NMR solution structure of plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica

The solution structure of a divergent plastocyanin (PC) from the photosynthetic prokaryote Prochlorothrix hollandica was determined by homonuclear 1H NMR spectroscopy. Nineteen structures were calculated from 1222 distance restraints, yielding a family of structures having an average rmsd of 0.42 +/- 0.08 A for backbone atoms and 0.71 +/- 0.07 A for heavy atoms to the mean structure. No distance constraint was violated by more than 0.26 A in the structure family. Despite the low number of conserved residues shared with other PC homologues, the overall folding pattern of P. hollandica PC is similar to other PCs, in that the protein forms a two-sheet beta-barrel tertiary structure. The greatest variability among the backbone structures is seen in the loop region from residues 47-60. The differences seen in the P. hollandica PC homologue likely arise due to a small deletion of 2-4 residues compared to the PC consensus; this yields a less extended loop containing a short alpha-helix from residues Ala52-Leu55. Additionally, the protein has an altered hydrophobic patch thought to be important in binding reaction partners. Whereas the backbone structure is very similar within the loops of the hydrophobic region, the presence of two unique residues (Tyr12 and Pro14) yields a structurally different hydrophobic surface likely important in binding P. hollandica Photosystem I.     

Structure of the fMet-tRNA(fMet)-binding domain of B. stearothermophilus initiation factor IF2

The three-dimensional structure of the fMet-tRNA(fMet) -binding domain of translation initiation factor IF2 from Bacillus stearothermophilus has been determined by heteronuclear NMR spectroscopy. Its structure consists of six antiparallel beta-strands, connected via loops, and forms a closed beta-barrel similar to domain II of elongation factors EF-Tu and EF-G, despite low sequence homology. Two structures of the ternary complexes of the EF-Tu small middle dotaminoacyl-tRNA small middle dot GDP analogue have been reported and were used to propose and discuss the possible fMet-tRNA(fMet)-binding site of IF2.     

Solution structure of neurotoxin I from the sea anemone Stichodactyla helianthus. A nuclear magnetic resonance, distance geometry, and restrained molecular dynamics study

The three-dimensional structure of the sea anemone polypeptide Stichodactyla helianthus neurotoxin I in aqueous solution has been determined using distance geometry and restrained molecular dynamics simulations based on NMR data acquired at 500 MHz. A set of 470 nuclear Overhauser enhancement values was measured, of which 216 were used as distance restraints in the structure determination along with 15 dihedral angles derived from coupling constants. After restrained molecular dynamics refinement, the eight structures that best fit the input data form a closely related family. They describe a structure that consists of a core of twisted, four-stranded, antiparallel beta-sheet encompassing residues 1-3, 19-24, 29-34, and 40-47, joined by three loops, two of which are well defined by the NMR data. The third loop, encompassing residues 7-16, is poorly defined by the data and is assumed to undergo conformational averaging in solution. Pairwise root mean square displacement values for the backbone heavy atoms of the eight best structures are 1.3 +/- 0.2A when the poorly defined loop is excluded and 3.6 +/- 1.0A for all backbone atoms. Refinement using restrained molecular dynamics improved the quality of the structures generated by distance geometry calculations with respect to the number of nuclear Overhauser enhancements violated, the size of the total distance violations and the total potential energies of the structures. The family of structures for S. heliathus neurotoxin I is compared with structures of related sea anemone proteins that also bind to the voltage-gated sodium channel.     

Characterization of the nucleic acid binding region of the intermediate filament protein vimentin by fluorescence polarization

Employing deletion mutant proteins and fluorescein-labeled oligodeoxyribonucleotides in a fluorescence polarization assay, the nucleic acid binding site of the intermediate filament (IF) subunit protein vimentin was localized to the middle of the arginine-rich, non-alpha-helical, N-terminal head domain. While deletion of the first few N-terminal residues (up to amino acid 17) had almost no effect, deletions of residues 25-64 or 25-68 essentially abolished the binding of nucleic acids by the respective proteins. Proteins with smaller deletions, of residues 25-39 or 43-68, were still able to bind nucleic acids quite well at low ionic strength, but only the proteins containing the first DNA-binding wing (residues 27-39) retained the ability to stably bind nucleic acids at physiological ionic strength. These results were confirmed by data obtained with two synthetic peptides whose sequences correspond to the smaller deletions. Nitration experiments showed that one or more of the tyrosines in the head domain are responsible for the stable binding by intercalation. Interestingly, the residues responsible for binding nucleic acids can be deleted without major influence on the in vivo polymerization properties of the mutant proteins. Only the protein with the largest internal deletion, of residues 25-68, failed to form filaments in vivo. Since the N-terminal head domains of IF proteins are largely exposed on the filament surface, but nevertheless essential for filament assembly, these results support the model that the middle of the head domain of vimentin may loop out from the filament surface and thus be available for interactions with other cellular structures or molecules.     

