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At least 145 records · Page 8Linked to original sources
PubMed1996

The solution structure of the first zinc finger domain of SWI5: a novel structural extension to a common fold.

BACKGROUND: The 2Cys-2His (C2-H2) zinc finger is a protein domain commonly used for sequence-specific DNA recognition. The zinc fingers of the yeast transcription factors SWI5 and ACE2 share strong sequence homology, which extends into a region N-terminal to the first finger, suggesting that the DNA-binding domains of these two proteins include additional structural elements. RESULTS: Structural analysis of the zinc fingers of SWI5 reveals that a 15 residue region N-terminal to the finger motifs forms part of the structure of the first finger domain, adding a beta strand and a helix not previously observed in other zinc finger structures. Sequence analysis suggests that other zinc finger proteins may also have this structure. Biochemical studies show that this additional structure increases DNA-binding affinity. CONCLUSIONS: The structural analysis presented reveals a novel zinc finger structure in which additional structural elements have been added to the C2-H2 zinc finger fold. This additional structure may enhance stability and has implications for DNA recognition by extending the potential DNA-binding surface of a single zinc finger domain.

Amino Acid Sequence↗
PubMed1996

Structure and dynamics of a designed helix-loop-helix dimer in dilute aqueous trifluoroethanol solution. A strategy for NMR spectroscopic structure determination of molten globules in the rational design of native-like proteins.

BACKGROUND: The overwhelming majority of engineered amino acid sequences designed to fold into well defined tertiary structures show the hallmarks of molten globules. Although imperfectly folded, the structures of these polypeptides are of considerable interest in assessing the predictive power of design strategies and in understanding the structural basis for the formation of proteins with native-like properties. This paper describes a strategy for the structural characterization of molten globules by NMR spectroscopy applied to the study of SA-42, a polypeptide with 42 amino acids that folds into a hairpin helix-loop-helix dimer. RESULTS: The 1H NMR spectrum of SA-42 was assigned in several mixtures of water and trifluoroethanol (TFE) (0-30 vol%) and small amounts of TFE were shown to have a significant effect on the spectrum. The secondary and supersecondary structures of SA-42 were determined. In aqueous solution a helix-loop-helix dimer is formed, but in 30 vol% of TFE the population of hairpin dimers are negligible and SA-42 is monomeric, folding into two non-interacting helical segments. In solutions containing less than 3 vol% of TFE the structure is very similar to that in water and the structural information may be used to develop the motif in aqueous solution. Less well ordered amino acid residue sidechains in the hydrophobic core were identified. Helix distortion in the tetrahelix bundle was found to be small. CONCLUSIONS: Detailed information about molten globule structures in aqueous solution can be obtained from NMR spectroscopy if the spectra are assigned in dilute TFE solution. On the basis of the NMR spectroscopic analysis, the solution structure of SA-42 was found to be close to the designed one. A route for developing native-like properties in SA-42 is suggested based on the identification by NMR spectroscopy of some less well ordered amino acid sidechains in the hydrophobic core and on the observed structural rigidity of the two helices.

Amino Acid Sequence↗
PubMed1992

Solution structure of neuronal bungarotoxin determined by two-dimensional NMR spectroscopy: calculation of tertiary structure using systematic homologous model building, dynamical simulated annealing, and restrained molecular dynamics.

Neuronal bungarotoxin has previously been shown, using two-dimensional 1H NMR spectroscopy, to have a triple-stranded antiparallel beta-sheet structure which dimerizes in solution [Oswald, R.E., Sutcliffe, M.J., Bamberger, M., Loring, R.H., Braswell, E., & Dobson, C.M. (1991) Biochemistry 30, 4901-4909]. In this paper, structural calculations are described which use the 582 experimentally measured NOE restraints in conjunction with 27 phi-angle restraints from J-value measurements. The positions of the N-terminal region and C-terminal region were poorly defined in the calculated structures with respect to the remainder of the structure. The region of the structure containing the triple-stranded beta-sheet was, however, well defined and similar to that found in the structure of homologous alpha-bungarotoxin (45% amino acid identity). The experimental restraints did not result in a well-defined dimer interface region because of the small number of NOEs which could be identified in this region. An approach was therefore adopted which produced model structures based to varying degrees on the alpha-bungarotoxin structure. Fourteen different structures were generated in this manner and subsequently used as starting points for refinement using dynamical simulated annealing followed by restrained molecular dynamics. This approach, which combines NMR data and homologous model building, has enabled a family of structures to be proposed for the dimeric molecule. In particular, Phe49 has been identified as possibly playing an important role in dimer formation, this residue in one chain interacting with the corresponding residue in the adjacent chain.

