NMR-derived Distance Restraints Research Articles

NAExplor: A Software Tool for Managing and Analyzing Nucleic Acid Structure Files

NAExplor is a software tool for converting coordinates files between the software packages AMBER, CHARMM, and XPLOR. In addition, it manages the conversion of NMR-derived distance restraints information from the MARDIGRAS program into the appropriate file formats used for input in AMBER, CHARMM, and XPLOR. Analyses of H-H distances in nucleic acid structures and calculations of torsion angles for nucleic acid backbone and riboses are also possible.

NMR studies of a viral protein that mimics the regulators of complement activation

Vaccinia virus complement control protein (VCP) is a 243-residue protein that is similar in sequence to the regulators of complement activation; its role is to defend the virus against attack by the host complement system. A fragment of this protein spanning the two complement protein (CP)-modules (residues 126 to 243) which make up the C-terminal half of VCP has been expressed in Pichia pastoris. A 15N-labelled sample was purified for the purposes of structure determination and measurements of dynamics in solution using NMR. Structures were calculated on the basis of 1767 NMR-derived distance and angle restraints, with a longer than normal high-temperature simulated annealing (SA) protocol which improved convergence. The viral CP-modules are structurally very similar to the 15th and 16th CP-modules of human factor H (fH; average r.m.s.d., for invariant Trp and Cys, four pair-wise comparisons,=1.2 Å) but less similar to the fifth CP-module of fH (average r.m.s.d.=2.2 Å). In the VCP fragment, the orientation of one module with respect to the other is clearly defined by the experimental data, and T 1 measurements are consistent with only limited flexibility at the module-module interface. The r.m.s.d. over all of the 118 residues (backbone atoms) is 0.73 Å. The intermodular orientation is better defined than, and significantly different from, that observed in a CP-module pair from fH (re-calculated using the extended SA protocol). In VCP the long axis of the second module is tilted by 59(±4)° with respect to the first module (50(±13)° in the fH pair), and twisted with respect to the first module by 22(±6)° (223(±17)° in fH). The differences between the human and viral proteins may be rationalised in terms of the lack of hydrogen-bond stabilised secondary structure in the N-terminal portion of fH module 16, and the number and type of amino acid side-chains which make up the interface. A similar intermodular interface may be predicted between the third and fourth module of human C4 binding protein and, probably, between the third and fourth modules of the guinea pig acrosomal matrix protein 67; but the formulation of general rules for predicting the structure of interfaces between CP-modules awaits further experimental data.

Determination of the Solution Structures of Conantokin-G and Conantokin-T by CD and NMR Spectroscopy

Conantokin-G and conantokin-T are two paralytic polypeptide toxins originally isolated from the venom of the fish-hunting cone snails of the genus Conus. Conantokin-G and conantokin-T are the only naturally occurring peptidic compounds which possess N-methyl-D-aspartate receptor antagonist activity, produced by a selective non-competitive antagonism of polyamine responses. They are also structurally unusual in that they contain a disproportionately large number of acid labile post-translational gamma-carboxyglutamic acid (Gla) residues. Although no precise structural information has previously been published for these peptides, early spectroscopic measurements have indicated that both conantokin-G and conantokin-T form alpha-helical structures, although there is some debate whether the presence of calcium ions is required for these peptides to adopt this fold. We now report a detailed structural study of synthetic conantokin-G and conantokin-T in a range of solution conditions using CD and 1H NMR spectroscopy. The three-dimensional structures of conantokin-T and conantokin-G were calculated from 1H NMR-derived distance and dihedral restraints. Both conantokins were found to contain a mixture of alpha- and 310 helix, that give rise to curved and straight helical conformers. Conantokin-G requires the presence of divalent cations (Zn2+, Ca2+, Cu2+, or Mg2+) to form a stable alpha-helix, while conantokin-T adopts a stable alpha-helical structure in aqueous conditions, in the presence or absence of divalent cations (Zn2+, Ca2+, Cu2+, or Mg2+).

Solution structure of the DNA-binding domain of a human papillomavirus E2 protein: evidence for flexible DNA-binding regions.

