Biomedical subjects

Irina D Pogozheva

Publications and source records attributed to Irina D Pogozheva.

12 recordsLinked to original sources

PubMed2006

OPM: orientations of proteins in membranes database.

SUMMARY: The Orientations of Proteins in Membranes (OPM) database provides a collection of transmembrane, monotopic and peripheral proteins from the Protein Data Bank whose spatial arrangements in the lipid bilayer have been calculated theoretically and compared with experimental data. The database allows analysis, sorting and searching of membrane proteins based on their structural classification, species, destination membrane, numbers of transmembrane segments and subunits, numbers of secondary structures and the calculated hydrophobic thickness or tilt angle with respect to the bilayer normal. All coordinate files with the calculated membrane boundaries are available for downloading. AVAILABILITY: http://opm.phar.umich.edu.

Computer Graphics↗

PubMed2006

Positioning of proteins in membranes: a computational approach.

A new computational approach has been developed to determine the spatial arrangement of proteins in membranes by minimizing their transfer energies from water to the lipid bilayer. The membrane hydrocarbon core was approximated as a planar slab of adjustable thickness with decadiene-like interior and interfacial polarity profiles derived from published EPR studies. Applicability and accuracy of the method was verified for a set of 24 transmembrane proteins whose orientations in membranes have been studied by spin-labeling, chemical modification, fluorescence, ATR FTIR, NMR, cryo-microscopy, and neutron diffraction. Subsequently, the optimal rotational and translational positions were calculated for 109 transmembrane, five integral monotopic and 27 peripheral protein complexes with known 3D structures. This method can reliably distinguish transmembrane and integral monotopic proteins from water-soluble proteins based on their transfer energies and membrane penetration depths. The accuracies of calculated hydrophobic thicknesses and tilt angles were approximately 1 A and 2 degrees, respectively, judging from their deviations in different crystal forms of the same proteins. The hydrophobic thicknesses of transmembrane proteins ranged from 21.1 to 43.8 A depending on the type of biological membrane, while their tilt angles with respect to the bilayer normal varied from zero in symmetric complexes to 26 degrees in asymmetric structures. Calculated hydrophobic boundaries of proteins are located approximately 5 A lower than lipid phosphates and correspond to the zero membrane depth parameter of spin-labeled residues. Coordinates of all studied proteins with their membrane boundaries can be found in the Orientations of Proteins in Membranes (OPM) database:http://opm.phar.umich.edu/.

Amino Acids↗

PubMed2006

Molecular mechanism of the constitutive activation of the L250Q human melanocortin-4 receptor polymorphism.

The Melanocortin-4 Receptor is a G-protein coupled receptor that has been physiologically linked to participate in the regulation of energy homeostasis. The Melanocortin-4 Receptor is stimulated by endogenous melanocortin agonists derived from the pro-opiomelanocortin gene transcript and antagonized by the endogenous antagonist agouti-related protein. Central administration of melanocortin agonists has been demonstrated to decrease food intake and conversely, treatment with antagonists resulted in increased food intake. Deletion of the Melanocortin-4 Receptor gene from the mouse genome results in an obese and hyperphagic phenotype. Polymorphisms of the human Melanocortin-4-Receptor have been found in severely obese individuals, suggesting that Melanocortin-4 Receptor malfunction might be involved in human obesity and obesity-associated diabetes. Herein, we have performed experiments to understand the molecular mechanisms associated with the L250Q human Melanocortin-4-Receptor polymorphism discovered in an extremely obese woman. This L250Q human Melanocortin-4-Receptor has been pharmacologically characterized to result in a constitutively active receptor. The fact that a constitutively active human Melanocortin-4-Receptor mutation was found in an obese person is a physiologic contradiction, as chronic activation of the human Melanocortin-4-Receptor and subsequently high cyclic adenosine monophosphate levels should theoretically result in a normal or lean phenotype. In this study, we demonstrated that agouti-related protein acts as an inverse agonist at this constitutively active receptor, and we propose a mechanism by which agouti-related protein might contribute to the obese phenotype in the L250Q patient. In addition, using receptor mutagenesis, pharmacology, and computer modeling approaches, we investigated the molecular mechanism by which modification of the L250 residue results in constitutive activation of the human Melanocortin-4-Receptor.

