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R Osman

Publications and source records attributed to R Osman.

At least 37 records · Page 2Linked to original sources
PubMed1996

A refined model of the thyrotropin-releasing hormone (TRH) receptor binding pocket. Novel mixed mode Monte Carlo/stochastic dynamics simulations of the complex between TRH and TRH receptor.

Previous mutational and computational studies of the thyrotropin-releasing hormone (TRH) receptor identified several residues in its binding pocket [see accompanying paper, Perlman et al. (1996) Biochemistry 35, 7643-7650]. On the basis of the initial model constructed with standard energy minimization techniques, we have conducted 15 mixed mode Monte Carlo/stochastic dynamics (MC-SD) simulations to allow for extended sampling of the conformational states of the ligand and the receptor in the complex. A simulated annealing protocol was adopted in which the complex was cooled from 600 to 310 K in segments of 30 ps of the MC-SD simulations for each change of 100 K. Analysis of the simulation results demonstrated that the mixed mode MC-SD protocol maintained the desired temperature in the constant temperature simulation segments. The elevated temperature and the repeating simulations allowed for adequate sampling of the torsional space of the complex with successful conservation of the general structure and good helicity of the receptor. For the analysis of the interaction between TRH and the binding pocket, TRH was divided into four groups consisting of pyroGlu, His, ProNH2, and the backbone. The pairwise interaction energies of the four separate portions of TRH with the corresponding residues in the receptor provide a physicochemical basis for the understanding of ligand-receptor complexes. The interaction of pyroGlu with Tyr106 shows a bimodal distribution that represents two populations: one with a H-bond and another without it. Asp195 was shown to compete with pyroGlu for the H-bond to Tyr106. Simulations in which Asp195 was interacting with Arg283, thus removing it from the vicinity of Tyr106, resulted in a stable H-bond to pyroGlu. In all simulations His showed a van der Waals attraction to Tyr282 and a weak electrostatic repulsion from Arg 306. The ProNH2 had a strong and frequent H-bonding interaction with Arg306. The backbone carbonyls show a frequent H-bonding interaction with the OH group of Tyr282 and strong, often multiple, interactions with Arg306. Three structures, which maintained these interactions simultaneously, were selected as candidates for ligand-receptor complexes. These show persistent interactions of TRH with Ile 109 and Ile 116 in HX 3 and with Tyr310 and Ser313 in HX 7, which will be tested to refine the structure of the ligand-receptor complex. The superposition of the three structures shows the extent of structural flexibility of the receptor and the ligand in the complex. The backbone of TRH inside the receptor is in an alpha-helical conformation, suggesting that the receptor, through its interaction with the ligand, provides the energy required for the conformational change in the ligand from an extended to the folded form.

Amino Acid Sequence↗
PubMed1996

Molecular and cellular biology of thyrotropin-releasing hormone receptors.

Thyrotropin-releasing hormone (TRH) receptor (TRH-R) complementary DNAs have been cloned from several species. The deduced amino acid sequences are compatible with TRH-R being a seven-transmembrane-spanning G protein-coupled receptor. These complementary DNAs and reagents derived from them have permitted detailed study of TRH-R biology at the molecular and cellular levels. Studies that have been performed since 1990 are reviewed in this article under the following headings: TRH-R gene, tissue distribution of TRH-R, primary structure of TRH-Rs, three-dimensional structure of the TRH-R binding pocket, TRH-R and G proteins, TRH-R activation, TRH desensitization, TRH-R endocytosis, and regulation of TRH-R number. It is evident that many new insights into the structure, function, and regulation of TRH-Rs have been gained in the last several years but that our understanding of these processes is incomplete. We look forward to even greater progress in the future.

Amino Acid Sequence↗
PubMed1996

Molecular dynamics (MD) simulation of DNA dodecamer with 5-hydroxy-6-cytosinyl radical.

