RESUMO
Obscurin is a giant muscle protein that connects the sarcomere with the sarcoplasmic reticulum, and has poorly understood structural and signalling functions. Increasingly, obscurin variants are implicated in the pathophysiology of cardiovascular diseases. The Arg4344Gln variant (R4344Q) in obscurin domain Ig58, initially discovered in a patient with hypertrophic cardiomyopathy, has been reported to reduce binding to titin domains Z8-Z9, impairing obscurin's Z-disc localization. An R4344Q knock-in mouse developed a cardiomyopathy-like phenotype with abnormal Ca2+-handling and arrhythmias, which were attributed to an enhanced affinity of a putative interaction between obscurin Ig58 and phospholamban (PLN) due to the R4344Q variant. However, the R4344Q variant is found in 15% of African Americans, arguing against its pathogenicity. To resolve this apparent paradox, we quantified the influence of the R4344Q variant (alongside another potentially pathogenic variant: Arg4444Trp (R4444W)) on binding to titin Z8-Z9, novex-3 and PLN using pull-down assays and microscale thermophoresis and characterized the influence on domain stability using differential scanning fluorimetry. We found no changes in titin binding and thermostability for both variants and modestly increased affinities of PLN for R4344Q and R4444W. While we could not confirm the novex-3/obscurin interaction, the PLN/obscurin interaction relies on the transmembrane region of PLN and is not reproducible in mammalian cells, suggesting it is an in vitro artefact. Without clear clinical evidence for disease involvement, we advise against classifying these obscurin variants as pathogenic.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Cardiomiopatia Hipertrófica/genética , Conectina/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Animais , Proteínas de Ligação ao Cálcio/ultraestrutura , Cardiomiopatia Hipertrófica/patologia , Conectina/ultraestrutura , Humanos , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Ligação Proteica/genética , Conformação Proteica , Mapas de Interação de Proteínas/genética , Proteínas Serina-Treonina Quinases/ultraestrutura , Estabilidade Proteica , Fatores de Troca de Nucleotídeo Guanina Rho/ultraestrutura , Sarcômeros/genética , Sarcômeros/metabolismo , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/genéticaRESUMO
The guanine nucleotide exchange factor GEF-H1 (also known as ARHGEF2) is identified as a member of the Dbl family of GEFs. It regulates RhoA-dependent cell signaling pathways and plays important roles in biological processes. GEF-H1 contains an N-terminal zinc finger domain, a Dbl-homologous (DH) domain followed by a Pleckstrin homology (PH) domain, and a C-terminal domain. The specific roles of its PH domain are poorly understood. Here we report the crystal structure of human GEF-H1 PH domain to 2.45 Å resolution. A conserved surface is formed by ß8, ß9, ß10, and it may mediate protein-protein interactions. Although the folding resembles other PH domains that have defined structures, superposition of different PH domains clearly shows that the loop between ß6/ß7 and the loop between ß3/ß4 are so close that they will prevent its binding with phosphoinositide due to steric hindrance, and this has been proved by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). Our studies provide a structural framework for further work on the function of GEF-H1.
Assuntos
Fosfatidilinositóis/química , Fatores de Troca de Nucleotídeo Guanina Rho/química , Sequência de Aminoácidos , Proteínas Sanguíneas/química , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Fosfoproteínas/química , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Troca de Nucleotídeo Guanina Rho/ultraestruturaRESUMO
M10 is the most C-terminal immunoglobulin (Ig) domain of the giant protein titin and a frequent target of disease-linked mutations. Currently, it is the only known muscle Ig domain able to interact with two alternative ligands-obscurin and obscurin-like-1 (Obsl1)-in different sarcomeric subregions. Obscurin and Obsl1 use their homologous N-terminal Ig domain (O1 in obscurin and OL1 in Obsl1) to bind M10 in a mutually exclusive manner. We present here the X-ray structure of the human titin:obscurin M10:O1 complex extending our previous work on the M10:OL1 interaction. Similar to M10:OL1, the M10:O1 complex displays a chevron-shaped antiparallel Ig-Ig architecture held together by a conserved molecular interface, which we validated by isothermal titration calorimetry and sorting experiments in neonatal rat cardiomyocytes. O1, although structurally related to OL1 and M10, both members of the intermediate set (I-set) Ig family, presents an intriguing switch of its ßA' strand. This leads to structural differences between the complexes, particularly for the "open side" of the chevron-shaped assembly. A bioinformatics analysis reveals that the ßA'-switch observed for O1 is rare and that it is involved in mediating protein-protein interactions. Molecular dynamics simulations also suggest that this topological alteration substantially increases local flexibility compared to the conventional I-set Ig domains. The O1/OL1 Ig domains are candidate discriminatory structural modules potentially directing the binding of specific additional partners at the M-band. Cellular sorting experiments in neonatal rat cardiomyocytes are consistent with the view that the titin:obscurin/Obsl1 complexes might be a platform for higher-order interactions.