Crystal structures of Nova-1 and Nova-2 K-homology RNA-binding domains.

BACKGROUND: Nova-1 and Nova-2 are related neuronal proteins that were initially cloned using antisera obtained from patients with the autoimmune neurological disease paraneoplastic opsoclonus-myoclonus ataxia (POMA). Both of these disease gene products contain three RNA-binding motifs known as K-homology or KH domains, and their RNA ligands have been identified via binding-site selection experiments. The KH motif structure has been determined previously using NMR spectroscopy, but not using X-ray crystallography. Many proteins contain more than one KH domain, yet there is no published structural information regarding the behavior of such multimers. RESULTS: We have obtained the first X-ray crystallographic structures of KH-domain-containing proteins. Structures of the third KH domains (KH3) of Nova-1 and Nova-2 were determined by multiple isomorphous replacement and molecular replacement at 2.6 A and 2.0 A, respectively. These highly similar RNA-binding motifs form a compact protease-resistant domain resembling an open-faced sandwich, consisting of a three-stranded antiparallel beta sheet topped by three alpha helices. In both Nova crystals, the lattice is composed of symmetric tetramers of KH3 domains that are created by two dimer interfaces. CONCLUSIONS: The crystal structures of both Nova KH3 domains are similar to the previously determined NMR structures. The most significant differences among the KH domains involve changes in the positioning of one or more of the alpha helices with respect to the betasheet, particularly in the NMR structure of the KH1 domain of the Fragile X disease protein FMR-1. Loop regions in the KH domains are clearly visible in the crystal structure, unlike the NMR structures, revealing the conformation of the invariant Gly-X-X-Gly segment that is thought to participate in RNA-binding and of the variable region. The tetrameric arrangements of the Nova KH3 domains provide insights into how KH domains may interact with each other in proteins containing multiple KH motifs.     

Validation of helical tilt angles in the solution NMR structure of the Z domain of Staphylococcal protein A by combined analysis of residual dipolar coupling and NOE data

Staphylococcal protein A (SpA) is a virulence factor from Staphylococcus aureus that is able to bind to immunoglobulins. The 3D structures of its immunoglobulin (Ig) binding domains have been extensively studied by NMR and X-ray crystallography, and are often used as model structures in developing de novo or ab initio strategies for predicting protein structure. These small three-helix-bundle structures, reported in free proteins or Ig-bound complexes, have been determined previously using medium- to high-resolution data. Although the location and relative orientation of the three helices in most of these published 3D domain structures are consistent, there are significant differences among the reported structures regarding the tilt angle of the first helix (helix 1). We have applied residual dipolar coupling data, together with nuclear Overhauser enhancement and scalar coupling data, in refining the NMR solution structure of an engineered IgG-binding domain (Z domain) of SpA. Our results demonstrate that the three helices are almost perfectly antiparallel in orientation, with the first helix tilting slightly away from the other two helices. We propose that this high-accuracy structure of the Z domain of SpA is a more suitable target for theoretical predictions of the free domain structure than previously published lower-accuracy structures of protein A domains.     

Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping.

The crystal structure of cyanovirin-N (CV-N), a protein with potent antiviral activity, was solved at 1.5 A resolution by molecular replacement using as the search model the solution structure previously determined by NMR. The crystals belong to the space group P3221 with one monomer of CV-N in each asymmetric unit. The primary structure of CV-N contains 101 residues organized in two domains, A (residues 1 to 50) and B (residues 51 to 101), with a high degree of internal sequence and structural similarity. We found that under the conditions of the crystallographic experiments (low pH and 26 % isopropanol), two symmetrically related monomers form a dimer by domain swapping, such that domain A of one monomer interacts with domain B' of its crystallographic symmetry mate and vice versa. Because the two swapped domains are distant from each other, domain swapping does not result in additional intramolecular interactions. Even though one of the protein sample solutions that was used for crystallization clearly contained 100 % monomeric CV-N molecules, as judged by various methods, we were only able to obtain crystals containing domain-swapped dimers. With the exception of the unexpected phenomenon of domain swapping, the crystal structure of CV-N is very similar to the NMR structure, with a root-mean-square deviation of 0.55 A for the main-chain atoms, the best agreement reported to date for structures solved using both techniques.     