Molecular dynamics (MD) simulation of DNA dodecamer d (CGCGAATTC*GCG)2 with a primary radiation damage represented by 5-hydroxy-6-cytosinyl radical (C*) in position 9 was performed with AMBER 4.0 force field with periodic boundary conditions for the solvent. The temperature, potential energy of the system, energetic contributions from groups and RMS deviation from original positions were examined throughout the course of the simulation up to 140 ps. The stabilized structure (after 100 ps) was distorted and bent near the damaged site, which is similar to that observed in the MD of DNA with thymine glycol (Miaskiewicz, K. et al (1994) Radiat. Protection Dosimetry, 52, 149-153). The results suggest that a small and local damage in DNA may cause a large and global conformational change in DNA. Water molecules form two layers at distance 2.5 A and 5.5 A around the DNA. The MD simulation is a new approach to study radiation damages in molecular level.

Base Sequence↗
PubMed1996

Restricted analogues provide evidence of a biologically active conformation of thyrotropin-releasing hormone.

Thyrotropin-releasing hormone (TRH) is a tripeptide (< Glu-His-Pro-NH2) that signals through a G protein-coupled receptor. TRH is a highly flexible molecule that can assume many conformations in solution. To attempt to delineate the biologically active conformation of TRH, we synthesized a pair of conformationally restricted cyclohexyl/Ala2-TRH analogues. The diastereomeric analogues use a lactam ring to restrict two of the six free torsional angles of TRH and constrain the X-Pro-NH2 peptide bond to trans. Unrestricted cyclohexyl/Ala2-TRH exhibited a 650-fold lower affinity than TRH for TRH receptor and was 430-fold less potent than TRH in stimulating inositol phosphate second messenger formation. One diastereomer exhibited higher affinity and potency than the unrestricted analogue despite the presence of the methylene bridge and fused ring, whereas the other showed lower affinity and potency. Computer simulations predicted that the positions of the cyclohexyl/Ala2 and Pro-NH2 moieties relative to < glutamate were different in the two analogues and that the conformation of the higher affinity analogue is different from that of trans-TRH in solution but is superimposable on that of trans-TRH found in a model of the TRH/TRH receptor complex. These experimental findings identify a favored relative position of < glutamate and Pro-NH2 in the more active conformation of two diastereomeric analogues of TRH and provide independent support for the model of the TRH/TRH receptor complex.

Amino Acid Sequence↗
PubMed1995

Molecular dynamics simulations of the effects of ring-saturated thymine lesions on DNA structure.

The effect of thymine lesions produced by radiation or oxidative damage on DNA structure was studied by molecular dynamics simulations of native and damaged DNA. Thymine in position 7 of native dodecamer d(CGCGAATTCGCG)2 was replaced by one of the four thymine lesions 5-hydroxy-5,6-dihydrothymine, 6-hydroxy-5,6-dihydrothymine (thymine photohydrate), 5,6-dihydroxy-5,6-dihydro-thymine (thymine glycol), and 5,6-dihydrothymine. Simulations were performed with Assisted Model Building with Energy Refinement force field. Solvent was represented by a rectangular box of water with periodic boundary conditions applied. A constant temperature and constant volume protocol was used. The observed level of distortions of DNA structure depends on the specific nature of the lesion. The 5,6-dihydrothymine does not cause distinguishable perturbations to DNA. Other lesions produce a dramatic increase in the rise parameter between the lesion and the 5' adjacent adenine. These changes are accompanied by weakening of Watson-Crick hydrogen bonds in the A6-T19 base pair on the 5' side of the lesion. The lesioned bases also show negative values of inclination relative to the helical axis. No changes in the pattern of backbone torsional angles are observed with any of the lesions incorporated into DNA. The structural distortions in DNA correlate well with known biological effects of 5,6-dihydrothymine and thymine glycol on such processes as polymerase action or recognition by repair enzymes.

Base Sequence↗
PubMed1995

Distinct roles for arginines in transmembrane helices 6 and 7 of the thyrotropin-releasing hormone receptor.

