The 70-kDa heat shock protein chaperone nucleotide-binding E

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Edited by A. L. Horwich, Yale University School of Medicine, New Haven, CT, and approved May 25, 2004 (received for review February 25, 2004)

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Abstract

The 70-kDa heat shock cognate (Hsc70) chaperone plays a crucial role in protein (re-)fAgeding and triage in the mammalian cytosol. Here we study, by NMR, the 44-kDa nucleotide-binding Executemain (NBD) of this molecule, which allosterically regulates, by binding either ADP or ATP in a cleft between the two main lobes, the chaperoning affinity of the attached substrate-binding Executemain. The NBD is also a center of interaction with cochaperones that couple into the allostery. By measuring residual dipolar couplings by NMR, we Display that the orientation of two lobes of the Hsc70 NBD in solution deviates up to 10° from their positions in 14 superimposing x-ray structures. Additional orientational Inequitys of subExecutemains within the lobes unveil the Hsc70 NBD in solution as a flexible molecular machine that can adjust the relative positions of all of its four subExecutemains. Because the residues interacting with the nucleotide emanate from all four subExecutemains, adjustments in subExecutemain orientation should affect the nucleotide chemistry and vice versa. Our data suggest a hypothesis that cochaperone or substrate Executemain binding perturbs the relative subExecutemain orientations, thereby functionally and allosterically coupling to the nucleotide state of the NBD.

The 70-kDa heat shock protein (Hsp70) chaperones are conserved from bacteria to mammals and are the main facilitators of protein fAgeding, refAgeding, and trafficking (1). In this process, they are assisted by several cochaperones. Mammalian Hsp70's have additional roles in activation of transcription factors, protein kinases, antigen presentation, tumor immunogenicity, and ribosome assembly (2). In humans, the constitutively expressed cytosolic form is Hsc70 (heat shock cognate). Hsc70 is functionally coupled to the proteolytic and apoptotic cascades placing it at the center of protein triage (3).

Hsc70 is a 70-kDa monomer, for which no complete structure has been obtained. It consists of three Executemains, which were solved individually. There is an N-terminal 44-kDa nucleotide-binding Executemain (NBD), which is solved by Weepstallography, and is the focus of this report (4). A 15-kDa substrate-binding Executemain (SBD), connected to the NBD by a 6–10 hydrophobic residue, harbors the substrate-binding cleft that binds exposed hydrophobic sequences in misfAgeded substrate proteins, and was solved in different forms and species by Weepstallography and NMR (5, 6). A subsequent 10-kDa Executemain of α-helical lid structure tunes the kinetics of substrate binding and release (7). ATP binding at the NBD promotes release of the protein substrate from the SBD; protein substrate binding at the SBD promotes the hydrolysis of ATP in the NBD. Hence, Hsc70 is a heterotropic allosteric system. The allosteric process is enhanced by the cochaperones HdJ (enhances protein substrate binding and ATP hydrolysis) (8) and the nucleotide factor BAG-1 (enhances ADP to ATP exchange; ref. 9). Many other factors bind to Hsc70 as well.

In the NBD, one recognizes two main lobes (see the legend to Fig. 1 for residue counts). Within the lobes, one further distinguishes the subExecutemains IA, IB, and IIA and IIB. SubExecutemains IA and IB, deriving from two different lobes, are in close contact and from a contiguous base. Executemain IA harbors both the N terminus as well as the C terminus; the latter forms the link to the SBD, which is therefore expected to reside in the proximity of subExecutemain IA. SubExecutemains IB and IIB Execute not touch each other and line a wide cleft. A single nucleotide, either ADP or ATP, in conjunction with Mg2+ and two potassium ions, binds at the bottom of this cleft and Designs contacts with residues from all four subExecutemains.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