NMR structure of the complex between the zinc finger protein NCp10 of Moloney murine leukemia virus and the single-stranded pentanucleotide d(ACGCC): comparison with HIV-NCp7 complexes.

The structure of the 56 amino acid nucleocapsid protein NCp10 of retrovirus MoMuLV, which contains a single CX(2)CX(4)HX(4)C-type zinc finger, has been determined previously by NMR. The important role of NCp10 (or NCp7 for HIV-1) in the retroviral life cycle seems mainly related to their preferential binding to single-stranded nucleic acids. We report here the structure of the complex formed between the biologically active (14-53)NCp10 and the oligonucleotide d(ACGCC) in aqueous solution determined by 2D (1)H NMR based methods. The aromatic residue Trp(35) of NCp10 directs nucleic acid complexation as shown by its complete fluorescence quenching upon addition of d(ACGCC). (1)H and (31)P NMR studies support the insertion of Trp(35) between the G(3) and C(4) bases. A total of 577 NOE distance restraints, of which 40 were intermolecular, were used for the structure determination. The zinc finger provides a well-defined surface for the binding of d(ACGCC) through hydrophobic interactions and tryptophan stacking on the guanine. This latter interaction was also observed in the NMR-derived structures of the complexes between NCp7, which contains two successive zinc fingers, and single-stranded DNA and RNA, supporting the proposal for a major role played by aromatic residues of NCp proteins in nucleic acid recognition. Upon binding to the nucleotide a new loop in NCp10 that participates in the intermolecular interaction is formed. Additional interactions provided by positively charged residues surrounding the zinc finger appear necessary for tight binding. The structure of the complex NCp10-d(ACGCC) gives a structural explanation for the loss of virus infectivity following point mutations in the finger domain.     

Toward genomic identification of β-barrel membrane proteins: Composition and architecture of known structures

The amino acid composition and architecture of all beta-barrel membrane proteins of known three-dimensional structure have been examined to generate information that will be useful in identifying beta-barrels in genome databases. The database consists of 15 nonredundant structures, including several novel, recent structures. Known structures include monomeric, dimeric, and trimeric beta-barrels with between 8 and 22 membrane-spanning beta-strands each. For this analysis the membrane-interacting surfaces of the beta-barrels were identified with an experimentally derived, whole-residue hydrophobicity scale, and then the barrels were aligned normal to the bilayer and the position of the bilayer midplane was determined for each protein from the hydrophobicity profile. The abundance of each amino acid, relative to the genomic abundance, was calculated for the barrel exterior and interior. The architecture and diversity of known beta-barrels was also examined. For example, the distribution of rise-per-residue values perpendicular to the bilayer plane was found to be 2.7 +/- 0.25 A per residue, or about 10 +/- 1 residues across the membrane. Also, as noted by other authors, nearly every known membrane-spanning beta-barrel strand was found to have a short loop of seven residues or less connecting it to at least one adjacent strand. Using this information we have begun to generate rapid screening algorithms for the identification of beta-barrel membrane proteins in genomic databases. Application of one algorithm to the genomes of Escherichia coli and Pseudomonas aeruginosa confirms its ability to identify beta-barrels, and reveals dozens of unidentified open reading frames that potentially code for beta-barrel outer membrane proteins.     

Predicting transmembrane beta-barrels and interstrand residue interactions from sequence

Transmembrane beta-barrel (TMB) proteins are embedded in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. The cellular location and functional diversity of beta-barrel outer membrane proteins (omps) makes them an important protein class. At the present time, very few nonhomologous TMB structures have been determined by X-ray diffraction because of the experimental difficulty encountered in crystallizing transmembrane proteins. A novel method using pairwise interstrand residue statistical potentials derived from globular (nonouter membrane) proteins is introduced to predict the supersecondary structure of transmembrane beta-barrel proteins. The algorithm transFold employs a generalized hidden Markov model (i.e., multitape S-attribute grammar) to describe potential beta-barrel supersecondary structures and then computes by dynamic programming the minimum free energy beta-barrel structure. Hence, the approach can be viewed as a "wrapping" component that may capture folding processes with an initiation stage followed by progressive interaction of the sequence with the already-formed motifs. This approach differs significantly from others, which use traditional machine learning to solve this problem, because it does not require a training phase on known TMB structures and is the first to explicitly capture and predict long-range interactions. TransFold outperforms previous programs for predicting TMBs on smaller (相似文献   

Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease

Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease (SNase110) have been studied by various biophysical and NMR methods. Samples of G-88W- and V-66W-mutant SNase110, namely G-88W110 and V-66W110, in aqueous solution and SNase110 in 2.0 M TMAO are adopted in this study. The unfolding transitions and folded conformations of the three SNase fragments were detected by far- and near-ultraviolet circular dichroism and intrinsic tryptophan fluorescence measurements. The tertiary structures and internal motions of the fragments were determined by NMR spectroscopy. Both G-88W and V-66W single mutations as well as a small organic osmolyte (Trimethylamine N-oxide, TMAO) can fold the fragment into a native-like conformation. However, the tertiary structures of the three fragments exhibit different degrees of folding stability and compactness. G-88W110 adopts a relatively rigid structure representing a most stable native-like beta-subdomain conformation of the three fragments. V-66W110- and TMAO-stabilized SNase110 produce less compact structures having a less stable "beta-barrel" structural region. The different folding status accounts for the different backbone dynamic and urea-unfolding transition features of the three fragments. The G-20I/G-29I-mutant variants of the three fragments have provided the evidence that the folding status is correlated closely to the packing of the beta-strands in the beta-barrel of the fragments. The native-like beta-barrel structural region acts as a nonlocal nucleus for folding the fragment. The tertiary folding of the three fragments is initiated by formation of the local nucleation sites at two beta-turn regions, I-18-D-21 and Y-27-Q-30, and developed by the formation of a nonlocal nucleation site at the beta-barrel region. The formation of beta-barrel and overall structure is concerted, but the level of cooperativity is different for the three 1-110 residues SNase fragments.     

Solution conformation on bovine growth hormone releasing factor by 1H NMR and molecular modeling.

The structure of bovine growth hormone releasing factor (bGHRF) consisting of 44 amino acids has been studied in CD and 1H nuclear magnetic resonance (NMR) spectroscopy in conjunction with molecular modeling. Since bGHRF does not have an ordered structure in water alone, a 30% 2,2,2-trifluoroethanol (TFE) aqueous solvent was used to induce considerable alpha-helical structures, which corresponds to a helical content of approximately 62% as determined by circular dichroism (CD). The secondary structure was obtained from nuclear Overhauser enhancement and 3J(HN alpha) coupling constant in 30% TFE solution. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculation. A set of 267 interproton distances derived from nuclear Overhauser effect correlation spectroscopy (NOESY) experiments and coupling constants were used. From the initial random conformations, 50 distance geometry structures with minimal violations were selected for further refinement. The 14 best structures were obtained after simulated annealing calculation with energy minimization. The structure of bGHRF in 30% TFE solution was characterized by one alpha-helix (residues 8-19), two poorly constrained helices (residues 23-27 and residues 31-34) and a beta I(III)-turn fragment (residues 20-23; phi(i+1) = -53.1 degrees, psi(i+1) = -19.6 degrees, phi(i+2) = -59.9 degrees, psi(i+2) = -20.6 degrees) connected by the segments of less defined structures in N-terminal and omega-shaped flexible C-terminal determined from NOESY cross peaks between helical segment (residues 14-18) and tail fragment (residues 42-44). The obtained structure will play an important role toward the understanding of the structural and functional role of the GHRF.     

Sites of nucleic acid binding in type I-IV intermediate filament subunit proteins

