/toxicidade , Citoproteção/efeitos dos fármacos , DNA/biossíntese , Doxorrubicina/toxicidade , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Epitélio/efeitos dos fármacos , Etoposídeo/toxicidade , Folículo Piloso/citologia , Humanos , Indóis/síntese química , Indóis/química , Camundongos , Camundongos SCID , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Proteína do Retinoblastoma/metabolismo , Couro Cabeludo/transplante , Sulfonamidas/síntese química , Sulfonamidas/química , Transplante HeterólogoRESUMO
BACKGROUND: Angiogenesis, the formation of new vessels from the existing vasculature, is a critical process during early development as well as in a number of disease processes. Tie2 (also known as Tek) is an endothelium-specific receptor tyrosine kinase involved in both angiogenesis and vasculature maintenance. RESULTS: We have determined the crystal structure of the Tie2 kinase domain to 2.2 A resolution. The structure contains the catalytic core, the kinase insert domain (KID), and the C-terminal tail. The overall fold is similar to that observed in other serine/threonine and tyrosine kinase structures; however, several unique features distinguish the Tie2 structure from those of other kinases. The Tie2 nucleotide binding loop is in an inhibitory conformation, which is not seen in other kinase structures, while its activation loop adopts an "activated-like" conformation in the absence of phosphorylation. Tyr-897, located in the N-terminal domain, may negatively regulate the activity of Tie2 by preventing dimerization of the kinase domains or by recruiting phosphatases when it is phosphorylated. CONCLUSION: Regulation of the kinase activity of Tie2 is a complex process. Conformational changes in the nucleotide binding loop, activation loop, C helix, and the C-terminal tail are required for ATP and substrate binding.
Assuntos
Receptores Proteína Tirosina Quinases/química , Substituição de Aminoácidos , Vasos Sanguíneos/anormalidades , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Genes Dominantes , Humanos , Ligação de Hidrogênio , Espectrometria de Massas , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor TIE-2 , Proteínas Recombinantes de Fusão/química , Relação Estrutura-Atividade , Domínios de Homologia de srcRESUMO
Recombinant human interleukin-5 (rhIL-5) has been crystallized by the hanging drop vapor diffusion method using 0.1 M-Tris.HCl buffer (pH 8.5) containing 0.2 to 0.25 M-sodium acetate and 26 to 30% PEG 4000 at 22 degrees C. The parallel-piped crystals belong to the space group C2 with unit cell dimensions of a = 122.1 A, b = 36.11 A, c = 56.42 A, beta = 98.59 degrees. They diffract to at least 2.0 A resolution on a rotating anode X-ray source. The molecular mass weight of the protein and the volume of the unit cell suggest that the asymmetric unit contains one intermolecular disulfide-bonded homodimer.
Assuntos
Interleucina-5/química , Cristalização , Escherichia coli , Humanos , Espectrometria de Massas , Proteínas Recombinantes/química , Difração de Raios XRESUMO
Crystals of the catalytic domain of human fibroblast collagenase have been grown in the presence and absence of an inhibitor. Crystals of the inhibitor complex grew from 0.2 M ammonium sulfate and 15 to 30% PEG 8000 at 22 degrees C as bipyramids in the space group P6(2) or P6(4). Crystals of the unligated enzyme grew as rods in the space group P4(1)2(1)2 or P4(3)2(1)2 from 1.0 to 2.0 M sodium formate at 4 degrees C. Both crystal forms grew quite slowly over a period of months, but ultimately yielded crystals that diffracted beyond 2.5 A. The collagenase samples used in these studies were heterogeneous at the amino terminus. Three major species (full length, N-1 and N-2) were identified by mass spectrometry and Edman sequencing. Analysis of dissolved crystals revealed the native crystal form selectively crystallized as the N-2 species; however, no selectivity of N-terminal forms was observed for crystals of the inhibitor complex.
