Biomedical subjects

Harel Weinstein

Publications and source records attributed to Harel Weinstein.

36 records · Page 2Linked to original sources

PubMed2004

Certain 1,4-disubstituted aromatic piperidines and piperazines with extreme selectivity for the dopamine D4 receptor interact with a common receptor microdomain.

We previously demonstrated that, in the D4 dopamine receptor, the aromatic microdomain that spans the interface of the second and third transmembrane segments influences the high-affinity interactions with the D4-selective ligand L750,667 [3-[[4-(4-iodophenyl) piperazin-1-yl]methyl]-1H-pyrrolo[2,3-b]pyridine] and the D2-selective ligands methylspiperone, aripiprazole, and its congener OPC4392 [7-[3-(4-(2,3-dimethylphenyl) piperazinyl) propoxy] 2-(1H)-quinolinone] (Schetz et al., 2000). Here we tested a variety of 1,4-disubstituted aromatic piperidines/piperazines (1,4-DAPs) with different subtype selectivities and functional properties against a panel of D4 receptor mutations in the aromatic microdomain to ascertain whether these ligands recognize this common site. Mutant D4 receptors were constructed by substituting the nonconserved amino acid(s) from the corresponding locations in the D2 receptor. The D4-L2.60W, D4-F2.61V, and D4-LM3.28-3.29FV substitutions result in alterations of the relative position of members of the aromatic microdomain. From these results and molecular models of the ligand-receptor complexes, we conclude that 9 of the 11 D4-selective 1,4-DAPs, including L750,667, have a common pattern of ligand-receptor recognition that depends upon favorable interactions with the phenylalanine at position 2.61 (D4-F2.61V, 20-96-fold decrease). Like methylspiperone, aripiprazole, and OPC4392, the two D4-selective 1,4-DAPs that are insensitive to the D4-F2.61V mutation are sensitive to aromatics at position 2.60 (D4-L2.60W, 7-20-fold increase), and they all have longer spacer arms that permit their tethered aromatics to adopt alternative orientations in the binding-site crevice. All 11 of the D4-selective 1,4-DAPs were sensitive to the D4-LM3.28-3.29FV mutation (13-494-fold decrease) but not the moderately D2-selective methylspiperone. The inferences suggest that subtype selectivity involves two different modes of interaction with the microdomain for the D4-selective 1,4-DAPs and a third mode for D2-selective 1,4-DAPs.

Animals↗

PubMed2004

Quantitative information management for the biochemical computation of cellular networks.

Understanding complex protein networks within cells requires the ability to develop quantitative models and to numerically compute the properties and behavior of the networks. To carry out such computational analysis, it is necessary to use modeling tools and information management systems (IMSs) where the quantitative data, associated to its biological context, can be stored, curated, and reliably retrieved. We have focused on the biochemical computation of cellular interactions and developed an IMS that stores both quantitative information on the cellular components and their interactions, and the basic reactions governing those interactions. This information can be used to construct pathways and eventually large-scale networks. This system, SigPath, is available on the Internet (http://www.sigpath.org). Key features of the approach include (i) the use of background information (for example, names of molecules, aliases, and accession codes) to ease data submission and link this quantitative database with other qualitative databases, (ii) a strategy to allow refinement of information over time by multiple users, (iii) the development of a data representation that stores both qualitative and quantitative information, and (iv) features to assist contributors and users in assembling custom quantitative models from the information stored in the IMS. Currently, models assembled in SigPath can be automatically exported to several computing environments, such as Kinetikit/Genesis, Virtual Cell, Jarnac/JDesigner, and JSim. We anticipate that, when appropriately populated, such a system will be useful for large-scale quantitative studies of cell-signaling networks and other cellular networks. SigPath is distributed under the GNU General Public License.

Animals↗

PubMed2004

Molecular dynamics simulations of transducin: interdomain and front to back communication in activation and nucleotide exchange.

