The cannabinoid receptor: computer-aided molecular modeling and docking of ligand

A three-dimensional model of human cannabinoid receptor is constructed using computer-aided molecular modeling techniques. The helices of bacteriorhodopsin were used as the initial template to construct the transmembrane helices. The extracellular and intracellular loops were added using the SYBYL molecular modeling package. The extracellular N terminus was modeled on the basis of its similarity to rat oncomodulin. Similarly, the C terminus was constructed on the basis of similarity to bovine prothrombin fragment 1. The final structure was refined by several runs of minimization and dynamics calculation using the CHARMm package. delta 9-Tetra hydrocannabinol was docked into the internal cavity using the AUTODOCK program. Our study snows that there may be a calcium-binding site in the extracellular N terminus of this receptor. The ligand binds mainly to a hydrophobic site, which consists of residues Met-240, Trp-241 (TMH-4), Trp-356, Leu-359, Leu-360 (TMH-6), and Ala-283 (TMH-5). Its phenolic hydroxyl group forms a hydrogen bond with the carboxy group of Ala-198 (TMH-3). The results of modeling agree well with experimental QSAR studies.     

The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates

The crystal structure of the recombinant form of rat liver fatty acid-binding protein was completed to 2.3 A and refined to an R factor of 19.0%. The structural solution was obtained by molecular replacement using superimposed polyalanine coordinates of six intracellular lipid-binding proteins as a search probe. The entire amino acid sequence of rat liver fatty acid-binding protein along with an amino-terminal formyl-methionine was modeled in the crystal structure. In addition, the crystal was obtained in the presence of oleic acid, and the initial electron density clearly showed two fatty acid molecules bound within a central cavity. The carboxylate of one fatty acid molecule interacts with arginine 122 and is shielded from free solvent. It has an overall bent conformation. The more solvent-exposed carboxylate of the other oleate is located near the helix-turn-helix that caps one end of the beta-barrel, while the acyl chain lies in the interior. The cavity contains both polar and nonpolar residues but also shows extensive hydrophobic character around the nonpolar atoms of the ligands. The primary and secondary oleate binding sites appear to be totally interdependent, mainly because favorable hydrophobic interactions form between both aliphatic chains.     

A detailed molecular model for human aromatase

Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P-450arom (human aromatase) based on that of P-450cam, the only cytochrome P-450 enzyme for which the crystal structure is known. The predicted structure is found to be in good agreement with current experimental data; both direct, from site-directed mutagenesis studies, and indirect, from the consideration of the structures and activities of known substrates and inhibitors.     

Structure of a sarcoplasmic calcium-binding protein from amphioxus refined at 2.4 A resolution

The three-dimensional structure of a sarcoplasmic Ca(2+)-binding protein from the protochordate amphioxus has been determined at 2.4 A resolution using multiple-isomorphous-replacement techniques. The refined model includes all 185 residues, three calcium ions, and one water molecule. The final crystallographic R-factor is 0.199. Bond lengths and bond angles in the molecules have root-mean-square deviations from ideal values of 0.015 A and 2.8 degrees, respectively. The overall structure is highly compact and globular with a predominantly hydrophobic core, unlike the extended dumbbell-shaped structures of calmodulin or troponin C. There are four distinct domains with the typical helix-loop-helix Ca(2+)-binding motif (EF hand). The conformation of the pair of EF hands in the N-terminal half of the protein is unusual due to the presence of an aspartate residue in the twelfth position of the first Ca(2+)-binding loop, rather than the usual glutamate. The C-terminal half of the molecule contains one Ca(2+)-binding domain with a novel helix-loop-helix conformation and one Ca(2+)-binding domain that is no longer functional because of amino acid changes. The overall structure is quite similar to a sarcoplasmic Ca(2+)-binding protein from sandworm, although there is only about 12% amino acid sequence identity between them. The similarity of the structures of these two proteins suggests that all sarcoplasmic Ca(2+)-binding proteins will have the same general conformation, even though there is very little conservation of primary structure among the proteins from various species.     

