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PubMed2004

Dual spatial folds and different local structures of the HIV-1 immunogenic crown in various virus isolates.

Local and global structural properties of the HIV-1 principal neutralizing epitope were studied in terms of NMR spectroscopy data reported in literature for the HIV-Haiti and HIV-RF isolates. To this effect, the NMR-based method comprising a probabilistic model of protein conformation in conjunction with the molecular mechanics and quantum chemical computations was used for determining the ensembles of conformers matching the NMR requirements and energy criteria. As a matter of record, the high resolution 3D structure models were constructed for the HIV-Haiti and HIV-RF immunogenic crowns, and their geometric parameters were collated with the ones of conformers derived previously for describing the conformational features of immunogenic tip of gp120 from Thailand and MN HIV-1 strains. The HIV-1 neutralization site was demonstrated to constitute in water solution highly flexible system sensitive to its environment. This inference is completely valid for the geometric space of dihedral angles where statistically significant differences in local structures of simulated conformers have been found for all virus isolates of interest. In spite of this fact, the stretch analyzed was shown to manifest a certain conservatism in the space of atomic coordinates, building up in four HIV-1 isolates two spatial folds similar to those observed in crystal for the V3 loop peptides bound to different neutralizing Fabs. The results are discussed in the light of literature data on HIV-1 neutralizing epitope structure.

Amino Acid Sequence↗

PubMed2001

The N-terminal region of human progesterone B-receptors: biophysical and biochemical comparison to A-receptors.

To understand the basis for functional differences between the two human progesterone receptors (PR), we have carried out a detailed biochemical and biophysical analysis of the N-terminal region of each isoform. Extending our previous work on the A-isoform (Bain, D. L, Franden, M. A., McManaman, J. L., Takimoto, G. S., and Horwitz, K. B. (2000) J. Biol. Chem. 275, 7313-7320), here we present studies on the N-terminal region of the B-isoform (NT-B) and compare its properties to its A-receptor counterpart (NT-A). As seen previously with NT-A, NT-B is quantitatively monomeric in solution, yet undergoes N-terminal-mediated assembly upon DNA binding. Limited proteolysis, microsequencing, and sedimentation analyses indicate that the B-isoform exists in a non-globular, extended conformation very similar to that of NT-A. Additionally, the 164 amino acids unique to the B-isoform (BUS) appear to be in a more extended conformation relative to sequences common to both receptors and do not exist as an independent structural domain. However, sedimentation studies of NT-A and NT-B show differences in the ensemble distribution of their conformational states. We hypothesize that isoform-specific functional differences are not due to structural differences, per se. Rather, the transcriptional element BUS, or possibly other transcription factors, causes a redistribution of the conformational ensemble by stabilizing a more functionally active set of conformations in NT-B.

Humans↗

PubMed1997

Multiconformational NMR analysis of sandostatin (octreotide): equilibrium between beta-sheet and partially helical structures.

This paper reports a detailed conformational analysis by 1H NMR (DMSO-d6, 300 K) and molecular modeling of the octapeptide D-Phe1-Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys7+ ++-Thr8-ol (disulfide bridged) known as sandostatin (or SMS 201-995 or octreotide) with both somatostatin-like and opioid-like bioactivities. This is the initial report on sandostatin showing that attempts to explain all NMR data using a single average conformation reveal several important inconsistencies including severe violations of mutually exclusive backbone-to-backbone NOEs. The inconsistencies are solved by assuming an equilibrium between antiparallel beta-sheet structures and conformations in which the C-terminal residues form a 3(10) helix-like fold (helical ensemble). This conformational equilibrium is consistent with previous X-ray diffraction investigations which show that sandostatin can adopt both the beta-sheet and the 3(10) helix-like secondary structure folds. In addition, indications of a conformational equilibrium between beta-sheet and helical structures are also found in solvent systems different from DMSO-d6 and for other highly bioactive analogs of sandostatin. In these cases a proper multiconformational NMR refinement is important in order to avoid conformational averaging artifacts. Finally, using the known models for somatostatin-like and opioid-like bioactivities of sandostatin analogs, the present investigation shows the potentials of the proposed structures for the design of novel sandostatin-based conformationally restricted peptidomimetics. These analogs are expected to refine the pharmacophore models for sandostatin bioactivities.