The thyrotropin-releasing hormone (TRH) receptor (TRH-R) is a member of the seven-transmembrane region, G protein-coupled receptor family. Arg-283 and Arg-306, in transmembrane helices 6 and 7, respectively, are putatively in positions homologous to those of residues that are important for agonist and antagonist binding in receptors for neurotransmitters. These arginines were mutated and the mutant receptors were transiently expressed in COS-1 cells. The affinity of the R306K TRH-R was similar to that of the wild-type (WT) TRH-R, whereas no specific binding was detected in cells expressing R306A, R306E, or R306L TRH-Rs. Because TRH stimulated inositol phosphate (IP) formation to similar maximal extents in cells expressing WT and Arg-306 mutant TRH-Rs, relative potencies were used to estimate the relative affinities of the receptors. The EC50 values for stimulation of R306A, R306E, and R306L TRH-Rs were 1500-, 1200-, and 3000-fold higher than that for the WT TRH-R. No specific binding was measurable in COS-1 cells expressing R283K, R283H, or R283A TRH-Rs, whereas maximal TRH stimulation of IP formation was to levels 64%, 42%, or < 1%, respectively, of that in cells expressing WT TRH-Rs; for R283K and R283H TRH-Rs, EC50 values were 6300- and 50,000-fold higher, respectively, than that for the WT TRH-R. In AtT-20 cells stably expressing R283A TRH-Rs, the binding affinity was 39,000-fold lower than that of the WT TRH-R and the number of receptors was estimated to be 0.88 x 10(6)/cell, but TRH did not stimulate IP formation. Thus, in the TRH-R, Arg-306 appears to be important for binding but not for activation, whereas Arg-283 appears to be important for binding and activation.

Animals↗
PubMed1994

A model of the thyrotropin-releasing hormone (TRH) receptor binding pocket. Evidence for a second direct interaction between transmembrane helix 3 and TRH.

The receptor for thyrotropin-releasing hormone (TRH) is a member of the seven-transmembrane-spanning, GTP-binding protein-coupled receptor family. We showed that tyrosine at position 106 in transmembrane helix 3 of the TRH receptor directly binds the ring carbonyl of the pyroglutamyl moiety of TRH (Perlman, J. H., Thaw, C. N., Laakkonen L., Bowers, C. Y., Osman, R., and Gershengorn, M. C. (1994) J. Biol. Chem. 269, 1610-1613). We now show that asparagine at position 110 of transmembrane helix 3 directly interacts with the ring N-H of the TRH pyroglutamyl moiety. Based on these findings and evidence that two transmembrane arginines are important in binding, we developed a three-dimensional model of the TRH receptor binding pocket using molecular modeling and simulation programs. The model places the binding pocket for TRH within the transmembrane domains of the receptor and predicts that multiple hydrogen-bonding interactions are involved in binding TRH. To our knowledge, this is the first model, at an atomic level of detail, of the interaction of a peptide ligand with a GTP-binding protein-coupled receptor.

Animals↗
PubMed1994

Energetic basis for structural preferences in 5/6-hydroxy-5,6-dihydropyrimidines: products of ionizing and ultraviolet radiation action on DNA bases.

The structures of all diastereoisomers of 5/6-hydroxy-5,6-dihydropyrimidines have been optimized with ab initio quantum chemical calculations using a 6-31G basis set. The energies of the optimized structures were calculated at the MP2/6-31G* level. The hydroxyl group prefers an equatorial over an axial orientation at the C(5) position of pyrimidines by 3-4 kcal/mol. At the C(6) position, the axial orientation of hydroxyl is preferred by 3-4 kcal/mol. The factors responsible for the different preferences result from dipolar intramolecular interactions between the hydroxyl and C(4) = O(4) on the one hand, and the N(1)-H(1) on the other hand. As a consequence of these structural preferences, the pseudo axial positions at C(5) and C(6), which are perpendicular to the molecular plane, can be occupied by different substituents. These pseudo axial groups are expected to be a major source of distortions to DNA structure with more bulky groups having a greater effect. This may constitute a structural basis for interpretation of experimental results on the biological consequences of pyrimidine lesions. The conclusions drawn from the calculations correlate well with experimental observations on the biological activities of thymine lesions.