Crystal structure of intact elongation factor EF-Tu from Escherichia coli in GDP conformation at 2.05 A resolution

The crystal structure of intact elongation factor Tu (EF-Tu) from Escherichia coli in GDP-bound conformation has been determined using a combination of multiple isomorphous replacement (MIR) and multiwavelength anomalous diffraction (MAD) methods. The current atomic model has been refined to a crystallographic R factor of 20.3 % and free R-factor of 26.8 % in the resolution range of 10-2.05 A. The protein consists of three domains: domain 1 has an alpha/beta structure; while domain 2 and domain 3 are beta-barrel structures. Although the global fold of the current model is similar to those of published structures, the secondary structural assignment has been improved due to the high quality of the current model. The switch I region (residues 40-62) is well ordered in this structure. Comparison with the structure of EF-Tu in GDP-bound form from Thermus aquaticus shows that although the individual domain structures are similar in these two structures, the orientation of domains changes significantly. Interactions between domains 1 and 3 in our E. coli EF-Tu-GDP complex are quite different from those of EF-Tu with bound GTP from T. aquaticus, due to the domain rearrangement upon GTP binding. The binding sites of the Mg2+ and guanine nucleotide are revealed in detail. Two water molecules that co-ordinate the Mg2+ have been identified to be well conserved in the GDP and GTP-bound forms of EF-Tu structures, as well as in the structure of Ras p21 with bound GDP. Comparisons of the Mg2+ binding site with other guanine nucleotide binding proteins in GDP-bound forms show that the Mg2+ co-ordination patterns are well preserved among these structures.     

Structure of the insect troponin complex

Isolated troponin-tropomyosin complex from Lethocerus indicus asynchronous flight muscle forms paracrystals on a positively charged lipid monolayer. Single particle analysis was carried out on individual complexes selected from electron micrographs of negatively stained paracrystals. By a combination of correlation and classification techniques, different average projections of the object were obtained. An initial three-dimensional model was calculated by determining the Euler angles for the different views using a common line approach. This starting model was then used as a reference for the further three-dimensional refinement of the model using the original data set. The refined model of the troponin complex has a diameter of approximately 90 A and a volume corresponding with a molecular mass of about 120 kDa for the globular domain. The resolution of the reconstruction was determined to be 32 A using the differential phase residual method and 26 A using the Fourier shell correlation criterion.     

Homology model for oncostatin M based on NMR structural data

Oncostatin M (OM) is a member of the cytokine family which regulates the proliferation and differentiation of a variety of cell types and includes interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and granulocyte-colony stimulating factor (G-CSF). This family of proteins adopts a four-helix bundle fold with up-up-down-down topology and contains intramolecular disulfide bonds. Since an X-ray or NMR structure for OM is not currently available, a homology model for OM was determined from the X-ray structures of human growth hormone (hGH), LIF, and G-CSF where the alignment was based on secondary structure instead of sequence. The OM secondary structure was determined from NMR structural data, and the secondary structures for hGH, LIF, and G-CSF were obtained from the reported X-ray structures. The resulting homology model was refined using sequential NOE distance 13C restraints, chemical shift information, and a conformational database.     

Solution structure of the superactive monomeric des-[Phe(B25)] human insulin mutant: elucidation of the structural basis for the monomerization of des-[Phe(B25)] insulin and the dimerization of native insulin

The three-dimensional solution structure of des-[Phe(B25)] human insulin has been determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Thirty-five structures were calculated by distance geometry from 581 nuclear Overhauser enhancement-derived distance constraints, ten phi torsional angle restraints, the restraints from 16 helical hydrogen bonds, and three disulfide bridges. The distance geometry structures were optimized using simulated annealing and restrained energy minimization. The average root-mean-square (r.m.s.) deviation for the best 20 refined structures is 1.07 angstroms for the backbone and 1.92 angstroms for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are more well defined, with r.m.s. deviations of 0.64 angstroms for the backbone and 1.51 angstroms for all atoms. It is found that the des-[Phe(B25)] insulin is a monomer under the applied conditions (4.6 to 4.7 mM, pH 3.0, 310 K), that the overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer of native insulin are preserved, and that the conformation-averaged NMR solution structure is close to the structure of molecule 1 in the hexamer. The structure reveals that the lost ability of des-[Phe(B25)] insulin to self-associate is caused by a conformational change of the C-terminal region of the B-chain, which results in an intra-molecular hydrophobic interaction between Pro(B28) and the hydrophobic region Leu(B11)-Leu(B15) of the B-chain alpha-helix. This interaction interferes with the inter-molecular hydrophobic interactions responsible for the dimerization of native insulin, depriving the mutant of the ability to dimerize. Further, the structure displays a series of features that may explain the high potency of the mutant on the basis of the current model for the insulin-receptor interaction. These features are: a change in conformation of the C-terminal region of the B-chain, the absence of strong hydrogen bonds between this region and the rest of the molecule, and a relatively easy accessibility to the Val(A3) residue.     