Amino Acid Sequence↗
PubMed1998

High-resolution crystal structures of human hemoglobin with mutations at tryptophan 37beta: structural basis for a high-affinity T-state,.

The high-resolution X-ray structures of the deoxy forms of four recombinant hemoglobins in which Trp37(C3)beta is replaced with Tyr (betaW37Y), Ala (betaW37A), Glu (betaW37E), or Gly (betaW37G) have been refined and analyzed with superposition methods that partition mutation-induced perturbations into quaternary structure changes and tertiary structure changes. In addition, a new cross-validation statistic that is sensitive to local changes in structure (a "local Rfree" parameter) was used as an objective measure of the significance of the tertiary structure changes. No significant mutation-induced changes in tertiary structure are detected at the mutation site itself for any of the four mutants studied. Instead, disruption of the intersubunit contacts associated with Trp37(C3)beta results in (1) a change in quaternary structure at the alpha1beta2 interface, (2) alpha subunit tertiary structure changes that are centered at Asp94(G1)alpha-Pro95(G2)alpha, (3) beta subunit tertiary structure changes that are located between residues Asp99(G1)beta and Asn102(G4)beta, (4) increased mobility of the alpha subunit COOH-terminal dipeptide, and (5) shortening of the Fe-Nepsilon2His(F8) bond in the alpha and beta subunits of the betaW37G and betaW37E mutants. In each case, the magnitude of the change in a particular structural parameter increases in the order betaW37Y < betaW37A < betaW37E approximately betaW37G, which corresponds closely to the degree of functional disruption documented in the preceding papers.

Amino Acid Substitution↗
PubMed1999

High resolution solution structure of apo calcyclin and structural variations in the S100 family of calcium-binding proteins.

The three-dimensional solution structure of apo rabbit lung calcyclin has been refined to high resolution through the use of heteronuclear NMR spectroscopy and 13C, 15N-enriched protein. Upon completing the assignment of virtually all of the 15N, 13C and 1H NMR resonances, the solution structure was determined from a combination of 2814 NOE-derived distance constraints, and 272 torsion angle constraints derived from scalar couplings. A large number of critical inter-subunit NOEs (386) were identified from 13C-select, 13C-filtered NOESY experiments, providing a highly accurate dimer interface. The combination of distance geometry and restrained molecular dynamics calculations yielded structures with excellent agreement with the experimental data and high precision (rmsd from the mean for the backbone atoms in the eight helices: 0.33 A). Calcyclin exhibits a symmetric dimeric fold of two identical 90 amino acid subunits, characteristic of the S100 subfamily of EF-hand Ca(2+)-binding proteins. The structure reveals a readily identified pair of putative sites for binding of Zn2+. In order to accurately determine the structural features that differentiate the various S100 proteins, distance difference matrices and contact maps were calculated for the NMR structural ensembles of apo calcyclin and rat and bovine S100B. These data show that the most significant variations among the structures are in the positioning of helix III and in loops, the regions with least sequence similarity. Inter-helical angles and distance differences for the proteins show that the positioning of helix III of calcyclin is most similar to that of bovine S100B, but that the helix interfaces are more closely packed in calcyclin than in either S100B structure. Surprisingly large differences were found in the positioning of helix III in the two S100B structures, despite there being only four non-identical residues, suggesting that one or both of the S100B structures requires further refinement.

Animals↗
PubMed1995

Global structure-acute toxicity relationships for mice using structural parameter ratios: new approach to molecular design and screening.

A method for a preliminary survey of the relationship between molecular structure and performance was described using 1506 random data of structure-acute toxicity for mice (intravenously dosed). The structural patterns of the weakest toxic structures (111) were extracted from the data and the patterns discriminated for 64.2% of the other structures (1395). As for the 826 structures of strongest toxicity, 78.3% were discriminated by these structural patterns. These results were obtained by using structural parameter ratios to describe the structural patterns and the exhaustive elimination process to select the best parameter ratio from many candidates. The results were summarized in the form of a chart which can be used for practical screening for the weakest toxic structures.

Animals↗
PubMed1995

A new approach to the evaluation of protein secondary structure predictions at the level of the elements of secondary structure.