Modeling the structure of Pyrococcus furiosus rubredoxin by homology to other X-ray structures.

The three-dimensional structure of rubredoxin from the hyperthermophilic archaebacterium, Pyrococcus furiosus, has been modeled from the X-ray crystal structures of three homologous proteins from Clostridium pasteurianum, Desulfovibrio gigas, and Desulfovibrio vulgaris. All three homology models are similar. When comparing the positions of all heavy atoms and essential hydrogen atoms to the recently solved crystal structure (Day, M. W., et al., 1992, Protein Sci. 1, 1494-1507) of the same protein, the homology model differ from the X-ray structure by 2.09 A root mean square (RMS). The X-ray and the zinc-substituted NMR structures (Blake, P. R., et al., 1992b, Protein Sci. 1, 1508-1521) show a similar level of difference (2.05 A RMS). On average, the homology models are closer to the X-ray structure than to the NMR structures (2.09 vs. 2.42 A RMS).     

Solution structures of human and porcine beta-microseminoprotein

Beta-microseminoprotein (MSP) is a small cysteine-rich protein (molecular mass about 10 kDa) first isolated from human seminal plasma and later identified in several other organisms. The function of MSP is not known, but a recent study has shown MSP to bind CRISP-3, a protein present in neutrophilic granulocytes. The amino acid sequence is highly variable between species raising the question of the evolutionary conservation of the 3D structure. Here we present NMR solution structures of both the human and the porcine MSP. The two proteins (sequence identity 51%) have a very similar 3D structure with the secondary structure elements well conserved and with most of the amino acid substitutions causing a change of charge localized to one side of the molecule. MSP is a beta-sheet-rich protein with two distinct domains. The N-terminal domain is composed of a four-stranded beta-sheet, with the strands arranged according to the Greek key-motif, and a less structured part. The C-terminal domain contains two two-stranded beta-sheets with no resemblance to known structural motifs. The two domains, connected to each other by the peptide backbone, one disulfide bond, and interactions between the N and C termini, are oriented to give the molecule a rather extended structure. This global fold differs markedly from that of a previously published structure for porcine MSP, in which the two domains have an entirely different orientation to each other. The difference probably stems from a misinterpretation of ten specific inter-domain NOEs.     

Solution structure of murine epidermal growth factor determined by NMR spectroscopy and refined by energy minimization with restraints.

The solution structure of murine epidermal growth factor (mEGF) at pH 3.1 and a temperature of 28 degrees C has been determined from NMR data, using distance geometry calculations and restrained energy minimization. The structure determination is based on 730 conformational constraints derived from NMR data, including 644 NOE-derived upper bound distance constraints, constraints on the ranges of 32 dihedral angles based on measurements of vicinal coupling constants, and 54 upper and lower bound constraints associated with nine hydrogen bonds and the three disulfide bonds. The distance geometry interpretation of the NMR data is based on previously published sequence-specific 1H resonance assignments [Montelione et al. (1988) Biochemistry 27, 2235-2243], supplemented here with individual assignments for some side-chain amide, methylene, and isopropyl methyl protons. The molecular architecture of mEGF is the same as that described previously [Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5226-5230], but the structure is overall more precisely determined by a more extensive set of NMR constraints. Analysis of proton NMR line widths, amide proton exchange rates, and side-chain 3J(H alpha-H beta) coupling constants provides evidence for internal motion in several regions of the mEGF molecule. Because mEGF is one member of a large family of homologous growth factors and protein domains for which X-ray crystal structures are not yet available, the atomic coordinates resulting from the present structure refinement (which we have deposited in the Brookhaven Protein Data Bank) are important data for understanding the structures of EGF-like proteins and for further detailed comparisons of these structures with mEGF.     

