Four-dimensional heteronuclear triple-resonance NMR spectroscopy of interleukin-1 beta in solution.

A method is presented that dramatically improves the resolution of protein nuclear magnetic resonance (NMR) spectra by increasing their dimensionality to four. The power of this technique is demonstrated by the application of four-dimensional carbon-13--nitrogen-15 (13C-15N)--edited nuclear Overhauser effect (NOE) spectroscopy to interleukin-1 beta, a protein of 153 residues. The NOEs between NH and aliphatic protons are first spread out into a third dimension by the 15N chemical shift of the amide 15N atom and subsequently into a fourth dimension by the 13C chemical shift of the directly bonded 13C atoms. By this means ambiguities in the assignment of NOEs between NH and aliphatic protons that are still present in the three-dimensional 15N-edited NOE spectrum due to extensive chemical shift overlap and degeneracy of aliphatic resonances are completely removed. Consequently, many more approximate interproton distance restraints can be obtained from the NOE data than was heretofore possible, thereby expanding the horizons of three-dimensional structure determination by NMR to larger proteins.

Alpha helix-RNA major groove recognition in an HIV-1 rev peptide-RRE RNA complex.

The solution structure of a human immunodeficiency virus type-1 (HIV-1) Rev peptide bound to stem-loop IIB of the Rev response element (RRE) RNA was solved by nuclear magnetic resonance spectroscopy. The Rev peptide has an alpha-helical conformation and binds in the major groove of the RNA near a purine-rich internal loop. Several arginine side chains make base-specific contacts, and an asparagine residue contacts a G.A base pair. The phosphate backbone adjacent to a G.G base pair adopts an unusual structure that allows the peptide to access a widened major groove. The structure formed by the two purine-purine base pairs of the RRE creates a distinctive binding pocket that the peptide can use for specific recognition.

Novel mode of ligand binding by the SH2 domain of the human XLP disease gene product SAP/SH2D1A.

BACKGROUND: The Src homology 2 (SH2) domains of cytoplasmic signaling proteins generally bind phosphotyrosine (pTyr) sites in the context of carboxy-terminal residues. SAP (also known as SH2D1A or DSHP), the product of the gene that is mutated in human X-linked lymphoproliferative (XLP) disease, comprises almost exclusively a single SH2 domain, which may modulate T-cell signaling by engaging T-cell co-activators such as SLAM, thereby blocking binding of other signaling proteins that contain SH2 domains. The SAP-SLAM interaction can occur in a phosphorylation-independent manner. RESULTS: To characterize the interaction between SAP and SLAM, we synthesized peptides corresponding to the SAP-binding site at residue Y281 in SLAM. Both phosphorylated and non-phosphorylated versions of an 11-residue SLAM peptide bound SAP, with dissociation constants of 150 nM and 330 nM, respectively. SLAM phosphopeptides that were truncated either at the amino or carboxyl terminus bound with high affinity to SAP, suggesting that the SAP SH2 domain recognizes both amino-terminal and carboxy-terminal sequences relative to the pTyr residue. These results were confirmed by nuclear magnetic resonance (NMR) studies on (15)N- and (13)C-labeled SAP complexed with three SLAM peptides: an amino-terminally truncated phosphopeptide, a carboxy-terminally truncated phosphopeptide and a non-phosphorylated Tyr-containing full-length peptide. CONCLUSIONS: The SAP SH2 domain has a unique specificity. Not only does it bind peptides in a phosphorylation-independent manner, it also recognizes a pTyr residue either preceded by amino-terminal residues or followed by carboxy-terminal residues. We propose that the three 'prongs' of a peptide ligand (the amino and carboxyl termini and the pTyr) can engage the SAP SH2 domain, accounting for its unusual properties. These data point to the flexibility of modular protein-interaction domains.

Field gradient techniques in NMR spectroscopy.