For many purposes, such as the prediction of the class of protein folds, the existence of an element of secondary structure rather than its precise position and length must be defined correctly. However, most methods for the evaluation of secondary structure prediction consider success in terms of the percentage of individual amino acids predicted correctly. In this paper the success in predicting elements of secondary structure is discussed. The number of overlapping residues in the predicted and observed secondary structures were considered as a function of the total number of amino acids in the observed and predicted secondary structures. A matrix search procedure was used to remove the ambiguity which similar studies may have had in defining the equivalent secondary structures between predicted and observed structures. In this study a loop was treated in the same way as an alpha-helix and a beta-strand. To describe the accuracy at the level of elements of secondary structure, a set of parameters was defined, similar to those used commonly at the level of individual amino acids. This approach was used to assess the methods of Chou and Fasman (1974b, Biochemistry, 13, 222-245), Lim (1974b, J. Mol. Biol., 88, 873-894) and Garnier et al. (1978, J. Mol. Biol., 120, 97-120). It was found that these methods were much poorer at the secondary structure level than at the amino acid level. This approach can be used generally for secondary structure prediction methods.

Amino Acids↗
PubMed1991

The three-dimensional structure of a DNA hairpin in solution two-dimensional NMR studies and structural analysis of d(ATCCTATTTATAGGAT).

The hairpin formed by d(ATCCTATTTATAGGAT) was studied by means of two-dimensional NMR spectroscopy and conformational analysis. Almost all 1H resonances of the stem region could be assigned, while the 1H and 31P spectra of the loop region were interpreted completely; this includes the stereospecific assignment of the H5' and H5" resonances. The derivation of the detailed loop structure was carried out in a stepwise fashion including some improved and new methods for structure determination from NMR data. In the first step, the mononucleotide structures were examined. The conformational space available to the mononucleotide was scanned systematically by varying the glycosidic torsion angle and pseudorotational parameters. Each generated conformer was tested against the experimental J coupling constants and NOE parameters. In the following stage, the structures of dinucleotides and longer fragments were derived. Inter-residue distances between protons were calculated by means of a procedure in which the simulated NOEs, obtained via a relaxation-matrix approach, were fitted to the experimental NOEs without the introduction of a molecular model. In addition, the backbone torsion angles beta, gamma and epsilon were deduced from homocoupling and heterocoupling constants. These data served as constraints in the next step, in which the loop sequence was subjected to a multi-conformer generation procedure. The resulting structures were tested against the mentioned constraints and disregarded if these constraints were violated. This yielded a family of structures for the loop region, confined to a relatively narrow conformational space. A representative conformation was subsequently docked on a B-type stem which fulfilled the structural constraints (derived from the NMR experiments for the stem region) to yield the hairpin structure. Results obtained from subsequent restrained-molecular-mechanics as well as free-molecular-mechanics calculations are in accordance with those obtained by means of the analysis described above. The structure of the hairpin loop is a compactly folded conformation and the first base of the central TTTA region forms a Hoogsteen T-A pair with the fourth base. This Hoogsteen base pair is stacked upon the sixth base pair of the B-type double-helical stem. The second base of the loop is folded into the minor groove, whereas the third base of the loop is partly stacked on the first and fourth bases. The phosphate backbone exhibits a sharp turn between the third and fourth nucleotides of the loop. The peculiar structure of this hairpin loop is discussed in relation to loop folding in DNA and RNA hairpins and in relation to a general model for loop folding.

Base Sequence↗
PubMed2003

Automated construction of structural motifs for predicting functional sites on protein structures.

Structural genomics initiatives are beginning to rapidly generate vast numbers of protein structures. For many of the structures, functions are not yet determined and high-throughput methods for determining function are necessary. Although there has been extensive work in function prediction at the sequence level, predicting function at the structure level may provide better sensitivity and predictive value. We describe a method to predict functional sites by automatically creating three dimensional structural motifs from amino acid sequence motifs. These structural motifs perform comparably well with manually generated structural motifs and perform better than sequence motifs. Automatically generated structural motifs can be used for structural-genomic scale function prediction on protein structures.

Amino Acid Motifs↗
PubMed2001

Protein anatomy: structure and function of peptide fragments corresponding to the secondary structure units of barnase.

