High-resolution solution structure of the 18 kDa substrate-binding domain of the mammalian chaperone protein Hsc70.

The three-dimensional structure for the substrate-binding domain of the mammalian chaperone protein Hsc70 of the 70 kDa heat shock class (HSP70) is presented. This domain includes residues 383-540 (18 kDa) and is necessary for the binding of the chaperone with substrate proteins and peptides. The high-resolution NMR solution structure is based on 4150 experimental distance constraints leading to an average root-mean-square precision of 0.38 A for the backbone atoms and 0.76 A for all atoms in the beta-sandwich sub-domain. The protein is observed to bind residue Leu539 in its hydrophobic substrate-binding groove by intramolecular interaction. The position of a helical latch differs dramatically from what is observed in the crystal and solution structures of the homologous prokaryotic chaperone DnaK. In the Hsc70 structure, the helix lies in a hydrophobic groove and is anchored by a buried salt-bridge. Residues involved in this salt-bridge appear to be important for the allosteric functioning of the protein. A mechanism for interdomain allosteric modulation of substrate-binding is proposed. It involves large-scale movements of the helical domain, redefining the location of the hinge area that enables such motions.

A protein structure from nuclear magnetic resonance data. lac repressor headpiece.

A procedure is described to determine the three-dimensional structure of biomolecules from nuclear magnetic resonance data. This procedure combines model building with a restrained molecular dynamics algorithm, in which distance information from nuclear Overhauser effects is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or headpiece (amino acid residues 1 to 51) of the lac repressor from Escherichia coli, for which no crystal structure is available. The relative orientation of the three helices of the headpiece is similar to that of the three homologous helices found in the cI repressor of bacteriophage lambda.

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor.

Stromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium-resolution structure of a 20-kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed beta-sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine-containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166-residue protein fragment complexed with a 4-residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 A. The structure has good stereochemical properties, including its backbone torsion angles. The beta-sheet and alpha-helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X-ray crystallographic model of Lovejoy B et al., 1994b, Biochemistry 33:8207-8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 A when three loop segments are disregarded. The hydroxamate-substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal S'1 subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic S'2 surface subsite, and a third hydrophobic side chain (P'3) lies at the surface.

Spatial arrangement of the three alpha helices in the solution conformation of E. coli lac repressor DNA-binding domain.

The relative orientations of the 3 helices in the DNA-binding domain ('headpiece') of lac repressor have been determined using distance constraints obtained from 2-dimensional 1H nuclear Overhauser enhancement spectra. The relative orientations of its helices is similar to that of the central 3 helices in the DNA-binding domain of the lambda repressor of the bacteriophage lambda.

Determination of protein structures from nuclear magnetic resonance data using a restrained molecular dynamics approach: the lac repressor DNA binding domain.

A procedure is described to determine from NMR data the three-dimensional structure of biomolecules. This procedure combines model building with a restrained Molecular Dynamics algorithm, in which distance information from NOEs is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or "headpiece" (amino acids 1-51) of the lac repressor from E. coli, for which no crystal structure is available. The spatial structure of the headpiece is discussed in terms of known physical and biochemical data and of its DNA binding properties.

NMR methods for determining the structures of enzyme/inhibitor complexes as an aid in drug design.

Four approaches are described for providing detailed structural information on large enzyme/inhibitor complexes to aid in the design of improved enzyme inhibitors. In one approach, proton NMR spectra are simplified by isotope-editing procedures in which only those protons that are attached to isotopically labeled nuclei (e.g. 13C or 15N) and their scalar or dipolar coupled partners are observed. Using this strategy, the conformation of an inhibitor bound to porcine pepsin can be determined and structural information on the active site obtained. In another approach, two-dimensional nuclear Overhauser effect (2D NOE) difference spectra are obtained by subtracting NOE spectra of two enzyme/inhibitor complexes prepared with either a protonated or a deuterated inhibitor. Only NOEs arising from protons of the inhibitor substituted with deuterium appear in the 2D NOE difference spectra as illustrated for a pepsin/inhibitor complex. In a third strategy, deuterated enzymes are employed to eliminate the many proton NMR signals of the enzyme and allow the selective detection of the resonances corresponding to the bound ligand as demonstrated for CTP bound to CMP-3-deoxy D-manno-octulosonic acid (KDO) synthetase. Finally, a fourth approach is described using heteronuclear three-dimensional NMR spectroscopy in which homonuclear 2D NMR spectra are edited with respect to the heteronuclear chemical shifts. Using these methods the complete three-dimensional structures of large enzyme/inhibitor complexes can potentially be obtained. Examples of the spectral simplification that can be achieved using 3D NMR are given for 15N-labeled CMP-KDO synthetase complexed with an inhibitor and CTP.

