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1.
Nature ; 557(7704): 196-201, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29720648

RESUMO

The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.


Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/química , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Quinolinas/química , Quinolinas/farmacologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Regulação Alostérica/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Escherichia coli/química , Hidrocarbonetos/química , Hidrocarbonetos/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Modelos Moleculares , Domínios Proteicos/efeitos dos fármacos
2.
Mol Cell ; 63(1): 4-6, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27392144

RESUMO

Abdul Rehman and colleagues identify a sixth family of deubiquitinase enzymes that are highly conserved throughout eukaryotes and show a remarkable selectivity for cleaving extended Lys-48-linked polyubiquitin chains.


Assuntos
Eucariotos , Poliubiquitina , Enzimas Desubiquitinantes , Humanos
3.
Nature ; 550(7677): 534-538, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29045385

RESUMO

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.


Assuntos
Aminopiridinas/química , Aminopiridinas/farmacologia , Indazóis/química , Indazóis/farmacologia , Fenóis/química , Fenóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Ubiquitina/metabolismo , Animais , Ligação Competitiva , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Especificidade por Substrato , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/deficiência , Peptidase 7 Específica de Ubiquitina/metabolismo
4.
J Bacteriol ; 198(14): 2001-2015, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27161118

RESUMO

UNLABELLED: De novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5'-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuX-guaB-guaA deletion in vivo In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility. IMPORTANCE: De novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient for S. aureus survival in the absence of de novo guanine biosynthesis. These data suggest targeting the de novo guanine biosynthesis pathway may have therapeutic utility in the treatment of S. aureus infections.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Guanina/biossíntese , Purinas/metabolismo , Riboswitch , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Animais , Proteínas de Bactérias/genética , Feminino , Humanos , Camundongos , Staphylococcus aureus/genética
5.
Proc Natl Acad Sci U S A ; 109(14): 5299-304, 2012 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-22431598

RESUMO

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.


Assuntos
Nucleotídeos/metabolismo , Proteínas Son Of Sevenless/metabolismo , Proteínas ras/metabolismo , Sítios de Ligação , Linhagem Celular , Humanos , Ligantes , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Proteínas ras/química
6.
Mol Microbiol ; 69(6): 1373-84, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18573176

RESUMO

SUMMARY: An unusual regulatory mechanism involving two response regulators, CheY1 and CheY2, but no CheZ phosphatase, operates in the chemotactic signalling chain of Sinorhizobium meliloti. Active CheY2-P, phosphorylated by the cognate histidine kinase, CheA, is responsible for flagellar motor control. In the absence of any CheZ phosphatase activity, the level of CheY2-P is quickly reset by a phospho-transfer from CheY2-P first back to CheA, and then to CheY1, which acts as a phosphate sink. In studying the mechanism of this phosphate shuttle, we have used GFP fusions to show that CheY2, but not CheY1, associates with CheA at a cell pole. Cross-linking experiments with the purified proteins revealed that both CheY2 and CheY2-P bind to an isolated P2 ligand-binding domain of CheA, but CheY1 does not. The dissociation constants of CheA-CheY2 and CheA-CheY2-P indicated that both ligands bind with similar affinity to CheA. Based on the NMR structures of CheY2 and CheY2-P, their interactions with the purified P2 domain were analysed. The interacting surface of CheY2 comprises its C-terminal beta4-alpha4-beta5-alpha5 structural elements, whereas the interacting surface of CheY2-P is shifted towards the loop connecting beta5 and alpha5. We propose that the distinct CheY2 and CheY2-P surfaces interact with two overlapping sites in the P2 domain that selectively bind either CheY2 or CheY2-P, depending on whether CheA is active or inactive.


Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia , Proteínas Quinases/metabolismo , Sinorhizobium meliloti/fisiologia , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Fusão Gênica Artificial , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteínas Quinases/química , Proteínas Quinases/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética
7.
Nat Commun ; 9(1): 1162, 2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29563501

RESUMO

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.