Globular proteins can be decomposed into several modules or secondary structure units. It is useful to investigate the functions of such structural units in order to understand the folding units of proteins. In our previous work, barnase was divided into six peptide fragments corresponding to modules, and some of them were shown to have RNA-binding and RNase activity [Yanagawa, et al. (1993) J. Biol. Chem. 268, 5861-5865]. Barnase mutant proteins obtained by permutation of the structural units also had RNase activity [Tsuji, T. et al. (1999) J. Mol. Biol. 286, 1581-1596]. Here we investigated the structure and function of peptide fragments corresponding to secondary structure units of barnase. The results of circular dichroism spectroscopy indicated that some of the peptide fragments form helical structures in aqueous solutions containing over 30% 2,2,2-trifluoroethanol, and the S6 (94-110) peptide fragment is induced to form a beta-sheet structure in the presence of RNA. The S6 peptide fragment forms aggregate complexes with RNA. Electron microscopic analysis showed that the aggregate complexes were comprised of filaments. These results indicate that not only modules but also secondary structure units dissected from a globular protein have functional and structure-forming capabilities.

Amino Acid Sequence↗
PubMed1997

A structural census of genomes: comparing bacterial, eukaryotic, and archaeal genomes in terms of protein structure.

Representative genomes from each of the three kingdoms of life are compared in terms of protein structure, in particular, those of Haemophilus influenzae (a bacteria), Methanococcus jannaschii (an archaeon), and yeast (a eukaryote). The comparison is in the form of a census (or comprehensive accounting) of the relative occurrence of secondary and tertiary structures in the genomes, which particular emphasis on patterns of supersecondary structure. Comparison of secondary structure shows that the three genomes have nearly the same overall secondary-structure content, although they differ markedly in amino acid composition. Comparison of super-secondary structure, using a novel "frequent-words" approach, shows that yeast has a preponderance of consecutive strands (e.g. beta-beta-beta patterns), Haemophilus, consecutive helices (alpha-alpha-alpha), and Methanococcus, alternating helix-strand structures (beta-alpha-beta). Yeast also has significantly more helical membrane proteins than the other two genomes, with most of the differences concentrated in proteins containing two transmembrane segments. Comparison of tertiary structure (by sequence matching and domain-level clustering) highlights the substantial duplication in each genome (approximately 30% to 50%), with the degree of duplication following similar patterns in all three. Many sequence families are shared among the genomes, with the degree of overlap between any two genomes being roughly similar. In total, the three genomes contain 148 of the approximately 300 known protein folds. Forty-five of these 148 that are present in all three genomes are especially enriched in mixed super-secondary structures (alpha/beta). Moreover, the five most common of these 45 (the "top-5") have a remarkably similar super-secondary structure architecture, containing a central sheet of parallel strands with helices packed onto at least one face and beta-alpha-beta connections between adjacent strands. These most basic molecular parts, which, presumably, were present in the last common ancestor to the three Kingdoms, include the TIM-barrel, Rossmann, flavodoxin, thiamin-binding, and P-loop-hydrolase folds.

Archaea↗
PubMed1993

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: an alternative method for generating a high-resolution solution structure.

A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTATAATG).d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of < 0.3 A between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD < 0.5 A) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.

Base Composition↗
PubMed1993

The three-dimensional structure of acyl-coenzyme A binding protein from bovine liver: structural refinement using heteronuclear multidimensional NMR spectroscopy.

The 3D structure of bovine recombinant acyl-coenzyme A binding protein has been determined using multidimensional heteronuclear magnetic resonance spectroscopy in a study that combines investigations of 15N-labeled and unlabeled protein. The present structure determination is a refinement of the structure previously determined (Andersen, K.V. and Poulsen, F.M. (1992) J. Mol. Biol., 226, 1131-1141). It is based on 1096 distance restraints and 124 dihedral angle restraints of which 69 are for phi-angles and 8 for chiral centers and 47 for prochiral centers. The new experimental input for the structure determination has provided an increase of 263 distance restraints, 5 phi-angle restraints, and 32 chi-angle restraints in 2 chiral centers, and 31 prochiral centers restraining an additional 23 chi 1, 8 chi 2, and 1 chi 3 angles. The increase of 300 distance and dihedral angle restraints representing an additional 30% of input parameters for the structure determination has been shown to be in agreement with the first structure. A set of 29 structures has been calculated and each of the structures has been compared to a mean structure to give an atomic root mean square deviation of 0.44 +/- 0.12 A (1 A is 0.1 nm) for the backbone atoms C, C alpha, and N in the four alpha-helices A1, residues 4-15, A2, residues 21-36, A3, residues 51-62 and A4, residues 65-84. The loop-region of residues Gly45-Lys50 could not be defined by the restraints obtained by NMR. The program PRONTO has been used for the spectrum analysis, assignment of the individual nuclear Overhauser effects, the integration of the cross peaks, and the measurement of the coupling constants. The programs DIANA, X-PLOR and INSIGHT have been used in the structure calculations and evaluations.

