RESUMEN
During protein biosynthesis, nascent polypeptide chains that emerge from the ribosomal exit tunnel encounter ribosome-associated chaperones, which assist their folding to the native state. Here we present a 2.7 A crystal structure of Escherichia coli trigger factor, the best-characterized chaperone of this type, together with the structure of its ribosome-binding domain in complex with the Haloarcula marismortui large ribosomal subunit. Trigger factor adopts a unique conformation resembling a crouching dragon with separated domains forming the amino-terminal ribosome-binding 'tail', the peptidyl-prolyl isomerase 'head', the carboxy-terminal 'arms' and connecting regions building up the 'back'. From its attachment point on the ribosome, trigger factor projects the extended domains over the exit of the ribosomal tunnel, creating a protected folding space where nascent polypeptides may be shielded from proteases and aggregation. This study sheds new light on our understanding of co-translational protein folding, and suggests an unexpected mechanism of action for ribosome-associated chaperones.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Biosíntesis de Proteínas , Pliegue de Proteína , Proteínas/metabolismo , Ribosomas/metabolismo , Cristalización , Cristalografía por Rayos X , Escherichia coli/metabolismo , Haloarcula marismortui , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Genéticos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Unión Proteica , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas/química , Ribosomas/químicaRESUMEN
The AAA+ protein ClpB cooperates with the DnaK chaperone system to solubilize and refold proteins from an aggregated state. The substrate-binding site of ClpB and the mechanism of ClpB-dependent protein disaggregation are largely unknown. Here we identified a substrate-binding site of ClpB that is located at the central pore of the first AAA domain. The conserved Tyr251 residue that lines the central pore contributes to substrate binding and its crucial role was confirmed by mutational analysis and direct crosslinking to substrates. Because the positioning of an aromatic residue at the central pore is conserved in many AAA+ proteins, a central substrate-binding site involving this residue may be a common feature of this protein family. The location of the identified binding site also suggests a possible translocation mechanism as an integral part of the ClpB-dependent disaggregation reaction.
Asunto(s)
Chaperonas Moleculares/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Modelos Químicos , Datos de Secuencia Molecular , Mutagénesis , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Tirosina/metabolismoRESUMEN
The ribosome-associated Trigger Factor (TF) cooperates with the DnaK system to assist the folding of newly synthesized polypeptides in Escherichia coli. TF unifies two functions in one to promote proper protein folding in vitro. First, as a chaperone it binds to unfolded protein substrates, thereby preventing aggregation and supporting productive folding. Second, TF catalyzes the cis/trans isomerization of peptidyl-prolyl bonds, which can be a rate-limiting step in protein folding. Here, we investigated whether the peptidyl-prolyl cis/trans isomerase (PPIase) function is essential for the folding activity of TF in vitro and in vivo by separating these two TF activities through site-directed mutagenesis of the PPIase catalytic center. Of the four different TF variants carrying point mutations in the PPIase domain, only the exchange of the conserved residue Phe-198 to Ala (TF F198A) abolished the PPIase activity of TF toward both a tetrapeptide and the model protein substrate RNase T1 in vitro. In contrast, all other activities of TF F198A tested were comparable with wild type TF. TF F198A retained a similar binding specificity toward membrane-bound peptides, assisted the refolding of denatured d-glyceraldehyde-3-phosphate dehydrogenase in vitro, and associated with nascent polypeptides in an in vitro transcription/translation system. Importantly, expression of the TF F198A encoding gene complemented the synthetic lethality of DeltatigDeltadnaK cells and prevented global protein misfolding at temperatures between 20 and 34 degrees C in these cells. We conclude that the PPIase activity is not required for the function of TF in folding of newly synthesized proteins.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Citosol/química , Citosol/metabolismo , Activación Enzimática , Proteínas de Escherichia coli/genética , Mutagénesis Sitio-Dirigida , Péptidos/química , Isomerasa de Peptidilprolil/genética , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Pliegue de Proteína , Chaperonina 60/metabolismo , Proteínas de Escherichia coli/química , Proteínas del Choque Térmico HSP40 , Proteínas de Choque Térmico/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Especificidad por SustratoRESUMEN