The three-dimensional structure of the DNA-binding domain of the E2 protein from human papillomavirus-31 was determined by using multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. A total of 1429 NMR-derived distance and dihedral angle restraints were obtained for each of the 83-residue subunits of this symmetric dimer. The average root mean square deviations of 20 structures calculated using a distance geometry-simulated annealing protocol are 0.59 and 0.90 angstroms for the backbone and all heavy atoms, respectively, for residues 2-83. The structure of the human virus protein free in solution consists of an eight-stranded beta-barrel and two pairs of alpha-helices. Although the overall fold of the protein is similar to the crystal structure of the bovine papillomavirus-1 E2 protein when complexed to DNA, several small but interesting differences were observed between these two structures at the subunit interface. In addition, a beta-hairpin that contacts DNA in the crystal structure of the protein-DNA complex is disordered in the NMR structures, and steady-state 1H-15N heteronuclear NOE measurements indicate that this region is highly mobile in the absence of DNA. The recognition helix also appears to be flexible, as evidenced by fast amide exchange rates. This phenomenon has also been observed for a number of other DNA-binding proteins and may constitute a common theme in protein/DNA recognition.

Predictability of designing specific binding interactions for DNA minor groove ligands from NMR spectroscopy and molecular modeling: a copper(II)-activated DNA cleaver based on Hoechst 33258.

Analogs of Hoechst 33258 have been designed and synthesized to incorporate a 3,4-catecholic or a 3,4,5-trihydroxyphenyl function in place of the 4-phenolic group of the template molecule. Molecular design was based on the three-dimensional models of the solution structures of the Hoechst 33258 and its m-hydroxy isomer which had been derived from high-field NMR spectroscopic analysis. The predicted solution structure of the catecholic analog as its minor groove complex with duplex d(CGCGAATTCGCG)2 was confirmed by direct high-resolution NMR spectroscopy combined with molecular dynamics, using NMR-derived distance restraints. While the 3,4-catecholic analog was unable to cleave DNA in the presence of Cu(II) ions, the 3,4,5-trihydroxyphenyl analog of Hoechst 33258 was found to be an effective cleaver of DNA at low concentration when activated by copper(II) ions, a difference ascribed to the inaccessibility for copper chelation of the 3,4-dihydroxy site in its minor groove complex with DNA.

Solution structure of a band 3 peptide inhibitor bound to aldolase: a proposed mechanism for regulating binding by tyrosine phosphorylation.

Human erythrocyte band 3 inhibits glycolytic enzymes, including aldolase, by binding these cytoplasmic enzymes at its N-terminus. Phosphorylation of Y8 disrupts inhibition, and there is evidence that in vivo glycolysis levels in erythrocytes are regulated in part by a phosphorylation/dephosphorylation signaling pathway. The structural basis for control by phosphorylation has been investigated by NMR studies on a complex between aldolase and a synthetic peptide corresponding to the first 15 residues of band 3 (MEELQDDYEDMMEEN-NH2). The structure of this band 3 peptide (B3P) when it is bound to rabbit muscle aldolase was determined using the exchange-transferred nuclear Overhauser effect (ETNOE). Two hundred NMR structures for B3P were generated by simulated annealing molecular dynamics with NMR-derived distance restraints and excluding electrostatic terms. Twenty structures were further refined against a force field including full partial charges. The important conformational feature of B3P in the bound state is a folded loop structure involving residues 4-9 and M12 that surrounds Y8 and is stabilized by a hydrophobic cluster with the ring of Y8 sandwiched between the methyl groups of L4 and M12. Differential line broadening indicates that this loop structure binds aldolase in a relatively specific manner, while terminal regions are structurally heterogeneous. To better understand B3P inhibition of aldolase and the mechanism of phosphorylation control, a complex was modeled by docking B3P into the active site of aldolase and optimizing the fit using restrained molecular dynamics and energy minimization. The B3P loop is complementary in conformation to the beta-barrel central core containing the aldolase active site residues. Binding is electrostatic in nature with numerous ionic and hydrogen-bonding interactions involving several conserved lysine and arginine residues of aldolase. How phosphorylation of band 3 could disrupt inhibition was considered by modeling a phosphoryl moiety onto Y8 of B3P. An energetic analysis with respect to rigid phosphate rotation suggests that aldolase inhibition is reversed primarily because of electrostatic repulsion between B3P residues that destabilizes the B3P loop formed in the complex. This proposed intramolecular mechanism for blocking protein--protein association by electrostatic repulsion with the phosphoryl group may be applicable to other protein--protein signaling complexes.

Three-dimensional solution structure of the N-terminal receiver domain of NTRC.

NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a member of the family of receiver domains of two-component regulatory systems. Using 3D and 4D NMR spectroscopy, we have completed the 1H, 15N, and 13C assignments and determined the solution structure of the N-terminal receiver domain of the NTRC protein. Determination of the three-dimensional structure was carried out with the program X-PLOR (Brünger, 1992) using a total of 915 NMR-derived distance and dihedral angle restraints. The resultant family of structures has an average root mean square deviation of 0.81 A from the average structure for the backbone atoms involved in well-defined secondary structure. The structure is comprised of five alpha-helices and a five-stranded parallel beta-sheet, in a (beta/alpha)5 topology. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY in both the Mg(2+)-free and Mg(2+)-bound forms [Stock, A.M., Mottonen, J. M., Stock, J. B., & Schutt, C. E. (1989) Nature 337, 745-749; Volz, K., & Matsumura, P. (1991) J. Biol. Chem. 296, 15511-15519; Stock, A. M., Martinez-Hackert, E., Rasmussen, B. F., West, A. H., Stock, J. B., Ringe, D., & Petsko, G. A. (1993) Biochemistry 32, 13375-13380; Bellsolell, L., Prieto, J., Serrano, L., & Coll, M. (1994) J. Mol. Biol. 238, 489-495] reveals a very similar fold, with the only significant difference occurring in the positioning of helix 4 relative to the rest of the protein. Examination of the conformation of consensus residues of the receiver domain superfamily [Volz, K. (1993) Biochemistry 32, 11741-11753] in the structures of the NTRC receiver domain and CheY establishes the structural importance of residues whose side chains are involved in hydrogen bonding or hydrophobic core interactions. The importance of some nonconsensus residues which may be conserved for their ability to fulfill helix capping roles is also discussed.

Three-dimensional Structure of the Human Immunodeficiency Virus Type 1 Matrix Protein

The HIV-1 matrix protein forms an icosahedral shell associated with the inner membrane of the mature virus. Genetic analyses have indicated that the protein performs important functions throughout the viral life-cycle, including anchoring the transmembrane envelope protein on the surface of the virus, assisting in viral penetration, transporting the proviral integration complex across the nuclear envelope, and localizing the assembling virion to the cell membrane. We now report the three-dimensional structure of recombinant HIV-1 matrix protein, determined at high resolution by nuclear magnetic resonance (NMR) methods. The HIV-1 matrix protein is the first retroviral matrix protein to be characterized structurally and only the fourth HIV-1 protein of known structure. NMR signal assignments required recently developed triple-resonance ( 1H, 13C, 15N) NMR methodologies because signals for 91% of 132 assigned H a protons and 74% of the 129 assignable backbone amide protons resonate within chemical shift ranges of 0.8 p.p.m. and 1 p.p.m., respectively; A total of 636 nuclear Overhauser effect-derived distance restraints were employed for distance geometry-based structure calculations, affording an average of 13.0 NMR-derived distance restraints per residue for the experimentally constrained amino acids. An ensemble of 25 refined distance geometry structures with penalties (sum of the squares of the distance violations) of 0.32 Å 2 or less and individual distance violations under 0.06 Å was generated; best-fit superposition of ordered backbone heavy atoms relative to mean atom positions afforded root-mean-square deviations of 0.50 (±0.08) Å. The folded HIV-1 matrix protein structure is composed of five α-helices, a short 3 10 helical stretch, and a three-strand mixed β-sheet. Helices I to III and the 3 10 helix pack about a central helix (IV) to form a compact globular domain that is capped by the β-sheet. The C-terminal helix (helix V) projects away from the β-sheet to expose carboxyl-terminal residues essential for early steps in the HIV-1 infectious cycle. Basic residues implicated in membrane binding and nuclear localization functions cluster about an extruded cationic loop that connects β-strands 1 and 2. The structure suggests that both membrane binding and nuclear localization may be mediated by complex tertiary structures rather than simple linear determinants.

Solution structure of the TR1C fragment of skeletal muscle troponin-C.

Residues 12-87 (TR1C fragment) of turkey skeletal muscle troponin-C comprises two helix-loop-helix calcium-binding motifs which are the regulatory calcium-binding sites in the N-terminal domain of the protein. We have used the combined distance geometry-simulated annealing protocol DGII (Havel, T. F. (1991) Prog. Biophys. Mol. Biol. 56, 43-78) to determine the structure of this 76-residue polypeptide in solution from 475 1H NMR-derived distance restraints. The nuclear Overhauser enhancement-derived distance constraints used in the DGII protocol were supplemented by introducing generic hydrogen bond distance restraints for slowly exchanging amide hydrogens in regular secondary structure elements, by restricting the available phi angle space to -180 degrees to 0 degrees for all residues except glycines, and by tailoring the distance boundaries used for quantitating the nuclear Overhauser enhancement intensities to correspond to characteristic distances found in helices. This improved the geometry of the four helices in the resulting structures. The relative positions of helices A and B which flank calcium-binding loop 1, helix D which follows calcium-binding loop 2, and the beta-sheet between the two calcium-binding loops were well defined and had an overall root-mean-square deviation for 20 converged structures of 1.4 +/- 0.2 A for backbone atoms. The structure and relative orientations of these regions are very similar to these of the corresponding regions of the protein in the crystal structure of intact turkey skeletal troponin C (Herzberg, O., and James, M. N. G. (1988) Nature 313, 653-659). The structure of helix C was well defined, but its relative position to the other helices was not defined. It occupied a range of positions in the set of 20 DGII structures, the average of which was quite similar to the orientation of helix C in the x-ray structure. The overall structure of the apo regulatory domain of troponin-C is therefore not affected by the loss of the N-helix, or the low pH conditions used for the x-ray structure, but may be more flexible in regions known to be involved in contacts with other skeletal muscle regulatory proteins.