A combination of enzymatic and chemical ladder sequencing of photo-cross-linked protein-single-stranded oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry was employed to identify the amino acid residues responsible for the stable binding of nucleic acids in several intermediate filament (IF) subunit proteins. The IF proteins studied included the type I and type II cytokeratins K8, K18, and K19; the type III proteins desmin, glial fibrillary acidic protein (GFAP), peripherin, and vimentin; and the type IV neurofilament triplet protein L (NF-L). The site of nucleic acid binding was localized to the non-alpha-helical, amino-terminal head domain of all of the IF proteins tested. GFAP, which has the shortest head domain of the proteins tested, cross-linked via only two amino acid residues. One of these residues was located within a conserved nonapeptide domain that has been shown to be required for filament formation. One or more cross-linked residues were found in a similar location in the other proteins studied. The major binding site for nucleic acids for most of the proteins appears to be localized within the middle of the head domain. The two exceptions to this generalization are GFAP, which lacks these residues, and NF-L, in which a large number of cross-linked residues were found scattered throughout the first half of the head domain. Control experiments were also done with two bacteriophage ssDNA-binding proteins, as well as actin and tubulin. The single sites of cross-linkage observed with the bacteriophage proteins, Phe(183) for the T4 gene 32 protein and Phe(73) for the M13 gene 5 protein, were in good agreement with literature data. Actin and tubulin could not be cross-linked to the oligonucleotide. Aside from the insight into the biological activity of IF proteins that these data provide, they also demonstrate that this analytical method can be employed to study a variety of protein-nucleic acid interactions.     

Mapping the fMet-tRNA(f)(Met) binding site of initiation factor IF2

The interaction between fMet-tRNA(f)(Met) and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the specificity of this interaction are localized within the acceptor hexanucleotide fMet-3'ACCAAC of the initiator tRNA and a fairly small area at the surface of the beta-barrel structure of the 90-amino acid C-terminal domain of IF2 (IF2 C-2). A weak but specific interaction between IF2 C-2 and formyl-methionyl was also demonstrated. The surface of IF2 C-2 interacting with fMet-tRNA(f)(Met) has been mapped using two independent approaches, site- directed mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side-chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNA(f)(Met), and the surface lined with residues K702-S660, along which the acceptor arm of the initiator tRNA spans in the direction 3' to 5'.     

Solution structure of the R3H domain from human Smubp-2

The R3H domain is a conserved sequence motif, identified in over 100 proteins, that is thought to be involved in polynucleotide-binding, including DNA, RNA and single-stranded DNA. In this work the 3D structure of the R3H domain from human Smubp-2 was determined by NMR spectroscopy. It is the first 3D structure determination of an R3H domain. The fold presents a small motif, consisting of a three-stranded antiparallel beta-sheet and two alpha-helices, which is related to the structures of the YhhP protein and the C-terminal domain of the translational initiation factor IF3. The similarities are non-trivial, as the amino acid identities are below 10%. Three conserved basic residues cluster on the same face of the R3H domain and could play a role in nucleic acid recognition. An extended hydrophobic area at a different site of the molecular surface could act as a protein-binding site. A strong correlation between conservation of hydrophobic amino acids and side-chain solvent protection indicates that the structure of the Smubp-2 R3H domain is representative of R3H domains in general.     

Solution structure of a recombinant mouse major urinary protein.

Major urinary proteins (MUPs) form an ensemble of protein isoforms which are expressed and secreted by sexually mature male mice only. They belong to the lipocalin superfamily and share with other members of this family the capacity to bind hydrophobic molecules, some of which are odorants. MUPs, either associated with or free of their natural ligands, play an important role in the reproductive cycle of these rodents by acting as pheromones. In fact, they are able to interact with receptors in the vomeronasal organ of the female mice, inducing hormonal and physiological responses by an as yet unknown mechanism. In order to investigate the structural and dynamical features of these proteins in solution, one of the various wild-type isoforms (rMUP: 162 residues) was cloned and subsequently isotopically labeled. The complete 1H, 13C and 15N resonance assignment of that isoform, achieved by using a variety of multidimensional heteronuclear NMR experiments, has been reported recently. Here, we describe the refined high-resolution three-dimensional solution structure of rMUP in the native state, obtained by a combination of distance geometry and energy minimization calculations based on 2362 NOE-derived distance restraints. A comparison with the crystal structure of the wild-type MUPs reveals, aside from minor differences, a close resemblance in both secondary structure and overall topology. The secondary structure of the protein consists of eight antiparallel beta-strands forming a single beta-sheet and an alpha-helix in the C-terminal region. In addition, there are several helical and hairpin turns distributed throughout the protein sequence, mostly connecting the beta-strands. The tertiary fold of the beta-sheet creates a beta-barrel, common to all members of the lipocalin superfamily. The shape of the beta-barrel resembles a calyx, lined inside by mostly hydrophobic residues that are instrumental for the binding and transport of small nonpolar ligand molecules.     

Alpha-cobratoxin: proton NMR assignments and solution structure.