Assuntos
Enzimas Desubiquitinantes/química , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Proteína de Replicação A/metabolismo , Coloração e Rotulagem/métodos , Biocatálise , Domínio Catalítico , Cisteína/química , Cisteína/metabolismo , Enzimas Desubiquitinantes/classificação , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/química , Lisina/metabolismo , Sondas Moleculares , Proteína de Replicação A/genética , Sumoilação , Ubiquitinação
8.
J Med Chem ; 60(24): 10056-10070, 2017 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-29166018

RESUMO

USP7 is a deubiquitinase implicated in destabilizing the tumor suppressor p53, and for this reason it has gained increasing attention as a potential oncology target for small molecule inhibitors. Herein we describe the biophysical, biochemical, and computational approaches that led to the identification of 4-(2-aminopyridin-3-yl)phenol compounds described by Kategaya ( Nature 2017 , 550 , 534 - 538 ) as specific inhibitors of USP7. Fragment based lead discovery (FBLD) by NMR combined with virtual screening and re-mining of biochemical high-throughput screening (HTS) hits led to the discovery of a series of ligands that bind in the "palm" region of the catalytic domain of USP7 and inhibit its catalytic activity. These ligands were then optimized by structure-based design to yield cell-active molecules with reasonable physical properties. This discovery process not only involved multiple techniques working in concert but also illustrated a unique way in which hits from orthogonal screening approaches complemented each other for lead identification.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Aminopiridinas/química , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Simulação por Computador , Cristalografia por Raios X , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Espectroscopia de Ressonância Magnética/métodos , Oxidiazóis/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/metabolismo
9.
Biomol NMR Assign ; 10(2): 345-9, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27386854

RESUMO

The deubiquitinase Ubiquitin Specific Protease 7 (USP7) is part of the regulatory cascade of proteins that modulates the activity of the tumor suppressor protein p53. Deubiquitination of its target Murine Double Minute 2 (MDM2) leads to increased proteosomal degradation of p53. Consequently, USP7 has emerged as an attractive oncology target because its inhibition stabilizes p53, thereby promoting p53-dependent apoptosis in cancer cells. Here we report the backbone resonance assignment for the 40.5 kDa catalytic domain of USP7.


Assuntos
Domínio Catalítico , Ressonância Magnética Nuclear Biomolecular , Ubiquitina Tiolesterase/química , Sequência de Aminoácidos , Humanos , Ubiquitina Tiolesterase/metabolismo , Peptidase 7 Específica de Ubiquitina
10.
Sci Rep ; 6: 35179, 2016 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-27734922

RESUMO

Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI-II, a model acyclic cystine-knot peptide. Even though synthetic methods have been used to generate EETI-II, recombinant methods that allow efficient large scale biosynthesis of EETI-II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI-II in high yields and to near homogeneity. We also uncover that EETI-II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co-incubation with a cell-penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI-II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine-knot peptides, and hence enable future exploration of their utility in drug discovery and delivery.


Assuntos
Cistina/metabolismo , Peptídeos Cíclicos/metabolismo , Transporte Proteico/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/metabolismo , Ciclização/fisiologia , Ciclotídeos/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Camundongos , Células NIH 3T3 , Proteínas de Plantas/metabolismo
11.
Structure ; 24(8): 1335-1345, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27452404

RESUMO

The deubiquitinating enzyme USP7 has a pivotal role in regulating the stability of proteins involved in fundamental cellular processes of normal biology and disease. Despite the importance of USP7, the mechanisms underlying substrate recognition and catalytic activation are poorly understood. Here we present structural, biochemical, and biophysical analyses elucidating the molecular mechanism by which the C-terminal 19 amino acids of USP7 (residues 1084-1102) enhance the ubiquitin cleavage activity of the deubiquitinase (DUB) domain. Our data demonstrate that the C-terminal peptide binds the activation cleft in the catalytic domain and stabilizes the catalytically competent conformation of USP7. Additional structures of longer fragments of USP7, as well as solution studies, provide insight into full-length USP7, the role of the UBL domains, and demonstrate that both substrate recognition and deubiquitinase activity are highly regulated by the catalytic and noncatalytic domains of USP7, a feature that could be essential for the proper function of multi-domain DUBs.


Assuntos
Ubiquitina Tiolesterase/química , Ubiquitina/química , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Peptidase 7 Específica de Ubiquitina
12.
Sci Rep ; 6: 39374, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-27996029