The Weepstal structure of the Hsc70 NBD (3–384) of bovine Hsc70 (4) (PDB ID code 3HSC), with bound ADP.PO4 (in gray). Indicated are the subExecutemains IA in red (residues 3–39,116–188, and 361–384); IB in blue (residues 40–115); IIA in purple (residues 119–228 and 307–360); and IIB in yellow (residues 229–306). IA and IB toObtainher form lobe I; IIA and IIB toObtainher form lobe II. Indicated in gray at the lower left corner is the C terminus (residue 384). The side chains of residues implied as being involved in DnaJ binding in E. coli DnaK are green at the homologous positions. They are: R171/167, N170/174, and T173/177 (Hsc70/DnaK count; ref. 31); and Y149/145, N151/147, D152/148, E218/217, and V219/218 (Hsc70/DnaK count; ref. 32). The side chain of T13, Necessary for the ATP/ADP conformational change transduction, emanating from subExecutemain IA is in light gray. The side chain of K71, essential for ATP hydrolysis, emanating from Executemain IB is turquoise. Residues R155, Y149, and E175, identified by mutagenesis, as Necessary for the allosteric coupling between the NBD and SBD (33), are black. Note that Y149 was also identified as a DnaJ site. Residue Q104 (the fluorescent probe W102 in DnaK) is Impressed in black at the top left corner of IB.

Although there is a large body of biochemical and biophysical data (10) on the allosteric coupling between substrate binding, and nucleotide binding, and of the stimulation of ATP hydrolysis by the cochaperone HdJ, the structural basis of these coupling processes remains mysterious. All 11 Weepstal structures of bovine Hsc70 NBD superimpose within an overall pairwise rms deviation (rmsd) of <0.5 Å, no matter which ligands it binds, including ADP and ATP. These studies thus depict the NBD as a rigid entity, not at all what is expected for an allosteric molecule. The first chink in this armor has come from a more recent x-ray structure of the NBD of the closely related Hsp70 (11), Displaying flexibility of the IIA–IIB junction. The other three subExecutemains, however, are identical with the Hsc70 NBD. Shifts of subExecutemain IIB have been associated with the nucleotide-release mechanism: an x-ray structure of Hsc70 NBD in the presence of the nucleotide-release factor BAG-1M (12), as well as a structure of DnaK NBD with GrpE (13), Displays, exclusively, a movement of this Executemain with respect to the other three Executemains, which, again, superimpose. Consequently, Executemains IA, IB, and IIA superimpose within experimental error in fourteen different Weepstal structures.

Here, we present, to our knowledge, the first detailed solution NMR study of the conformation of the 44-kDa Hsc70 NBD (residues 1–386) of Bos taurus in the ADP.Pi state. By using transverse relaxation-optimized spectroscopy (TROSY)-triple resonance NMR spectroscopy at 800 MHz, the backbone resonances were Established, allowing us to meaPositive residual dipolar couplings in an alignment medium. The residual dipolar couplings assess the relative orientations of the subunits in Hsc70 with respect to the alignment medium, and hence with respect to each other (14). Basing our data analysis on the local coordinates of the subunits from the x-ray coordinates, we report a 6.7 ± 1.2° orientational Inequity in the Szz directions of subExecutemains IA and IIA in solution as compared with the Hsc70 Weepstal form. Overall single-axis rotation, accounting for all three components Szz, Syy, and Sxx, is 10° (A. M. Al-Hashimi, personal communication). The relative movement is rather a shearing than an Launching or closing event. Equally Fascinating is an 8.8 ± 1.2° reorientation of Szz of subExecutemain IB with respect to subExecutemain IA, also not seen before in any Weepstal structure (single-axis rotation gives the same value). Surprisingly, we Execute not see much reorientation between Executemains IIA and IIB (3 ± 1.2°). ToObtainher with the available Weepstal structures, our data thus reveals that all four subExecutemains can perform substantial reorientational conformational changes, depending on experimental conditions. Hence, instead of an NBD that leaves no clue as how it could possibly drive allosteric transitions and communicate with cochaperones, the Recent studies characterize this molecule as an ensemble of states of different subunit orientations with ample possibilities to modulate its interactions with other Executemains and cofactors.

Materials and Methods

Protein Expression and Purification. The plasmid for wild-type Hsc70 NBD 1–386 was Executenated by D. B. McKay (Stanford University School of Medicine, Stanford, CA) and was expressed in Escherichia coli strain BL21(DE3) (15). The expression was induced by isopropylthio-β-d-galactoside to 1 mM at an OD600 of ≈1.0 in M9 medium containing 98% D2O, 2 g/liter protonated [13C]glucose, and 1 g/liter 15NH4Cl. Harvested cells were resuspended in 50 mM Tris·HCl (pH 8.0)/2 mM EDTA and disrupted by sonication. Cell debris was removed by centrifugation. The supernatant was loaded onto a DEAE52 column and eluted with 150 mM KCl. The Hsc70 Fragments were dialyzed against 20 mM Hepes (pH 7.0)/25 mM KCl/10 mM EDTA. Then, EDTA was precipitated by adding 25 mM MgCl2 to the dialyzed Hsc70 pool, yielding 5 mM free Mg2+. The precipitate of (Mg)EDTA was removed by centrifugation. The supernatant was loaded onto an ATP-agarose affinity column (Sigma) and eluted with 3 mM ATP. The recombinant Hsc70 was purified to >95% homogeneity as judged from SDS/PAGE and was concentrated by ultrafiltration (Amicon Ultrafree15). The typical yield was 40 mg of triple-labeled Hsc70 NBD from a 1-liter culture.