Hormones↗

PubMed2006

Conformational transitions in protein-protein association: binding of fasciculin-2 to acetylcholinesterase.

The neurotoxin fasciculin-2 (FAS2) is a picomolar inhibitor of synaptic acetylcholinesterase (AChE). The dynamics of binding between FAS2 and AChE is influenced by conformational fluctuations both before and after protein encounter. Submicrosecond molecular dynamics trajectories of apo forms of fasciculin, corresponding to different conformational substates, are reported here with reference to the conformational changes of loop I of this three-fingered toxin. This highly flexible loop exhibits an ensemble of conformations within each substate corresponding to its functions. The high energy barrier found between the two major substates leads to transitions that are slow on the timescale of the diffusional encounter of noninteracting FAS2 and AChE. The more stable of the two apo substates may not be the one observed in the complex with AChE. It seems likely that the more stable apo form binds rapidly to AChE and conformational readjustments then occur in the resulting encounter complex.

Acetylcholinesterase↗

PubMed1999

The effect of inhibitor binding on the structural stability and cooperativity of the HIV-1 protease.

The effects of the peptide inhibitor acetyl pepstatin on the structural stability of the HIV-1 protease have been measured by high sensitivity calorimetric techniques. At 25 degrees C and pH 3.6, acetyl pepstatin binds to HIV-1 protease with an affinity of 1.6 x 10(7 )M-1 and an enthalpy of 7.3 +/- 0.5 kcal/mol, indicating that binding is not favored enthalpically and that the favorable Gibbs energy originates from a large positive entropy. Since the binding of acetyl pepstatin is associated with a negative change in heat capacity (-450 cal/K*mol) the association reaction becomes enthalpically favored at temperatures higher than 40 degrees C. The presence of the inhibitor stabilizes the dimeric structure of the protease in a fashion that can be quantitatively described by a set of thermodynamic linkage equations. The combination of titration and differential scanning calorimetry provides an accurate way of determining binding constants for high affinity inhibitors that cannot be determined by titration calorimetry alone. A structure-based thermodynamic analysis of the binding process indicates that the stabilization effect is not distributed uniformly throughout the protease molecule. The binding of the inhibitor selectively stabilizes those conformational states in which the binding site is formed, triggering a redistribution of the state probabilities in the ensemble of conformations populated under native conditions. As a result, the stability constants for individual residues do not exhibit the same change in magnitude upon inhibitor binding. Residues in certain areas of the protein are affected significantly whereas residues in other areas are not affected at all. In particular, inhibitor binding has a significant effect on those regions that define the binding site, especially the flap region which becomes structurally stable as a result of the additional binding free energy. The induced stabilization propagates to regions not in direct contact with the inhibitor, particularly to the strand between residues Pro9 and Ala22 and the helix between Arg87 and Gly94. On the other hand, the stability of the strand between Asp60 and Leu76 is not significantly affected by inhibitor binding. The structural distribution of binding effects define cooperative pathways within the protease molecule. Proteins 1999;36:147-156.

Algorithms↗

PubMed1997

Structure-based statistical thermodynamic analysis of T4 lysozyme mutants: structural mapping of cooperative interactions.

