Erik R. P. Zuiderweg

Heteronuclear three-dimensional nmr spectroscopy. A strategy for the simplification of homonuclear two-dimensional NMR spectra

Recently, three-dimensional Fourier transform techniques have been suggested (I3) as a possible method to resolve the spectra of complicated systems. Analogous to the comparison of 2D with 1 D NMR, three-dimensional NMR spectroscopy allows an increase in resolution and offers additional correlations for the elucidation of scalarand dipolar-coupled spin networks as compared to 2D NMR. Homonuclear 3D NMR experiments have been reported which emp loy tailored soft pulses (I, 2) to excite only parts of the proton spectral window or which use the narrow frequency band of J couplings for the third dimension (3) in order to reduce the experimental accumulation time. In this communication, we propose heteronuclear 3D NMR spectroscopy to aid in the simplification of two-dimensional proton NMR spectra of isotopically labeled compounds. For proteins, labels can easily be introduced by adding isotopically labeled nutrients to the growth med ium of bacterial expression systems (e.g., Ref. (4)). The experiments described comprise a combination of heteronuclear mu ltiple-quantum correlation (HMQC) (5, 6) with homonuclear COSY and NOESY and may therefore be named HMQC-COSY and HMQC-NOESY. Heteronuclear 3D experiments hold considerable promise for application to larger systems because one of the coherence transfer steps involves scalar couplings much larger than the ‘H linewidths. F igure 1 shows the pulse sequences of the HMQC-COSY and HMQC-NOESY experiments. Analogous to the HMQC experiment, heteronuclear mu ltiple-quantum coherence created by the first two RF pulses evolves during tI effectively with the single-quantum heteronuclear chemical shift. This coherence is converted to proton single-quantum coherence ant iphase with respect to the heteronucleus by the second hetero pulse, which is refocused at the end of the second 7 period. On ly those protons which are coupled to the heteronucleus are frequency labeled during tl . Therefore, the further frequency labeling during t2 under the homonuclear Hamiltonian needs to cover only the heteronuclear coupled protons, thus allowing nonselective RF pulses to be used throughout the sequence. Protons not coupled to the heteronuclei, which may be considered axial peaks in ol, are suppressed by an inversion of the first hetero pulse concomitant with an inversion of the receiver phase (6). Depending on the experiment, the signals arising from heteronuclear coupled protons are converted to COSY (Fg 1A) or NOESY (Fii 1B) type responses. In order to enhance sen-

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° 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.

Chemical Manipulation of Hsp70 ATPase Activity Regulates Tau Stability

Alzheimers disease and other tauopathies have recently been clustered with a group of nervous system disorders termed protein misfolding diseases. The common element established between these disorders is their requirement for processing by the chaperone complex. It is now clear that the individual components of the chaperone system, such as Hsp70 and Hsp90, exist in an intricate signaling network that exerts pleiotropic effects on a host of substrates. Therefore, we have endeavored to identify new compounds that can specifically regulate individual components of the chaperone family. Here, we hypothesized that chemical manipulation of Hsp70 ATPase activity, a target that has not previously been pursued, could illuminate a new pathway toward chaperone-based therapies. Using a newly developed high-throughput screening system, we identified inhibitors and activators of Hsp70 enzymatic activity. Inhibitors led to rapid proteasome-dependent tau degradation in a cell-based model. Conversely, Hsp70 activators preserved tau levels in the same system. Hsp70 inhibition did not result in general protein degradation, nor did it induce a heat shock response. We also found that inhibiting Hsp70 ATPase activity after increasing its expression levels facilitated tau degradation at lower doses, suggesting that we can combine genetic and pharmacologic manipulation of Hsp70 to control the fate of bound substrates. Disease relevance of this strategy was further established when tau levels were rapidly and substantially reduced in brain tissue from tau transgenic mice. These findings reveal an entirely novel path toward therapeutic intervention of tauopathies by inhibition of the previously untargeted ATPase activity of Hsp70.

Structural insights into substrate binding by the molecular chaperone DnaK

How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire α-helical domain, DnaK(1–507), retains the ability to support λ phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393–507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand β3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the β-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the β-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.

Heteronuclear three-dimensional NMR spectroscopy of isotopically labelled biological macromolecules

Due to the development of two-dimensional Fourier transformation techniques (for reviews see Bax, 1982; Ernst et al. 1987), NMR spectroscopy has become a powerful tool for determining the 3D structures of small proteins (MW ≤ 10 kDa); for reviews see Wuthrich, 1986; Clore & Gronenborn, 1987. For larger molecules, however, the amount of detailed structural information that can be obtained using homonuclear 2D NMR techniques is limited because of the vast number of overlapping signals. In order to extend the capabilities of NMR to the study of larger systems, new approaches are required.

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 Alzheimers and cancer. Recent approaches to combat these diseases through interference with the Hsp70 allosteric mechanism are discussed.

Three-dimensional 13C-resolved proton NOE spectroscopy of uniformly 13C-labeled proteins for the NMR assignment and structure determination of larger molecules

The currently accepted protocol for the determination of the solution structures of proteins by NMR consists of the assignment of the ‘H resonances, the identification of NOE distance constraints on the basis of the assignments, and the computation of the three-dimensional structure using the NMR-derived distance constraints (for a review, see Ref. (I)). Recently, by the use of isotope-edited two-dimensional NMR experiments on selectively labeled proteins (2-6)) or heteronuclear three-dimensional NMR experiments ( 7) on proteins that have been uniformly labeled with i5N (8-I]), the complete assignments of the backbone protons for proteins up to MW 20,000 have become possible. However, in order to determine the three-dimensional structures of these larger proteins, distance constraints derived from long-range NOES between (assigned) aliphatic protons are necessary. These NOES are very difficult if not impossible to obtain from conventional 2D NOESY spectra due to extensive overlap in the aliphatic region of the spectra. Thus, although the (backbone) proton NMR signals may be assigned for larger systems such as T4 lysozyme and staphylococcal nuclease (5, 6)) the 3D structures cannot be determined. Here, we describe an approach to resolve the spectral overlap in the aliphatic part of the NOESY data. The method involves the use of 13C heteronuclear three-dimensional NMR spectroscopy in which the NOESY spectra are simplified by editing with respect to the 13C frequencies. In this study, a sample of 3.8 mM T4 lysozyme ( 164 amino acid residues, 18.7 kDa) that was uniformly enriched with both 13C and 15N at a level greater than 95% was employed. The pulse sequence used was

Functional dynamics in the active site of the ribonuclease binase

Binase, a member of a family of microbial guanyl-specific ribonucleases, catalyzes the endonucleotic cleavage of single-stranded RNA. It shares 82% amino acid identity with the well-studied protein barnase. We used NMR spectroscopy to study the millisecond dynamics of this small enzyme, using several methods including the measurement of residual dipolar couplings in solution. Our data show that the active site of binase is flanked by loops that are flexible at the 300-μs time scale. One of the catalytic residues, His-101, is located on such a flexible loop. In contrast, the other catalytic residue, Glu-72, is located on a β-sheet, and is static. The residues Phe-55, part of the guanine base recognition site, and Tyr-102, stabilizing the base, are the most dynamic. Our findings suggest that binase possesses an active site that has a well-defined bottom, but which has sides that are flexible to facilitate substrate access/egress, and to deliver one of the catalytic residues. The motion in these loops does not change on complexation with the inhibitor d(CGAG) and compares well with the maximum kcat (1,500 s−1) of these ribonucleases. This observation indicates that the NMR-measured loop motions reflect the opening necessary for product release, which is apparently rate limiting for the overall turnover.

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.

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].