NMR-derived Distance Constraints Research Articles

Solution Structure of Prosurvival Mcl-1 and Characterization of Its Binding by Proapoptotic BH3-only Ligands

The B cell lymphoma-2 (Bcl-2) homologs myeloid cell leukemia-1 (Mcl-1) and A1 are prosurvival factors that selectively bind a subset of proapoptotic Bcl homology (BH) 3-only proteins. To investigate the molecular basis of the selectivity, we determined the solution structure of the C-terminal Bcl-2-like domain of Mcl-1. This domain shares features expected of a prosurvival Bcl-2 protein, having a helical fold centered on a core hydrophobic helix and a surface-exposed hydrophobic groove for binding its cognate partners. A number of residues in the binding groove differentiate Mcl-1 from its homologs, and in contrast to other Bcl-2 homologs, Mcl-1 has a binding groove in a conformation intermediate between the open structures characterized by peptide complexes and the closed state observed in unliganded structures. Mutagenesis of potential binding site residues was used to probe the contributions of groove residues to the binding properties of Mcl-1. Although mutations in Mcl-1 had little impact on binding, a single mutation in the BH3-only ligand Bad enabled it to bind both Mcl-1 and A1 while retaining its binding to Bcl-2, Bcl-xL, and Bcl-w. Elucidating the selective action of certain BH3-only ligands is required for delineating their mode of action and will aid the search for effective BH3-mimetic drugs.

Exploration of the conformational space of oxytocin and arginine-vasopressin using the electrostatically driven Monte Carlo and molecular dynamics methods.

Conformational analysis of the neurohypophyseal hormones oxytocin (OT) and arginine-vasopressin (AVP) has been carried out using two different computational approaches and three force fields, namely by the Electrostatically Driven Monte Carlo (EDMC) method, with the Empirical Conformational Energy Program for Peptides (ECEPP/3) force field or with the ECEPP/3 force field plus a hydration-shell model, and by simulated-annealing molecular dynamics with the Consistent Valence Force Field (CVFF). The low-energy conformations obtained for both hormones were classified using the minimal-tree clustering algorithm and characterized according to the locations of beta-turns in the cyclic moieties. Calculations with the CVFF force field located conformations with a beta-turn at residues 3 and 4 as the lowest energy ones both for OT and for AVP. In the ECEPP/3 force field the lowest energy conformation of OT contained a beta-turn at residues 2 and 3, conformations with this location of the turn being higher in energy for AVP. The latter difference can be attributed to the difference in the size of the side chain in position 3 of the sequences: the bulkier phenylalanine residue of AVP in combination with the bulky Tyr2 residue hinders the formation of a turn at residues 2 and 3. Conformations of OT and AVP with a turn at residues 3,4 were in the best agreement with the x-ray structures of deaminooxytocin and pressinoic acid (the cyclic moiety of vasopressin), respectively, and with the nmr-derived distance constraints. Generally, the low-energy conformations obtained with the hydration-shell model were in a better agreement with the experimental data than the conformations calculated in vacuo. It was found, however, that the obtained low-energy conformations do not satisfy all of the nmr-derived distance constraints and the nuclear Overhauser effect pattern observed in nmr studies can be fully explained only by assuming a dynamic equilibrium between conformations with beta-turns at residues 2,3, 3,4, and 4,5. The low-energy structures of OT with a beta-turn at residues 2,3 have the disulfide ring conformations close to the model proposed recently for a potent bicyclic antagonist of OT [M. D. Shenderovich et al. (1994) Polish Journal of Chemistry, Vol. 25, pp. 921-927], although the native hormone differs from the bicyclic analogue by the conformation of the C-terminal tripeptide. This finding confirms the hypothesis of different receptor-bound conformations of agonists and antagonists of OT.

Three-dimensional structure of moenomycin A--a potent inhibitor of penicillin-binding protein 1b.