Agouti-Related Protein↗

PubMed2005

Homology modeling of opioid receptor-ligand complexes using experimental constraints.

Opioid receptors interact with a variety of ligands, including endogenous peptides, opiates, and thousands of synthetic compounds with different structural scaffolds. In the absence of experimental structures of opioid receptors, theoretical modeling remains an important tool for structure-function analysis. The combination of experimental studies and modeling approaches allows development of realistic models of ligand-receptor complexes helpful for elucidation of the molecular determinants of ligand affinity and selectivity and for understanding mechanisms of functional agonism or antagonism. In this review we provide a brief critical assessment of the status of such theoretical modeling and describe some common problems and their possible solutions. Currently, there are no reliable theoretical methods to generate the models in a completely automatic fashion. Models of higher accuracy can be produced if homology modeling, based on the rhodopsin X-ray template, is supplemented by experimental structural constraints appropriate for the active or inactive receptor conformations, together with receptor-specific and ligand-specific interactions. The experimental constraints can be derived from mutagenesis and cross-linking studies, correlative replacements of ligand and receptor groups, and incorporation of metal binding sites between residues of receptors or receptors and ligands. This review focuses on the analysis of similarity and differences of the refined homology models of mu, delta, and kappa-opioid receptors in active and inactive states, emphasizing the molecular details of interaction of the receptors with some representative peptide and nonpeptide ligands, underlying the multiple modes of binding of small opiates, and the differences in binding modes of agonists and antagonists, and of peptides and alkaloids.

Analgesics, Opioid↗

PubMed2005

Exploring the active site of benzaldehyde lyase by modeling and mutagenesis.

Benzaldehyde lyase (BAL) is a thiamin diphosphate-dependent enzyme, which catalyzes the breakdown of (R)-benzoin to benzaldehyde. In essence, this is the reverse of the carboligation reaction catalyzed by benzoylformate decarboxylase (BFD). Here, we describe the first steps towards understanding the factors influencing BFD to form a CC bond under conditions wherein BAL will cleave the same bond. What are the similarities and differences between these two enzymes that result in the different catalytic activities? The X-ray structures of BFD and pyruvate decarboxylase (PDC) were used as templates for modeling benzaldehyde lyase. The model shows that a glutamine residue, Gln113, replaces the active site histidines of BFD and PDC. Replacement of the Gln113 by alanine or histidine reduced the value of k(cat) for lyase activity by more than 200-fold. The residues in BFD interacting with the phenyl ring of benzoylformate have similarly positioned counterparts in BAL but Ser26, the residue known to interact with the carboxylate group of benzoylformate, has been replaced by an alanine (Ala28). The BAL A28S variant exhibited 7% of WT activity in the BAL assay but, in the most intriguing result, this variant was able to catalyze the decarboxylation of benzoylformate. Conversely, the BFD S26A variant was unable to cleave benzoin.

Aldehyde-Lyases↗

PubMed2005

Interactions of human melanocortin 4 receptor with nonpeptide and peptide agonists.

Specific interactions of human melanocortin-4 receptor (hMC4R) with its nonpeptide and peptide agonists were studied using alanine-scanning mutagenesis. The binding affinities and potencies of two synthetic, small-molecule agonists (THIQ, MB243) were strongly affected by substitutions in transmembrane alpha-helices (TM) 2, 3, 6, and 7 (residues Glu(100), Asp(122), Asp(126), Phe(261), His(264), Leu(265), and Leu(288)). In addition, a I129A mutation primarily affected the binding and potency of THIQ, while F262A, W258A, Y268A mutations impaired interactions with MB243. By contrast, binding affinity and potency of the linear peptide agonist NDP-MSH were substantially reduced only in D126A and H264A mutants. Three-dimensional models of receptor-ligand complexes with their agonists were generated by distance-geometry using the experimental, homology-based, and other structural constraints, including interhelical H-bonds and two disulfide bridges (Cys(40)-Cys(279), Cys(271)-Cys(277)) of hMC4R. In the models, all pharmacophore elements of small-molecule agonists are spatially overlapped with the corresponding key residues (His(6), d-Phe(7), Arg(8), and Trp(9)) of the linear peptide: their charged amine groups interact with acidic residues from TM2 and TM3, similar to His(6) and Arg(6) of NDP-MSH; their substituted piperidines mimic Trp(9) of the peptide and interact with TM5 and TM6, while the d-Phe aromatic rings of all three agonists contact with Leu(133), Trp(258), and Phe(261) residues.

