RESUMEN
The tryptophan synthase bienzyme complex is the most extensively documented example of substrate channeling in which the oligomeric unit has been described at near atomic resolution. Transfer of the common metabolite, indole, between the alpha- and the beta-sites occurs by diffusion along a 25-A-long interconnecting tunnel within each alphabeta-dimeric unit of the alpha(2)beta(2) oligomer. The control of metabolite transfer involves allosteric interactions that trigger the switching of alphabeta-dimeric units between open and closed conformations and between catalytic states of low and high activity. This allosteric signaling is triggered by covalent transformations at the beta-site and ligand binding to the alpha-site. The signals are transmitted between sites via a scaffolding of structural elements that includes a monovalent cation (MVC) binding site and salt bridging interactions of betaLys 167 with betaAsp 305 or alphaAsp 56. Through the combined strategies of site-directed mutations of these amino acid residues and cation substitutions at the MVC site, this work examines the interrelationship of the MVC site and the alternative salt bridges formed between Lys beta167 with Asp beta305 or Asp alpha56 to the regulation of channeling. These experiments show that both the binding of a MVC and the formation of the Lys beta167-Asp alpha56 salt bridge are important to the transmission of allosteric signals between the sites, whereas, the salt bridge between betaK167 and betaD305 appears to be only of minor significance to catalysis and allosteric regulation. The mechanistic implications of these findings both for substrate channeling and for catalysis are discussed.
Asunto(s)
Mutagénesis Sitio-Dirigida , Sales (Química)/química , Triptófano Sintasa/química , Triptófano Sintasa/genética , Alanina/genética , Regulación Alostérica/genética , Asparagina/genética , Ácido Aspártico/genética , Cationes Monovalentes/química , Deuterio/química , Dimerización , Activación Enzimática/genética , Cinética , Lisina/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Potasio/química , Compuestos de Amonio Cuaternario/química , Salmonella typhimurium/enzimología , Sodio/química , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Treonina/genética , VolumetríaRESUMEN
The intracellular degradation of many proteins is mediated in an ATP-dependent manner by large assemblies comprising a chaperone ring complex associated coaxially with a proteolytic cylinder, e.g., ClpAP, ClpXP, and HslUV in prokaryotes, and the 26S proteasome in eukaryotes. Recent studies of the chaperone ClpA indicate that it mediates ATP-dependent unfolding of substrate proteins and directs their ATP-dependent translocation into the ClpP protease. Because the axial passageway into the proteolytic chamber is narrow, it seems likely that unfolded substrate proteins are threaded from the chaperone into the protease, suggesting that translocation could be directional. We have investigated directionality in the ClpA/ClpP-mediated reaction by using two substrate proteins bearing the COOH-terminal ssrA recognition element, each labeled near the NH(2) or COOH terminus with fluorescent probes. Time-dependent changes in both fluorescence anisotropy and fluorescence resonance energy transfer between donor fluorophores in the ClpP cavity and the substrate probes as acceptors were measured to monitor translocation of the substrates from ClpA into ClpP. We observed for both substrates that energy transfer occurs 2--4 s sooner with the COOH-terminally labeled molecules than with the NH(2)-terminally labeled ones, indicating that translocation is indeed directional, with the COOH terminus of the substrate protein entering ClpP first.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/fisiología , Adenosina Trifosfato/análogos & derivados , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/fisiología , Adenosina Trifosfato/metabolismo , Transporte Biológico , Endopeptidasa Clp , Escherichia coli/enzimología , Polarización de Fluorescencia , Cinética , Muramidasa/metabolismo , ARN Bacteriano/metabolismo , Especificidad por SustratoRESUMEN
The bacterial protein CIpA, a member of the Hsp100 chaperone family, forms hexameric rings that bind to the free ends of the double-ring serine protease ClpP. ClpA directs the ATP-dependent degradation of substrate proteins bearing specific sequences, much as the 19S ATPase 'cap' of eukaryotic proteasomes functions in the degradation of ubiquitinated proteins. In isolation, ClpA and its relative ClpX can mediate the disassembly of oligomeric proteins; another similar eukaryotic protein, Hsp104, can dissociate low-order aggregates. ClpA has been proposed to destabilize protein structure, allowing passage of proteolysis substrates through a central channel into the ClpP proteolytic cylinder. Here we test the action of ClpA on a stable monomeric protein, the green fluorescent protein GFP, onto which has been added an 11-amino-acid carboxy-terminal recognition peptide, which is responsible for recruiting truncated proteins to ClpAP for degradation. Fluorescence studies both with and without a 'trap' version of the chaperonin GroEL, which binds non-native forms of GFP, and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Chaperoninas/metabolismo , Proteínas de Escherichia coli , Proteínas de Choque Térmico/metabolismo , Pliegue de Proteína , Serina Endopeptidasas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/metabolismo , Chaperonina 60/metabolismo , Deuterio , Endopeptidasa Clp , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Espectrometría de Masas , Mutagénesis Sitio-Dirigida , Serina Endopeptidasas/genéticaRESUMEN
Chaperone rings play a vital role in the opposing ATP-mediated processes of folding and degradation of many cellular proteins, but the mechanisms by which they assist these life and death actions are only beginning to be understood. Ring structures present an advantage to both processes, providing for compartmentalization of the substrate protein inside a central cavity in which multivalent, potentially cooperative interactions can take place between the substrate and a high local concentration of binding sites, while access of other proteins to the cavity is restricted sterically. Such restriction prevents outside interference that could lead to nonproductive fates of the substrate protein while it is present in non-native form, such as aggregation. At the step of recognition, chaperone rings recognize different motifs in their substrates, exposed hydrophobicity in the case of protein-folding chaperonins, and specific "tag" sequences in at least some cases of the proteolytic chaperones. For both folding and proteolytic complexes, ATP directs conformational changes in the chaperone rings that govern release of the bound polypeptide. In the case of chaperonins, ATP enables a released protein to pursue the native state in a sequestered hydrophilic folding chamber, and, in the case of the proteases, the released polypeptide is translocated into a degradation chamber. These divergent fates are at least partly governed by very different cooperating components that associate with the chaperone rings: that is, cochaperonin rings on one hand and proteolytic ring assemblies on the other. Here we review the structures and mechanisms of the two types of chaperone ring system.