RESUMO

Accumulation of amyloid-ß (Aß) peptides and amyloid plaque deposition in brain is postulated as a cause of Alzheimer's disease (AD). The precise pathological species of Aß remains elusive although evidence suggests soluble oligomers may be primarily responsible for neurotoxicity. Crenezumab is a humanized anti-Aß monoclonal IgG4 that binds multiple forms of Aß, with higher affinity for aggregated forms, and that blocks Aß aggregation, and promotes disaggregation. To understand the structural basis for this binding profile and activity, we determined the crystal structure of crenezumab in complex with Aß. The structure reveals a sequential epitope and conformational requirements for epitope recognition, which include a subtle but critical element that is likely the basis for crenezumab's versatile binding profile. We find interactions consistent with high affinity for multiple forms of Aß, particularly oligomers. Of note, crenezumab also sequesters the hydrophobic core of Aß and breaks an essential salt-bridge characteristic of the ß-hairpin conformation, eliminating features characteristic of the basic organization in Aß oligomers and fibrils, and explains crenezumab's inhibition of aggregation and promotion of disaggregation. These insights highlight crenezumab's unique mechanism of action, particularly regarding Aß oligomers, and provide a strong rationale for the evaluation of crenezumab as a potential AD therapy.

13.
J Lab Autom ; 21(1): 64-75, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26077161

RESUMO

Acoustic droplet ejection (ADE) as a means of transferring library compounds has had a dramatic impact on the way in which high-throughput screening campaigns are conducted in many laboratories. Two Labcyte Echo ADE liquid handlers form the core of the compound transfer operation in our 1536-well based ultra-high-throughput screening (uHTS) system. Use of these instruments has promoted flexibility in compound formatting in addition to minimizing waste and eliminating compound carryover. We describe the use of ADE for the generation of assay-ready plates for primary screening as well as for follow-up dose-response evaluations. Custom software has enabled us to harness the information generated by the ADE instrumentation. Compound transfer via ADE also contributes to the screening process outside of the uHTS system. A second fully automated ADE-based system has been used to augment the capacity of the uHTS system as well as to permit efficient use of previously picked compound aliquots for secondary assay evaluations. Essential to the utility of ADE in the high-throughput screening process is the high quality of the resulting data. Examples of data generated at various stages of high-throughput screening campaigns are provided. Advantages and disadvantages of the use of ADE in high-throughput screening are discussed.


Assuntos
Tecnologia Biomédica/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Acústica , Tecnologia Biomédica/instrumentação , Interpretação Estatística de Dados , Avaliação Pré-Clínica de Medicamentos/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Bibliotecas de Moléculas Pequenas , Software , Soluções
14.
BMC Bioinformatics ; 6: 91, 2005 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-15819976

RESUMO

BACKGROUND: We have developed the program PERMOL for semi-automated homology modeling of proteins. It is based on restrained molecular dynamics using a simulated annealing protocol in torsion angle space. As main restraints defining the optimal local geometry of the structure weighted mean dihedral angles and their standard deviations are used which are calculated with an algorithm described earlier by Doker et al. (1999, BBRC, 257, 348-350). The overall long-range contacts are established via a small number of distance restraints between atoms involved in hydrogen bonds and backbone atoms of conserved residues. Employing the restraints generated by PERMOL three-dimensional structures are obtained using standard molecular dynamics programs such as DYANA or CNS. RESULTS: To test this modeling approach it has been used for predicting the structure of the histidine-containing phosphocarrier protein HPr from E. coli and the structure of the human peroxisome proliferator activated receptor gamma (Ppar gamma). The divergence between the modeled HPr and the previously determined X-ray structure was comparable to the divergence between the X-ray structure and the published NMR structure. The modeled structure of Ppar gamma was also very close to the previously solved X-ray structure with an RMSD of 0.262 nm for the backbone atoms. CONCLUSION: In summary, we present a new method for homology modeling capable of producing high-quality structure models. An advantage of the method is that it can be used in combination with incomplete NMR data to obtain reasonable structure models in accordance with the experimental data.


Assuntos
Biologia Computacional/métodos , Algoritmos , Proteínas de Bactérias/genética , Gráficos por Computador , Simulação por Computador , Cristalografia por Raios X , Bases de Dados de Proteínas , Enterococcus faecalis/metabolismo , Escherichia coli/metabolismo , Histidina/química , Humanos , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Moleculares , Modelos Estatísticos , Modelos Teóricos , Conformação Molecular , PPAR gama/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Análise de Sequência de Proteína , Software
15.
J Mol Biol ; 338(2): 287-97, 2004 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-15066432