High-resolution four-dimensional HMQC-NOESY-HSQC spectroscopy.

Practical optimization of the 4D [(1)H, (13)C, (13)C, (1)H] HMQC-NOESY-HSQC experiment in terms of distribution of resolution over the indirect dimensions is analyzed in detail. Recommendations for an optimal experiment are based on computer simulations assessing the effective resolution of the experiment, defined as the percentage of all possible NOE cross peaks that can be assigned unambiguously on the basis of the spectral data alone. Using actual (13)C-(1)H spectra of an 18-kDa chaperone protein, the analysis shows that experiments with the best effective resolution are also among the most sensitive ones. When combined with an efficient aliasing scheme that reduces indirect spectral space 124-fold, a 4D experiment that yields unambiguous assignments for 41% of all possible NOE cross peaks can be recorded in 28 h. A high-resolution experiment, which can be recorded in 8 days, yields 61% unambiguous assignments and can be analyzed more easily using standard NMR display software. The predictions are verified with experimental 4D spectra from which 1850 NOEs (914 long-range) were extracted for the 18-kDa chaperone protein.

An iterative fitting procedure for the determination of longitudinal NMR cross-correlation rates.

We present a method to measure (15)N-(1)H dipolar/(15)N CSA longitudinal cross-correlation rates in protonated proteins. The method depends on the measurement of four observables: the cumulative proton-proton cross relaxation rates, the (15)N R(1) relaxation rate, the multiexponential decay of 2N(Z)H(N)(Z) spin-order, and multiexponential buildup of 2N(Z)H(N)(Z) spin-order. The (15)N-(1)H dipolar/(15)N CSA longitudinal cross-correlation rate is extracted from these measurements by an iterative fitting procedure to the solution of differential equations describing the coupled relaxation dynamics of the z-magnetization of the (15)N nucleus, the two-spin-order 2N(Z)H(N)(Z), and a two-spin-order term 2N(Z)H(Q)(Z) describing the interaction with remote protons. The method is applied to the microbial ribonuclease binase. The method can also extract longitudinal cross-correlation rates for those amide protons that are involved in rapid solvent exchange. The experiment that serves for extracting proton-proton cross-relaxation rates is a modification of 3D (15)N-resolved NOESY-HSQC. The experiment restores the solvent magnetization to its equilibrium state during data detection for all phase cycling steps and all values of NOE mixing times and is recommended for use in standard applications as well.

Protein heteronuclear NMR assignments using mean-field simulated annealing.

A computational method for the assignment of the NMR spectra of larger (21 kDa) proteins using a set of six of the most sensitive heteronuclear multidimensional nuclear magnetic resonance experiments is described. Connectivity data obtained from HNC alpha, HN(CO)C alpha, HN(C alpha)H alpha, and H alpha (C alpha CO)NH and spin-system identification data obtained from CP-(H)CCH-TOCSY and CP-(H)C(C alpha CO)NH-TOCSY were used to perform sequence-specific assignments using a mean-field formalism and simulated annealing. This mean-field method reports the resonance assignments in a probabilistic fashion, displaying the certainty of assignments in an unambiguous and quantitative manner. This technique was applied to the NMR data of the 172-residue peptide-binding domain of the E. coli heat-shock protein, DnaK. The method is demonstrated to be robust to significant amounts of missing, spurious, noisy, extraneous, and erroneous data.

Magnetization transfer via residual dipolar couplings: application to proton-proton correlations in partially aligned proteins.