Refinement of 3D structure of bovine lens alpha A-crystallin

In absence of 3D structures for alpha-crystallin subunits, alpha A and alpha B, we utilized a number of experimental and molecular modeling techniques to generate working 3D models of these polypeptides (Farnsworth et al., 1994. In Molecular Modeling: From Virtual Tools to Real Problems (Eds. Kumosinski, T.F. and Liebman, M.N.) ACS Symposium Series 576, Ch. 9:123-134, 1994, ACS Books, Washington DC). The refinement of the initial bovine alpha A model was achieved using a more accurate estimation of secondary structure by new/updated methods for analyzing the far UV-CD spectra and by neural network secondary structure predictions in combination with database searches. The spectroscopic study reveals that alpha-crystallin is not an all beta-sheet protein but contains approximately 17% alpha-helices, approximately 33% beta-structures and approximately 50% turns and coils. The refinement of the alpha A structure results in an elongate, asymmetric amphipathic molecule. The hydrophobic N-terminal domain imparts the driving force for subunit aggregation while the more flexible, polar C-terminal domain imparts aggregate solubility. In our quaternary structure of the aggregate, the monomer is the minimal cooperative subunit. In bovine alpha A, the highly negatively charged C-terminal domain has three small positive areas which may participate in dimer or tetramer formation of independently expressed C-terminal domains. The electrostatic potential of positive areas is modulated and become more negative with phosphorylation and ATP binding. The refined bovine alpha A model was used to construct alpha A models for the human, chick and dogfish shark. A high degree of conservation of the three dimensional structure and the electrostatic potential was observed. Our proposed open micellar quaternary structure correlates well with experimental data accumulated over the past several decades. The structure is also predictive of the more recent data.     

Techniques for assessing protein and glucose kinetics

The three-dimensional structure of cytochrome-c552 from Thermus thermophilus has been determined by the multiple anomalous dispersion technique using synchrotron radiation and refined to a resolution of 1.28 A. Data collection at 90 K and the recording of three data sets (f'-minimum: 7125 eV, f"-maximum: 7138 eV and reference for scaling: 10,077 eV) resulted in an initial electron density of very high quality at 2.1 A, which was readily interpretable for model building. The model was refined to an R value of 19.1% (Rfree=22.4%) at 1.28 A resolution using a fourth data set collected at a photon energy of 11,810 eV. Comparison of this thermophilic cytochrome with its mesophilic mitochondrial or bacterial counterparts reveals significant structural differences which are discussed with respect to their importance for thermostability and binding between this cytochrome and its corresponding ba3-oxidase. Amino acid sequence similarities to other class I cytochromes are very weak and entirely limited to the region around the CXXCH motif close to the N terminus. The N-terminal two-thirds of cytochrome-c552 cover spatial regions around the heme prosthetic group that are similar to those observed for other cytochromes. The actual secondary structural elements that are responsible for that shielding do not, however, correlate well to other structures. Only the N-terminal helix (containing the heme binding cysteine residues) aligns reasonably well with other class I cytochromes. The most striking differences that distinguish the present structure from all other class I cytochromes is the C-terminal one-third of the molecule that wraps around the remainder of the structure as a stabilizing clamp, the existence of an extended beta-sheet covering one edge of the heme and the lack of any internal water molecule.     

Structural details of a calcium-induced molecular switch: X-ray crystallographic analysis of the calcium-saturated N-terminal domain of troponin C at 1.75 A resolution

We have solved and refined the crystal and molecular structures of the calcium-saturated N-terminal domain of troponin C (TnC) to 1.75 A resolution. This has allowed for the first detailed analysis of the calcium binding sites of this molecular switch in the calcium-loaded state. The results provide support for the proposed binding order and qualitatively, for the affinity of calcium in the two regulatory calcium binding sites. Based on a comparison with the high-resolution apo-form of TnC we propose a possible mechanism for the calcium-mediated exposure of a large hydrophobic surface that is central to the initiation of muscle contraction within the cell.     

