RESUMO
Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform specificity.
Assuntos
Ciclofilinas/química , Ciclofilinas/metabolismo , Família Multigênica , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoKalpha (alpha1 and alpha2) and ChoKbeta isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoKalpha1 and ChoKbeta isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoKalpha1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoKbeta was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoKalpha isoforms (alpha1 and alpha2) compared with ChoKbeta. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoKalpha2 (equivalent to Leu-419 of ChoKalpha1), or the corresponding residue Phe-352 of ChoKbeta, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.
Assuntos
Colina Quinase/antagonistas & inibidores , Colina Quinase/química , Inibidores Enzimáticos/química , Hemicolínio 3/química , Domínio Catalítico , Colina Quinase/genética , Colina Quinase/metabolismo , Colinérgicos/química , Colinérgicos/metabolismo , Inibidores Enzimáticos/metabolismo , Hemicolínio 3/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isoenzimas , Espectrometria de Massas , Mutação de Sentido Incorreto , FosforilaçãoRESUMO
The AMP-activated protein kinase (AMPK) is a highly conserved trimeric protein complex that is responsible for energy homeostasis in eukaryotic cells. Here, a 1.9 A resolution crystal structure of the isolated kinase domain from the alpha2 subunit of human AMPK, the first from a multicellular organism, is presented. This human form adopts a catalytically inactive state with distorted ATP-binding and substrate-binding sites. The ATP site is affected by changes in the base of the activation loop, which has moved into an inhibited DFG-out conformation. The substrate-binding site is disturbed by changes within the AMPKalpha2 catalytic loop that further distort the enzyme from a catalytically active form. Similar structural rearrangements have been observed in a yeast AMPK homologue in response to the binding of its auto-inhibitory domain; restructuring of the kinase catalytic loop is therefore a conserved feature of the AMPK protein family and is likely to represent an inhibitory mechanism that is utilized during function.
Assuntos
Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Biocatálise , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Alinhamento de SequênciaRESUMO
The Eph family of receptor tyrosine kinases has drawn growing attention due to their role in regulating diverse biological phenomena. However, pharmacological interrogation of Eph kinase function has been hampered by a lack of potent and selective Eph kinase inhibitors. Here we report the discovery of compounds 6 and 9 using a rationally designed kinase-directed library which potently inhibit Eph receptor tyrosine kinases, particularly EphB2 with cellular EC(50)s of 40nM. Crystallographic data of EphA3 and EphA7 in complex with the inhibitors show that they bind to the 'DFG-out' inactive kinase conformation and provide valuable information for structure-based design of second generation inhibitors.
Assuntos
Química Farmacêutica/métodos , Inibidores Enzimáticos/farmacologia , Receptores da Família Eph/antagonistas & inibidores , Trifosfato de Adenosina/química , Benzamidas/síntese química , Benzamidas/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X/métodos , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Humanos , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Niacinamida/análogos & derivados , Niacinamida/síntese química , Niacinamida/farmacologia , Fosforilação , Receptores da Família Eph/química , Relação Estrutura-AtividadeRESUMO
Calpains are calcium activated cysteine proteases found throughout the animal, plant, and fungi kingdoms; 14 isoforms have been described in the human genome. Calpains have been implicated in multiple models of human disease; for instance, calpain 1 is activated in the brains of individuals with Alzheimer's disease, and the digestive tract specific calpain 9 is down-regulated in gastric cancer cell lines. We have solved the structures of human calpain 1 and calpain 9 protease cores using crystallographic methods; both structures have clear implications for the function of non-catalytic domains of full-length calpains in the calcium-mediated activation of the enzyme. The structure of minicalpain 1 is similar to previously solved structures of the protease core. Auto-inhibition in this system is most likely through rearrangements of a central helical/loop region near the active site cysteine, which occlude the substrate binding site. However, the structure of minicalpain 9 indicates that auto-inhibition in this enzyme is mediated through large intra-domain movements that misalign the catalytic triad. This disruption is reminiscent of the full-length inactive calpain conformation. The structures of the highly conserved, ubiquitously expressed human calpain 1 and the more tissue specific human calpain 9 indicate that although there are high levels of sequence conservation throughout the calpain family, isolated structures of family members are insufficient to explain the molecular mechanism of activation for this group of proteins.