Calorimetry↗
PubMed1994

Electrostatic attraction by surface charge does not contribute to the catalytic efficiency of acetylcholinesterase.

Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.

Acetylcholinesterase↗
PubMed1994

Structure-function studies of DNA damage using ab initio quantum mechanics and molecular dynamics simulation.

Studies of ring-saturated pyrimidine base lesions are used to illustrate an integrated modeling approach that combines quantum-chemical calculations with molecular dynamics simulation. Electronic structure calculations on the lesions in isolation reveal strong conformational preferences due to interactions between equatorial substituents to the pyrimidine ring. Large distortions of DNA should result when these interactions force the methyl group of thymine to assume an axial orientation, as is the case for thymine glycol but not for dihydrothymine. Molecular dynamics simulations of the dodecamer d(CGCGAATTCGCG)2 with and without a ring-saturated thymine lesion at position T7 support this conclusion. Implications of these studies for recognition of thymine lesions by endonuclease III are also discussed.

Base Sequence↗
PubMed1994

Hydrogen bonding interaction of thyrotropin-releasing hormone (TRH) with transmembrane tyrosine 106 of the TRH receptor.

Thyrotropin-releasing hormone (TRH, pyroglutamic acid-histidine-proline-amide) binds to a seven-transmembrane-spanning, G protein-coupled receptor. We tested the hypothesis that Tyr106 of the third transmembrane helix of the TRH receptor (TRH-R) binds pyroglutamyl of TRH by mutating Tyr106 to Phe and replacing the ring carbonyl of the TRH pyroglutamyl moiety with a methylene group ([Pro1]TRH). Compared to the affinity of wild-type TRH-R for TRH, the affinities of [Phe106]TRH-R for TRH and of wild-type TRH-R for [Pro1]TRH were 100,000- and 110,000-fold lower, respectively. The affinity of [Phe106]TRH-R for [Pro1]TRH was only 16-fold lower than that for TRH, demonstrating a lack of additivity of the effects of these changes in the receptor and ligand. These data provide compelling evidence that the hydroxyl group of Tyr106 of the TRH-R binds the TRH pyroglutamyl carbonyl group. To our knowledge, this represents the highest affinity, non-covalent bond yet observed between single functional groups of a GPCR and ligand and is the first delineation of a direct binding interaction between a residue in the transmembrane core of a GPCR and a specific moiety of a peptide agonist.

Amino Acid Sequence↗
PubMed1993

Ab initio theoretical study of the structures of thymine glycol and dihydrothymine.

The structures of all diastereoisomers of 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol) an 5,6-dihydrothymine, two important DNA lesions, have been optimized with ab initio quantum chemical methods at a 6-31 G level of calculations. The methyl group on C5 of thymine glycol shows a strong preference for a pseudo axial orientation. In contrast, in 5,6-dihydrothymine a pseudo equatorial methyl is preferred. Consequently, the thymine glycol lesion is much more bulky than 5,6-dihydrothymine. This observation may explain the different biological consequences observed for the two lesions.

DNA↗
PubMed1993

Role of threonine 342 in helix 7 of the 5-hydroxytryptamine type 1D receptor in ligand binding: an indirect mechanism for receptor selectivity.

Recent mutations of the 5-hydroxytryptamine (5-HT)1B and 5-HT1D receptor subtypes suggest that a threonine in the seventh transmembrane helix may be responsible for the selectivity of these receptors. A molecular dynamics simulation of a three-dimensional model of the 5-HT1D receptor interacting with a selective agonist, sumatriptan, shows that, although Thr342 in helix 7 does not have a direct interaction with sumatriptan, it contributes to the selectivity of this receptor through an indirect mechanism. The hydrogen bond between O gamma-H of Thr342 and the backbone C = O of Phe338 stabilizes a bent conformation of the helix that is formed due to the interaction between sumatriptan and Asp339 at one end and Tyr346 at the other end. The indirect mechanism may explain the small change in the affinity for the selective agonist sumatriptan of the receptor in which Thr342 was mutated to asparagine.