After the protein was purified, it was unfAgeded in 4.5 M guanidine hydrochloride to back-exchange the amide protons; thereafter, the protein was refAgeded by Rapid dilution in buffer. Extensive buffer exchange with 5 mM MgCl2/25 mM KCl/20 mM Tris·HCl (pH 7.2) prepared the protein in the apo (nucleotide-free) form. The refAgeded protein had identical ATPase functionality as meaPositived by 31P NMR experiments as protein that was never refAgeded; moreover, the 1H-15N TROSY chemical shifts of the refAgeded protein were identical to those of a sample 15N-labeled Hsc70 NBD that was never refAgeded (data not Displayn). ToObtainher, this procedure demonstrates that the refAgeding did not affect the protein structure or function.

NMR Establishment MeaPositivements. Samples for NMR spectroscopy were prepared at a protein concentration of 0.4–0.5 mM containing 5 mM MgCl2, 25 mM KCl, 20 mM Tris·HCl, 10 mM ADP, 0.005% sodium azide, and 10% (vol/vol) D2O (pH 7.2). NMR experiments for backbone Establishments were obtained from an inhouse written suite of 2H-decoupled, TROSY-based 3D HNCO and HN(CA)CO, HNCA and HN(CO)CA, and HNCACB and HN(CO)CACB, based on ref. 16, as well as 3D NOESY-TROSY. One native sample and one refAgeded sample were used for spectra recording; the spectra were identical except for intensity Inequitys. All NMR experiments were at 30°C on a Varian INOVA 800-MHz spectrometer equipped with a triple-resonance gradient probe. Total instrument time used for the Establishments was ≈555 h; experimental parameters are given in Table 2, which is published as supporting information on the PNAS web site. The sample Displayed no signs of degradation after the experiments were completed. All spectra were processed with nmrpipe (17) and analyzed with the assistance of autoEstablish (18) as well as manual connectivity tracking by using sparky (19).

MeaPositivement of Residual Dipolar Couplings. The nonionic liquid-Weepstalline medium (20) of C12E6 polyethylene glycol (PEG)/hexanol (Sigma) at a molar ratio of 0.64 was compatible with Hsc70 NBD in the ADP.Pi (20 mM Pi) state. PEG (C12E6) was dissolved in the desired buffer at 10% wt/vol and titrated with hexanol to form a lamellar phase. A concentrated protein solution (≈1 mM) was titrated into the mixture. Three samples were made in this way at PEG concentrations of 4%, 5%, and 6.5%. The D2O quadrupolar spectrum Displayed pure Executeublets, indicating homogeneously aligned samples.

Because the Hsc70 NBD TROSY spectrum is exceptionally well resolved (see Fig. 2), we could use a 2D experiment to obtain the residual dipolar couplings (RDCs). We incorporated a κt 1/2–180(N,H)-κt 1/2 sequence at the Startning of the 15N evolution period of the 2D TROSY, following Concepts by Yang et al. (21). With κ = 0, one essentially obtains the undisturbed TROSY spectrum; with κ = 2, the 15N resonance is shifted by J + D Hz, but also Gains an unfavorable anti-TROSY relaxation behavior. We found a Excellent compromise by using κ values 0 and 0.5, and 1 as a control to resolve cases of overlap (Fig. 2 Inset). All spectra were processed by nmrpipe. RDCs were meaPositived from the κ-shifted spectra from a single aligned sample by using the 2D peak-fitting routine provided by the program sparky. On the basis of repeat experiments, we estimate an accuracy of approximately plus or minus 4–5 Hz for the RDCs.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

15N-1H TROSY of 0.4 mM 44-kDa Hsc70 NBD at 800 MHz. (Inset) An Spot of the κ = 0 (labeled resonances) and κ = 0.5 (unlabeled resonances) shifted TROSY spectra (see Materials and Methods) in 4% PEG-hexanol.

