Jocelyne Fiaux

NMR analysis of a 900k GroEL-GroES complex

Biomacromolecular structures with a relative molecular mass (Mr) of 50,000 to 100,000 (50K–100K) have been generally considered to be inaccessible to analysis by solution NMR spectroscopy. Here we report spectra recorded from bacterial chaperonin complexes ten times this size limit (up to Mr 900K) using the techniques of transverse relaxation-optimized spectroscopy and cross-correlated relaxation-enhanced polarization transfer. These techniques prevent deterioration of the NMR spectra by the rapid transverse relaxation of the magnetization to which large, slowly tumbling molecules are otherwise subject. We tested the resolving power of these techniques by examining the isotope-labelled homoheptameric co-chaperonin GroES (Mr 72K), either free in solution or in complex with the homotetradecameric chaperonin GroEL (Mr 800K) or with the single-ring GroEL variant SR1 (Mr 400K). Most amino acids of GroES show the same resonances whether free in solution or in complex with chaperonin; however, residues 17–32 show large chemical shift changes on binding. These amino acids belong to a mobile loop region of GroES that forms contacts with GroEL. This establishes the utility of these techniques for solution NMR studies that should permit the exploration of structure, dynamics and interactions in large macromolecular complexes.

Metabolic flux responses to pyruvate kinase knockout in Escherichia coli.

The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.

Metabolic-Flux Profiling of the Yeasts Saccharomyces cerevisiae and Pichia stipitis

ABSTRACT The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance. The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts. The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.

Hsp110 Is a Nucleotide-activated Exchange Factor for Hsp70

Hsp110 proteins constitute a subfamily of the Hsp70 chaperones and are potent nucleotide exchange factors (NEFs) for canonical Hsp70s of the eukaryotic cytosol. Here, we show that the NEF activity of the yeast Hsp110 homologue Sse1 itself is controlled by nucleotide. Nucleotide binding results in formation of a stabilized conformation of Sse1 that is required for association with the yeast Hsp70 Ssa1. The interaction triggers release of bound ADP from Ssa1, but nucleotide persists bound to Sse1 in the complex. Surprisingly, removal of this nucleotide does not affect the integrity of the complex. Instead, rebinding of ATP to the Hsp70 prompts the dissociation of the complex. Our data demonstrate that in contrast to previously characterized NEFs for Hsp70 chaperones, the NEF activity of Sse1 requires nucleotide binding and let us propose a new model for Hsp110 function.

Insights into the structural dynamics of the Hsp110-Hsp70 interaction reveal the mechanism for nucleotide exchange activity

Hsp110 proteins are relatives of canonical Hsp70 chaperones and are expressed abundantly in the eukaryotic cytosol. Recently, it has become clear that Hsp110 proteins are essential nucleotide exchange factors (NEFs) for Hsp70 chaperones. Here, we report the architecture of the complex between the yeast Hsp110, Sse1, and its cognate Hsp70 partner, Ssa1, as revealed by hydrogen–deuterium exchange analysis and site-specific cross-linking. The two nucleotide-binding domains (NBDs) of Sse1 and Ssa1 are positioned to face each other and form extensive contacts between opposite lobes of their NBDs. A second contact with the periphery of the Ssa1 NBD lobe II is likely mediated via the protruding C-terminal α-helical subdomain of Sse1. To address the mechanism of catalyzed nucleotide exchange, we have compared the hydrogen exchange characteristics of the Ssa1 NBD in complex with either Sse1 or the yeast homologs of the NEFs HspBP1 and Bag-1. We find that Sse1 exploits a Bag-1-like mechanism to catalyze nucleotide release, which involves opening of the Ssa1 NBD by tilting lobe II. Thus, Hsp110 proteins use a unique binding mode to catalyze nucleotide release from Hsp70s by a functionally convergent mechanism.

Dissection of Central Carbon Metabolism of Hemoglobin-Expressing Escherichia coli by 13C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses

ABSTRACT Escherichia coli MG1655 cells expressingVitreoscilla hemoglobin (VHb), Alcaligenes eutrophus flavohemoprotein (FHP), the N-terminal hemoglobin domain of FHP (FHPg), and a fusion protein which comprises VHb and theA. eutrophus C-terminal reductase domain (VHb-Red) were grown in a microaerobic bioreactor to study the effects of low oxygen concentrations on the central carbon metabolism, using fractional13C-labeling of the proteinogenic amino acids and two-dimensional [13C, 1H]-correlation nuclear magnetic resonance (NMR) spectroscopy. The NMR data revealed differences in the intracellular carbon fluxes between E. coli cells expressing either VHb or VHb-Red and cells expressingA. eutrophus FHP or the truncated heme domain (FHPg).E. coli MG1655 cells expressing either VHb or VHb-Red were found to function with a branched tricarboxylic acid (TCA) cycle. Furthermore, cellular demands for ATP and reduction equivalents in VHb- and VHb-Red-expressing cells were met by an increased flux through glycolysis. In contrast, in E. coli cells expressingA. eutrophus hemeproteins, the TCA cycle is running cyclically, indicating a shift towards a more aerobic regulation. Consistently, E. coli cells displaying FHP and FHPg activity showed lower production of the typical anaerobic by-products formate, acetate, and d-lactate. The implications of these observations for biotechnological applications are discussed.

The endoplasmic reticulum Grp170 acts as a nucleotide exchange factor of Hsp70 via a mechanism similar to that of the cytosolic Hsp110.

Grp170 and Hsp110 proteins constitute two evolutionary distinct branches of the Hsp70 family that share the ability to function as nucleotide exchange factors (NEFs) for canonical Hsp70s. Although the NEF mechanism of the cytoplasmic Hsp110s is well understood, little is known regarding the mechanism used by Grp170s in the endoplasmic reticulum. In this study, we compare the yeast Grp170 Lhs1 with the yeast Hsp110 Sse1. We find that residues important for Sse1 NEF activity are conserved in Lhs1 and that mutations in these residues in Lhs1 compromise NEF activity. As previously reported for Sse1, Lhs1 requires ATP to trigger nucleotide exchange in its cognate Hsp70 partner Kar2. Using site-specific cross-linking, we show that the nucleotide-binding domain (NBD) of Lhs1 interacts with the NBD of Kar2 face to face, and that Lhs1 contacts the side of the Kar2 NBD via its protruding C-terminal α-helical domain. To directly address the mechanism of nucleotide exchange, we have compared the hydrogen-exchange characteristics of a yeast Hsp70 NBD (Ssa1) in complex with either Sse1 or Lhs1. We find that Lhs1 and Sse1 induce very similar changes in the conformational dynamics in the Hsp70. Thus, our findings demonstrate that despite some differences between Hsp110 and Grp170 proteins, they use a similar mechanism to trigger nucleotide exchange.

Structural Analysis of the Ribosome-associated Complex (RAC) Reveals an Unusual Hsp70/Hsp40 Interaction

Yeast Zuotin and Ssz are members of the conserved Hsp40 and Hsp70 chaperone families, respectively, but compared with canonical homologs, they atypically form a stable heterodimer termed ribosome-associated complex (RAC). RAC acts as co-chaperone for another Hsp70 to assist de novo protein folding. In this study, we identified the molecular basis for the unusual Hsp70/Hsp40 pairing using amide hydrogen exchange (HX) coupled with mass spectrometry and mutational analysis. Association of Ssz with Zuotin strongly decreased the conformational dynamics mainly in the C-terminal domain of Ssz, whereas Zuotin acquired strong conformational stabilization in its N-terminal segment. Deletion of the highly flexible N terminus of Zuotin abolished stable association with Ssz in vitro and caused a phenotype resembling the loss of Ssz function in vivo. Thus, the C-terminal domain of Ssz, the N-terminal extension of Zuotin, and their mutual stabilization are the major structural determinants for RAC assembly. We furthermore found dynamic changes in the J-domain of Zuotin upon complex formation that might be crucial for RAC co-chaperone function. Taken together, we present a novel mechanism for converting Zuotin and Ssz chaperones into a functionally active dimer.

Proton–proton Overhauser NMR spectroscopy with polypeptide chains in large structures

The use of 1H–1H nuclear Overhauser effects (NOE) for structural studies of uniformly deuterated polypeptide chains in large structures is investigated by model calculations and NMR experiments. Detailed analysis of the evolution of the magnetization during 1H–1H NOE experiments under slow-motion conditions shows that the maximal 1H–1H NOE transfer is independent of the overall rotational correlation time, even in the presence of chemical exchange with the bulk water, provided that the mixing time is adjusted for the size of the structure studied. 1H–1H NOE buildup measurements were performed for the 472-kDa complex of the 72-kDa cochaperonin GroES with a 400-kDa single-ring variant of the chaperonin GroEL (SR1). These experiments demonstrate that multidimensional NOESY experiments with cross-correlated relaxation-enhanced polarization transfer and transverse relaxation-optimized spectroscopy elements can be applied to structures of molecular masses up to several hundred kilodaltabs, which opens new possibilities for studying functional interactions in large maromolecular assemblies in solution.