Binding Sites↗
PubMed1992

Thyrotropin-releasing hormone binding to the mouse pituitary receptor does not involve ionic interactions. A model for neutral peptide binding to G protein-coupled receptors.

Thyrotropin-releasing hormone, TRH (< Glu-His-Proamide), and [N tau-Me-His]TRH (MeTRH) are present as neutral and positively charged forms at physiologic pH, and it was possible that they bind to the TRH receptor (TRH-R) as charged (protonated) species. Binding affinities of TRH and MeTRH to endogenous rat TRH-Rs and to transfected wild type mouse TRH-Rs decreased below pH 7.1. Half-maximal decreases in binding occurred at the approximate pK alpha values of these ligands. Asp to Ala mutations in extracellular loop 1, TM-4, and TM-5 did not decrease binding affinity, but an Asp to Ala mutation in TM-2 caused the affinity to decrease 8-fold. The pH dependences of binding of MeTRH, however, were similar in wild type and all mutant receptors and were consistent with the protonated form of MeTRH binding less well. Thus, the binding of TRH to its receptor does not involve ionic interactions and may be a prototype for binding of neutral peptide ligands to G protein-coupled receptors.

Amino Acid Sequence↗
PubMed1992

On the use of the transmembrane domain of bacteriorhodopsin as a template for modeling the three-dimensional structure of guanine nucleotide-binding regulatory protein-coupled receptors.

The molecular architecture of bacteriorhodopsin (BR) is commonly regarded as a structural template for the three-dimensional structure of membrane receptors that are functionally coupled to guanine nucleotide-binding regulatory proteins (GPCR). More recently, specific molecular models of such GPCR were constructed on the basis of the functional and structural relation of rhodopsin to BR as well as the sequence homology between rhodopsin and the GPCR. Such models of GPCR leave unresolved the difficulty caused by the apparent lack of any significant degree of sequence homology between the seven transmembrane helices (TMH) of BR and the portions in the sequence of the various GPCR that are considered to constitute their transmembrane domains. Evolutionary arguments offered in favor of the structural relation between BR and the opsins, and hence the GPCR, prompted our investigation of the possibility that the sequence homology, including any similarity in the distribution of kink-inducing proline residues among the helices, might have been obscured by the assumption that the TMH maintained their sequential order from BR in the evolution of the mammalian proteins. With a definition of the TMH in the neurotransmitter GPCR guided by hydropathicity predictions, and additional criteria used to define the span of each helix, optimal alignment of each pair of sequences was determined with no gaps allowed in the matching. The resulting alignment proposed here reveals considerable homology between the TMH in BR and those in GPCR, if the sequential order of the helices is ignored. These findings suggest the possibility that exon shuffling could have occurred in the proposed evolution of the GPCR gene from BR and point to a modification of the BR template to account for the correct packing of the helices in the tertiary structures of GPCR. These findings could guide the construction of three-dimensional models of the neurotransmitter GPCR on the basis of specific interhelical interactions observed in BR.

Amino Acid Sequence↗
PubMed1991

Molecular mechanisms of radiation induced DNA damage: H-abstraction and beta-cleavage.

Quantum mechanical simulations of hydrogen abstraction by hydroxyl radical from methanol and ethanol yield barriers that agree very well with those measured experimentally. Analysis of the multiconfigurational wavefunction indicates that the strength of the C-H bond is the electronic parameter that has a major contribution to the barrier for H-abstraction. Similar analysis applied to 2-deoxy-D-ribose shows that the strength of a C-H bond together with the steric accessibility of the hydrogen determine that H4 is the most susceptible hydrogen for abstraction by a hydroxyl radical. Quantum mechanical simulations of beta-cleavage show that a concerted mechanism in which a water molecule assists in the bond breaking process is more likely than a SN1 mechanism. However, the polar transition state suggests that the environment of the DNA and the surrounding water will have an important effect on the reaction.

DNA↗