Assuntos
Colagenases/ultraestrutura , Cristalografia por Raios X , Fibroblastos/enzimologia , Humanos , Substâncias Macromoleculares , Espectrometria de Massas , Inibidores de Metaloproteinases de Matriz , Peso Molecular , Proteínas RecombinantesRESUMO
The human papillomaviruses (HPVs) are ubiquitous human pathogens that cause a wide variety of benign and pre-malignant epithelial tumours. Of the almost 100 different types of HPV that have been characterized to date, approximately two dozen specifically infect genital and oral mucosa. Mucosal HPVs are most frequently sexually transmitted and, with an incidence roughly twice that of herpes simplex virus infection, are considered one of the most common sexually transmitted diseases throughout the world. A subset of genital HPVs, termed 'high-risk' HPVs, is highly associated with the development of genital cancers including cervical carcinoma. The absence of a simple monolayer cell culture system for analysis and propagation of the virus has substantially retarded progress in the development of diagnostic and therapeutic strategies for HPV infection. In spite of these difficulties, great progress has been made in the elucidation of the molecular controls of virus gene expression, replication and pathogenesis. With this knowledge and some important new tools, there is great potential for the development of improved diagnostic and prognostic tests, prophylactic and therapeutic vaccines, and traditional antiviral medicines.
Assuntos
Antivirais/uso terapêutico , Papillomaviridae , Infecções por Papillomavirus/tratamento farmacológico , Infecções Tumorais por Vírus/tratamento farmacológico , HumanosAssuntos
Colagenases/química , Conformação Proteica , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Metaloproteinase 8 da Matriz , Inibidores de Metaloproteinases de Matriz , Metaloendopeptidases/antagonistas & inibidores , Modelos Estruturais , Dados de Sequência Molecular , Oligopeptídeos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/químicaRESUMO
Collagenase is a member of the matrix metalloproteinase (MMP) family of enzymes. Aberrant regulation of this family has been implicated in pathologies such as arthritis and metastasis. Two crystal forms of the catalytic (19-kDa) domain of human fibroblast collagenase have been determined using collagenase complexed with a peptide-based inhibitor (CPLX) as a starting model [Lovejoy et al. (1994) Science 263, 375]. The first crystal form (CF1) contains one molecule in the asymmetric unit and has been determined at 1.9-A resolution with an R factor of 19.8%. The second crystal form (CF2) contains two molecules (A and B) in the asymmetric unit and has been determined at 2.1-A resolution with an R factor of 19.7%. The catalytic domain of collagenase is spherical with an active site cleft that contains a ligated catalytic zinc ion. Collagenase shares some structural homology with the bacterial zinc proteinase, thermolysin [Matthews et al. (1972) Nature, New Biol. 238, 37], and the crayfish digestive peptidase, astacin [Bode et al. (1992) Nature 358, 164]. The amino terminus (Leu 102 to Gly 105) of CF1 and CF2 molecules A and B differs from the conformation found in CPLX by bending away from the molecule and interacting with the active site cleft of symmetry-related molecules. In this alternative conformation, both the mainchain nitrogen and carbonyl oxygen of Leu 102 ligate the symmetry-related catalytic zinc. Although there are structural differences in the active site clefts of CF1, CF2, and CPLX, a number of complex-stabilizing interactions are conserved. The structure of collagenase will be useful for developing compounds that selectively inhibit individual members of the closely related matrix metalloproteinase family.
Assuntos
Colagenases/química , Fibroblastos/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/farmacologia , Colagenases/metabolismo , Cristalização , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Espectrometria de Massas , Inibidores de Metaloproteinases de Matriz , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Conformação Proteica , Proteínas Recombinantes/química , Termolisina/antagonistas & inibidores , Termolisina/química , Termolisina/metabolismo , Zinco/farmacologiaRESUMO
Orotidine 5'-phosphate decarboxylase produces the largest rate enhancement that has been reported for any enzyme. The crystal structure of the recombinant Saccharomyces cerevisiae enzyme has been determined in the absence and presence of the proposed transition state analog 6-hydroxyuridine 5'-phosphate, at a resolution of 2.1 A and 2.4 A, respectively. Orotidine 5'-phosphate decarboxylase folds as a TIM-barrel with the ligand binding site near the open end of the barrel. The binding of 6-hydroxyuridine 5'-phosphate is accompanied by protein loop movements that envelop the ligand almost completely, forming numerous favorable interactions with the phosphoryl group, the ribofuranosyl group, and the pyrimidine ring. Lysine-93 appears to be anchored in such a way as to optimize electrostatic interactions with developing negative charge at C-6 of the pyrimidine ring, and to donate the proton that replaces the carboxylate group at C-6 of the product. In addition, H-bonds from the active site to O-2 and O-4 help to delocalize negative charge in the transition state. Interactions between the enzyme and the phosphoribosyl group anchor the pyrimidine within the active site, helping to explain the phosphoribosyl group's remarkably large contribution to catalysis despite its distance from the site of decarboxylation.