In recent years NMR spectroscopy has emerged as a powerful technique for studying the structure and dynamics of biomolecules in solution. The development of shielded pulsed-field gradient coils for high-resolution NMR spectroscopy has led to significant improvements in a large number of experiments which are used to provide such information. Experiments enhanced with pulsed-field gradients have fewer artifacts, suffer far less from problems of solvent suppression and have reduced phase cycles relative to their non-gradient counterparts. The whole array of NMR experiments available for macromolecular structure determination is likely to benefit substantially from the incorporation of gradient pulses.

New developments in isotope labeling strategies for protein solution NMR spectroscopy.

The development of novel isotope labeling strategies for proteins has facilitated the study of the structure and dynamics of these molecules. In addition, the recent emergence of alternative methods of bacterial expression for obtaining isotopically labeled proteins permits the study of new classes of important proteins by solution NMR methods.

Solution NMR spectroscopy beyond 25 kDa.

Improvements in NMR instrumentation, higher magnetic field strengths, novel NMR experiments and new deuterium-labeling strategies have significantly increased the scope of structural problems that can now be addressed by solution NMR methods. To date, a number of structures of proteins of 30 kDa have been solved using multidimensional 15N,13C,2H NMR techniques, and this molecular weight limit will probably be surpassed in the near future.

Contributions to conformational entropy arising from bond vector fluctuations measured from NMR-derived order parameters: application to protein folding.

The relation between order parameters derived from NMR spin relaxation experiments and the contribution to conformational entropy from ns-ps timescale bond vector dynamics is investigated by considering a number of simple models describing bond vector motion. In a few cases both classical and quantum mechanical derivations are included to establish the validity of obtaining order parameter-entropy relations using classical mechanics only. For these cases it is found that classical and quantum mechanical derivations give very similar results so long as the square of the order parameter of the bond vector is less than approximately 0.95. For a given change in order parameter, the change in conformational entropy is sensitive to the model employed, with the absolute value of the entropy change increasing with the number of degrees of freedom in the model. The entropy-order parameter profile calculated from a 1.12 ns molecular dynamics trajectory of fully hydrated Escherichia coli ribonuclease HI is well fit using a simple expression based on a model assuming bond vector diffusion in a cone, suggesting that it may well be possible to extract meaningful entropy changes reflecting changes in ps-ns time scale motions from changes in NMR-derived order parameters. Contributions to the conformational entropy change associated with a folding-unfolding transition of an SH3 domain and calculated from changes in rapid N-HN backbone dynamics are presented.

Comprehensive NOE characterization of a partially folded large fragment of staphylococcal nuclease Delta131Delta, using NMR methods with improved resolution.

Comprehensive NOE results from detailed structural characterization of a 131 residue partially folded fragment of staphylococcal nuclease (Delta131Delta) made possible by NMR methods with improved resolution are presented. The resulting NOE patterns reflect sampling of both alpha and beta regions of phi, phi conformational space, yet demonstrate significant preferences for both native-like and non-native-like turn and potentially helical conformations. Together with data from studies of the unfolded state of the drkN SH3 domain, NOE patterns observed for partially folded or unfolded proteins are summarized. It is surprising that few long-range NOEs were observed in Delta131Delta. The two longest-range NOEs are both native-like; one of these, an (i,i+5) NOE, provides evidence for a Schellman capping motif for helix termination. Many aliphatic-aliphatic and aliphatic-amide NOEs, which are not normally observed in folded proteins, were detected. We have ruled out significant contributions from spin-diffusion for a number of these NOEs and suggest that one source may be sampling of non-prolyl cis peptide bond configurations in the disordered state of Delta131Delta.

A study of protein side-chain dynamics from new 2H auto-correlation and 13C cross-correlation NMR experiments: application to the N-terminal SH3 domain from drk.