Amino Acid Sequence↗

PubMed2005

Receptor-antagonist interactions in the complexes of agouti and agouti-related protein with human melanocortin 1 and 4 receptors.

The molecular interactions between human melanocortin receptor-1 and -4 (hMC1R and hMC4R) and their endogenous antagonists, agouti signaling protein (ASIP) and agouti-related protein (AGRP), were assessed by studying the effects of site-directed mutations on the binding affinity of (125)I-ASIP[90-132(L89Y)] and (125)I-AGRP(86-132). Mutations of homologous residues from transmembrane helices (TMHs) 3 and 6 and extracellular loop (EL) 3 (D121A, T124A, F257A, and F277M in hMC1R and D126A, I129A F261A, and M281F in hMC4R) impaired binding of both antagonists to hMC4R and binding of the ASIP fragment to hMC1R. However, the mutations in TMH2 (E94A in hMC1R and E100A in hMC4R), TMH7 (F280A in hMC1R and F284A in hMC4R), and EL2 (Y183S, H184S, and D184H in hMC1R) only significantly affected binding of the ASIP fragment. The dependence of agonist binding on the dithiothreitol concentration followed a monophasic curve for wild-type hMC4R and its C40A, C271A, and C279A mutants and a biphasic curve for hMC1R, suggesting the presence of at least one structurally and functionally essential disulfide bond in both wild-type receptors and the hMC4R mutants. Models of complexes of both receptors with the ASIP fragment and hMC4R with the AGRP fragment were calculated using constraints from the experimental structures of rhodopsin and AGRP fragments, a set of deduced hydrogen bonds, supplemented by two proposed disulfide bridges and receptor-ligand contacts, derived from our mutagenesis data. In the models of the ASIP fragment complexed with both receptors, the core ligand tripeptide, Arg-Phe-Phe, positioned between TMHs 3 and 6, is shifted toward TMHs 2 and 7 relative to its position in the AGRP-hMC4R model, while the N-terminal loop and two central disulfides of the antagonists interact with EL2 of the receptors.

Agouti Signaling Protein↗

PubMed2004

Complex of an active mu-opioid receptor with a cyclic peptide agonist modeled from experimental constraints.

Site-directed mutagenesis and design of Zn(2+)-binding centers have been used to determine a set of specific tertiary interactions between the mu-opioid receptor, a rhodopsin-like G protein-coupled receptor (GPCR), and its cyclic peptide agonist ligand, Tyr(1)-c(S-Et-S)[d-Cys(2)-Phe(3)-d-Pen(4)]NH(2) (JOM6). The binding affinity of the tetrapeptide is strongly dependent on the nature of its first and third residues and on substitutions at positions 213, 216, 237, 300, 315, and 318 of the mu-opioid receptor. His(1) and His(3) analogues of the ligand were able to form metal-binding complexes with the V300C and G213C/T315C receptor mutants, respectively. Direct contact of the Phe(3) residue of JOM6 with Gly(213), Asp(216), Thr(315), and Trp(318) of the receptor was suggested by the binding affinities of His(3)-, Nle(3)-, Leu(3)-, Aci(3)-, Delta(E)Phe(3)-, and Delta(Z)Phe(3)-substituted peptides with the G213C/T315C, D216V, T315C, and W318L mutants. The improved binding affinity of the free carboxylate analogue of JOM6 for binding to the E229D mutant revealed an interaction between the C-terminal group of the peptide and Glu(229) of the receptor. The experimental constraints that were obtained were applied for distance geometry modeling of the mu-receptor in complex with the tetrapeptide agonist ligand, JOM6. The active conformation of the opioid receptor was calculated using the crystal structure of "inactive" rhodopsin and published engineered and intrinsic metal-binding sites and disulfide bonds that allow or facilitate activation of GPCRs. Interhelical H-bonds existing in the mu-receptor were applied as additional distance constraints. The calculated model of the receptor-ligand complex can serve as a prototype of the active state for all rhodopsin-like GPCRs. It displays a strongly shifted transmembrane helix 6 (TM6) and reorientation of the conserved Trp(293) residue in TM6 upon its interaction with the agonist. Importantly, the binding pockets of the active and inactive states are not identical, which implies distinct interaction modes of agonists and antagonists. In the active state, the binding pocket of the mu-receptor is complementary to the previously proposed receptor-bound conformation of JOM6.