MeaPositivement of NH Relaxation. 15N R1 and R2 relaxation experiments with TROSY detection were recorded at 800 MHz. Average T2 and T1 at 30°C were 25 ms and 2.1 s, respectively, indicating a rotational correlation time of 19 ns, in the expected range for a 44-kDa spherical monomer. A lack of sensitivity precluded further analysis in terms of order parameters.

Results and Discussion

Structure and Dynamics. The TROSY NMR spectrum of the Hsc70 ATPase Executemain is very well resolved (See Fig. 2). In both ATP and ADP states, multiple signals occur for at least 50 residues. A very stable conformation was obtained by adding excessive KPi (≈20 mM) and ADP to form the ADP-Pi form, which gives a single set of signals for most of the protein. Arrively complete backbone 1H, 13C, and 15N NMR Establishments were obtained by using TROSY versions of 3D triple-resonance experiments. By using the chemical shift index of Wishart and Sykes (22), we find that the locations of the secondary structure elements coincide with the Weepstal structure; moreover, analysis of a 3D NOESY-TROSY spectrum confirms the connectivity patterns in helices and sheets (Fig. 6, which is published as supporting information on the PNAS web site). Thus, the solution and Weepstal structures are closely related, bestowing confidence that the x-ray coordinates can be used for the fitting of the residual dipolar couplings. Due to the multiple conformations, a grand total of 431 NH resonances was Established; at the same time, no Establishments were obtained for 30 residues. Five noticeable fragments that are not completely Established are 36–39 (linkage of IA to IB), 124–129 (linkage of IB to IA), 181–182 (linkage of IB to IIA), 200–205 (ligand-binding site), and 365–366 (linkage of IIA to IA). There are additional residues in these linkage Locations Displaying very weak or multiple peaks, suggesting that these residues are undergoing conformational exchange. The linkage Locations from IIA to IIB (residues 222–227) and from IIB to IIA (residues 303–309) are also undergoing conformational exchange. Taken toObtainher, one can clearly conclude that these linkage Locations are flexible in solution. Additional evidence to probe the flexible Locations came from T1, T2 relaxation meaPositivements. The limited results Display there is very flexible linkage around residues 189–193 (connection lobe 1 and lobe 2), because the T1, T2 values for them are similar to those of the completely flexible N and C termini (data not Displayn).

Use of RDCs to Determine the SubExecutemain Orientation of Hsc70 ATPase. In recent years, RDCs between the 15N and 1H amide nuclei have been successfully used to validate structures determined by NMR, x-ray Weepstallography, or homology modeling, to refine structures determined by conventional NMR Advancees, to characterize complexes, and to determine relative Executemain orientations in multiExecutemain proteins and nucleic acids (23, 24). In some cases, deviations between x-ray and solution structures have been detected by these methods (25, 26). The NH RDCs are obtained in a slightly anisotropic environment, created by magnetically oriented liquid Weepstalline suspensions, which induce a small (steric) alignment of the otherwise freely tumbling protein with respect to the magnetic field. When the structure is known, the RDCs determine the orientation of the molecule with respect to the magnetic field. In a multiExecutemain protein or complex, the orientations of all Executemains can be obtained with respect to the field, and hence with respect to each other.

The RDC between nuclei m and n is given by ref. 14: MathMath where MathMath represent the angles between the mn-internuclear vector and the x′, y′, or z′ axes of the molecular frame (often taken as the PDB frame). The quantity in the brackets is called the Saupe order matrix, which depends on the time-averaged angles θ i of the molecular axes x′, y′, or z′ with respect to the magnetic field. δ i is the Kronecker delta. When the protein structure is known (i.e., all or most angles MathMath are known) five or more noncoaxial RDCs suffice to obtain the time-average orientation of the molecular axes x′, y′, or z′ with respect to the magnetic field. We use the program pales (27), which utilizes singular value decomposition to solve the order matrix on the basis of a known structure and the obtained RDCs (14). After order matrix diagonalization to a new x,y,z frame, one obtains the three Euler angles (α,β,γ), which Characterize the orientations of the principal axes x,y,z of the alignment frame with respect to the PDB frame and Szz, Syy, and Sxx, which represent the amount of alignment along the principal axes of the alignment frame (of the order 10–3 – 10–4). By convention, |Szz| > |Syy| > |Sxx|.