Assuntos
Orotidina-5'-Fosfato Descarboxilase/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Saccharomyces cerevisiae/enzimologiaRESUMO
Crystals of the Saccharomyces cerevisiae pyrimidine biosynthetic enzyme orotidine 5'-phosphate decarboxylase (ODCase) were grown by the hanging-drop vapor-diffusion technique at 277 K using polyethylene glycol 4000 as the precipitant. Crystals of native and selenomethionyl ODCase diffract to less than 2.2 A and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 90.1, b = 116.2, c = 117.0 A. Crystals of ODCase grown in the presence of the postulated transition-state analog inhibitor 6-hydroxyuridine 5'--phosphate (BMP) diffract to less than 2.5 A and belong to space group P2(1), with unit-cell parameters a = 79.9, b = 80.0, c = 98.2 A, beta = 108.6 degrees.
Assuntos
Orotidina-5'-Fosfato Descarboxilase/química , Saccharomyces cerevisiae/enzimologia , Cristalização , Cristalografia por Raios X , Escherichia coli , Orotidina-5'-Fosfato Descarboxilase/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Selenometionina/metabolismoRESUMO
Two different systems for recording crystallization results are described. Both programs are written in Microsoft Excel. Results can be entered either as text or numerical scores. In each system, one can search or query on the results, macromolecule name, experimental parameters, etc. and correlate these to specific crystallization conditions or trends in the crystallizations. Such information is valuable when optimizing crystal growth conditions and determining new crystallization parameters to test. XTAL RES is a Macintosh program that is linked to PCRYSTAL (software used to enter details of the crystallization experiments) through an Oracle database on a VAX network. A second system, the Electronic Notebook, was designed as a 'stand-alone' Excel application running under Microsoft Windows.
RESUMO
Nitric oxide (NO) is postulated to play a key role in the pathophysiology of renal failure in sepsis. Whether the renal effects of increased NO are beneficial or harmful remains unclear. In a porcine model of lipopolysaccharide (LPS)-induced shock, we evaluated the effect of LPS on glomerular filtration rate (GFR) and renal blood flow (RBF). We then administered the nonselective nitric oxide synthase (NOS) inhibitor N(G)-L-arginine methyl ester (L-NAME), and compared its effects on GFR and RBF with those of S-methylisothiourea (SMT), a selective NOS inhibitor, and those of saline. We postulated that SMT, by maintaining constitutive NO, would be more beneficial than either L-NAME or saline. LPS infusion decreased mean arterial pressure (MAP), and increased cardiac output, RBF, and medullary NO content. The increased RBF was diverted to the medulla. There was no evidence of renal dysfunction in the saline-resuscitated group. Both NOS inhibitors increased MAP but decreased RBF, but only L-NAME reduced GFR and increased sodium excretion and renal oxygen extraction. We conclude that NO in endotoxemia is beneficial because it maintains RBF and GFR. Additionally, selective NOS inhibition did not offer any advantages over saline resuscitation.