The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc

BACKGROUND: Integrase mediates a crucial step in the life cycle of the human immunodeficiency virus (HIV). The enzyme cleaves the viral DNA ends in a sequence-dependent manner and couples the newly generated hydroxyl groups to phosphates in the target DNA. Three domains have been identified in HIV integrase: an amino-terminal domain, a central catalytic core and a carboxy-terminal DNA-binding domain. The amino-terminal region is the only domain with unknown structure thus far. This domain, which is known to bind zinc, contains a HHCC motif that is conserved in retroviral integrases. Although the exact function of this domain is unknown, it is required for cleavage and integration. RESULTS: The three-dimensional structure of the amino-terminal domain of HIV-2 integrase has been determined using two-dimensional and three-dimensional nuclear magnetic resonance data. We obtained 20 final structures, calculated using 693 nuclear Overhauser effects, which display a backbone root-mean square deviation versus the average of 0.25 A for the well defined region. The structure consists of three alpha helices and a helical turn. The zinc is coordinated with His 12 via the N epsilon 2 atom, with His16 via the N delta 1 atom and with the sulfur atoms of Cys40 and Cys43. The alpha helices form a three-helix bundle that is stabilized by this zinc-binding unit. The helical arrangement is similar to that found in the DNA-binding domains of the trp repressor, the prd paired domain and Tc3A transposase. CONCLUSION: The amino-terminal domain of HIV-2 integrase has a remarkable hybrid structure combining features of a three-helix bundle fold with a zinc-binding HHCC motif. This structure shows no similarity with any of the known zinc-finger structures. The strictly conserved residues of the HHCC motif of retroviral integrases are involved in metal coordination, whereas many other well conserved hydrophobic residues are part of the protein core.     

Membrane-induced secondary structures of neuropeptides: a comparison of the solution conformations adopted by agonists and antagonists of the mammalian tachykinin NK1 receptor

We present what we believe to be the first documented example of an inducement of distinctly different secondary structure types onto agonists and antagonists selective for the same G-coupled protein receptor using the same membrane-model matrix wherein the induced structures are consistent with those suggested to be biologically active by extensive analogue studies and conventional binding assays. 1H NMR chemical shift assignments for the mammalian NK1 receptor-selective agonists alpha-neurokinin (NKA) and beta-neurokinin (NKB) as well as the mammalian NK1 receptor-selective antagonists [d-Pro2,d-Phe7,d-Trp9]SP and [d-Arg1, d-Pro2,d-Phe7,d-His9]SP have been determined at 600 MHz in sodium dodecyl sulfate (SDS) micelles. The SDS micelle system simulates the membrane-interface environment the peptide experiences when in the proximity of the membrane-embedded receptor, allowing for conformational studies that are a rough approximation of in vivo conditions. Two-dimensional NMR techniques were used to assign proton resonances, and interproton distances were estimated from the observed nuclear Overhauser effects (NOEs). The experimental distances were used as constraints in a molecular dynamics and simulated annealing protocol using the modeling package DISCOVER to generate three-dimensional structures of the two agonists and two antagonists when present in a membrane-model environment to determine possible prebinding ligand conformations. It was determined that (1) NKA is helical from residues 6 to 9, with an extended N-terminus; (2) NKB is helical from residues 4 to 10, with an extended N-terminus; (3) [d-Pro2,d-Phe7,d-Trp9]SP has poorly defined helical properties in the midregion and a beta-turn structure in the C-terminus (residues 6-9); and (4) [d-Arg1,d-Pro2, d-Phe7,d-His9]SP has a helical structure in the midregion (residues 4-6) and a well-defined beta-turn structure in the C-terminus (residues 6-10). Attempts have been made to correlate the observed conformational differences between the agonists and antagonists to their binding potencies and biological activity.     

Application of in-situ hybridization techniques to study human preimplantation embryos: a review

The three-dimensional solution structure of recombinant bovine myristoylated recoverin in the Ca(2+)-free state has been refined using an array of isotope-assisted multidimensional heteronuclear NMR techniques. In some experiments, the myristoyl group covalently attached to the protein N-terminus was labeled with C and the protein was unlabeled or vice versa; in others, both were C-labeled. This differential labeling strategy was essential for structural refinement and can be applied to other acylated proteins. Stereospecific assignments of 41 pairs of beta-methylene protons and 48 methyl groups of valine and leucine were included in the structure refinement. The refined structure was constructed using a total of 3679 experimental NMR restraints, comprising 3242 approximate interproton distance restraints (including 153 between the myristoyl group and the polypeptide), 140 distance restraints for 70 backbone hydrogen bonds, and 297 torsion angle restraints. The atomic rms deviations about the average minimized coordinate positions for the secondary structure region of the N-terminal and C-terminal domains are 0.44 +/- 0.07 and 0.55 +/- 0.18 A for backbone atoms, and the 1.09 +/- 0.07 and 1.10 +/- 0.15 A for all heavy atoms, respectively. The refined structure allows for a detailed analysis of the myristoyl binding pocket. The myristoyl group is in a slightly bent conformation: the average distance between C1 and C14 atoms of the myristoyl group is 14.6 A. Hydrophobic residues Leu28, Trp31, and Tyr32 from a cluster that interacts with the front end of the myristoyl (C1-C8), whereas residues Phe49, Phe56, Tyr86, Val87, and Leu90 interact with the tail end (C9-C14). The relatively deep hydrophobic pocket that binds the myristoyl group (C14:0) could also accommodate other naturally occurring acyl groups such as C12:0, C14:1, C14:2 chains.     