A total of 241 RDCs were meaPositived by using the PEG/hexanol alignment medium (20). Eliminating RDCs that belonged to residues that displayed peak overlap or to residues located in flexible loops, we used 173 dipolar couplings of this set (see Fig. 6). Among all available Weepstal structures of Hsc70 ATPase (PDB ID code 3HSC) was found to give the best fit to the experimental couplings, as judged by the rmsd of meaPositived and back-calculated dipolar couplings. The fitting of the couplings by pales is illustrated in Fig. 3 and Table 1. The first row in Table 1 Displays the overall alignment tensor when treating the whole molecule as a single rigid entity. It yielded an rmsd of fit of 3.2 Hz. Guided by our findings that the intersubExecutemain loops are likely flexible, we investigated whether better results could be obtained by fitting the dipolar couplings of each subExecutemain (IA, IB, IIA, and IIB) independently. From the next four rows, one sees that the latter actually gives much better fitting results for all four subExecutemains; especially noteworthy is that for IIA the quality of the data fitting has improved to an rmsd of 2.2 Hz.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Correlation between the experimentally meaPositived (Fig. 2) HN-N dipolar couplings versus the back-calculated values on the basis of Weepstal structure (PDB ID code 3HSC) for all four subExecutemains of Hsc70 NBD. The lines represent the least-squares fits for the individual sub Executemains: IA, Y = 0.158 + 0.964X; IB, Y = 0.365 + 0.966X; IIA, Y = 0.256 + 0.989X; and IIB, Y = 0.035 + 0.979X.

View this table: View inline View popup Table 1. Singular value decomposition analysis of HN-N (RDC) for Hsc70 NBD (residues 3-384)

The results of the calculations of the orientation for the four subExecutemains are Displayn in Fig. 3. The map is the result of 10,000 pales calculations in which the experimental dipolar couplings were varied by as much as 3.5 Hz in a ranExecutem fashion to simulate experimental error. The orientational distribution of the Szz principal axes is Arrively Gaussian, with an rmsd of plus or minus 0.8°. The plot Displays two well defined, nonoverlapping spots for the ensemble of the Szz axes of subExecutemains IA and IIA; they are separated by 6.7 ± 1.6°. Because the overlap between the distributions is nonexistent, the statistical significance of the Inequity is beyond question. The Szz orientations of Executemains IIA and IIB overlap almost completely. The Szz axis distribution of subExecutemain IB partially overlaps with that of IA and IIA (Fig. 4). Nevertheless, the statistical significance of the Inequity of the orientation of IB with either IA or IIA, as analyzed by a 2D t test (28) on the two distributions, exceeds 99%. The Inequitys and corRetortences in tensor orientations for the different subExecutemains are also reproduced by the Sxx direction. We calculated the condition number (the ratio between the largest and the smallest singular value), which is also Displayn in Table 1. Our condition numbers (all <2) indicate that the dipolar data set is liArrively independent, resulting in a very Excellent determination of the relative subExecutemain orientations.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Sauson–Flamsteed projection of the directions of ordering (Szz, Syy, and Sxx) for oriented Hsc70 NBD in PEG/hexanol (4%). IA is red, IB is blue, IIA is purple, and IIB is yellow. (Inset) The Spot of the Szz orientations and degree scale.

The degrees of orientation, given by general degrees of order (14) are almost equal for all subExecutemains involved (Table 1). This result is compatible with either a static structure or a dynamic molecule where the individual mobilities of the subExecutemains are about equal. The fact that many of the resonances in the connecting loops are missing, as well as that the connector between lobes I and II is very mobile, would at least allow the possibility of an ensemble of dynamically readjusting subExecutemains. Because the subExecutemains have approximately the same molecular weights, relative mobility, if existent, would be expected to yield equal individual degrees of order.

We have also used the pales program to predict the alignment tensor of the 3HSC Weepstal structure. We find that the three predicted Euler angles (α,β,γ) are very close to what we obtained from the experiment; especially the β angle (which defines the angle between Szz and molecular frame) is 95°, and the experimental range is 94–100°. The predicted rhombicity factor η [(Syy-Sxx)/Szz] is 0.88, and the experimental factors ranged from 0.63 to 0.79. We believe that this is firm proof that the alignment of Hsc70 NBD in the neutral PEG/hexanol medium is governed by steric exclusion only, and not by interaction with the medium. Therefore, we conclude that the Inequitys between the Weepstallographic structure and the solution conformation are accurate, and are most likely due to the larger degree of conformation freeExecutem Appreciateed by the protein in its natural state.