Two new NMR experiments are presented for measuring side-chain dynamics in proteins. The first method, requiring 15N, 13C, approximately 50% 2H-labeled protein, measures 2H T1 and T1p spin relaxation times at side-chain positions. A second experiment permits the straightforward measurement of 13C-1H dipole-dipole cross-correlation relaxation rates at 13C beta positions in 15N, 13C-labeled molecules. An excellent correlation is observed between order parameters, describing the amplitude of motion at these sites, obtained on the basis of 2H relaxation and dipole-dipole cross-correlation relaxation rates. Together these experiments provide a powerful approach for selecting appropriate motional models. The methods are applied to study the side-chain motional properties of the N-terminal SH3 domain from the signaling protein drk.

What is the average conformation of bacteriophage T4 lysozyme in solution? A domain orientation study using dipolar couplings measured by solution NMR.

Lysozyme from T4 bacteriophage is comprised of two domains that are both involved in binding substrate. Although wild-type lysozyme has been exclusively crystallized in a closed form that is similar to the peptidoglycan-bound conformation, a more open structure is thought to be required for ligand binding. To determine the relative arrangement of domains within T4 lysozyme in the solution state, dipolar couplings were measured in several different dilute liquid crystalline media by solution NMR methods. The dipolar coupling data were analyzed with a domain orientation procedure described previously that utilizes high- resolution X-ray structures. The cleft between the domains is significantly larger in the average solution structure than what is observed in the X-ray structure of the ligand-free form of the protein (approximately 17 degrees closure from solution to X-ray structures). A comparison of the solution domain orientation with X-ray-derived structures in the protein data base shows that the solution structure resembles a crystal structure obtained for the M6I mutant. Dipolar couplings were also measured on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-domain disulfide bond (T4SS). In this case, the inter-domain solution structure was found to be more closed than was observed in the crystal (approximately 11 degrees). Direct refinement of lysozyme crystal structures with the measured dipolar couplings using the program CNS, establishes that this degree of closure can be accommodated whilst maintaining the inter-domain cystine bond. The differences between the average solution conformations obtained using dipolar couplings and the crystal conformations for both forms of lysozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and the importance of alternative methods to X-ray crystallography for evaluating inter-domain structure.

Ligand-induced structural changes to maltodextrin-binding protein as studied by solution NMR spectroscopy.

Solution NMR studies on the physiologically relevant ligand-free and maltotriose-bound states of maltodextrin-binding protein (MBP) are presented. Together with existing data on MBP in complex with beta-cyclodextrin (non-physiological, inactive ligand), these new results provide valuable information on changes in local structure, dynamics and global fold that occur upon ligand binding to this two-domain protein. By measuring a large number of different one-bond residual dipolar couplings, the domain conformations, critical for biological function, were investigated for all three states of MBP. Structural models of the solution conformation of MBP in a number of different forms were generated from the experimental dipolar coupling data and X-ray crystal structures using a quasi-rigid-body domain orientation algorithm implemented in the structure calculation program CNS. Excellent agreement between relative domain orientations in ligand-free and maltotriose-bound solution conformations and the corresponding crystal structures is observed. These results are in contrast to those obtained for the MBP/beta-cyclodextrin complex where the solution state is found to be approximately 10 degrees more closed than the crystalline state. The present study highlights the utility of residual dipolar couplings for orienting protein domains or macromolecules with respect to each other.

Contributions to protein entropy and heat capacity from bond vector motions measured by NMR spin relaxation.

