EZ-ASSIGN, a program for exhaustive NMR chemical shift assignments of large proteins from complete or incomplete triple-resonance data.

For several of the proteins in the BioMagResBank larger than 200 residues, 60 % or fewer of the backbone resonances were assigned. But how reliable are those assignments? In contrast to complete assignments, where it is possible to check whether every triple-resonance Generalized Spin System (GSS) is assigned once and only once, with incomplete data one should compare all possible assignments and pick the best one. But that is not feasible: For example, for 200 residues and an incomplete set of 100 GSS, there are 1.6 × 10260 possible assignments. In "EZ-ASSIGN", the protein sequence is divided in smaller unique fragments. Combined with intelligent search approaches, an exhaustive comparison of all possible assignments is now feasible using a laptop computer. The program was tested with experimental data of a 388-residue domain of the Hsp70 chaperone protein DnaK and for a 351-residue domain of a type III secretion ATPase. EZ-ASSIGN reproduced the hand assignments. It did slightly better than the computer program PINE (Bahrami et al. in PLoS Comput Biol 5(3):e1000307, 2009) and significantly outperformed SAGA (Crippen et al. in J Biomol NMR 46:281-298, 2010), AUTOASSIGN (Zimmerman et al. in J Mol Biol 269:592-610, 1997), and IBIS (Hyberts and Wagner in J Biomol NMR 26:335-344, 2003). Next, EZ-ASSIGN was used to investigate how well NMR data of decreasing completeness can be assigned. We found that the program could confidently assign fragments in very incomplete data. Here, EZ-ASSIGN dramatically outperformed all the other assignment programs tested.

Allostery in the Hsp70 chaperone proteins.

Heat shock 70-kDa (Hsp70) chaperones are essential to in vivo protein folding, protein transport, and protein re-folding. They carry out these activities using repeated cycles of binding and release of client proteins. This process is under allosteric control of nucleotide binding and hydrolysis. X-ray crystallography, NMR spectroscopy, and other biophysical techniques have contributed much to the understanding of the allosteric mechanism linking these activities and the effect of co-chaperones on this mechanism. In this chapter these findings are critically reviewed. Studies on the allosteric mechanisms of Hsp70 have gained enhanced urgency, as recent studies have implicated this chaperone as a potential drug target in diseases such as Alzheimer's and cancer. Recent approaches to combat these diseases through interference with the Hsp70 allosteric mechanism are discussed.

Heat shock protein 70 kDa chaperone/DnaJ cochaperone complex employs an unusual dynamic interface.

The heat shock protein 70 kDa (Hsp70)/DnaJ/nucleotide exchange factor system assists in intracellular protein (re)folding. Using solution NMR, we obtained a three-dimensional structure for a 75-kDa Hsp70-DnaJ complex in the ADP state, loaded with substrate peptide. We establish that the J domain (residues 1-70) binds with its positively charged helix II to a negatively charged loop in the Hsp70 nucleotide-binding domain. The complex shows an unusual "tethered" binding mode which is stoichiometric and saturable, but which has a dynamic interface. The complex represents part of a triple complex of Hsp70 and DnaJ both bound to substrate protein. Mutagenesis data indicate that the interface is also of relevance for the interaction of Hsp70 and DnaJ in the ATP state. The solution complex is completely different from a crystal structure of a disulfide-linked complex of homologous proteins [Jiang, et al. (2007) Mol Cell 28:422-433].

Chemical screens against a reconstituted multiprotein complex: myricetin blocks DnaJ regulation of DnaK through an allosteric mechanism.

DnaK is a molecular chaperone responsible for multiple aspects of bacterial proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors we screened plant-derived extracts against a reconstituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK's intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaK to DnaJ. Together, these results highlight a "gray box" screening approach, which might facilitate the identification of inhibitors of other protein-protein interactions.

Binding of a small molecule at a protein-protein interface regulates the chaperone activity of hsp70-hsp40.

Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that plays multiple roles in protein homeostasis. In these various tasks, the activity of Hsp70 is shaped by interactions with co-chaperones, such as Hsp40. The Hsp40 family of co-chaperones binds to Hsp70 through a conserved J-domain, and these factors stimulate ATPase and protein-folding activity. Using chemical screens, we identified a compound, 115-7c, which acts as an artificial co-chaperone for Hsp70. Specifically, the activities of 115-7c mirrored those of a Hsp40; the compound stimulated the ATPase and protein-folding activities of a prokaryotic Hsp70 (DnaK) and partially compensated for a Hsp40 loss-of-function mutation in yeast. Consistent with these observations, NMR and mutagenesis studies indicate that the binding site for 115-7c is adjacent to a region on DnaK that is required for J-domain-mediated stimulation. Interestingly, we found that 115-7c and the Hsp40 do not compete for binding but act in concert. Using this information, we introduced additional steric bulk to 115-7c and converted it into an inhibitor. Thus, these chemical probes either promote or inhibit chaperone functions by regulating Hsp70-Hsp40 complex assembly at a native protein-protein interface. This unexpected mechanism may provide new avenues for exploring how chaperones and co-chaperones cooperate to shape protein homeostasis.

Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate.

DnaK is the canonical Hsp70 molecular chaperone protein from Escherichia coli. Like other Hsp70s, DnaK comprises two main domains: a 44-kDa N-terminal nucleotide-binding domain (NBD) that contains ATPase activity, and a 25-kDa substrate-binding domain (SBD) that harbors the substrate-binding site. Here, we report an experimental structure for wild-type, full-length DnaK, complexed with the peptide NRLLLTG and with ADP. It was obtained in aqueous solution by using NMR residual dipolar coupling and spin labeling methods and is based on available crystal structures for the isolated NBD and SBD. By using dynamics methods, we determine that the NBD and SBD are loosely linked and can move in cones of +/-35 degrees with respect to each other. The linker region between the domains is a dynamic random coil. Nevertheless, an average structure can be defined. This structure places the SBD in close proximity of subdomain IA of the NBD and suggests that the SBD collides with the NBD at this area to establish allosteric communication.

Allostery in Hsp70 chaperones is transduced by subdomain rotations.

Hsp70s (heat shock protein 70 kDa) are central to protein folding, refolding, and trafficking in organisms ranging from archaea to Homo sapiens under both normal and stressed cellular conditions. Hsp70s are comprised of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). The nucleotide binding site in the NBD and the substrate binding site in the SBD are allosterically linked: ADP binding promotes substrate binding, while ATP binding promotes substrate release. Hsp70s have been linked to inhibition of apoptosis (i.e., cancer) and diseases associated with protein misfolding such as Alzheimer's, Parkinson's, and Huntington's. It has long been a goal to characterize the nature of allosteric coupling in these proteins. However, earlier studies of the isolated NBD could not show any difference in overall conformation between the ATP state and the ADP state. Hence the question: How is the state of the nucleotide communicated between NBD and SBD? Here we report a solution NMR study of the 44-kDa NBD of Hsp70 from Thermus thermophilus in the ADP and AMPPNP states. Using the solution NMR methods of residual dipolar coupling analysis, we determine that significant rotations occur for different subdomains of the NBD upon exchange of nucleotide. These rotations modulate access to the nucleotide binding cleft in the absence of a nucleotide exchange factor. Moreover, the rotations cause a change in the accessibility of a hydrophobic surface cleft remote from the nucleotide binding site, which previously has been identified as essential to allosteric communication between NBD and SBD. We propose that it is this change in the NBD surface cleft that constitutes the allosteric signal that can be recognized by the SBD.

High-throughput screen for small molecules that modulate the ATPase activity of the molecular chaperone DnaK.

DnaK is a molecular chaperone of Escherichia coli that belongs to a family of conserved 70-kDa heat shock proteins. The Hsp70 chaperones are well known for their crucial roles in regulating protein homeostasis, preventing protein aggregation, and directing subcellular traffic. Given the complexity of functions, a chemical method for controlling the activities of these chaperones might provide a useful experimental tool. However, there are only a handful of Hsp70-binding molecules known. To build this area, we developed a robust, colorimetric, high-throughput screening (HTS) method in 96-well plates that reports on the ATPase activity of DnaK. Using this approach, we screened a 204-member focused library of molecules that share a dihydropyrimidine core common to known Hsp70-binding leads and uncovered seven new inhibitors. Intriguingly, the candidates do not appear to bind the hydrophobic groove that normally interacts with peptide substrates. In sum, we have developed a reliable HTS method that will likely accelerate discovery of small molecules that modulate DnaK/Hsp70 function. Moreover, because this family of chaperones has been linked to numerous diseases, this platform might be used to generate new therapeutic leads.

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.

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.