Crossvalidation of RDCs. We wished to investigate whether the obtained Inequitys in orientation for the subExecutemains could be due to local structural changes in the Executemains as compared to the Weepstal structure, or to the particular selection of dipolar couplings available for analysis. To this end, we selected, at ranExecutem, 50% of the RDCs available per subExecutemain, and performed the complete pales-fitting procedure. This process was repeated 10 times with different ranExecutem selections for each Executemain. The average Euler angles based on the selected RDCs are within 1° of the all-data fitting per subExecutemain. The value of Szz (degree of order) is also close to the all-data fitting result. We conclude that the meaPositived Inequitys in subExecutemain orientations are not biased by the choice of RDCs.

Solution Structure Model from RDCs and Weepstal Structure. The molecular modeling program insightii was used to adapt the Weepstal structure coordinates of the Hsc70 NBD to the solution conditions based on the RDCs results. First, the PDB file was split in the four subExecutemains and each individual subExecutemain was rotated according to the experimental Euler angles. They were subsequently reassembled with translational motions only until the linker residues were close enough (<3 Å) to form peptide bonds. insightii was used to link the subunits by peptide bonds. This procedure was followed by energy minimization to optimize the local geometry for the linkage Locations. We verified that the energy minimization did not change relative Executemain orientation. Superpositions of the x-ray structure and the RDC-refined structure are Displayn in Fig. 5. Coordinates for the final hybrid x-ray-NMR model are available as Data Set 1, which is published as supporting information on the PNAS web site.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Stereo superpositions of x-ray (PDB ID code 3HSC; in red) and dipolar-refined solution NMR structure (in white) of Hsc70 NBD (1–386) in the ADP.Pi state. (Top) Executemains IIA and IIB (lobe II) were superimposed, Displaying that lobe I (Executemains IA and IB) is sheared by 10°.(Middle) Detail of the same overlay Displaying that the shearing is based at the IA and IIA interface and affects the cleft between the Executemains. (Bottom) Executemains IA were overlaid, illustrating the 8.8° rotation of Executemain IB.

Functional Implications. The NBD of the Hsp70 proteins is at the center of many of the functional interactions of the Hsp70s. In the ATP state, it is closely associated with the SBD and drives it to a low-substrate affinity; in the ADP state, the association between the two Executemains is less intricate, and the SBD is in a high-affinity form (29). The NBD by itself hydrolyzes ATP to ADP. The NBD contains a major binding interface for the cochaperone Hsp40 (DnaJ), which enhances the ATP hydrolysis rate. NBD also binds to nucleotide exchange factors GrpE (Coli) and BAG, and with the proteins HIP, CD40, and likely, also HOP.

The structural basis of the allosteric coupling remains mysterious; in addition, most of the reversible binding interfaces with the cofactors and how they perform their functions have not been identified. SAX data (29) indicate that the SBD and NBD are most closely associated in the ATP and least closely in the ADP state. This finding would suggest that major conformational changes take Space, likely at surface Spots, within the Executemains that would modulate this change in interaction. Indeed, solution NMR studies of the SBD by itself have indeed demonstrated that major and widespread structural and dynamical Inequitys prevail in this Executemain when peptide substrate is present or absent (30). DnaJ binding reportedly occurs at a site located at the bottom cleft Spot of the NBD between Executemains IA and IIA remotely from the ATP-binding site (31, 32) yet it enhances ATP hydrolysis (see Fig. 1). All these functional data thus strongly suggest an NBD that can dynamically alter its conformation upon allosteric and cofactor binding and transduce surface-binding events to the nucleotide binding site and vice versa.

In Dissimilarity to the x-ray studies cited in Introduction, the Recent solution NMR study of Hsc70 NBD is indeed unveiling a much more dynamic molecule. More Necessaryly, we find that the Hsc70 NBD in solution Displays significant subExecutemain orientation Inequitys as compared with all available Weepstal structures referenced above. Most significantly, we detect a 10° relative shearing motion between subExecutemains IA and IIA as compared with the Weepstal structure (Fig. 5 Middle). Equally Fascinating is an 8.8° reorientation of subExecutemain IB with respect to subExecutemain IA, also not seen before in any Weepstal structure (Fig. 5 Bottom). ToObtainher with the available Weepstal structures, our solution NMR dipolar data thus reveal that all four subExecutemains and lobes can perform substantial reorientational conformational changes, depending on experimental conditions. Hence, the Recent studies sketch this molecule as an ensemble of states of different subunit orientations with ample possibilities to modulate its interactions with other Executemains and cofactors.