The first three-dimensional structure of moenomycin A in aqueous solution based on NMR-derived distance constraints and molecular dynamics simulations is presented. The antibiotic moenomycin A was obtained from the FlavomycinR complex by ultrafiltration, chromatography on a DEAE-cellulose ion exchanger and reverse-phase chromatography in 98% purity. In contrast to the previously reported behaviour, the compound gave rise to well-resolved NMR spectra in standard solvents. Using several two-dimensional experiments, a complete assignment of proton and carbon chemical shifts was achieved in (CD3)2SO, CD3OD and H2O/D2O (9:1, pH 7.3). A total of 175 interproton distances were determined from 600-MHz rotating-frame NOE (ROE) spectra and were used as restraints in molecular dynamics calculations. These restraints included 84 ROEs between protons of the moenocinol part leading to a very well-defined structure of the lipid part of the molecule. The relative orientation of the subunits was determined by 66 ROEs among different sugar rings and between the sugar rings and moenocinol. As a result of the molecular dynamics calculation, rings D, E, and F as well as the moenocinol part are very well-defined (average rms deviation over all heavy atoms 0.48 A) whereas rings A, B and C display a higher degree of conformational flexibility which might be an artefact due to the lower number of ROEs in this part of the molecule. A three-dimensional pharmacophore hypothesis comprising functional groups of rings E and F and the carboxyl group of glyceric acid is presented on the basis of the degradation and derivatization studies of Welzel and coworkers [Welzel, P., Kunisch, F., Kruggel, F., Stein, H., Scherkenbeck, J., Hiltmann, A., Duddeck, H., Müller, D., Maggio, J. E., Fehlhaber, H.-W., Seibert, G., van Heijenoort, Y. & van Heijenoort, J. (1987) Moenomycin A: Minimum structural requirements for biological activity, Tetrahedron 43, 585-598; Welzel, P. (1993) Transglycosylase inhibition, in Antibiotics and antiviral compounds--Chemical synthesis and modifications (Krohn, K., Kirst, H. A. & Maag, H., eds) pp. 373-378, VCH Weinheim].

Simulated annealing with restrained molecular dynamics using a flexible restraint potential: theory and evaluation with simulated NMR constraints.

A new functional representation of NMR-derived distance constraints, the flexible restraint potential, has been implemented in the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137-168) for molecular structure generation. In addition, flat-bottomed restraint potentials for representing dihedral angle and vicinal scalar coupling constraints have been introduced into CONGEN. An effective simulated annealing (SA) protocol that combines both weight annealing and temperature annealing is described. Calculations have been performed using ideal simulated NMR constraints, in order to evaluate the use of restrained molecular dynamics (MD) with these target functions as implemented in CONGEN. In this benchmark study, internuclear distance, dihedral angle, and vicinal coupling constant constraints were calculated from the energy-minimized X-ray crystal structure of the 46-amino acid polypeptide crambin (ICRN). Three-dimensional structures of crambin that satisfy these simulated NMR constraints were generated using restrained MD and SA. Polypeptide structures with extended backbone and side-chain conformations were used as starting conformations. Dynamical annealing calculations using extended starting conformations and assignments of initial velocities taken randomly from a Maxwellian distribution were found to adequately sample the conformational space consistent with the constraints. These calculations also show that loosened internuclear constraints can allow molecules to overcome local minima in the search for a global minimum with respect to both the NMR-derived constraints and conformational energy. This protocol and the modified version of the CONGEN program described here are shown to be reliable and robust, and are applicable generally for protein structure determination by dynamical simulated annealing using NMR data.

Conformation of CD4‐derived cyclic hexapeptides by nmr and molecular dynamics

Two cyclic hexapeptides, cyclo[Ala1-D-Ala2-Ser3-Phe4-Gly5-Ser6] and cyclo[Ala1-Gly2-Ser3-Phe4-Gly5-Ser6], derived from the loop portion of the C'C" ridge of CD4, were characterized by high-resolution nmr spectroscopy and simulated annealing studies. In DMSO-d6 both of these peptides display a single conformer on the nmr time scale with two intramolecular H-bond (1<--4) stabilized beta-turns at positions 2-3 and 5-6. The nmr derived distance constraints were used in simulated annealing calculations to generate the solution structures. These structures adopt energetically comparable conformational substates that are not resolvable on the nmr time scale. In aqueous solution, the H-bond stabilized beta-turn conformation for cyclo[Ala-D-Ala-Ser-Phe-Gly-Ser] is no longer the predominant structural form. Structures generated using molecular dynamics simulations with no experimental constraints were compared with those from nmr analysis. The correlation between these two sets of structures allows the use of molecular simulations as a predictive tool for the conformational analysis of small peptides.

G protein-bound conformation of mastoparan-X, a receptor-mimetic peptide.