The solution structure of alpha-cobratoxin, a neurotoxin purified from the venom of the snake Naja naja siamensis, at pH 3.2 is reported. Sequence-specific assignments of the NMR resonances was attained by a combination of a generalized main-chain-directed strategy and of the sequential method. The NMR data show the presence of a triple-stranded beta-sheet (residues 19-25, 36-41, and 52-57), a short helix, and turns. An extensive number of NOE cross peaks were identified in the NOESY NMR maps. These were applied as distance constraints in a molecular modeling protocol which includes distance geometry and dynamical simulated annealing calculations. A single family of structures is observed which fold in such a way that three major loops emerge from a globular head. The solution and crystal structures of alpha-cobratoxin are very similar. This is in clear contrast to results reported for alpha-bungarotoxin where significant differences exist.     

Three-dimensional structure of potato carboxypeptidase inhibitor in solution. A study using nuclear magnetic resonance, distance geometry, and restrained molecular dynamics

The solution conformation of potato carboxypeptidase inhibitor (CPI) has been investigated by 1H NMR spectroscopy. The spectrum is assigned in a sequential manner by using two-dimensional NMR techniques to identify through-bond and through-space (less than 5 A) connectivities. A set of 309 approximate interproton distance restraints is derived from the two-dimensional nuclear Overhauser enhancement spectra and used as the basis of a three-dimensional structure determination by a combination of metric matrix distance geometry and restrained molecular dynamics calculations. A total of 11 converged distance geometry structures were computed and refined by using restrained molecular dynamics. The average atomic root mean square (rms) difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.3 A for residues 2-39 and 0.9 +/- 0.2 A for residues 5-37. The corresponding values for all atoms are 1.9 +/- 0.3 and 1.4 +/- 0.2 A, respectively. The larger values for residues 2-38 relative to those for residues 5-37 arise from the fact that the positions of the N- (residues 1-4) and C- (residues 38-39) terminal tails are rather poorly determined, whereas those of the core of the protein (residues 5-37) are well determined by the experimental interproton distance data. The computed structures are very close to the X-ray structure of CPI in its complex with carboxypeptidase, and the backbone atomic rms difference between the mean of the computed structures and the X-ray structure is only 1.2 A. Nevertheless, there are some real differences present which are evidenced by significant deviations between the experimental upper interproton distance limits and the corresponding interproton distances derived from the X-ray structure. These principally occur in two regions, residues 18-20 and residues 28-30, the latter comprising part of the region of secondary contacts between CPI and carboxypeptidase in the X-ray structure.     

Biophysical approaches to membrane protein structure determination.

Recently, there have been several technical advances in the use of solution and solid-state NMR spectroscopy to determine the structures of membrane proteins. The structures of several isolated transmembrane (TM) helices and pairs of TM helices have been solved by solution NMR methods. Similarly, the complete folds of two TM beta-barrel proteins with molecular weights of 16 and 19 kDa have been determined by solution NMR in detergent micelles. Solution NMR has also provided a first glimpse at the dynamics of an integral membrane protein. Structures of individual TM helices have also been determined by solid-state NMR. A combination of NMR with site-directed spin-label electron paramagnetic resonance or Fourier transform IR spectroscopy allows one to assemble quite detailed protein structures in the membrane.     

Myxoma virus immunomodulatory protein M156R is a structural mimic of eukaryotic translation initiation factor eIF2alpha

Phosphorylation of the translation initiation factor eIF2 on Ser51 of its alpha subunit is a key event for regulation of protein synthesis in all eukaryotes. M156R, the product of the myxoma virus M156R open reading frame, has sequence similarity to eIF2alpha as well as to a family of viral proteins that bind to the interferon-induced protein kinase PKR and inhibit phosphorylation of eIF2alpha. In this study, we demonstrate that, like eIF2alpha. M156R is an efficient substrate for phosphorylation by PKR and can compete with eIF2alpha. To gain insights into the substrate specificity of the eIF2alpha kinases, we have determined the nuclear magnetic resonance (NMR) structure of M156R, the first structure of a myxoma virus protein. The fold consists of a five-stranded antiparallel beta-barrel with two of the strands connected by a loop and an alpha-helix. The similarity between M156R and the beta-barrel structure in the N terminus of eIF2alpha suggests that the viral homologs mimic eIF2alpha structure in order to compete for binding to PKR. A homology-modeled structure of the well-studied vaccinia virus K3L was generated on the basis of alignment with M156R. Comparison of the structures of the K3L model, M156R, and human eIF2alpha indicated that residues important for binding to PKR are located at conserved positions on the surface of the beta-barrel and in the mobile loop, identifying the putative PKR recognition motif.