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 alpha-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.

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 alpha-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.

Large-scale purification of ribosome-nascent chain complexes for biochemical and structural studies.

Here we present a method to purify large amounts of highly pure and stably arrested ribosome-nascent chain complexes (RNCs) from Escherichia coli cells. It relies on the combined use of translation-arrest sequences to generate nascent polypeptides of specified length and subsequent tag purification of the RNCs. Moreover, we adapted this method for the in vivo production of RNCs with specific isotope labeling of the nascent chains for nuclear magnetic resonance (NMR) studies. This method opens therefore possibilities for a wide range of biochemical and structural studies exploring conformations of nascent chains during the early steps of protein folding and targeting.

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.

Use of biosynthetic fractional 13C-labeling for backbone NMR assignment of proteins.

We describe a simple approach to classify amino acid residue types in NMR spectra of proteins for supporting the backbone resonance assignments. It makes use of the differences in biosynthetic pathways of the 20 amino acids in Escherichia coli. Therefore, it is distinct from the parameters routinely exploited in the backbone resonance assignment such as chemical shifts and spin topology information. The combination of biosynthetically directed fractional 13C-labeling and uniform 15N-labeling enables us to obtain both residue-type specific information and sequential connectivities from a single protein sample. The residue-type classification exploiting biosynthetic pathways can be used for accelerating the conventional backbone assignment procedure.

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.

Direct NMR observation of a substrate protein bound to the chaperonin GroEL.

The reaction cycle and the major structural states of the molecular chaperone GroEL and its cochaperone, GroES, are well characterized. In contrast, very little is known about the nonnative states of the substrate polypeptide acted on by the chaperonin machinery. In this study, we investigated the substrate protein human dihydrofolate reductase (hDHFR) while bound to GroEL or to a single-ring analog, SR1, by NMR spectroscopy in solution under conditions where hDHFR was efficiently recovered as a folded, enzymatically active protein from the stable complexes upon addition of ATP and GroES. By using the NMR techniques of transverse relaxation-optimized spectroscopy (TROSY), cross-correlated relaxation-induced polarization transfer (CRIPT), and cross-correlated relaxation-enhanced polarization transfer (CRINEPT), bound hDHFR could be observed directly. Measurements of the buildup of hDHFR NMR signals by different magnetization transfer mechanisms were used to characterize the dynamic properties of the NMR-observable parts of the bound substrate. The NMR data suggest that the bound state includes random coil conformations devoid of stable native-like tertiary contacts and that the bound hDHFR might best be described as a dynamic ensemble of randomly structured conformers.

Uniform and residue-specific 15N-labeling of proteins on a highly deuterated background.

A general method for stable-isotope labeling of large proteins is introduced and applied for studies of the E. coli GroE chaperone proteins by solution NMR. In addition to enabling the residue-specific (15)N-labeling of proteins on a highly deuterated background, it is also an efficient approach for uniform labeling. The method meets the requirements of high-level deuteration, minimal cross-labeling and high protein yield, which are crucial for NMR studies of structures with sizes above 150 kDa. The results obtained with the new protocol are compared to other strategies for protein labeling, and evaluated with regard to the influence of external factors on the resulting isotope labeling patterns. Applications with the GroE system show that these strategies are efficient tools for studies of structure, dynamics and intermolecular interactions in large supramolecular complexes, when combined with TROSY- and CRINEPT-based experimental NMR schemes.

Solution NMR techniques for large molecular and supramolecular structures.

Transverse relaxation-optimized spectroscopy (TROSY) or generation of heteronuclear multiple quantum coherences during the frequency labeling period and TROSY during the acquisition period have been combined either with cross-correlated relaxation-induced polarization transfer (CRIPT) or cross-correlated relaxation-enhanced polarization transfer (CRINEPT) to obtain two-dimensional (2D) solution NMR correlation spectra of (15)N,(2)H-labeled homo-oligomeric macromolecules with molecular weights from 110 to 800 kDa. With the experimental conditions used, the line widths of the TROSY-components of the (1)H- and (15)N-signals were of the order of 60 Hz at 400 kDa, whereas, for structures of size 800 kDa, the line widths were about 75 Hz for (15)N and 110 Hz for (1)H. This paper describes the experimental schemes used and details of their setup for individual measurements. The performance of NMR experiments with large structures depends critically on the choice of the polarization transfer times, the relaxation delays between subsequent recordings, and the water-handling routines. Optimal transfer times for 2D [(15)N,(1)H]-CRIPT-TROSY experiments in H(2)O solutions were found to be 6 ms for a molecular weight of approximately 200 kDa, 2.8 ms for 400 kDa, and 1.4 ms for 800 kDa. These data validate theoretical predictions of inverse proportionality between optimal transfer time and size of the structure. The proton longitudinal relaxation times in H(2)O solution were found to be of the order of 0.8 s for structure sizes around 200 kDa, 0.4 s at 400 kDa, and 0.3 s at 800 kDa, which enabled the use of recycle times below 1 s. Since improper water handling results in severe signal loss, the water resonance was kept along the z-axis during the entire duration of the experiments by adjusting each water flip-back pulse individually.

Metabolic-flux profiling of the yeasts Saccharomyces cerevisiae and Pichia stipitis.

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.

NMR analysis of a 900K GroEL GroES complex.

Biomacromolecular structures with a relative molecular mass (M(r)) 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 M(r) 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 (M(r) 72K), either free in solution or in complex with the homotetradecameric chaperonin GroEL (M(r) 800K) or with the single-ring GroEL variant SR1 (M(r) 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.