SPROUT: a program for structure generation

SPROUT is a new computer program for constrained structure generation that is designed to generate molecules for a range of applications in molecular recognition. It uses artificial intelligence techniques to moderate the combinatorial explosion that is inherent in structure generation. The program is presented here for the design of enzyme inhibitors. Structure generation is divided into two phases: (i) primary structure generation to produce molecular graphs to fit the steric constraints; and (ii) secondary structure generation which is the process of introducing appropriate functionality to the graphs to produce molecules that satisfy the secondary constraints, e.g., electrostatics and hydrophobicity. Primary structure generation has been tested on two enzyme receptor sites; the p-amidino-phenyl-pyruvate binding site of trypsin and the acetyl pepstatin binding site of HIV-1 protease. The program successfully generates structures that resemble known substrates and, more importantly, the predictive power of the program has been demonstrated by its ability to suggest novel structures.     

Postexposure treatment with aminophylline protects against phosgene-induced acute lung injury

Our model of the human m1 muscarinic receptor has been refined on the basis of the recently published projection map of bovine rhodopsin. The refined model has a slightly different helix arrangement, which reveals the presence of an extra hydrophobic pocket located between helices 3, 4 and 5. The interaction of series of agonists and antagonists with the m1 muscarinic receptor has been studied experimentally by site-directed mutagenesis. In order to account for the observed results, three-dimensional models of m1 ligands docked in the target receptor are proposed. Qualitatively, the obtained models are in good agreement with the experimental observations. Agonists and partial agonists have a relatively small size. They can bind to the same region of the receptor using, however, different anchoring receptor residues. Antagonists are usually larger molecules, filling almost completely the same pocket as agonists. They can usually produce much stronger interactions with aromatic residues. Experimental data combined with molecular modelling studies highlight how subtle and diverse receptor-ligand interactions could be.     

Improved contractility and coronary flow in isolated hearts after 1-day hypothermic preservation with isoflurane is not dependent on K(ATP) channel activation

S8 is one of the core ribosomal proteins. It binds to 16 S RNA with high affinity and independently of other ribosomal proteins. It also acts as a translational repressor in Escherichia coli by binding to its own mRNA. The structure of Thermus thermophilus S8 has been determined by the method of multiple isomorphous replacement at 2.9 A resolution and refined to a crystallographic R-factor of 16.2% (Rfree 27.5%). The two domains of the structure have an alpha/beta fold and are connected by a long protruding loop. The two molecules in the asymmetric unit of the crystal interact through an extensive hydrophobic core and form a tightly associated dimer, while symmetry-related molecules form a joint beta-sheet of mixed type. This type of protein-protein interaction could be realized within the ribosomal assembly. A comparison of the structures of T. thermophilus and Bacillus stearothermophilus S8 shows that the interdomain loop is eight residues longer in the former and reveals high structural conservation of an extensive region, located in the C-terminal domain. From mutational studies this region was proposed earlier to be involved in specific interaction with RNA. On the basis of these data and on the comparison of the two structures of S8, it is proposed that the three-dimensional structure of specific RNA binding sites in ribosomal proteins is highly conserved among different species.     

Review: Casein micelle structure; an examination of models

The casein micelle system of bovine milk is unique in that protein aggregates of similar spherical shape but extreme variability of size are formed by the self-assembly of three major nonidentical subunits. The monomeric subunits appear to be approximately the same size and shape with similar amphiphilic natures, the chief difference in properties being in the carbohydrate-containing kappa-casein which acts to stabilize the system against precipitation by calcium ion. Micelle models with kappa-casein exclusively in the interior lack a stabilization mechanism and can be eliminated. Statistical considerations of a chain polymer model also lead to its rejection. Electron microscopy reveals spherical submicellar aggregates which at present can be accounted for by only three models. Of these three, the experimental data are predicted only by one in which, alphas 1-, beta-, and kappa-casein subunits are associated into spherical soap micelle-like particles with the kappa-casein segregated into one portion, giving these submicelles an amphiphilic nature. The alphas 1- and beta-caseins are hydrophobic while the kappa-casein portion of the submicelle surface is hydrophilic. Of particular interest is the ability of this micelle model to explain the formation of a minimum micelle which is larger than a submicellar particle.