Amino Acid Sequence↗
PubMed1989

Structure of the L-leucine-binding protein refined at 2.4 A resolution and comparison with the Leu/Ile/Val-binding protein structure.

The three-dimensional X-ray structure of the leucine-binding protein (36,900 Mr and 346 residues), an active transport component of Escherichia coli, has been determined by the method of molecular replacement, using the refined structure of the Leu/Ile/Val-binding protein (344 residues) as the model structure. The two amino acid-binding proteins have 80% sequence identity and, although both crystallize in the same space group, they have very different unit cell dimensions. The rotation function yielded one significant peak, which subsequently led to a single self-consistent translation function solution. The model was first refined by the constrained least-squares method, with each of the two domains of the molecule treated separately to allow for any small change in the relative orientation of the two domains. The model was then modified in order to reflect the 72 changes in amino acid side-chains and two insertions in going from the Leu/Ile/Val-binding protein sequence to that of the L-leucine-binding protein. Final structure refinement, using the restrained least-squares technique, resulted in an R-factor of 0.20 for 13,797 reflections to a resolution of 2.4 A. The model is comprised of 2600 protein atoms and 91 solvent molecules. The L-leucine-binding protein structure is, as expected, very similar to the Leu/Ile/Val-binding protein structure; both are in the unliganded conformation with the cleft between the two domains wide open and easily accessible. The superimposing of the structures yields a root-mean-square difference of 0.68 A in the alpha-carbon atoms of the 317 equivalent residues. The five regions of the leucine-binding protein structure that differ by more than 1.6 A from the Leu/Ile/Val-binding protein structure are far from the major portion of the ligand-binding site, which is located in one domain of the bilobate protein. Between the structures, there are three differences in the amino acid side-chains that form the major portion of the substrate-binding sites. These substitutions, by themselves, fail to clearly explain the differences in the specificities for branched aliphatic amino acids.

Amino Acid Sequence↗
PubMed1992

Determination of a high-quality nuclear magnetic resonance solution structure of the bovine pancreatic trypsin inhibitor and comparison with three crystal structures.

A high-quality three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution was determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and compared to the three available high-resolution X-ray crystal structures. A newly collected input of 642 distance constraints derived from nuclear Overhauser effects and 115 dihedral angle constraints was used for the structure calculations with the program DIANA, followed by restrained energy minimization with the program AMBER. The BPTI solution structure is represented by a group of 20 conformers with an average root-mean-square deviation (RMSD) relative to the mean solution structure of 0.43 A for backbone atoms and 0.92 A for all heavy atoms of residues 2 to 56. The pairwise RMSD values of the three crystal structures relative to the mean solution structure are 0.76 to 0.85 A for the backbone atoms and 1.24 to 1.33 A for all heavy atoms of residues 2 to 56. Small local differences in backbone atom positions between the solution structure and the X-ray structures near residues 9, 25 to 27, 46 to 48 and 52 to 58, and conformational differences for individual amino acid side-chains were analyzed for possible correlations with intermolecular protein-protein contacts in the crystal lattices, using the pairwise RMSD values among the three crystal structures as a reference.

Amino Acid Sequence↗
PubMed1992

Determination of the nuclear magnetic resonance solution structure of the DNA-binding domain (residues 1 to 69) of the 434 repressor and comparison with the X-ray crystal structure.