The backbone dynamics of both folded and unfolded states of staphylococcal nuclease (SNase) and the N-terminal SH3 domain from drk (drkN SH3) are studied at two different temperatures. A simple method for obtaining order parameters, describing the amplitudes of motion of bond vectors, from NMR relaxation measurements of both folded and unfolded proteins is presented and the data obtained for 15N-NH bond vectors in both the SNase and drkN SH3 systems analyzed with this approach. Using a recently developed theory relating the amplitude of bond vector motions to conformational entropy, the entropy change between the folded and unfolded forms of SNase is calculated on a per residue basis. It is noteworthy that the region of the molecule with the smallest entropy change includes those residues showing native-like structure in the unfolded form of the molecule, as established by NOE-based experiments. Order parameters of backbone 15N-NH bond vectors show significantly larger changes with temperature in the unfolded states of both proteins relative to the corresponding folded forms. The differential temperature dependence is interpreted in terms of differences in the heat capacities of folded and unfolded polypeptide chains. The contribution to the heat capacity of the unfolded chain from rapid 15N-NH bond vector motions is calculated and compared with estimates of the heat capacity of the backbone unit, -CHCONH-, obtained from calorimetric data. Methyl dynamics measured at 14 and 30 degrees C establish that the amplitudes of side-chain motions in the folded SH3 domain are more sensitive to changes in temperature than the backbone dynamics, suggesting that over this temperature range side-chain ps to ns time-scale motions contribute more to the heat capacity than backbone motions for this protein.

Similarities between the spectrin SH3 domain denatured state and its folding transition state.

We have expanded our description of the energy landscape for folding of the SH3 domain of chicken alpha-spectrin by a detailed structural characterization of its denatured state ensemble (DSE). This DSE is significantly populated under mildly acidic conditions in equilibrium with the folded state. Evidence from heteronuclear nuclear magnetic resonance (NMR) experiments on (2)H, (15)N-labeled protein suggests the presence of conformers whose residual structure bears some resemblence to the structure of the folding transition state of this protein. NMR analysis in a mutant with an engineered, non-native alpha-helical tendency shows a significant amount of local non-native structure in the mutant, while the overall characteristics of the DSE are unchanged. Comparison with recent theoretical predictions of SH3 domain folding reactions reveals an interesting correlation with the predicted early events. Based on these results and recent data from other systems, we propose that the DSE of a protein will resemble the intermediate or transition state of its nearest rate-limiting step, as a consequence of simple energetic and kinetic principles.

Selective methyl group protonation of perdeuterated proteins.

Deuteration of aliphatic sites in proteins has shown great potential to increase the range of molecules amenable to study by NMR spectroscopy. One problem inherent in high-level deuterium incorporation is the loss of 1H-1H distance information obtainable from NOESY spectra of the labeled proteins. In the limit of perdeuteration, the available NH-NH NOEs are insufficient in many cases to define the three-dimensional structure of a folded protein. We describe here a method of producing proteins that retains all the advantages of perdeuteration, while enabling observation of many NOEs absent from spectra of fully deuterated samples. Overexpression of proteins in bacteria grown in 2H2O medium containing protonated pyruvate as the sole carbon source results in complete deuteration at C alpha and > 80% deuteration at C beta positions of nearly all amino acids. In contrast, the methyl groups of Ala, Val, Leu and Ile (gamma 2 only) remain highly protonated. This labeling pattern can be readily understood from analysis of bacterial pathways for pyruvate utilization and amino acid biosynthesis. As Ala, Val, Leu and Ile are among the most highly represented residue types in protein hydrophobic cores and at protein-protein interfaces, selectively methyl-protonated samples will be useful in many areas of structural analysis of larger molecules and molecular complexes by NMR.

Changes in side-chain and backbone dynamics identify determinants of specificity in RNA recognition by human U1A protein.

The ribonucleoprotein (RNP) domain is one of the most common eukaryotic protein domains, and is found in many proteins involved in recognition of a wide variety of RNAs. Two structures of RNA complexes of human U1A protein have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning how RNP domains discriminate between different RNAs. In this work, we extend the investigation of U1A-RNA recognition by comparing the dynamics of U1A protein both free and in complex with RNA. We have also investigated the trimolecular complex between two U1A proteins and the complete polyadenylation inhibition element to study the effect of RNA-dependent protein-protein interactions on protein conformational flexibility. We report that changes in backbone dynamics upon complex formation identify regions of the protein where conformational exchange processes are quenched in the RNA-bound conformation. Furthermore, amino acids whose side-chains experience significant changes in conformational flexibility coincide with residues particularly important for the specificity of the U1A protein/RNA interaction. This study adds a new dimension to the description of the coordinated changes in structure and dynamics that are critical to define the biological specificity of U1A and other RNP proteins.