RESUMO

The chemotactic signalling chain to the flagellar motor of Sinorhizobium meliloti features a new type of response regulator, CheY2. CheY2 activated by phosphorylation (CheY2-P) controls the rotary speed of the flagellar motor (instead of reversing the sense of rotation), and it is efficiently dephosphorylated by phospho-retrotransfer to the cognate kinase, CheA. Here, we report the NMR solution structures of the Mg(2+)-complex of inactive CheY2, and of activated CheY2-BeF(3), a stable analogue of CheY2-P, to an overall root mean square deviation of 0.042 nm and 0.027 nm, respectively. The 14 kDa CheY2 protein exhibits a characteristic open (alpha/beta)(5) conformation. Modification of CheY2 by BeF(3)(-) leads to large conformational changes of the protein, which are in the limits of error identical with those observed by phosphorylation of the active-centre residue Asp58. In BeF(3)-activated CheY2, the position of Thr88-OH favours the formation of a hydrogen bond with the active site, Asp58-BeF(3), similar to BeF(3)-activated CheY from Escherichia coli. In contrast to E.coli, this reorientation is not involved in a Tyr-Thr-coupling mechanism, that propagates the signal from the incoming phosphoryl group to the C-terminally located FliM-binding surface. Rather, a rearrangement of the Phe59 side-chain to interact with Ile86-Leu95-Val96 along with a displacement of alpha4 towards beta5 is stabilised in S.meliloti. The resulting, activation-induced, compact alpha4-beta5-alpha5 surface forms a unique binding domain suited for specific interaction with and signalling to a rotary motor that requires a gradual speed control. We propose that these new features of response regulator activation, compared to other two-component systems, are the key for the observed unique phosphorylation, dephosphorylation and motor control mechanisms in S.meliloti.


Assuntos
Proteínas de Bactérias/química , Berílio/química , Fluoretos/química , Conformação Proteica , Sinorhizobium meliloti/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Quimiotaxia/fisiologia , Escherichia coli/química , Escherichia coli/metabolismo , Flagelos/metabolismo , Modelos Moleculares , Proteínas Motores Moleculares/metabolismo , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Sinorhizobium meliloti/metabolismo
16.
Methods Mol Biol ; 305: 197-214, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15939999

RESUMO

Interaction between biological macromolecules or of macromolecules with low-molecular-weight ligands is a central paradigm in the understanding of function in biological systems. It is also the major goal in pharmaceutical research to find and optimize ligands that modulate the function of biological macromolecules. Both technological advances and new methods in the field of nuclear magnetic resonance (NMR) have led to the development of several tools by which the interaction of proteins or DNA and low molecular weight-ligands can be characterized at an atomic level. Information can be gained quickly and easily with ligand-based techniques. These need only small amounts of nonisotope labeled, and thus readily available target macromolecules. As the focus is on the signals stemming only from the ligand, no further NMR information regarding the target is needed. Techniques based on the observation of isotopically labeled biological macromolecules open the possibility to observe interactions of proteins with low-molecular-weight ligands, DNA or other proteins. With these techniques, the structure of high-molecular-weight complexes can be determined. Here, the resonance signals of the macromolecule must be identified beforehand, which can be time consuming but with the benefit of obtaining more information with respect to the target ligand complex.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , Proteínas/química , Proteínas/metabolismo , Sítios de Ligação , Isótopos de Carbono , Humanos , Técnicas In Vitro , Ligantes , Macrófagos/enzimologia , Modelos Moleculares , Isótopos de Nitrogênio , Elastase Pancreática/química , Elastase Pancreática/metabolismo , Albumina Sérica/química , Albumina Sérica/metabolismo
17.
FEBS Lett ; 588(23): 4487-96, 2014 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-25448598

RESUMO

Cyclotides belong to the family of cyclic cystine-knot peptides and have shown promise as scaffolds for protein engineering and pharmacological modulation of cellular protein activity. Cyclotides are characterized by a cystine-knotted topology and a head-to-tail cyclic polypeptide backbone. While they are primarily produced in plants, cyclotides have also been obtained by chemical synthesis. However, there is still a need for methods to generate cyclotides in high yields to near homogeneity. Here, we report a biomimetic approach which utilizes an engineered version of the enzyme Sortase A to catalyze amide backbone cyclization of the recombinant cyclotide MCoTI-II, thereby allowing the efficient production of active homogenous species in high yields. Our results provide proof of concept for using engineered Sortase A to produce cyclic MCoTI-II and should be generally applicable to generating other cyclic cystine-knot peptides.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Cistina/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/química , Aminoaciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ciclização , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/genética , Staphylococcus aureus/enzimologia
18.
Enzymes ; 33 Pt A: 15-39, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-25033799