Assuntos
Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/microbiologia , Modelos Animais de Doenças , Endotoxemia/complicações , Endotoxemia/metabolismo , Isotiurônio/análogos & derivados , Lipopolissacarídeos/efeitos adversos , Óxido Nítrico/fisiologia , Choque Séptico/complicações , Choque Séptico/metabolismo , Injúria Renal Aguda/tratamento farmacológico , Análise de Variância , Animais , Pressão Sanguínea/efeitos dos fármacos , Débito Cardíaco/efeitos dos fármacos , Endotoxemia/tratamento farmacológico , Endotoxemia/fisiopatologia , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Isotiurônio/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/farmacologia , Consumo de Oxigênio , Circulação Renal/efeitos dos fármacos , Ressuscitação/métodos , Choque Séptico/tratamento farmacológico , Choque Séptico/fisiopatologia , SuínosRESUMO
Potential advantages of C2-symmetric inhibitors designed for the symmetric HIV-1 protease include high selectivity, potency, stability, and bioavailability. Pseudo-C2-symmetric monools and C2-symmetric diols, containing central hydroxymethylene and (R,R)-dihydroxyethylene moieties flanked by a variety of hydrophobic P1/P1' side chains, were studied as HIV-1 protease inhibitors. The monools and diols were synthesized in 8-10 steps from D-(+)-arabitol and D-(+)-mannitol, respectively. Monools with ethyl or isobutyl P1/P1' side chains were weak inhibitors of recombinant HIV-1 protease (Ki > 10 microM), while benzyl P1/P1' side chains afforded a moderately potent inhibitor (apparent Ki = 230 nM). Diols were 100-10,000x more potent than analogous monools, and a wider range of P1/P1' side chains led to potent inhibition. Both classes of compounds exhibited lower apparent Ki values under high-salt conditions. Surprisingly, monool and diol HIV-1 protease inhibitors were potent inhibitors of porcine pepsin, a prototypical asymmetric monomeric aspartic protease. These results were evaluated in the context of the pseudosymmetric structure of monomeric aspartic proteases and their evolutionary kinship with the retroviral proteases. The X-ray crystal structure of HIV-1 protease complexed with a symmetric diol was determined at 2.6 A. Contrary to expectations, the diol binds the protease asymmetrically and exhibits 2-fold disorder in the electron density map. Molecular dynamics simulations were conducted beginning with asymmetric and symmetric HIV-1 protease/inhibitor model complexes. A more stable trajectory resulted from the asymmetric complex, in agreement with the observed asymmetric binding mode. A simple four-point model was used to argue more generally that van der Waals and electrostatic force fields can commonly lead to an asymmetric association between symmetric molecules.
Assuntos
Dipeptídeos/farmacologia , Glicóis/farmacologia , Inibidores da Protease de HIV/farmacologia , Protease de HIV/efeitos dos fármacos , HIV-1/enzimologia , Álcoois/química , Álcoois/metabolismo , Dipeptídeos/síntese química , Dipeptídeos/química , Dipeptídeos/metabolismo , Glicóis/síntese química , Glicóis/metabolismo , Protease de HIV/metabolismo , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Pepsina A/antagonistas & inibidores , Conformação Proteica , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Relação Estrutura-AtividadeRESUMO
Interleukin-5 (IL-5) is a lineage-specific cytokine for eosinophilpoiesis and plays an important part in diseases associated with increased eosinophils, such as asthma. Human IL-5 is a disulphide-linked homodimer with 115 amino-acid residues in each chain. The crystal structure at 2.4 A resolution reveals a novel two-domain structure, with each domain showing a striking similarity to the cytokine fold found in granulocyte macrophage and macrophage colony-stimulating factors, IL-2 (ref. 5), IL-4 (ref. 6), and human and porcine growth hormones. IL-5 is unique in that each domain requires the participation of two chains. The IL-5 structure consists of two left-handed bundles of four helices laid end to end and two short beta-sheets on opposite sides of the molecule. Surprisingly, the C-terminal strand and helix of one chain complete a bundle of four helices and a beta-sheet with the N-terminal three helices and one strand of the other chain. The structure of IL-5 provides a molecular basis for the design of antagonists and agonists that would delineate receptor recognition determinants critical in signal transduction. This structure determination extends the family of the cytokine bundle of four helices and emphasizes its fundamental significance and versatility in recognizing its receptor.
Assuntos
Interleucina-5/química , Animais , Sítios de Ligação , Simulação por Computador , Citocinas/química , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Difração de Raios XRESUMO
Interleukin-5 (IL-5) plays a key role in the proliferation and differentiation of eosinophils. To aid the solution of the crystallographic three-dimensional structure, we have expressed large quantities of recombinant human IL-5 (hIL-5) in a methionine auxotroph strain of Escherichia coli (DL41) grown on an enriched seleno-DL-methionine-containing medium. Cell densities of A650 = 10 have been achieved. The selenomethionyl-labelled hIL-5 (Se-hIL-5) has been purified and found to contain 3.6 selenium atoms/dimer, and 0.4 methionine residues/dimer. In a B-cell growth factor assay, the Se-hIL-5 is significantly more active than the non-labelled hIL-5. Electrospray mass spectrometry shows two major peaks, with relative molecular masses of 26,326 +/- 6 and 26,280 +/- 8 corresponding to the 4Se and 3Se/1S forms of hIL-5. Unlike the methionine-containing hIL-5, the N-terminal selenomethionine is neither oxidised nor carbamoylated and can only be resolved into two species in isoelectric focusing gel electrophoresis. Se-hIL-5 crystallises in the same space group and unit cell as hIL-5. Difference Fourier calculations identify two of the selenomethionines corresponding to Met107 in the dimer. However, the N-terminal is disordered in the crystal, and the N-terminal selenomethionines are not resolved in the difference Fourier.