Crystal structure of the N-terminal NC4 domain of collagen IX, a zinc binding member of the laminin-neurexin-sex hormone binding globulin (LNS) domain family

Collagen IX, located on the surface of collagen fibrils, is crucial for cartilage integrity and stability. The N-terminal NC4 domain of the alpha1(IX) chain is probably important in this because it interacts with various macromolecules such as proteoglycans and cartilage oligomeric matrix protein. At least 17 distinct collagen polypeptides carry an NC4-like unit near their N terminus, but this report, describing the crystal structure of NC4 at 1.8-A resolution, represents the first atomic level structure for these domains. The structure is similar to previously characterized laminin-neurexin-sex hormone binding globulin (LNS) structures, dominated by an antiparallel beta-sheet sandwich. In addition, a zinc ion was found in a position similar to that of the metal binding site of other LNS domains. A partial backbone NMR assignment of NC4 was obtained and utilized in NMR titration studies to investigate the zinc binding in solution state and to quantitate the affinity of metal binding. The K(d) of 11.5 mM suggests a regulatory rather than a structural role for zinc in solution. NMR titration with a heparin tetrasaccharide revealed the presence of a secondary binding site for heparin on NC4, showing structural and functional conservation with thrombospondin-1, but a markedly reduced affinity for the ligand. Also the overall arrangement of the N and C termini of NC4 resembles most closely the N-terminal domain of thrombospondin-1, distinguishing the two from the majority of the published LNS structures.     

Membrane Protein Structural Validation by Oriented Sample Solid-State NMR: Diacylglycerol Kinase

The validation of protein structures through functional assays has been the norm for many years. Functional assays perform this validation for water-soluble proteins very well, but they need to be performed in the same environment as that used for the structural analysis. This is difficult for membrane proteins that are often structurally characterized in detergent environments, although functional assays for these proteins are most frequently performed in lipid bilayers. Because the structure of membrane proteins is known to be sensitive to the membrane mimetic environment, such functional assays are appropriate for validating the protein construct, but not the membrane protein structure. Here, we compare oriented sample solid-state NMR spectral data of diacylglycerol kinase previously published with predictions of such data from recent structures of this protein. A solution NMR structure of diacylglycerol kinase has been obtained in detergent micelles and three crystal structures have been obtained in a monoolein cubic phase. All of the structures are trimeric with each monomer having three transmembrane and one amphipathic helices. However, the solution NMR structure shows typical perturbations induced by a micelle environment that is reflected in the predicted solid-state NMR resonances from the structural coordinates. The crystal structures show few such perturbations, especially for the wild-type structure and especially for the monomers that do not have significant crystal contacts. For these monomers the predicted and observed data are nearly identical. The thermostabilized constructs do show more perturbations, especially the A41C mutation that introduces a hydrophilic residue into what would be the middle of the lipid bilayer inducing additional hydrogen bonding between trimers. These results demonstrate a general technique for validating membrane protein structures with minimal data obtained from membrane proteins in liquid crystalline lipid bilayers by oriented sample solid-state NMR.     

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.     

Solution structure of the N-terminal A domain of the human voltage-gated Ca2+channel beta4a subunit

Ca2+ channel beta subunits regulate trafficking and gating (opening and closing) of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A-E) that are related to the large family of membrane associated guanylate-kinase (MAGUK) proteins. The crystal structures of the beta subunit core, B-D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N-terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4). Furthermore, this domain undergoes alternative splicing to create multiple N-terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human beta4a subunit, a splice variant that we have shown previously to have alpha1 subunit subtype-specific effects on Ca2+ channel trafficking and gating.     