Amino Acid Sequence↗

PubMed2004

Refinement of a homology model of the mu-opioid receptor using distance constraints from intrinsic and engineered zinc-binding sites.

Publication of the rhodopsin X-ray structure has facilitated the development of homology models of other G protein-coupled receptors. However, possible shifts of transmembrane (TM) alpha helices, expected variations in helical distortions, and differences in loop size necessitate experimental verification of these comparative models. To refine a rhodopsin-based homology model of the mu-opioid receptor (MOR), we experimentally determined structural-distance constraints from intrinsic and engineered metal-binding sites in the rat MOR. Investigating the relatively high intrinsic affinity of MOR for Zn(2+) (IC(50) approximately 30microM), we observed that mutation of His(319) (TM7) abolished Zn(2+) inhibition of ligand binding, while mutation of Asp(216) (extracellular loop 2) decreased the effect of Zn(2+), suggesting these residues participate in the intrinsic Zn(2+)-binding center of MOR. To verify the relative orientation of TM5 and TM6 and to examine whether a rhodopsin-like alpha aneurism is present in TM5, we engineered Zn(2+)-binding centers by mutating residues of TM5 and TM6 to Cys or His, making use of the native His(297) in TM6 as an additional Zn(2+)-coordination site. Inhibition of opioid ligand binding by Zn(2+) suggests that residues Ile(234) and Phe(237) in TM5 face the binding-site crevice and form a metal-binding center with His(297) and Val(300) in TM6. This observation is inconsistent with a rhodopsin-like structure, which would locate Ile(234) on the lipid-exposed side of TM5, too distant from other residues making up the Zn(2+)-binding site. Subsequent distance geometry refinement of the MOR model indicates that the rhodopsin-like alpha aneurism is likely absent in TM2 but present in TM5.

Amino Acid Sequence↗

PubMed2003

Highly conserved serine in the third transmembrane helix of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor activation.

The elucidation of the role of highly conserved polar amino acids in the transmembrane helices of G-protein-coupled receptors (GPCRs) is important in understanding the mechanism of receptor activation. To this end, the significance of a highly conserved serine residue in the third transmembrane alpha-helix (TM3) of the luteinizing hormone/human chorionic gonadotropin receptor (LH/hCGR) in regulating receptor activation was examined. Results showed that mutation of serine 431 to alanine (S431A) decreased the ability of the receptor to mediate cAMP production in response to hCG, suggesting that S431 stabilizes the active state of the receptor. Homology with other GPCRs suggests that S431 may participate in the coordination of a Na(+) ion. Since Na(+) has been found to stabilize the active state of the receptor in the presence of hCG, the possibility that S431 promotes receptor activation by mediating the effects of Na(+) was explored. Results showed that the regulation of hormone-induced receptor activation by S431 was independent of Na(+). A rhodopsin-based homology model of the TM region of the LH/hCGR was developed to identify other amino acids that might mediate the effects of Na(+) on receptor function. Results indicate that substitution of an Asp at position 556 with Tyr alters the ability of Na(+) to regulate receptor activation. The homology model is used to explain this result as well as to identify a mechanism through which S431 may regulate receptor signaling. Taken together, these studies provide novel insights into the mechanism of LH/hCG receptor activation.

Amino Acid Substitution↗

PubMed2003

Pharmacophore and receptor models for neurokinin receptors.