Mastoparans are a family of 14-residue peptide toxins from wasp venom which have been proposed to stimulate secretion from a variety of cells, by directly activating GTP-binding regulatory proteins (G proteins). In vitro studies have shown that mastoparans activate G proteins by a mechanism remarkably similar to that used by agonist-bound receptors (Higashijima, T., Uzu, S., Nakajima, T., and Ross, E. M. (1988) J. Biol. Chem. 263, 6491-6494). Here, we report the conformation of mastoparan-X (INWKGIAAMAKKLL-NH2) when it is bound to the alpha subunits of recombinant G(i) and G(o), derived from an analysis of transferred nuclear Overhauser effects in a two-dimensional 1H NMR spectrum of mastoparan-X obtained in the presence of these G proteins. Restrained molecular dynamic simulations with NMR-derived distance constraints were used to determine conformations consistent with NMR data. The G(i)- and G(o)-bound conformations of mastoparan-X are very similar, and in both cases, a major part of the molecule adopts an amphiphilic alpha-helical conformation. The lysine residues are known to be crucial for activity, and it is thus likely that at least the polar face of the amphiphilic helix is in contact with the G proteins. These conclusions should be useful in the design of potent and selective analogs of mastoparan and in the development of models for receptor-G protein interaction.

Analysis of side‐chain conformational distributions in neutrophil peptide‐5 nmr structures

The side-chain conformations have been analyzed in the antimicrobial peptide, Neutrophil Peptide-5 (NP-5), whose structure was independently generated from nmr-derived distance constraints using a distance geometry algorithm. The side-chain and peptide dihedral angle distributions in the nmr structures were compared with those constructed from a data base of high-resolution protein crystal structures. The side-chain conformational preferences for NP-5 in solution are significantly different from those observed in the crystal structure data base. These results indicate that the side-chain conformations are quite disordered for many of the residues of NP-5. The absence of a correlation between the width of the conformational distribution and surface accessibility suggests that the disorder may be due to limitations in the structural information extracted from the nmr data rather than to molecular motion. However, it is also observed that the degree of conformational disorder is only weakly correlated with the number of nuclear Overhauser enhancements to a given side chain. Possible reasons for this are discussed. Molecular mechanics refinement of these structures did not significantly change the side-chain populations. Anomolously wide distributions are observed for rotations about the peptide bonds and the disulfide bonds in the NP-5 distance geometry structures, which are improved by the refinement. The very high degree of order observed for the central dihedral angle of the disulfide bond in the high-resolution crystal data base suggests that the rotation about this bond in proteins is determined by the local potential.

Three-dimensional 13C-resolved proton NOE spectroscopy of uniformly 13C-labeled proteins for the NMR assignment and structure determination of larger molecules

The currently accepted protocol for the determination of the solution structures of proteins by NMR consists of the assignment of the ‘H resonances, the identification of NOE distance constraints on the basis of the assignments, and the computation of the three-dimensional structure using the NMR-derived distance constraints (for a review, see Ref. (I)). Recently, by the use of isotope-edited two-dimensional NMR experiments on selectively labeled proteins (2-6)) or heteronuclear three-dimensional NMR experiments ( 7) on proteins that have been uniformly labeled with i5N (8-I]), the complete assignments of the backbone protons for proteins up to MW 20,000 have become possible. However, in order to determine the three-dimensional structures of these larger proteins, distance constraints derived from long-range NOES between (assigned) aliphatic protons are necessary. These NOES are very difficult if not impossible to obtain from conventional 2D NOESY spectra due to extensive overlap in the aliphatic region of the spectra. Thus, although the (backbone) proton NMR signals may be assigned for larger systems such as T4 lysozyme and staphylococcal nuclease (5, 6)) the 3D structures cannot be determined. Here, we describe an approach to resolve the spectral overlap in the aliphatic part of the NOESY data. The method involves the use of 13C heteronuclear three-dimensional NMR spectroscopy in which the NOESY spectra are simplified by editing with respect to the 13C frequencies. In this study, a sample of 3.8 mM T4 lysozyme ( 164 amino acid residues, 18.7 kDa) that was uniformly enriched with both 13C and 15N at a level greater than 95% was employed. The pulse sequence used was

Three-dimensional structure of mouse epidermal growth factor determined by two-dimensional NMR.

The three-dimensional structures of mouse epidermal growth factor (53 residues) in pH 2.1 and pH 6.8 solutions were determined by two-dimensional NMR and distance geometry calculation. Ten structures that satisfied the NMR-derived distance constraints were obtained for each pHs using XPLOR program. The topology of the main chain was identical at both pHs, but the global shape at pH 6.8 was found to be more compact than that at pH 2.1.