High-resolution crystal structures of villin headpiece and mutants with reduced F-actin binding activity

Villin-type headpiece domains are approximately 70 amino acid modular motifs found at the C terminus of a variety of actin cytoskeleton-associated proteins. The headpiece domain of villin, a protein found in the actin bundles of the brush border epithelium, is of interest both as a compact F-actin binding domain and as a model folded protein. We have determined the high-resolution crystal structures of chicken villin headpiece (HP67) at 1.4 A resolution as well as two mutants, R37A and W64Y, at 1.45 and 1.5 A resolution, respectively. Replacement of R37 causes a 5-fold reduction in F-actin binding affinity in sedimentation assays. Replacement of W64 results in a much more drastic reduction in F-actin binding affinity without significant changes in headpiece structure or stability. The detailed comparison of these crystal structures with each other and to our previously determined NMR structures of HP67 and the 35-residue autonomously folding subdomain in villin headpiece, HP35, provides the details of the headpiece fold and further defines the F-actin binding site of villin-type headpiece domains.     

Probing the supramodular architecture of a multidomain protein: the structure of syntenin in solution

Full understanding of the mechanism of function of multidomain proteins is dependent on our knowledge of their supramodular architecture in solution. This is a nontrivial task for both X-ray crystallography and NMR, because intrinsic flexibility makes crystallization of these proteins difficult, while their size creates a challenge for NMR. Here, we describe synergistic application of data derived from X-ray crystallography and NMR residual dipolar couplings (RDCs) to address the question of the supramodular structure of a two-domain protein, syntenin. Syntenin is a 32 kDa molecule containing two PDZ domains and is involved in cytoskeleton-membrane organization. We show that the mutual disposition of the PDZ domains clearly differs from that seen in the crystal structure, and we provide evidence that N- and C-terminal fragments of syntenin, hitherto presumed to lack ordered structure, contain folded structural elements in the full-length protein in contact with the PDZ tandem.     

SPRY Domain-Containing SOCS Box Protein 2: Crystal Structure and Residues Critical for Protein Binding

The four mammalian SPRY (a sequence repeat in dual-specificity kinase splA and ryanodine receptors) domain-containing suppressor of cytokine signalling (SOCS) box proteins (SSB-1 to -4) are characterised by a C-terminal SOCS box and a central SPRY domain. The latter is a protein interaction module found in over 1600 proteins, with more than 70 encoded in the human genome. Here we report the crystal structure of the SPRY domain of murine SSB-2 and compare it with the SSB-2 solution structure and crystal structures of other B30.2/SPRY domain-containing family proteins. The structure is a bent β-sandwich, consisting of two seven-stranded β-sheets wrapped around a long loop that extends from the centre strands of the inner or concave β-sheet; it closely matches those of GUSTAVUS and SSB-4. The structure is also similar to those of two recently determined Neuralized homology repeat (NHR) domains (also known as NEUZ domains), with detailed comparisons, suggesting that the NEUZ/NHR domains form a subclass of SPRY domains. The binding site on SSB-2 for the prostate apoptosis response-4 (Par-4) protein has been mapped in finer detail using mutational analyses. Moreover, SSB-1 was shown to have a Par-4 binding surface similar to that identified for SSB-2. Structural perturbations of SSB-2 induced by mutations affecting its interaction with Par-4 and/or c-Met have been characterised by NMR. These comparisons, in conjunction with previously published dynamics data from NMR relaxation studies and coarse-grained dynamics simulation using normal mode analysis, further refine our understanding of the structural basis for protein recognition of SPRY domain-containing proteins.     

Solution structure and backbone dynamics of the catalytic domain of matrix metalloproteinase-2 complexed with a hydroxamic acid inhibitor

MMP-2 is a member of the matrix metalloproteinase family that has been implicated in tumor cell metastasis and angiogenesis. Here, we describe the solution structure of a catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020), determined by three-dimensional heteronuclear NMR spectroscopy. The catalytic domain, designated MMP-2C, has a short peptide linker replacing the internal fibronectin-domain insertion and is enzymatically active. Distance geometry-simulated annealing calculations yielded 14 converged structures with atomic root-mean-square deviations (r.m.s.d.) of 1.02 and 1.62 A from the mean coordinate positions for the backbone and for all heavy atoms, respectively, when 11 residues at the N-terminus are excluded. The structure has the same global fold as observed for other MMP catalytic domains and is similar to previously solved crystal structures of MMP-2. Differences observed between the solution and the crystal structures, near the bottom of the S1' specificity loop, appear to be induced by the large inhibitor present in the solution structure. The MMP-2C solution structure is compared with MMP-8 crystal structure bound to the same inhibitor to highlight the differences especially in the S1' specificity loop. The finding provides a structural explanation for the selectivity between MMP-2 and MMP-8 that is achieved by large inhibitors.     