Assuntos
Interleucina-5/análogos & derivados , Interleucina-5/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Selenometionina/análogos & derivados , Animais , Linfócitos B , Divisão Celular/efeitos dos fármacos , Cromatografia em Gel , Cromatografia por Troca Iônica , Cristalização , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Interleucina-5/química , Interleucina-5/farmacologia , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Selenometionina/química , Selenometionina/isolamento & purificação , Selenometionina/farmacologiaRESUMO
Analogues of peptides ranging in size from three to six amino acids and containing the hydroxyethylene dipeptide isosteres Phe psi Gly, Phe psi Ala, Phe psi NorVal, Phe psi Leu, and Phe psi Phe, where psi denotes replacement of CONH by (S)-CH(OH)CH2, were synthesized and studied as HIV-1 protease inhibitors. Inhibition constants (Ki) with purified HIV-1 protease depend strongly on the isostere in the order Phe psi Gly greater than Phe psi Ala greater than Phe psi NorVal greater than Phe psi Leu greater than Phe psi Phe and decrease with increasing length of the peptide analogue, converging to a value of 0.4 nM. Ki values are progressively less dependent on inhibitor length as the size of the P1' side chain within the isostere increases. The structures of HIV-1 protease complexed with the inhibitors Ala-Ala-X-Val-Val-OMe, where X is Phe psi Gly, Phe psi Ala, Phe psi NorVal, and Phe psi Phe, have been determined by X-ray crystallography (resolution 2.3-3.2 A). The crystals exhibit symmetry consistent with space group P6(1) with strong noncrystallographic 2-fold symmetry, and the inhibitors all exhibit 2-fold disorder. The inhibitors bind in similar conformations, forming conserved hydrogen bonds with the enzyme. The Phe psi Gly inhibitor adopts an altered conformation that places its P3' valine side chain partially in the hydrophobic S1' pocket, thus suggesting an explanation for the greater dependence of the Ki value on inhibitor length in the Phe psi Gly series. From the kinetic and crystallographic data, a minimal inhibitor model for tight-binding inhibition is derived in which the enzyme subsites S2-S2' are optimally occupied. The Ki values for several compounds are compared with their potencies as inhibitors of proteolytic processing in T-cell cultures chronically infected with HIV-1 (MIC values) and as inhibitors of acute infectivity (IC50 values). There is a rank-order correspondence, but a 20-1000-fold difference, between the values of Ki and those of MIC or IC50. IC50 values can approach those of Ki but are highly dependent on the conditions of the acute infectivity assay and are influenced by physiochemical properties of the inhibitors such as solubility.
Assuntos
Antivirais/síntese química , Inibidores da Protease de HIV , HIV-1/fisiologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Inibidores de Proteases/síntese química , Replicação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Western Blotting , Linhagem Celular , Etilenos/química , Etilenos/farmacologia , Protease de HIV/química , Protease de HIV/isolamento & purificação , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Indicadores e Reagentes , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Ligação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Relação Estrutura-Atividade , Especificidade por Substrato , Linfócitos T , Difração de Raios XRESUMO
Human interleukin-1 alpha, cloned and expressed in E. coli, has been purified and structurally characterized by various physiochemical methods, including mass spectrometry. The recombinant protein has been crystallized by the hanging drop vapor diffusion method using dimethyl sulfoxide as the precipitating agent. The space group is P2(1)2(1)2(1). Unit cell dimensions are a = 44.1, b = 57.1, and c = 61.7 A and alpha = beta = gamma = 90 degrees. The crystals diffract to beyond 1.7 A and are suitable for high resolution data collection. Native diffraction data were collected. Screens for heavy atom derivatives have been initiated.