The DNA-binding domain of the phage 434 repressor consisting of N-terminal residues 1 to 69 (434 repressor(1-69)), was expressed in Escherichia coli with natural isotope abundance, uniform 15N-labeling and biosynthetically directed fractional 13C-labeling in extent of about 10%. With these protein preparations the three-dimensional structure was determined in solution. The techniques used were nuclear magnetic resonance (n.m.r.) spectroscopy for the collection of conformational constraints, calculation of the protein structure from the n.m.r. data with the program DIANA and structure refinements by restrained energy minimization with a modified version of the program AMBER. A group of 20 conformers characterizes a well-defined structure for residues 1 to 63, with an average of 0-6 A for the root-mean-square deviations (RMSD) calculated for the backbone atoms of the individual conformers relative to the mean co-ordinates. The spatial structure of C-terminal residues 64 to 69 is not defined by the n.m.r. data. The molecular architecture of the 434 repressor(1-69) in solution includes five alpha-helices extending from residues 2 to 13, 17 to 24, 28 to 35, 45 to 52 and 56 to 60, which enclose a well-defined hydrophobic core. The n.m.r. structure is closely similar to the reported crystal structure of the 434 repressor(1-69), with an RMSD value of 1.1 A for the backbone atoms of residues 1 to 63. Small differences between the two structures in regions of the first helix and the loop between helices 3 and 4 were analyzed relative to possible correlations with protein-protein contacts in the crystal lattice and the different milieus of pH and ionic strength in the crystals and n.m.r. samples. Further systematic comparisons of local conformational features indicated that there are correlations between amino acid types, local precision of the structure determination by both techniques and local differences between the structures in the crystals and in solution. Overall, hydrophobic residues are most precisely characterized and agree most closely in the two environments.

Amides↗
PubMed1998

The structural basis of ankyrin-like repeat function as revealed by the solution structure of myotrophin.

BACKGROUND: Myotrophin is a 12.5 kDa protein that appears to have a key role in the initiation of cardiac hypertrophy, a central process in many heart diseases. Myotrophin primarily comprises ankyrin-like (ANK) repeats, the 33 amino acid motifs involved in a wide range of protein-protein interactions. As a first step in the structure-based search for cardiac hypertrophy antagonists and in order to gain insight into the molecular basis of action of the ubiquitous and multifunctional ANK repeat motif, we have determined the solution structure of myotrophin using multidimensional heteronuclear NMR spectroscopy. RESULTS: The myotrophin structure determination was based on 2786 experimental NMR restraints, and the precision of the coordinates for the final 45 simulated-annealing structures is 0.43 A for the backbone atoms and 0.87 A for all atoms. The structure of myotrophin is well defined and is ellipsoidal: approximately 46 A long and 21 A wide. The ANK repeats, which constitute the main part of the myotrophin structure, are characteristic of a hairpin-like protruding tip followed by a helix-turn-helix motif. The V-shaped helix-turn-helix of the ANK repeats stack sequentially in bundles and are stabilized by compact hydrophobic cores, whereas the protruding tips are less ordered. This arrangement is quite different to the continuous beta-sheet topology observed in the corresponding regions of another ANK protein, 53BP2, the structure of which was determined in complex with p53. CONCLUSIONS: The solution structure of myotrophin provides important insights into the structural and dynamic features of the ANK motif, and suggests that the protruding tips with highly variable sequences may be critical to facilitate diverse protein-protein recognition. The present structure also provides a molecular basis for the further functional characterization of myotrophin and the development of therapeutics for hypertrophy-related heart diseases.

Amino Acid Sequence↗
PubMed1991

Modeling the three-dimensional structure of the monocyte chemo-attractant and activating protein MCAF/MCP-1 on the basis of the solution structure of interleukin-8.

A model of the three-dimensional structure of the monocyte chemo-attractant and activating protein MCAF/MCP-1 is presented. The model is predicted based on the previously determined solution structure of interleukin-8 (IL-8/NAP-1) [Clore, G.M., Appella, E., Yamada, M., Matsushima, K. and Gronenborn, A.M. (1990) Biochemistry 29, 1689-1696]. Both proteins belong to a superfamily of cytokine proteins involved in cell-specific chemotaxis, host defense and the inflammatory response. The amino acid sequence identity between the two proteins is 24%. It is shown that the regular secondary structure elements of the parent structure can be retained in the modeled structure, such that the backbone hydrogen bonding pattern is very similar in the two structures. The polypeptide backbone is superimposable with an atomic r.m.s. difference of 0.9 A and all side chains can be modeled by transferring the parent side chain conformation to the new structure. Thus, the deduced structure, like the parent one, is a dimer and consists of a six-stranded antiparallel beta-sheet, formed by two three-stranded Greek keys, one from each monomer, upon which lie two symmetry-related antiparallel alpha-helices, approximately 24 A long and separated by approximately 14 A. All amino acid sequence changes can be accommodated within the parent polypeptide framework without major rearrangements. This is borne out by the fact that the IL-8/NAP-1 and modeled MCAF/MCP-1 structures have similar non-bonding energies. These results strongly suggest that both proteins and all other members of the superfamily most likely have the same tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