The recent development of a structural parameterization of the energetics of protein folding has permitted the incorporation of the functions that describe the enthalpy, entropy and heat capacity changes, i.e. the individual components of the Gibbs energy, into a statistical thermodynamic formalism that describes the distribution of conformational states under equilibrium conditions. The goal of this approach is to construct with the computer a large ensemble of conformational states, and then to derive the most probable population distribution, i.e. the distribution of states that best accounts for a wide array of experimental observables. This analysis has been applied to four different mutants of T4 lysozyme (S44A, S44G, V131A, V131G). It is shown that the structural parameterization predicts well the stability of the protein and the effects of the mutations. The entire set of folding constants per residue has been calculated for the four mutants. In all cases, the effect of the mutations propagates beyond the mutation site itself through sequence and three-dimensional space. This phenomenon occurs despite the fact that the mutations are at solvent-exposed locations and do not directly affect other interactions in the protein. These results suggest that single amino acid mutations at solvent-exposed locations, or other locations that cause a minimal perturbation, can be used to identify the extent of cooperative interactions. The magnitude and extent of these effects and the accuracy of the algorithm can be tested by means of NMR-detected hydrogen exchange.

Bacteriophage T4↗

PubMed2005

Structural changes involved in protein binding correlate with intrinsic motions of proteins in the unbound state.

Protein-protein binding usually involves structural changes that may extend beyond the rearrangements on a local scale, and cannot be explained by a classical lock-and-key mechanism. Several models have been advanced to explain the flexible binding of proteins such as the induced fit mechanism where the ligand is postulated to induce a conformational change at the interaction site upon binding, or the preexisting equilibrium hypothesis that assumes that protein samples an ensemble of conformations at equilibrium conditions and that the ligand binds selectively to an active conformation. We explored the equilibrium motions of proteins that exhibit relatively large (nonlocal) conformational changes upon protein binding using the Gaussian network model and the anisotropic network model of protein dynamics. For four complexes, LIR-1/HLA-A2, Actin/DNase I, CDK2/cyclin, and CDK6/p16(INK4a), the motions calculated for the monomer exhibiting the largest conformational change, in its unbound (free) form, correlate with the experimentally observed structural changes upon binding. This study emphasizes the preexisting equilibrium/conformational selection as a mechanism for protein-protein interaction and lends support the concept that proteins, in their native conformation, are predisposed to undergo conformational fluctuations that are relevant to, or even required for, their biological functions.

Actins↗

PubMed2005

Representing receptor flexibility in ligand docking through relevant normal modes.

Inspired by the current representation of the ligand-receptor binding process, a normal-mode-based methodology is presented to incorporate receptor flexibility in ligand docking and virtual screening. However, the systematic representation of the deformation space grows geometrically with the number of modes, and furthermore, midscale loop rearrangements like those found in protein kinase binding pockets cannot be accounted for with the first lowest-frequency modes. We thus introduced a measure of relevance of normal modes on a given region of interest and showed that only very few modes in the low-frequency range are necessary and sufficient to describe loop flexibility in cAMP-dependent protein kinase. We used this approach to generate an ensemble of representative receptor backbone conformations by perturbing the structure along a combination of relevant modes. Each ensemble conformation is complexed with known non-native binders to optimize the position of the binding-pocket side chains through a full flexible docking procedure. The multiple receptor conformations thus obtained are used in a small-scale virtual screening using receptor ensemble docking. We evaluated this algorithm on holo and apo structures of cAMP-dependent protein kinase that exhibit backbone rearrangements on two independent loop regions close to the binding pocket. Docking accuracy is improved, since the ligands considered in the virtual screening docked within 1.5 A to at least one of the structures. The discrimination between binders and nonbinders is also enhanced, as shown by the improvement of the enrichment factor. This constitutes a new step toward the systematic integration of flexible ligand-flexible receptor docking tools in structure-based drug discovery.

Algorithms↗

PubMed1999

Improved modeling of side-chains in proteins with rotamer-based methods: a flexible rotamer model.