NOE data demonstrating a compact unfolded state for an SH3 domain under non-denaturing conditions.

The N-terminal SH3 domain of drk (drkN SH3) is unstable, existing in equilibrium between a folded state (Fexch) and an unfolded state (Uexch) under non-denaturing buffer conditions. Using a15N/2H-labeled sample, long range amide NOEs can be observed in the Uexchstate as a result of reduced relaxation, in some cases correlating protons over 40 residues apart. These long range NOEs disappear upon addition of 2 M guanidinium chloride, demonstrating that there are substantial differences between the Uexchand the guanidine denatured states. Calculations using the long range NOEs of the Uexchstate yield highly compact structures having non-native turns and a non-native buried tryptophan residue. These structures agree with experimental stopped-flow fluorescence data and analytical ultracentrifugation results. Since protein stability depends on the structural and dynamic properties of both the folded and unfolded states, this study provides insights into the stability of the drkN SH3 domain. These results provide the first strong NOE-based evidence for compact unfolded states of proteins and suggest that some unfolded states under physiological conditions have specific interactions leading to compact structures.

Backbone and methyl dynamics of the regulatory domain of troponin C: anisotropic rotational diffusion and contribution of conformational entropy to calcium affinity.

The N-terminal domain (residues 1 to 90) of chicken skeletal troponin C (NTnC) regulates muscle contraction upon the binding of a calcium ion to each of its two calcium binding loops. In order to characterize the backbone dynamics of NTnC in the apo state (NTnC-apo), we measured and carefully analyzed 15N NMR relaxation parameters T1, T2 and NOE at 1H NMR frequencies of 500 and 600 MHz. The overall rotational correlation time of NTnC-apo at 29.6 degrees C is 4.86 (+/-0.15) ns. The experimental data indicate that the rotational diffusion of NTnC-apo is anisotropic with a diffusion anisotropy, D parallel/D perpendicular, of 1.10. Additionally, the dynamic properties of side-chains having a methyl group were derived from 2H relaxation data of CH2D groups of a partially deuterated sample. Based on the dynamic characteristics of TnC, two different levels of "fine tuning" of the calcium affinity are presented. Significantly lower backbone order parameters (S2), were observed for calcium binding site I relative to site II and the contribution of the bond vector fluctuations to the conformational entropy of sites I and II was calculated. The conformational entropy loss due to calcium binding (DeltaDeltaSp) differs by 1 kcal/mol between sites I and II. This is consistent with the different dissociation constants previously measured for sites I and II of 16 microM and 1. 7 microM, respectively. In addition to the direct role of binding loop dynamics, the side-chain methyl group dynamics play an indirect role through the energetics of the calcium-induced structural change from a closed to an open state. Our results show that the side-chains which will be exposed upon calcium binding have reduced motion in the apo state, suggesting that conformational entropic contributions can be used to offset the free energy cost of exposing hydrophobic groups. It is clear from this work that a complete determination of their dynamic characteristics is necessary in order to fully understand how TnC and other proteins are fine tuned to appropriately carry out their function.

Solution structure of recombinant human interleukin-6.

Interleukin-6 (IL-6) is a 185 amino acid cytokine which exerts multiple biological effects in vivo and whose dysregulation underlies several disease processes. The solution structure of recombinant human interleukin-6 has now been determined using heteronuclear three and four-dimensional NMR spectroscopy. The structure of the molecule was determined using 3044 distance and torsion restraints derived by NMR spectroscopy to generate an ensemble of 32 structures using a combined distance geometry/simulated annealing protocol. The protein contains five alpha-helices interspersed with variable-length loops; four of these helices constitute a classical four-helix bundle with the fifth helix located in the CD loop. There were no distance violations greater than 0.3 A in any of the final 32 structures and the ensemble has an average-to-the-mean backbone root-mean-square deviation of 0.50 A for the core four-helix bundle. Although the amino-terminal 19 amino acids are disordered in solution, the remainder of the molecule has a well defined structure that shares many features displayed by other long-chain four-helix bundle cytokines. The high-resolution NMR structure of hIL-6 is used to rationalize available mutagenesis data in terms of a heteromeric receptor complex.

Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein.

The global fold of maltose-binding protein in complex with the substrate beta-cyclodextrin was determined by solution NMR methods. The two-domain protein is comprised of a single polypeptide chain of 370 residues, with a molecular mass of 42 kDa. Distance information in the form of H(N)-H(N), H(N)-CH(3) and CH(3)-CH(3) NOEs was recorded on (15)N, (2)H and (15)N, (13)C, (2)H-labeled proteins with methyl protonation in Val, Leu, and Ile (C(delta1) only) residues. Distances to methyl protons, critical for the structure determination, comprised 77 % of the long-range restraints. Initial structures were calculated on the basis of 1943 NOEs, 48 hydrogen bond and 555 dihedral angle restraints. A global pair-wise backbone rmsd of 5.5 A was obtained for these initial structures with rmsd values for the N and C domains of 2.4 and 3.8 A, respectively. Direct refinement against one-bond (1)H(N)-(15)N, (13)C(alpha)-(13)CO, (15)N-(13)CO, two-bond (1)H(N)-(13)CO and three-bond (1)H(N)-(13)C(alpha) dipolar couplings resulted in structures with large numbers of dipolar restraint violations. As an alternative to direct refinement against measured dipolar couplings we have developed an approach where discrete orientations are calculated for each peptide plane on the basis of the dipolar couplings described above. The orientation which best matches that in initial NMR structures calculated from NOE and dihedral angle restraints exclusively is used to refine further the structures using a new module written for CNS. Modeling studies from four different proteins with diverse structural motifs establishes the utility of the methodology. When applied to experimental data recorded on MBP the precision of the family of structures generated improves from 5.5 to 2.2 A, while the rmsd with respect to the X-ray structure (1dmb) is reduced from 5.1 to 3.3 A.

Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin.

Protein function is often regulated by conformational changes that occur in response to ligand binding or covalent modification such as phosphorylation. In many multidomain proteins these conformational changes involve reorientation of domains within the protein. Although X-ray crystallography can be used to determine the relative orientation of domains, the crystal-state conformation can reflect the effect of crystal packing forces and therefore may differ from the physiologically relevant form existing in solution. Here we demonstrate that the solution-state conformation of a multidomain protein can be obtained from its X-ray structure using an extensive set of dipolar couplings measured by triple-resonance multidimensional NMR spectroscopy in weakly aligning solvent. The solution-state conformation of the 370-residue maltodextrin-binding protein (MBP) loaded with beta-cyclodextrin has been determined on the basis of one-bond (15)N-H(N), (15)N-(13)C', (13)C(alpha)-(13)C', two-bond (13)C'-H(N), and three-bond (13)C(alpha)-H(N) dipolar couplings measured for 280, 262, 276, 262, and 276 residues, respectively. This conformation was generated by applying hinge rotations to various X-ray structures of MBP seeking to minimize the difference between the experimentally measured and calculated dipolar couplings. Consistent structures have been derived in this manner starting from four different crystal forms of MBP. The analysis has revealed substantial differences between the resulting solution-state conformation and its crystal-state counterpart (Protein Data Bank accession code 1DMB) with the solution structure characterized by an 11(+/-1) degrees domain closure. We have demonstrated that the precision achieved in these analyses is most likely limited by small uncertainties in the intradomain structure of the protein (ca 5 degrees uncertainty in orientation of internuclear vectors within domains). In addition, potential effects of interdomain motion have been considered using a number of different models and it was found that the structures derived on the basis of dipolar couplings accurately represent the effective average conformation of the protein.