RESUMEN
V1-ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A3B3 complex and a central DF shaft. The nucleotide-free A3B3 complex of Enterococcus hirae, composed of three identical A1B1 heterodimers, showed a unique asymmetrical structure, probably due to the strong binding of the N-terminal barrel domain, which forms a crown structure. Here, we mutated the barrel region to weaken the crown, and performed structural analyses using high-speed atomic force microscopy and x-ray crystallography of the mutant A3B3. The nucleotide-free mutant A3B3 complex had a more symmetrical open structure than the wild type. Binding of nucleotides produced a closely packed spiral-like structure with a disrupted crown. These findings suggest that wild-type A3B3 forms a metastable (stressed) asymmetric structure composed of unstable A1B1 conformers due to the strong constraint of the crown. The results further the understanding of the principle of the cooperative transition mechanism of rotary motors.
Asunto(s)
Enterococcus hirae/enzimología , Estructura Cuaternaria de Proteína , ATPasas de Translocación de Protón Vacuolares/química , Sitios de Unión , Biocatálisis , Sistema Libre de Células/metabolismo , Cristalografía por Rayos X , Escherichia coli/citología , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía de Fuerza Atómica , Proteínas Mutantes/química , Mutación , Nucleótidos/química , Dominios Proteicos/genética , Multimerización de Proteína , Subunidades de Proteína/química , RotaciónRESUMEN
V1-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae A3B3DF (V1) complex, corresponding to the catalytic dwell state waiting for ATP hydrolysis. Here we present the crystal structures for two other dwell states obtained by soaking nucleotide-free V1 crystals in ADP. In the presence of 20 µM ADP, two ADP molecules bind to two of three binding sites and cooperatively induce conformational changes of the third site to an ATP-binding mode, corresponding to the ATP-binding dwell. In the presence of 2 mM ADP, all nucleotide-binding sites are occupied by ADP to induce conformational changes corresponding to the ADP-release dwell. Based on these and previous findings, we propose a V1-ATPase rotational mechanism model.
Asunto(s)
ATPasas de Translocación de Protón Vacuolares/metabolismo , Adenosina Difosfato , Adenilil Imidodifosfato , Cristalografía por Rayos X , Escherichia coli , Conformación Proteica , ATPasas de Translocación de Protón Vacuolares/químicaRESUMEN
Human reticulocalbin-1 (hRCN1) has six EF-hand motifs and binds Ca(2+). hRCN1 is a member of the CREC family localized in the secretory pathway, and its cellular function remains unclear. In this study, we established a new bacterial expression and purification procedure for hRCN1. We observed that hRCN1 binds Ca(2+) in a cooperative manner and the Ca(2+) binding caused an increase in the α-helix content of hRCN1. On the other hand, hRCN1 did not change the structure with Mg(2+) loading. hRCN1 is a monomeric protein, and its overall structure became more compact upon Ca(2+) binding, as revealed by gel-filtration column chromatography and small-angle X-ray scattering. This is the first report of conformational changes in the CREC family upon Ca(2+) binding. Our data suggest that CREC family member interactions with target proteins are regulated in the secretory pathway by conformational changes upon Ca(2+) binding.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Cromatografía en Gel , Escherichia coli/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Magnesio/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Dispersión de Radiación , Homología de Secuencia de AminoácidoRESUMEN
Chromosome 1 open reading frame 10 (C1orf10) is a recently identified gene encoding a protein with an S100 EF-hand calcium-binding motif, and its expression is known to be down-regulated in esophageal squamous cell carcinoma. In this study, to determine whether the loss of C1orf10 gene function could contribute to the development of oral squamous cell carcinoma (OSCC), we have evaluated the expression status of this gene by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and quantitative real-time PCR analysis. A high frequency of decrease in C1orf10 gene was detected not only in OSCC-derived cell lines but also in tumor tissues. Next, to define biological function of this gene in oral carcinogenesis, we transfected Clorf10 with an Ecdysone-inducible system in OSCC cell lines and analyzed the effects of its overexpression. Induction of C1orf10 expression resulted in a significant decline in the rate of cell proliferation, and in an arrest in the G1 phase of the cell cycle, with a down-regulation of Cyclin D1 expression. However, we could not detect significant difference in the percentage of apoptotic cells. Thus, our results suggest that the down-regulation of C1orf10 gene plays a role in oral carcinogenesis, and that its expression may negatively regulate OSCC cell proliferation by arresting the cell cycle.