Assuntos
Calpaína/química , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
X-linked lymphoproliferative disease is caused by mutations in the protein SAP, which consists almost entirely of a single SH2 domain. SAP interacts with the Tyr281 site of the T<-->B cell signaling protein SLAM via its SH2 domain. Interestingly, binding is not dependent on phosphorylation but does involve interactions with residues N-terminal to the Tyr. We have used 15N and 2H NMR relaxation experiments to investigate the motional properties of the SAP SH2 domain backbone amides and side-chain methyl groups in the free protein and complexes with phosphorylated and non-phosphorylated peptides derived from the Tyr281 site of SLAM. The most mobile methyl groups are in side-chains with large RMSD values between the three crystal structures of SAP, suggesting that fast time-scale dynamics in side-chains is associated with conformational plasticity. The backbone amides of two residues which interact with the C-terminal part of the peptides experience fast time-scale motions in the free SH2 domain that are quenched upon binding of either the phosphorylated or non-phosphorylated peptide. Of most importance, the mobility of methyl groups in and around the binding site for residues in the N-terminus of the peptide is significantly restricted in the complexes, underscoring the dominance of this interaction with SAP and demonstrating a correlation between changes in rapid side-chain motion upon binding with local binding energy.
Assuntos
Proteínas de Transporte/química , Peptídeos e Proteínas de Sinalização Intracelular , Fragmentos de Peptídeos/química , Domínios de Homologia de src , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Humanos , Cinética , Ligantes , Mecânica , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/metabolismo , Fosfotirosina/metabolismo , Ligação Proteica , Dobramento de Proteína , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Termodinâmica , Tirosina/metabolismoRESUMO
The significant increase in the demand for purified protein for crystallization and structural studies has made necessary the development of multi-sample methods for identifying solution conditions that affect protein stability and aggregation. Conditions that stabilize proteins can improve protein purification and crystallization. These methods can be used to identify small molecule compounds or inhibitors that interact with the purified proteins, and might serve as starting points for drug discovery. In this article three methods for measuring protein stability and aggregation are described and discussed: differential scanning fluorimetry (DSF), differential static light scattering (DSLS), and isothermal denaturation (ITD).
Assuntos
Bioquímica/métodos , Estabilidade Proteica , Humanos , Luz , Desnaturação Proteica , Estrutura Quaternária de Proteína , Espalhamento de RadiaçãoRESUMO
Protein kinase C eta (PKCeta) is one of several PKC isoforms found in humans. It is a novel PKC isoform in that it is activated by diacylglycerol and anionic phospholipids but not calcium. The crystal structure of the PKCeta-C2 domain, which is thought to mediate anionic phospholipid sensing in the protein, was determined at 1.75 A resolution. The structure is similar to that of the PKC epsilon C2 domain but with significant variations at the putative lipid-binding site. Two serine residues within PKC eta were identified in vitro as potential autophosphorylation sites. In the unphosphorylated structure both serines line the putative lipid-binding site and may therefore play a role in the lipid-regulation of the kinase.
Assuntos
Modelos Químicos , Modelos Moleculares , Proteína Quinase C/química , Proteína Quinase C/ultraestrutura , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
Ubiquitin-specific protease 8 (USP8) hydrolyzes mono and polyubiquitylated targets such as epidermal growth factor receptors and is involved in clathrin-mediated internalization. In 1182 residues, USP8 contains multiple domains, including coiled-coil, rhodanese, and catalytic domains. We report the first high-resolution crystal structures of these domains and discuss their implications for USP8 function. The amino-terminal domain is a homodimer with a novel fold. It is composed of two five-helix bundles, where the first helices are swapped, and carboxyl-terminal helices are extended in an antiparallel fashion. The structure of the rhodanese domain, determined in complex with the E3 ligase NRDP1, reveals the canonical rhodanese fold but with a distorted primordial active site. The USP8 recognition domain of NRDP1 has a novel protein fold that interacts with a conserved peptide loop of the rhodanese domain. A consensus sequence of this loop is found in other NRDP1 targets, suggesting a common mode of interaction. The structure of the carboxyl-terminal catalytic domain of USP8 exhibits the conserved tripartite architecture but shows unique traits. Notably, the active site, including the ubiquitin binding pocket, is in a closed conformation, incompatible with substrate binding. The presence of a zinc ribbon subdomain near the ubiquitin binding site further suggests a polyubiquitin-specific binding site and a mechanism for substrate induced conformational changes.
Assuntos
Endopeptidases/química , Endopeptidases/metabolismo , Tiossulfato Sulfurtransferase/química , Tiossulfato Sulfurtransferase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Complexos Multiproteicos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eletricidade Estática , Tiossulfato Sulfurtransferase/genética , Ubiquitina Tiolesterase , Ubiquitina-Proteína Ligases/genéticaRESUMO
The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas/química , Proteínas/metabolismo , Animais , Biologia Computacional , Cristalização , Humanos , Ligantes , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Temperatura , TermodinâmicaRESUMO
The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.