NMR structural refinement of a tandem G.A mismatched decamer d(CCAAGATTGG)2 via the hybrid matrix procedure

A complete relaxation matrix approach employing a matrix eigenvalue/eigenvector solution to the Bloch equations is used to evaluate the NMR solution structure of a tandemly positioned G.A double mismatch decamer oligodeoxyribonucleotide duplex, d(CCAAGATTGG)2. An iterative refinement method using a hybrid relaxation matrix combined with restrained molecular dynamics calculations is shown to provide structures having good agreement with the experimentally derived structures. Distances incorporated into the MD simulations have been calculated from the relaxation rate matrix evaluated from a hybrid NOESY volume matrix whose elements are obtained from the merging of experimental and calculated NOESY intensities. Starting from both A- and B-DNA and mismatch syn and anti models, it is possible to calculate structures that are in good atomic RMS agreement with each other (less than 1.6 A RMS) but differ from the reported crystal structure (greater than 3.6 A). Importantly, the hybrid matrix derived structures are in excellent agreement with the experimental solution conformation as determined by comparison of the 200-ms simulated and experimental NOESY spectra, while the crystallographic data provide spectra that are grossly different.     

Structure and self-association of the Rous sarcoma virus capsid protein

BACKGROUND: The capsid protein (CA) of retroviruses, such as Rous sarcoma virus (RSV), consists of two independently folded domains. CA functions as part of a polyprotein during particle assembly and budding and, in addition, forms a shell encapsidating the genomic RNA in the mature, infectious virus. RESULTS: The structures of the N- and C-terminal domains of RSV CA have been determined by X-ray crystallography and solution nuclear magnetic resonance (NMR) spectroscopy, respectively. The N-terminal domain comprises seven alpha helices and a short beta hairpin at the N terminus. The N-terminal domain associates through a small, tightly packed, twofold symmetric interface within the crystal, different from those previously described for other retroviral CAs. The C-terminal domain is a compact bundle of four alpha helices, although the last few residues are disordered. In dilute solution, RSV CA is predominantly monomeric. We show, however, using electron microscopy, that intact RSV CA can assemble in vitro to form both tubular structures constructed from toroidal oligomers and planar monolayers. Both modes of assembly occur under similar solution conditions, and both sheets and tubes exhibit long-range order. CONCLUSIONS: The tertiary structure of CA is conserved across the major retroviral genera, yet sequence variations are sufficient to cause change in associative behavior. CA forms the exterior shell of the viral core in all mature retroviruses. However, the core morphology differs between viruses. Consistent with this observation, we find that the capsid proteins of RSV and human immunodeficiency virus type 1 exhibit different associative behavior in dilute solution and assemble in vitro into different structures.     

X-ray crystal structures of a severely desiccated protein.

Unlike most protein crystals, form IX of bovine pancreatic ribonuclease A diffracts well when severely dehydrated. Crystal structures have been solved after 2.5 and 4 days of desiccation with CaSO4, at 1.9 and 2.0 A resolution, respectively. The two desiccated structures are very similar. An RMS displacement of 1.6 A is observed for main-chain atoms in each structure when compared to the hydrated crystal structure with some large rearrangements observed in loop regions. The structural changes are the result of intermolecular contacts formed by strong electrostatic interactions in the absence of a high dielectric medium. The electron density is very diffuse for some surface loops, consistent with a very disordered structure. This disorder is related to the conformational changes. These results help explain conformational changes during the lyophilization of protein and the associated phenomena of denaturation and molecular memory.