RESUMO
BACKGROUND: BAD protein (Bcl-2 antagonist of cell death) belongs to the BH3-only subfamily of proapoptotic proteins and is proposed to function as the sentinel of the cellular health status. Physiological activity of BAD is regulated by phosphorylation, association with 14-3-3 proteins, binding to membrane lipids and pore formation. Since the functional role of the BAD C-terminal part has not been considered so far, we have investigated here the interplay of the structure and function of this region. METHODS: The structure of the regulatory C-terminal part of human BAD was analyzed by CD spectroscopy. The channel-forming activity of full-length BAD and BAD peptides was carried out by lipid bilayer measurements. Interactions between proteins and peptides were monitored by the surface plasmon resonance technique. In aqueous solution, C-terminal part of BAD exhibits a well-ordered structure and stable conformation. In a lipid environment, the helical propensity considerably increases. The interaction of the C-terminal segment of BAD with the isolated BH3 domain results in the formation of permanently open pores whereby the phosphorylation of serine 118 within the BH3 domain is necessary for effective pore formation. In contrast, phosphorylation of serine 99 in combination with 14-3-3 association suppresses formation of channels. C-terminal part of BAD controls BAD function by structural transitions, lipid binding and phosphorylation. Conformational changes of this region upon membrane interaction in conjunction with phosphorylation of the BH3 domain suggest a novel mechanism for regulation of BAD. GENERAL SIGNIFICANCE: Multiple signaling pathways mediate inhibition and activation of cell death via BAD.
Assuntos
Bicamadas Lipídicas/química , Conformação Proteica , Estrutura Terciária de Proteína , Proteína de Morte Celular Associada a bcl/química , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ressonância de Plasmônio de Superfície , Água/química , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
The putative transcriptional regulator protein YvoA (BSU35030) from Bacillus subtilis was cloned and heterologously expressed in Escherichia coli. The protein was purified by immobilized metal-affinity chromatography and size-exclusion chromatography and subsequently crystallized. A complete native data set was collected to 2.50 A resolution. The crystals belonged to the monoclinic space group C2 and preliminary analysis of the diffraction data indicated the presence of approximately 12 molecules per asymmetric unit.
Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Difração de Raios X , Proteínas de Bactérias/metabolismo , Cromatografia em Gel , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Estrutura Terciária de ProteínaRESUMO
Carbon metabolism and regulation is poorly understood in mycobacteria, a genus that includes some major pathogenic species like Mycobacterium tuberculosis and Mycobacterium leprae. Here, we report the identification of a glucose kinase from Mycobacterium smegmatis. This enzyme serves in glucose metabolism and global carbon catabolite repression in the related actinomycete Streptomyces coelicolor. The gene, msmeg1356 (glkA), was found by means of in silico screening. It was shown that it occurs in the same genetic context in all so far sequenced mycobacterial species, where it is located in a putative tricistronic operon together with a glycosyl hydrolase and a putative malonyl-CoA transacylase. Heterologous expression of glkA in an Escherichia coli glucose kinase mutant led to the restoration of glucose growth, which provided in vivo evidence for glucose kinase function. GlkA(Msm) was subsequently overproduced in order to study its enzymatic features. We found that it can form a dimer and that it efficiently phosphorylates glucose at the expense of ATP. The affinity constant for glucose was with 9 mM about eight times higher and the velocity was about tenfold slower when compared to the parallel measured glucose kinase of S. coelicolor. Both enzymes showed similar substrate specificity, which consists in an ATP-dependent phosphorylation of glucose and no, or very inefficient, phosphorylation of the glucose analogues 2-deoxyglucose and methyl alpha-glucoside. Hence, our data provide a basis for studying the role of mycobacterial glucose kinase in vivo to unravel possible catalytic and regulatory functions.