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1.
Nature ; 513(7516): 124-7, 2014 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-25043059

RESUMO

Human GPR40 receptor (hGPR40), also known as free fatty-acid receptor 1 (FFAR1), is a G-protein-coupled receptor that binds long-chain free fatty acids to enhance glucose-dependent insulin secretion. Novel treatments for type-2 diabetes mellitus are therefore possible by targeting hGPR40 with partial or full agonists. TAK-875, or fasiglifam, is an orally available, potent and selective partial agonist of hGPR40 receptor, which reached phase III clinical trials for the potential treatment of type-2 diabetes mellitus. Data from clinical studies indicate that TAK-875, which is an ago-allosteric modulator of hGPR40 (ref. 3), demonstrates improved glycaemic control and low hypoglycaemic risk in diabetic patients. Here we report the crystal structure of hGPR40 receptor bound to TAK-875 at 2.3 Å resolution. The co-complex structure reveals a unique binding mode of TAK-875 and suggests that entry to the non-canonical binding pocket most probably occurs via the lipid bilayer. The atomic details of the extensive charge network in the ligand binding pocket reveal additional interactions not identified in previous studies and contribute to a clear understanding of TAK-875 binding to the receptor. The hGPR40-TAK-875 structure also provides insights into the plausible binding of multiple ligands to the receptor, which has been observed in radioligand binding and Ca(2+) influx assay studies. Comparison of the transmembrane helix architecture with other G-protein-coupled receptors suggests that the crystallized TAK-875-bound hGPR40 complex is in an inactive-like state.


Assuntos
Benzofuranos/química , Benzofuranos/farmacologia , Agonismo Parcial de Drogas , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Sulfonas/química , Sulfonas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Benzofuranos/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Ligantes , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Receptores Acoplados a Proteínas G/metabolismo , Homologia Estrutural de Proteína , Sulfonas/metabolismo , Propriedades de Superfície
2.
Nat Protoc ; 7(3): 453-66, 2012 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-22322218

RESUMO

X-ray crystal structures of human membrane proteins, although potentially of extremely great impact, are highly underrepresented relative to those of prokaryotic membrane proteins. One key reason for this is that human membrane proteins can be difficult to express at a level, and at a quality, suitable for structural studies. This protocol describes the methods that we use to overexpress human membrane proteins from clonal human embryonic kidney 293 (HEK293S) cells lacking N-acetylglucosaminyltransferase I (GnTI(-)), and was recently used in our 2.1-ŠX-ray crystal structure determination of human RhCG. Upon identification of highly expressing cell lines, suspension cell cultures are scaled up in a facile manner either using spinner flasks or cellbag bioreactors, resulting in a final purified yield of ∼0.5 mg of membrane protein per liter of medium. The protocol described here is reliable and cost effective, can be used to express proteins that would otherwise be toxic to mammalian cells and can be completed in 8-10 weeks.


Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Cristalografia por Raios X/métodos , Células HEK293/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Transfecção/métodos , Clonagem Molecular/métodos , Humanos , N-Acetilglucosaminiltransferases/deficiência
3.
Proc Natl Acad Sci U S A ; 107(21): 9638-43, 2010 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-20457942

RESUMO

In humans, NH(3) transport across cell membranes is facilitated by the Rh (rhesus) family of proteins. Human Rh C glycoprotein (RhCG) forms a trimeric complex that plays an essential role in ammonia excretion and renal pH regulation. The X-ray crystallographic structure of human RhCG, determined at 2.1 A resolution, reveals the mechanism of ammonia transport. Each monomer contains 12 transmembrane helices, one more than in the bacterial homologs. Reconstituted into proteoliposomes, RhCG conducts NH(3) to raise internal pH. Models of the erythrocyte Rh complex based on our RhCG structure suggest that the erythrocytic Rh complex is composed of stochastically assembled heterotrimers of RhAG, RhD, and RhCE.


Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Sistema do Grupo Sanguíneo Rh-Hr/química , Sistema do Grupo Sanguíneo Rh-Hr/metabolismo , Amônia/metabolismo , Transporte Biológico , Linhagem Celular , Cristalografia por Raios X , Eritrócitos/química , Eritrócitos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
4.
Nat Protoc ; 4(5): 619-37, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19360018

RESUMO

Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.


Assuntos
Cristalização/métodos , Cristalografia por Raios X , Proteínas de Membrana/química , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , Detergentes , Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Estrutura Terciária de Proteína , Solubilidade
5.
J Struct Funct Genomics ; 10(1): 9-16, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19031011

RESUMO

Persistent hurdles impede the successful determination of high-resolution crystal structures of eukaryotic integral membrane proteins (IMP). We designed a high-throughput structural genomics oriented pipeline that seeks to minimize effort in uncovering high-quality, responsive non-redundant targets for crystallization. This "discovery-oriented" pipeline sidesteps two significant bottlenecks in the IMP structure determination pipeline: expression and membrane extraction with detergent. In addition, proteins that enter the pipeline are then rapidly vetted by their presence in the included volume on a size-exclusion column--a hallmark of well-behaved IMP targets. A screen of 384 rationally selected eukaryotic IMPs in baker's yeast Saccharomyces cerevisiae is outlined to demonstrate the results expected when applying this discovery-oriented pipeline to whole-organism membrane proteomes.