Solution structure by 1H and dynamics by natural abundance 13C NMR of a receptor recognising peptide derived from a C-terminal fragment of neuropeptide Y

A peptide consisting of 20 amino acid residues, derived from a C-terminal fragment of neuropeptide Y (NPY) and showing high affinity to NPY receptors, was synthesised. Its sequence is PAADLARYRHYINLITRQRY-NH2, and the solution structure was calculated from NMR-derived distance and torsion angle restraints, obtained at 15 degrees C in a solvent mixture of water and 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, by using DIANA and restrained energy minimisation. The structure was found to consist of a well-defined alpha-helix in the centre, with a few residues at the termini having less well defined conformations. The spin-lattice and spin-spin relaxation rates of alpha-carbons have been determined on 13C at natural abundance. From 1D experiments the global rotational correlation time was determined and from 2D experiments the dynamics of each individual residue was obtained. The results demonstrate that the C alpha-H alpha vectors in the alpha-helix essentially follow the global motion. Towards the termini, contributions from local dynamics increase. This tendency is correlated to the increasing uncertainty of the structure towards the peptide ends. An effective molecular volume was calculated from the temperature dependence of the global rotational correlation time. This is well compatible with a monomeric peptide, solvated by water and 1,1,1,3,3,3-hexafluoro-2-propanol. The presence of peptide dimers was ruled out as being inconsistent with the relaxation data.

Molecular Dynamics with Weighted Time-averaged Restraints for a DNA Octamer: Dynamic Interpretation of Nuclear Magnetic Resonance Data

For conformationally flexible molecules in solution, NMR-derived distance restraints are time-averaged. In contrast to deriving structures from NMR distance constraints via conventional restrained molecular dynamics (rMD), the range of conformational flexibility may be better represented by MD simulations using weighted time-averaged restraints (MD-tar). This approach has been utilized for dynamic structure refinement of the DNA octamer [d(GTATAATG] · [d(CATATTAC)] which contains the Pribnow box consensus sequence. An improved set of distance restraints was calculated via complete relaxation matrix analysis utilizing the solution structure of the octamer which was determined previously by rMD as a starting model. MD-tar calculations were performed with the program AMBER4 under various conditions, some including explicit solvent. All trajectories generated via MD-tar exhibited reduced constraint energies and average deviations for the distances compared to standard rMD. Quantitative comparison with experimental data, i.e. two-dimensional NOE intensities and COSY-derived coupling constants yielded a significant improvement for MD-tar simulated structures relative to rMD-derived structures. The conformational envelope of the MD-tar structures is wider than that from rMD and even unrestrained MD. Explicit solvent force-fields tightened the conformational envelope, leading to even better agreement with experimental data. All MD-tar simulations exhibit sugar repuckering for basically all residues, yielding a minor population in the low N-region and one or more S-conformers. For most backbone torsion angles, one or more minor conformers were found, while the major conformations generally coincided with those obtained in standard rMD. Distributions of helical parameters for MD tar trajectories are rather symmetric but slightly broader than those for rMD. Average values and associated standard deviations are discussed with respect to sequence-dependent variations. All trajectories obtained with an explicit solvent force field exhibited a narrower minor groove compared to in vacuo calculations.

Solution structure of cyclosporin A and a nonimmunosuppressive analog bound to fully deuterated cyclophilin.