Side-chain modeling has a widespread application in many current methods for protein tertiary structure determination, prediction, and design. Of the existing side-chain modeling methods, rotamer-based methods are the fastest and most efficient. Classically, a rotamer is conceived as a single, rigid conformation of an amino acid sidechain. Here, we present a flexible rotamer model in which a rotamer is a continuous ensemble of conformations that cluster around the classic rigid rotamer. We have developed a thermodynamically based method for calculating effective energies for the flexible rotamer. These energies have a one-to-one correspondence with the potential energies of the rigid rotamer. Therefore, the flexible rotamer model is completely general and may be used with any rotamer-based method in substitution of the rigid rotamer model. We have compared the performance of the flexible and rigid rotamer models with one side-chain modeling method in particular (the self-consistent mean field theory method) on a set of 20 high quality crystallographic protein structures. For the flexible rotamer model, we obtained average predictions of 85.8% for chi1, 76.5% for chi1+2 and 1.34 A for root-mean-square deviation (RMSD); the corresponding values for core residues were 93.0%, 87.7% and 0.70 A, respectively. These values represent improvements of 7.3% for chi1, 8.1% for chi1+2 and 0.23 A for RMSD over the predictions obtained with the rigid rotamer model under otherwise identical conditions; the corresponding improvements for core residues were 6.9%, 10.5% and 0.43 A, respectively. We found that the predictions obtained with the flexible rotamer model were also significantly better than those obtained for the same set of proteins with another state-of-the-art side-chain placement method in the literature, especially for core residues. The flexible rotamer model represents a considerable improvement over the classic rigid rotamer model. It can, therefore, be used with considerable advantage in all rotamer-based methods commonly applied to protein tertiary structure determination, prediction, and design and also in predictions of free energies in mutational studies.

Amino Acids↗

PubMed2004

Testing a flexible-receptor docking algorithm in a model binding site.

Sampling receptor flexibility is challenging for database docking. We consider a method that treats multiple flexible regions of the binding site independently, recombining them to generate different discrete conformations. This algorithm scales linearly rather than exponentially with the receptor's degrees of freedom. The method was first evaluated for its ability to identify known ligands of a hydrophobic cavity mutant of T4 lysozyme (L99A). Some 200000 molecules of the Available Chemical Directory (ACD) were docked against an ensemble of cavity conformations. Surprisingly, the enrichment of known ligands from among a much larger number of decoys in the ACD was worse than simply docking to the apo conformation alone. Large decoys, accommodated in the larger cavity conformations sampled in the ensemble, were ranked better than known small ligands. The calculation was redone with an energy correction term that considered the cost of forming the larger cavity conformations. Enrichment improved, as did the balance between high-ranking large and small ligands. In a second retrospective test, the ACD was docked against a conformational ensemble of thymidylate synthase. Compared to docking against individual enzyme conformations, the flexible receptor docking approach improved enrichment of known ligands. Including a receptor conformational energy weighting term improved enrichment further. To test the method prospectively, the ACD database was docked against another cavity mutant of lysozyme (L99A/M102Q). A total of 18 new compounds predicted to bind this polar cavity and to change its conformation were tested experimentally; 14 were found to bind. The bound structures for seven ligands were determined by X-ray crystallography. The predicted geometries of these ligands all corresponded to the observed geometries to within 0.7A RMSD or better. Significant conformational changes of the cavity were observed in all seven complexes. In five structures, part of the observed accommodations were correctly predicted; in two structures, the receptor conformational changes were unanticipated and thus never sampled. These results suggest that although sampling receptor flexibility can lead to novel ligands that would have been missed when docking a rigid structure, it is also important to consider receptor conformational energy.

Algorithms↗

PubMedDate not supplied

Estimation of molecular similarity based on 4D-QSAR analysis: formalism and validation.

The 4D-QSAR paradigm has been used to develop a formalism to estimate molecular similarity measures as a function of conformation, alignment, and atom type. It is possible to estimate the molecular similarity of pairs of molecules based upon the entire ensemble of conformational states each molecule can adopt at a given temperature, normally room temperature. Molecular similarity can be measured in terms of the types of atoms composing each molecule leading to multiple measures of molecular similarity. Multiple measures of molecular similarity can also arise from using different alignment rules to perform relative molecular similarity, RMS, analysis. An alignment independent method of determining molecular similarity measures, referred to as absolute molecular similarity, AMS, analysis has been developed. Various sets and libraries of compounds, including the amino acids, are analyzed using 4D-QSAR molecular similarity analysis to demonstrate the features of the formalism. Exploration of molecular similarity as a function of chirality, identification of common embedded 3D pharmacophores in compounds, and elucidation of 3D-isosteric compounds from structurally diverse libraries are carried out in the application studies.