RESUMO

Despite decades of intense drug discovery efforts, to date no small molecules have been described that directly bind to Ras protein and effectively antagonize its function. In order to identify and characterize small-molecule binders to KRas, we carried out a fragment-based lead discovery effort. A ligand-detected primary nuclear magnetic resonance (NMR) screen identified 266 fragments from a library of 3285 diverse compounds. Protein-detected NMR using isotopically labeled KRas protein was applied for hit validation and binding site characterization. An area on the KRas surface emerged as a consensus site of fragment binding. X-ray crystallography studies on a subset of the hits elucidated atomic details of the ligand-protein interactions, and revealed that the consensus site comprises a shallow hydrophobic pocket. Comparison among the crystal structures indicated that the ligand-binding pocket is flexible and can be expanded upon ligand binding. The identified ligand-binding pocket is proximal to the protein-protein interface and therefore has the potential to mediate functional effects. Indeed, some ligands inhibited SOS1-dependent nucleotide exchange, although with weak potency. Several Ras ligands have been published in literature, the majority of which were discovered using NMR-based methods. Mapping of the ligand-binding sites revealed five areas on Ras with a high propensity for ligand binding and the potential of modulating Ras activity.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas ras/química , Proteínas ras/metabolismo , Animais , Sítios de Ligação , Humanos , Ligantes , Conformação Proteica
19.
Nat Struct Mol Biol ; 19(2): 171-5, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22245969

RESUMO

Addition and removal of ubiquitin or ubiquitin chains to and from proteins is a tightly regulated process that contributes to cellular signaling and protein stability. Here we show that phosphorylation of the human deubiquitinase DUBA (OTUD5) at a single residue, Ser177, is both necessary and sufficient to activate the enzyme. The crystal structure of the ubiquitin aldehyde adduct of active DUBA reveals a marked cooperation between phosphorylation and substrate binding. An intricate web of interactions involving the phosphate and the C-terminal tail of ubiquitin cause DUBA to fold around its substrate, revealing why phosphorylation is essential for deubiquitinase activity. Phosphoactivation of DUBA represents an unprecedented mode of protease regulation and a clear link between two major cellular signal transduction systems: phosphorylation and ubiquitin modification.


Assuntos
Endopeptidases/metabolismo , Processamento de Proteína Pós-Traducional , Serina/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Fosforilação , Conformação Proteica , Ubiquitina/metabolismo
20.
Methods Enzymol ; 493: 469-85, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21371602

RESUMO

The application of NMR in fragment-based lead discovery (FBLD) has quickly developed from a sensitive method for the identification of low-affinity binders to an important tool in the hit-to-lead process. NMR can play a constructive role in the process from identifying those fragments with the best potential toward a biochemically active compound to developing them into molecules with high affinity and selectivity to a given target protein. NMR hit-to-lead involves revising the lead identification process at the beginning of a fragment-based drug discovery project, the primary screen, and also looking toward protein-detected NMR methods in advancing compounds from fragment hit into and through fragment hit-to-lead. With the development of higher sensitivity cold NMR probes, ligand-based NMR methods can be successfully applied to a majority of projects found in a pharmaceutical pipeline. Having matured from the original concepts such as SAR by NMR (Shuker, S. B., Hajduk, P. J., Meadows, R. P., Fesik, S. W. (1996) Discovering high-affinity ligands for proteins: SAR by NMR. Science274 (5292), 1531-1534.), projects that base their lead matter on fragment hits are close to or already in the clinic (Woodhead, A. J., Angove, H., Carr, M. G., Chessari, G., Congreve, M., Coyle, J. E., Cosme, J., Graham, B., Day, P. J., Downham, R., Fazal, L., Feltell, R., et al. (2010) discovery of (2,4-dihydroxy-5-isopropylphenyl)-[5-(4-methylpiperazin-1-ylmethyl)-1,3-dihydroisoindol-2-yl]methanone (AT13387), a novel inhibitor of the molecular chaperone Hsp90 by fragment based drug design. J. Med. Chem.53, 5956-5969, Chessari, G., and Woodhead, A. J. (2009). From fragment to clinical candidate: A historical perspective. Drug Discov. Today14 (13-14), 668-675.). Generating new ideas toward new binding modes and mechanisms of action as well as new intellectual property will be the standard by which the success of FBLD will need to be measured. A strategy outlining the various steps involved in NMR hit-to-lead is provided. By means of a specific example, the workflow is described to guide the reader through the experimental setup.


Assuntos
Descoberta de Drogas/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica , Bibliotecas de Moléculas Pequenas , Automação Laboratorial , Sítios de Ligação , Desenho de Fármacos , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Relação Estrutura-Atividade
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