A novel three-dimensional NMR experiment is reported that allows the observation of correlations between amide and other protons via residual dipolar couplings in partially oriented proteins. The experiment is designed to permit quantitative measurement of the magnitude of proton-proton residual dipolar couplings in larger molecules and at higher degree of alignments. The observed couplings contain data valuable for protein resonance assignment, local protein structure refinement, and determination of low-resolution protein folds.

HCCH-TOCSY spectroscopy of 13C-labeled proteins in H2O using heteronuclear cross-polarization and pulsed-field gradients.

A pulsed-field gradient-enhanced, heteronuclear cross-polarization-driven, 3D HCCH-TOCSY experiment is described, which in a single scan can achieve nearly ideal solvent suppression for protein samples in H2O solution. The 3D experiment can be transformed without additional pre- or post-processing, thus leaving solute resonances at the solvent resonance position undisturbed and easily identifiable. As the gradients are used in combination with a 13C z-filter, only minimal relaxation losses are encountered as compared to non-gradient versions.

Heteronuclear three-dimensional NMR spectroscopy of the inflammatory protein C5a.

The utility of three-dimensional heteronuclear NMR spectroscopy for the assignment of 1H and 15N resonances of the inflammatory protein C5a (MW 8500), uniformly labeled with 15N, is demonstrated at a protein concentration of 0.7 mM. It is shown that dramatic simplification of the 2D nuclear Overhauser effect spectrum (NOESY) is obtained by editing with respect to the frequency of the 15N heteronucleus in a third dimension. The improved resolution in the 3D experiment largely facilitates the assignment of protein NMR spectra and allows for the determination of distance constraints from otherwise overlapping NOE cross peaks for purposes of 3D structure determination. The results show that 15N heteronuclear 3D NMR can facilitate the structure determination of small proteins and promises to be a useful tool for the study of larger systems that cannot be studied by conventional 2D NMR techniques.

Two-dimensional double quantum 1H NMR spectroscopy of proteins.

Two-dimensional double quantum 1H NMR spectra are recorded for a protein. The application of this technique to macromolecules is shown not to be impeded by the long preparation pulse sequence essential for this experiment. The identification of spin systems by analysis of the double quantum spectrum is illustrated. Since double quantum spectra do not contain diagonal peaks, connectivities between almost degenerate signals can be detected. By analysis of remote connectivities (i) it can be established whether amide and beta-protons belong to the same spin system or not, (ii) degeneracy of beta-proton chemical shifts can be demonstrated, and (iii) glycine amide protons can be distinguished from all others.

Solution structure of a DNA hairpin and its disulfide cross-linked analog.

The solution structures of a 21 base long DNA hairpin derived from the ColE1 cruciform, and an analog possessing a disulfide cross-link bridging the terminal bases, have been determined by NMR spectroscopy. The 8 bp long stem of these sequences adopts a B-form helix whereas the five base long single-stranded loop appears to be flexible and cannot be represented by a unique static conformation. NOESY cross-peak volumes, proton and phosphorus chemical shifts, and both homo- and heteronuclear coupling constants for the cross-linked hairpin are virtually identical to those measured for the unmodified sequence, even for the residues that are proximal to the cross-link. These results indicate that both hairpins are structurally isomorphous. Because this cross-link can be incorporated site specifically in a sequence independent manner, and does not appear to alter native conformation, it should prove broadly applicable in studies of DNA structure and function.

Study of protein dynamics in solution by measurement of (13)C (α)- (13)CO NOE and (13)CO longitudinal relaxation.

(13)C(α)-(13)CO homonuclear NOE and (13)CO T(1) relaxation were measured for a 20 kDa protein using tripleresonance pulse sequences. The experiments were sufficiently sensitive to obtain statistically significant differences in relaxation parameters over the molecule. The (13)C(α)-(13)CO cross-relaxation rate, obtained from these data, is directly proportional to an order parameter describing local motion and it is largely independent of the local correlation time. It is therefore a relatively straightforward observable for the identification of local dynamics.

Secondary structure of the lac repressor DNA-binding domain by two-dimensional 1H nuclear magnetic resonance in solution.