Assuntos
Proteínas de Membrana/química , Animais , Cristalografia por Raios X , Bases de Dados de Proteínas , Genômica , Humanos , Conformação Proteica , Proteoma/análise , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
BMC Struct Biol ; 8: 49, 2008 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-19014592

RESUMO

BACKGROUND: An accurate rendering of interior surfaces can facilitate the analysis of mechanisms at atomic-level detail, such as the transport of substrates in the ammonia channel. In molecular viewers, one must remove the exterior surface that obscures the channel surface by clipping the viewing plane or manually selecting the channel residues in order to display a partial surface. Neither method is entirely satisfactory, as unwanted additional pieces of surfaces are always generated. RESULTS: To cleanly visualize a channel surface, we present HOLLOW, a program that generates a "casting" of the interior volume of the protein as dummy atoms. We show that the molecular surface of the dummy atoms closely approximates the channel surface, where this complementary surface of the protein channel can be displayed without superfluous surfaces. CONCLUSION: The use of HOLLOW significantly simplifies the generation of channel surfaces, and other interior surfaces of protein structures. HOLLOW is written in PYTHON and is available at http://hollow.sourceforge.net.


Assuntos
Modelos Moleculares , Software , Gráficos por Computador , Estrutura Molecular , Conformação Proteica , Propriedades de Superfície
7.
Proc Natl Acad Sci U S A ; 104(1): 42-7, 2007 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-17190799

RESUMO

Ammonia conductance is highly regulated. A P(II) signal transduction protein, GlnK, is the final regulator of transmembrane ammonia conductance by the ammonia channel AmtB in Escherichia coli. The complex formed between AmtB and inhibitory GlnK at 1.96-A resolution shows that the trimeric channel is blocked directly by GlnK and how, in response to intracellular nitrogen status, the ability of GlnK to block the channel is regulated by uridylylation/deuridylylation at Y51. ATP and Mg(2+) augment the interaction of GlnK. The hydrolyzed product, adenosine 5'-diphosphate orients the surface of GlnK for AmtB blockade. 2-Oxoglutarate diminishes AmtB/GlnK association, and sites for 2-oxoglutarate are evaluated.


Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Escherichia coli/química , Nucleotidiltransferases/química , Proteínas PII Reguladoras de Nitrogênio/química , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/fisiologia , Ácidos Cetoglutáricos/metabolismo , Nucleotidiltransferases/fisiologia , Proteínas PII Reguladoras de Nitrogênio/fisiologia , Periplasma/metabolismo , Conformação Proteica , Processamento de Proteína Pós-Traducional , Compostos de Amônio Quaternário/metabolismo
9.
J Mol Biol ; 333(5): 993-1002, 2003 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-14583195

RESUMO

We investigated mechanical unfolding of Borrelia burgdorferi outer surface protein A (OspA), a Lyme disease antigen containing a unique single-layer beta-sheet, with atomic force microscopy (AFM). We mechanically stretched a monomeric unit, rather than a tandem repeat, by pulling it from its N and C-terminal residues without using intervening polymer as a spacer. We detected two peaks in the force-extension profile before the final rupture of a fully extended polypeptide, which we interpreted as unfolding of multiple substructures in OspA. The double-peaked unfolding curves are consistent with results of previous thermodynamic studies showing two cooperative units in OspA. The mechanical unfolding processes were reversible, and the two substructures refolded within one second. Mutations near the boundary of the two thermodynamic cooperative units reduced the height of the first unfolding peak to undetectable levels and marginally affected the second one, indicating that the boundary between the two mechanical substructures is related to that previously assigned between the thermodynamic cooperative units. Based on a "worm-like chain" analysis of our AFM data, we propose a model for mechanical unfolding of OspA, where nearly a half of the chain is stretched with minimal resistive force, followed by sequential breakdown of C-terminal and N-terminal substructures. Based on these results, we discuss similarities and differences between mechanical and thermodynamic unfolding reactions of OspA. This work demonstrates that AFM study of monomeric proteins can elucidate details of the intramolecular mechanics of protein substructures.


Assuntos
Antígenos de Superfície/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Lipoproteínas , Animais , Antígenos de Superfície/química , Proteínas da Membrana Bacteriana Externa/química , Vacinas Bacterianas , Borrelia burgdorferi/química , Borrelia burgdorferi/metabolismo , Humanos , Microscopia de Força Atômica , Mutação , Desnaturação Proteica , Estrutura Terciária de Proteína
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