Three neurokinin (NK) antagonist pharmacophore models (Models 1-3) accounting for hydrogen bonding groups in the 'head' and 'tail' of NK receptor ligands have been developed by use of a new procedure for treatment of hydrogen bonds during superimposition. Instead of modelling the hydrogen bond acceptor vector in the strict direction of the lone pair, an angle is allowed between the hydrogen bond acceptor direction and the ideal lone pair direction. This approach adds flexibility to hydrogen bond directions and produces more realistic RMS values. By using this approach, two novel pharmacophore models were derived (Models 2 and 3) and a hydrogen bond acceptor was added to a previously published NK2 pharmacophore model [Poulsen et al., J. Comput.-Aided Mol. Design, 16 (2002) 273] (Model 1). Model 2 as well as Model 3 are described by seven pharmacophore elements: three hydrophobic groups, three hydrogen bond acceptors and a hydrogen bond donor. Model 1 contains the same hydrophobic groups and hydrogen bond donor as Models 2 and 3, but only one hydrogen bond acceptor. The hydrogen bond acceptors and donor are represented as vectors. Two of the hydrophobic groups are always aromatic rings whereas the other hydrophobic group can be either aromatic or aliphatic. In Model 1 the antagonists bind in an extended conformation with two aromatic rings in a parallel displaced and tilted conformation. Model 2 has the same two aromatic rings in a parallel displaced conformation whereas Model 3 has the rings in an edge to face conformation. The pharmacophore models were evaluated using both a structure (NK receptor homology models) and a ligand based approach. By use of exhaustive conformational analysis (MMFFs force field and the GB/SA hydration model) and least-squares molecular superimposition studies, 21 non-peptide antagonists from several structurally diverse classes were fitted to the pharmacophore models. More antagonists could be fitted to Model 2 with a low RMS and a low conformational energy penalty than to Models 1 and 3. Pharmacophore Model 2 was also able to explain the NK1, NK2 and NK3 subtype selectivity of the compounds fitted to the model. Three NK 7TM receptor models were constructed, one for each receptor subtype. The location of the antagonist binding site in the three NK receptor models is identical. Compounds fitted to pharmacophore Model 2 could be docked into the NK1, NK2 and NK3 receptor models after adjustment of the conformation of the flexible linker connecting the head and tail. Models I and 3 are not compatible with the receptor models.

Amino Acid Sequence↗

PubMed2002

Interatomic potentials and solvation parameters from protein engineering data for buried residues.

Van der Waals (vdW) interaction energies between different atom types, energies of hydrogen bonds (H-bonds), and atomic solvation parameters (ASPs) have been derived from the published thermodynamic stabilities of 106 mutants with available crystal structures by use of an originally designed model for the calculation of free-energy differences. The set of mutants included substitutions of uncharged, inflexible, water-inaccessible residues in alpha-helices and beta-sheets of T4, human, and hen lysozymes and HI ribonuclease. The determined energies of vdW interactions and H-bonds were smaller than in molecular mechanics and followed the "like dissolves like" rule, as expected in condensed media but not in vacuum. The depths of modified Lennard-Jones potentials were -0.34, -0.12, and -0.06 kcal/mole for similar atom types (polar-polar, aromatic-aromatic, and aliphatic-aliphatic interactions, respectively) and -0.10, -0.08, -0.06, -0.02, and nearly 0 kcal/mole for different types (sulfur-polar, sulfur-aromatic, sulfur-aliphatic, aliphatic-aromatic, and carbon-polar, respectively), whereas the depths of H-bond potentials were -1.5 to -1.8 kcal/mole. The obtained solvation parameters, that is, transfer energies from water to the protein interior, were 19, 7, -1, -21, and -66 cal/moleA(2) for aliphatic carbon, aromatic carbon, sulfur, nitrogen, and oxygen, respectively, which is close to the cyclohexane scale for aliphatic and aromatic groups but intermediate between octanol and cyclohexane for others. An analysis of additional replacements at the water-protein interface indicates that vdW interactions between protein atoms are reduced when they occur across water.

Amino Acid Substitution↗