Trigger Factor (TF) is the first chaperone that interacts with nascent chains of cytosolic proteins in Escherichia coli. Although its chaperone activity requires association with ribosomes, TF is present in vivo in a 2-3 fold molar excess over ribosomes and a fraction of it is not ribosome-associated after cell lysis. Here we show that TF follows a three-state equilibrium. Size exclusion chromatography, crosslinking and analytical ultracentrifugation revealed that uncomplexed TF dimerizes with an apparent Kd of 18 microM. Dimerization is mediated by the N-terminal ribosome binding domain and the C-terminal domain of TF, whereas the central peptidyl prolyl isomerase (PPlase) and substrate binding domain does not contribute to dimerization. Crosslinking experiments showed that TF is monomeric in its ribosome-associated state. Quantitative analysis of TF binding to ribosomes revealed a dissociation constant for the TF-ribosome complex of approximately 1.2 microM. From these data we estimate that in vivo most of the ribosomes are in complex with monomeric TF. Uncomplexed TF, however, is in a monomer-dimer equilibrium with approximately two thirds of TF existing in a dimeric state.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Cromatografía en Gel , Reactivos de Enlaces Cruzados/química , Dimerización , Electroforesis en Gel de Poliacrilamida , Proteínas de Escherichia coli/genética , Glutaral/química , Cinética , Mutagénesis Sitio-Dirigida , Isomerasa de Peptidilprolil/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Ribosomas/metabolismo , Ultracentrifugación/métodosRESUMEN
During translation, the first encounter of nascent polypeptides is with the ribosome-associated chaperones that assist the folding process--a principle that seems to be conserved in evolution. In Escherichia coli, the ribosome-bound Trigger Factor chaperones the folding of cytosolic proteins by interacting with nascent polypeptides. Here we identify a ribosome-binding motif in the amino-terminal domain of Trigger Factor. We also show the formation of crosslinked products between Trigger Factor and two adjacent ribosomal proteins, L23 and L29, which are located at the exit of the peptide tunnel in the ribosome. L23 is essential for the growth of E. coli and the association of Trigger Factor with the ribosome, whereas L29 is dispensable in both processes. Mutation of an exposed glutamate in L23 prevents Trigger Factor from interacting with ribosomes and nascent chains, and causes protein aggregation and conditional lethality in cells that lack the protein repair function of the DnaK chaperone. Purified L23 also interacts specifically with Trigger Factor in vitro. We conclude that essential L23 provides a chaperone docking site on ribosomes that directly links protein biosynthesis with chaperone-assisted protein folding.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Reactivos de Enlaces Cruzados , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Espectrometría de Masas , Chaperonas Moleculares/química , Datos de Secuencia Molecular , Mutación/genética , Isomerasa de Peptidilprolil/química , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genéticaRESUMEN
Hsp70 chaperones assist protein folding processes through nucleotide-controlled cycles of substrate binding and release. In our effort to understand the structure-function relationship within the Hsp70 family of proteins, we characterized the Escherichia coli member of a novel Hsp70 subfamily, HscC, and identified considerable differences to the well studied E. coli homologue, DnaK, which together suggest that HscC is a specialized chaperone. The basal ATPase cycle of HscC had k(cat) and K(m) values that were 8- and 10,000-fold higher than for DnaK. The HscC ATPase was not affected by the nucleotide exchange factor of DnaK GrpE and stimulated 8-fold by DjlC, a DnaJ protein with a putative transmembrane domain, but not by other DnaJ proteins tested. Substrate binding dynamics and substrate specificity differed significantly between HscC and DnaK. These differences are explicable by distinct structural variations. HscC does not have general chaperone activity because it did not assist refolding of a denatured model substrate. In vivo, HscC failed to complement temperature sensitivity of DeltadnaK cells. Deletion of hscC caused a slow growth phenotype that was suppressed after several generations. Triple knock-outs of all E. coli genes encoding Hsp70 proteins (DeltadnaK DeltahscA DeltahscC) were viable, indicating that Hsp70 proteins are not strictly essential for viability. An extensive search for DeltahscC phenotypes revealed a hypersensitivity to Cd(2+) ions and UV irradiation, suggesting roles of HscC in the cellular response to these stress treatments. Together our data show that the Hsp70 structure exhibits an astonishing degree of adaptive variations to accommodate requirements of a specialized function.