A simple strategy involving 1H nuclear magnetic resonance (NMR) spectroscopy and complete protein deuteration was used to determine the structures of two receptor-bound drugs. A potent immunosuppressive, cyclosporin A (CsA) binds tightly to the ubiquitous and highly conserved 17.7-kDa immunophilin, cyclophilin (CyP). Fully deuterated CyP was produced by overexpressing the human CyP gene in Escherichia coli grown on deuterated algal hydrolysate in 98% D2O. As only the CsA molecule is protonated in the CsA-CyP complex, we were able to make a complete sequential assignment of the bound drug using standard two-dimensional proton NMR experiments. The structure determination was accomplished using dynamical simulated annealing calculations with a total of 124 NMR-derived distance and torsion angle restraints. Aside from binding CsA, CyP also acts as a peptidyl-prolyl cis-trans isomerase. Thus, much importance had been ascribed to the cis peptide bond present in the structures reported for free CsA in organic solvents and in crystal studies. Interestingly, CyP-bound CsA exists in an all-trans conformation with no detectable elements of regular secondary structure and no intramolecular hydrogen bonds. A nonactive CsA analog, MeAla6-CsA, was studied using the same CyP deuteration strategy. In addition to structural elucidation of the two bound drugs, we were able to differentiate between the bound and surface-exposed residues of the drugs and also validate our previous hypothesis that the single CyP tryptophan is located in the CsA-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Induced peptide conformations in different antibody complexes: molecular modeling of the three-dimensional structure of peptide-antibody complexes using NMR-derived distance restraints.

Intramolecular interactions in bound cholera toxin peptide (CTP3) in three antibody complexes were studied by two-dimensional transferred NOE spectroscopy. These measurements together with previously recorded spectra that show intermolecular interactions in these complexes were used to obtain restraints on interproton distances in two of these complexes (TE32 and TE33). The NMR-derived distance restraints were used to dock the peptide into calculated models for the three-dimensional structure of the antibody combining site. It was found that TE32 and TE33 recognize a loop comprising the sequence VPGSQHID and a beta-turn formed by the sequence VPGS. The third antibody, TE34, recognizes a different epitope within the same peptide and a beta-turn formed by the sequence IDSQ. Neither of these two turns was observed in the free peptide. The formation of a beta-turn in the bound peptide gives a compact conformation that maximizes the contact with the antibody and that has greater conformational freedom than alpha-helix or beta-sheet secondary structure. A total of 15 antibody residues are involved in peptide contacts in the TE33 complex, and 73% of the contact area in the antibody combining site consists of the side chains of aromatic amino acids. A comparison of the NMR-derived models for CTP3 interacting with TE32 and TE33 with the previously derived model for TE34 reveals a relationship between amino acid sequence and combining site structure and function. (a) The three aromatic residues that interact with the peptide in TE32 and TE33 complexes, Tyr 32L, Tyr 32H, and Trp 50H, are invariant in all light chains sharing at least 65% identity with TE33 and TE32 and in all heavy chains sharing at least 75% identity with TE33. Although TE34 differs from TE32 and TE33 in its fine specificity, these aromatic residues are conserved in TE34 and interact with its antigen. Therefore, we conclude that the role of these three aromatic residues is to participate in nonspecific hydrophobic interactions with the antigen. (b) Residues 31, 31c, and 31e of CDR1 of the light chain interact with the antigen in all three antibodies that we have studied. The amino acids in these positions in TE34 differ from those in TE32 and TE33, and they are involved in specific polar interactions with the antigen. (c) CDR3 of the heavy chain varies considerably both in length and in sequence between TE34 and the two other anti-CTP3 antibodies. These changes modify the shape of the combining site and the hydrophobic and polar interactions of CDR3 with the peptide antigen.

NMR-derived model for a peptide-antibody complex

The TE34 monoclonal antibody against cholera toxin peptide 3 (CTP3; VEVPGSQHIDSQKKA) was sequenced and investigated by two-dimensional transferred NOE difference spectroscopy and molecular modeling. The VH sequence of TE34, which does not bind cholera toxin, shares remarkable homology to that of TE32 and TE33, which are both anti-CTP3 antibodies that bind the toxin. However, due to a shortened heavy chain CDR3, TE34 assumes a radically different combining site structure. The assignment of the combining site interactions to specific peptide residues was completed by use of AcIDSQRKA, a truncated peptide analogue in which lysine-13 was substituted by arginine, specific deuteration of individual polypeptide chains of the antibody, and a computer model for the Fv fragment of TE34. NMR-derived distance restraints were then applied to the calculated model of the Fv to generate a three-dimensional structure of the TE34/CTP3 complex. The combining site was found to be a very hydrophobic cavity composed of seven aromatic residues. Charged residues are found in the periphery of the combining site. The peptide residues HIDSQKKA form a beta-turn inside the combining site. The contact area between the peptide and the TE34 antibody is 388 A2, about half of the contact area observed in protein-antibody complexes.