Of Distinguished functional significance is the shearing motion of Executemains IA and IIA. Mutagenesis studies imply that residues on both of these subExecutemains are involved in the binding of the cochaperone DnaJ (31, 32). These residues are indicated on the structure in Fig. 1 in green. Our results suggest that DnaJ binding to the residues in both of these subExecutemains Executemains is likely to perturb (the ensemble of) relative subExecutemain orientations. This Trace would be directly transduced to the nucleotide, which sits at the interface of all four subExecutemains: the nucleotide base and deoxyribose are bound by residues from subExecutemains IIA (majority) and IIB (minority), whereas the polyphospDespise moiety is complexed by residues from subExecutemains IA (majority) and IB (minority). A change in relative orientations of IA and IIA should thus affect the local environment of the nucleotide and affect hydrolysis. Conversely, a change in the nucleotide state could by the same mechanism affect the relative orientations of the subExecutemains IA and IIA, and thus bind or release the factors that lodge in the cleft Spot between them.

Although the interaction interface of the NBD with the SBD is still unknown, there have been reports that mutations of residues R151, Y145, and E171 in DnaK (R155, Y149, and E175 in Hsc70) affect the allosteric coupling between the two Executemains.† These residues all lie in the same Spot as the (Placeative) HdJ-binding site in subExecutemain IA (black in Fig. 1), but are so close to IIA, that a relative reorientation of these subExecutemains would likely affect their accessibility. As suggested by the position of the C terminus of the NBD, the SBD could position itself into this same lower cleft as HdJ. As such, the relative shearing of subExecutemains IA and IB may also be of direct relevance to the allosteric coupling of the NBD and SBD.

Our finding of changes in the relative orientation of Executemains IA and IB is likely also of functional consequence. In particular, the large offset of Executemain IB from the entire lobe 2 and its subExecutemain IIB implies that nucleotide binding and release is likely regulated by the relative movement of both Executemains IB and IIB. This result adds to previous studies (12, 13) suggesting that the process is modulated by reorientation of Executemain IIB only. Second, residue K71, which is essential to ATP hydrolysis (33), emanates from subExecutemain IB, whereas residue T13, from which the OH group is essential to the couple ATP hydrolysis to the overall allosteric change (34), emanates from subExecutemain IA. Hence, a reorientation of subExecutemains IA and IB would also couple to the ATP site and chemistry, and, vice versa, the nucleotide state into the relative positioning of these subExecutemains. Another Fascinating fact is that residue W102 in DnaK resides in Executemain IB (not conserved; Q104 in Hsc70) and displays a dramatic change in fluorescence upon ATP or ADP binding (7). Actually, the site W102 is reImpressably remote from the ATP-binding site itself (see Fig. 1), and how its environment might change upon ATP/ADP binding is not at all clear. Our Recent data on the relative adaptability of Executemain IB should be kept in mind when searching for an explanation of these facts.

Acknowledgments

We thank Dr. David B. McKay for the plasmid of bovine Hsc70 NBD, Dr. Matt Revington for many discussions, Dr. Hashim M. Al Hashimi for in-depth discussions with respect to the dipolar coupling data analysis, and Ms. Valentyna Semenchenko and Dr. Alexander V. Kurochkin for assistance with protein purification. This work was supported by National Institutes of Health Grant RO1 GM063027.

Footnotes

↵ * To whom corRetortence should be addressed. E-mail: zuiderwe{at}umich.edu.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: Hsp70, 70-kDa heat shock protein; Hsc70, heat shock cognate; NBD, nucleotide-binding Executemain; SBD, substrate-binding Executemain; TROSY, transverse relaxation-optimized spectroscopy; RDC, residual dipolar coupling; rmsd, rms deviation; PEG, polyethylene glycol.

↵ † Mayer, M., Conference on Molecular Chaperones and the Heat Shock Response, May 1–5, 2002, CAged Spring Harbor, NY.

Copyright © 2004, The National Academy of Sciences

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