Journal Article↗

PubMed2000

Structure of m-carboxyphenyl-alpha-D-galactopyranoside complexed to heat-labile enterotoxin at 1.3 A resolution: surprising variations in ligand-binding modes.

In the quest to develop drugs against traveller's diarrhoea and cholera, the structure of the B pentamer of heat-labile enterotoxin (LT) complexed with a new receptor-binding antagonist, m-carboxyphenyl-alpha-D-galactopyranoside, has been determined. The high resolution obtained for this structure allowed anisotropic refinement of the model. It was also now possible to confirm at a near-atomic resolution the structural similarity between the B subunits of LT and the closely related cholera toxin (CT), including the similarity in deviations of planarity of the same peptide unit in LT and CT. The structure of the LT complex clearly revealed different conformations for the m--carboxyphenyl moiety of the ligand in the five B subunits of LT, while the binding modes of the well defined galactopyranoside moieties were identical. In two binding sites the m-carboxyphenyl moiety displayed no significant electron density, demonstrating significant flexibility of this moiety. In a third binding site the m-carboxyphenyl moiety could be modelled unambiguously into the density. The two remaining binding sites were involved in crystal packing contacts and the density for the ligands in these two binding sites clearly revealed different binding modes, of which one conformation was identical to and one completely different from the conformation of m-carboxyphenyl-galactopyranoside in the third subunit. The multiple binding modes observed in the crystal may represent the ensemble of conformations of m-carboxyphenyl-alpha-D-galactopyranoside complexed to LT in solution.

Bacterial Toxins↗

PubMed1982

Conformational energy analysis of melanostatin.

Conformational energy calculations were carried out on the hypothalamic hormone melanostatin, a tripeptide with the primary structure H-L-Pro-L-Leu-Gly-NH2. The calculated lowest energy conformation was a type II beta bend, very similar to that reported in an X-ray crystal study. This conformation, however, was only one of 109 low-energy structures (less than or equal to 3 kcal/mol above the global minimum), indicating that the molecule in solution exists as an ensemble of conformations and is very flexible, in agreement with relaxation data from n.m.r. measurements. A statistical analysis yielded an average end-to-end distance of 6.8 A and a bend probability of 0.62, suggesting that, in nonpolar solvents, bend structures predominate within the statistical ensemble. The statistical analysis, however, also yielded a probability of only 0.11 for the occurrence of a 4 leads to 1 hydrogen bond. Hence, the calculations show that, although bend conformations predominate, bends would be difficult to observe in solution if the experiments were designed only to detect 4 leads to 1 hydrogen bonds.

Animals↗

PubMed1993

Solution structure of lariat RNA by 500 MHz NMR spectroscopy and molecular dynamics studies in water.

A 500 MHz NMR study of the lariat RNA tetramer 1 and pentamer 2 mimicking the naturally occurring lariat RNA is reported. The conformational properties of 1 and 2 were compared with those of a linear branched RNA tetramer 3, which show that the conformational features of the two lariat RNAs, 1 and 2, are quite constrained and significantly different from those observed for the linear branched RNA tetramer 3. The conformation of all sugar residues forming the lariat ring in 1 and 2 are locked in a rigid South-type conformation. All residues in both lariat RNAs have a high population of gamma+ (67-85%) and beta t (95-100%) rotamers except guanosine where the gamma+ population is low. The conformation around the glycosidic bond is anti for all residues except for guanosine where NOE data indicates an equilibrium of syn<-->anti. In both lariat RNAs, 1 and 2, the temperature dependent 1H and 31P chemical shifts as well as the oligomerization shifts, with respect to adenosine 2',3',5'-triethyl-phosphate (Sund et al., 1992, Tetrahedron 48, 695) suggests that the 3'-->5' linked U4 or C4 residue is stacked on guanosine. Subsequently, 1H-1H, 1H-31P and 13C-31P coupling constants derived torsional constraints were used for molecular dynamics study in water with counter sodium ions for a total of 226 ps. The MD simulations were first carried out with harmonic torsional constraints which were derived from J couplings (0-86 ps) and then completely without constraints (96-226 ps). The lack of any major changes in the conformation of the two lariat-RNA structures upon releasing the NMR constraints indicate that the conformers generated in the MD simulation in water agree well with the structural features suggested by experimental observables. This means that the ensemble of conformers generated during the MD trajectory of 226 ps are not artificially held in these conformations due to the NMR constraints, suggesting that these conformers can be considered to be good representatives of the actual NMR observed solution structures.