A recently proposed approach for spatial structure determination in noncrystalline proteins by nuclear magnetic resonance was applied to the lac repressor DNA-binding domain. On the basis of sequence-specific 1H NMR assignments, the location of alpha-helices in the amino acid sequence was determined from nuclear Overhauser enhancement data and from amide proton exchange studies. These investigations provide detailed experimental data on the structure of a noncrystalline DNA-binding protein. The results support the hypothesis advanced by others that sequence-specific interactions between lac repressor and DNA are mediated by a particular spatial arrangement of two alpha-helices common to various different DNA-binding proteins.

Sequence-specific resonance assignments in the 1H nuclear-magnetic-resonance spectrum of the lac repressor DNA-binding domain 1-51 from Escherichia coli by two-dimensional spectroscopy.

The assignment of the 1H nuclear magnetic resonance (NMR) spectrum of the DNA-binding domain 1-51 of lac repressor from Escherichia coli is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional NMR experiments at 360 MHz and 500 MHz. Individual assignments were obtained at 18 degrees C for the backbone protons of 44 out of the total of 51 amino acids residues, the exceptions being Met-1, Lys-2, Tyr-7, Arg-35, Glu-36, Lys-37 and Ile-48. Complete assignments of the non-labile hydrogen atoms of the side chain were obtained for 33 residues, and for Asn-46 and Asn-50 the delta amide protons were also identified. The chemical shifts for the assigned resonances at 18 degrees C are listed for an aqueous solution at pH 4.9 and at pH 6.8.

The peptide-binding domain of the chaperone protein Hsc70 has an unusual secondary structure topology.

Modern NMR methods were used to determine the secondary structure topology of the 18 kDa peptide binding domain of the chaperone protein Hsc70 in solution. This report constitutes the first experimental conformational information on this important domain of the class of Hsp70 proteins. The domain consists of two four-stranded antiparallel beta-sheets and a single alpha-helix. The topology does not resemble at all the topology observed in the human leukocyte antigen (HLA) proteins of the major histocompatibility complex. This is significant because such resemblance was predicted on the basis of limited amino acid homology, secondary structure prediction, and related function. Moreover, the exact meander-type beta-sheet topology identified in Hsc70 has to our best knowledge not been observed in any other known protein structure.

Comparison of model and nuclear magnetic resonance structures for the human inflammatory protein C5a.

The model structure previously proposed for human C5a, based upon the crystal structure of the homologous protein human C3a, is compared to the solution structure of human C5a recently determined by nuclear magnetic resonance (NMR) methods in our laboratory. The general folding and helix topography of the C5a protein were modeled very well. The N-terminus, which is disordered in the C3a crystal, was correctly predicted in the C5a model both as to its being a helix and as to its docking site on the rest of the molecule. On the other hand, the NMR data show that the biologically important C-terminal residues are disordered in solution, unlike the model and the C3a crystal structure where this region was helical.

Secondary structure of complement component C3a anaphylatoxin in solution as determined by NMR spectroscopy: differences between crystal and solution conformations.

Two-dimensional 1H NMR investigations were used to locate elements of regular secondary structure in the human complement protein C3a (the des-Arg77 derivative) in solution. The results were compared to a refined crystal structure based on the 3.2-A resolution structure of des-Arg77-C3a [Huber, R., Scholze, H., Paques, E. P. & Deisenhofer, J. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1389-1399]. In excellent agreement with the x-ray data, helices occur in the regions of residues 17-28 and 36-43 in solution. In contrast to the x-ray data, where a third long helix was found from residue 47 to residue 73, the solution data show a shorter helix in the region from residue 47 to residue 66, followed by a transition range at positions 67-70, leading into a six-residue carboxyl-terminal peptide in dynamic random coil conformation. At the amino terminus, a well-defined helix is observed in solution for the residues 8-15 region, which, like the carboxyl terminus, gradually changes to dynamic random coil toward the end of the polypeptide chain. This is at variance with the x-ray data as well, in which residues 13-15 are nonhelical and no electron density could be assigned to the first 12 residues due to disorder.