Base Sequence↗

PubMed1980

Conformational study of [Leu5]-enkephalin by laser Raman spectroscopy.

The Raman spectrum of [Leu5]-enkephalin in the solid state indicates the presence of a type I' beta bend, in agreement with X-ray data of Smith & Griffin (1978). In solution, this molecule takes on different conformations in different solvents. In DMSO-d6, it forms a beta-bend structure whereas in water it exists as an ensemble of conformations.

Dimethyl Sulfoxide↗

PubMed2001

Highly potent side-chain to side-chain cyclized enkephalin analogues containing a carbonyl bridge: synthesis, biology and conformation.

Six novel cyclic enkephalin analogues have been synthesized. Cyclization of the linear peptides containing basic amino acid residues in position 2 and 5 was achieved by treatment with bis(4-nitrophenyl)carbonate. It was found that some of the compounds exibit unusually high mu-opioid activity in the guinea pig ileum (GPI) assay. The 18-membered analogue cyclo(N(epsilon),N(beta)-carbonyl-D-Lys2,Dap5)-enkephalinamide turned out to be one of the most potent mu-agonists reported so far. NMR spectra of the peptides were recorded and structural parameters were determined. The conformational space was exhaustively examined for each of them using the electrostatically driven Monte Carlo method. Each peptide was finally described as an ensemble of conformations. A model of the bioactive conformation of this class of opioid peptides was proposed.

Animals↗

PubMed2005

Cyclic enkephalin and dermorphin analogues containing a carbonyl bridge.

Four cyclic enkephalin analogues and four cyclic dermorphin analogues have been synthesized. Cyclization of linear peptides containing basic amino acid residues of various side chain length in position 2 and 5 (enkephalin analogues) or 2 and 4 (dermorphin analogues) was achieved by treatment with bis-(4-nitrophenyl) carbonate to form a urea unit. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse activity was observed, depending on the size of the ring and the location of the urea unit. The conformation of two dermorphin analogues has been studied: one of high activity (IC(50) = 4.15 nM in the GPI assay) and a second of low activity (IC(50) = 6700 nM in the GPI assay). The conformational space of these peptides was examined using the EDMC method. Using data from the NMR spectra, each peptide was described as an ensemble of conformers. Biological activity was discussed in light of the structural data.

Amino Acid Sequence↗

PubMed2001

Assessing the effect of conformational averaging on the measured values of observables.

Experiment and computer simulation are two complementary tools to understand the dynamics and behavior of biopolymers in solution. One particular area of interest is the ensemble of conformations populated by a particular molecule in solution. For example, what fraction of a protein sample exists in its folded conformation? How often does a particular peptide form an alpha helix versus a beta hairpin? To address these questions, it is important to determine the sensitivity of a particular experiment to changes in the distribution of molecular conformations. Consequently, a general analytic formalism is proposed to determine the sensitivity of a spectroscopic observable to the underlying distribution of conformations. A particular strength of the approach is that it provides an expression for a weighted average across conformational substates that is independent of the averaging function used. The formalism is described and applied to experimental and simulated nuclear Overhauser enhancement (NOE) and 3J-coupling data on peptides in solution.

Computer Simulation↗