The dynamic events that underlie the nucleotide exchange process for the Galpha subunit of transducin (Galpha(t)) were studied with nanosecond time-scale molecular dynamics simulations. The modeled systems include the active and inactive forms of the wild-type Galpha(t) and three of its mutants (GDP-bound form only): F332A, A322S, and Q326A that are known to exhibit various degrees of enhancement of their basal and receptor-catalyzed rates of nucleotide exchange (150-fold, 70-fold and WT-like, respectively). The results of these computational experiments reveal a number of nucleotide-dependent structural and dynamic changes (involving the alpha(B)-alpha(C) loop, the inter-domain orientation of the helical and GTPase domains and the alpha(5) helix) that were not observed in the various crystal structures of Galpha(t). Notably, the results show the existence of a front to back communication device (involving the beta(2)-beta(3) hairpin, the alpha(1) helix and the alpha(5) helix), strategically located near all elements susceptible to be involved in receptor-mediated activation/nucleotide exchange. The wild-type simulations suggest that the dynamic interplay between the elements of this device would be critical for the activation of the Galpha(t) subunit. This inference is confirmed by the results of the computational experiments on the mutants that show that even in their GDP-bound forms, the A322S and F332A mutants acquire an "active-like" structure and dynamics phenotype. The same is not true for the Q326A mutant whose structural and dynamic properties remain similar to those of the GDP-bound WT. Taken together the results suggest a nucleotide exchange mechanism, analogous to that found in the Arf family GTPases, in which a partially activated state, achievable from a receptor-mediated action of the front to back communication device either by displacement of the C-terminal alpha(5) helix, of the N-terminal alpha(N) helix, or of the Gbetagamma subunit, could precede the dissociation of GDP from the native Galpha subunit.

Enzyme Activation↗

PubMed2004

Mechanistic insights from a refined three-dimensional model of integrin alphaIIbbeta3.

The integrin alpha(IIb)beta(3) plays an important role in platelet function, and abnormalities of this protein result in a serious bleeding disorder, known as Glanzmann thrombasthenia. Although crystallographic data exist for the related integrin alpha(V)beta(3), to date, there are no high resolution structures of integrin alpha(IIb)beta(3) available in the literature. Therefore, it is still unclear how specific elements of the alpha(IIb) subunit contribute to integrin alpha(IIb)beta(3) function. Here we describe a refined model of the alpha(IIb) N-terminal portion of integrin alpha(IIb)beta(3) obtained by using the alpha(V)beta(3) template combined with a new method for predicting the conformations of the unique alpha(IIb) loop regions comprising residues 71-85, 114-125, and 148-164. The refined model was probed based on a structural prediction that differentiates it from standard homology models: specifically, that Lys-118 of alpha(IIb) contacts Glu-171 of beta(3). To test this hypothesis experimentally, the mutant integrin chains alpha(IIb) K118C and beta(3) E171C were cotransfected into HEK 293 cells. We show that the cells expressed the mutants alpha(IIb)beta(3) on their surface as a disulfide-linked dimer, supporting the close proximity between alpha(IIb) Lys-118 and beta(3) Glu-171 predicted from the refined model. This validated model provides a specific structural context for the analysis and interpretation of structure-function relations of integrin alpha(IIb)beta(3). In addition, it suggests mechanistic hypotheses pertaining to both naturally occurring mutations responsible for Glanzmann thrombasthenia and to point mutations that affect ligand binding.

Alanine↗

PubMed2004

A knowledge-based scale for the analysis and prediction of buried and exposed faces of transmembrane domain proteins.

MOTIVATION: The dearth of structural data on alpha-helical membrane proteins (MPs) has hampered thus far the development of reliable knowledge-based potentials that can be used for automatic prediction of transmembrane (TM) protein structure. While algorithms for identifying TM segments are available, modeling of the TM domains of alpha-helical MPs involves assembling the segments into a bundle. This requires the correct assignment of the buried and lipid-exposed faces of the TM domains. RESULTS: A recent increase in the number of crystal structures of alpha-helical MPs has enabled an analysis of the lipid-exposed surfaces and the interiors of such molecules on the basis of structure, rather than sequence alone. Together with a conservation criterion that is based on previous observations that conserved residues are mostly found in the interior of MPs, the bias of certain residue types to be preferably buried or exposed is proposed as a criterion for predicting the lipid-exposed and interior faces of TMs. Applications to known structures demonstrates 80% accuracy of this prediction algorithm. AVAILABILITY: The algorithm used for the predictions is implemented in the ProperTM Web server (http://icb.med.cornell.edu/services/propertm/start).

Algorithms↗

PubMed2003

Probing conformational changes in neurotransmitter transporters: a structural context.

The Na+/Cl-dependent neurotransmitter transporters, a family of proteins responsible for the reuptake of neurotransmitters and other small molecules from the synaptic cleft, have been the focus of intensive research in recent years. The biogenic amine transporters, a subset of this larger family, are especially intriguing as they are the targets for many psychoactive compounds, including cocaine and amphetamines, as well as many antidepressants. In the absence of a high-resolution structure for any transporter in this family, research into the structure-function relationships of these transporters has relied on analysis of the effects of site-directed mutagenesis as well as of chemical modification of reactive residues. The aim of this review is to establish a structural context for the experimental study of these transporters through various computational approaches and to highlight what is known about the conformational changes associated with function in these transporters. We also present a novel numbering scheme to assist in the comparison of aligned positions between sequences of the neurotransmitter transporter family, a comparison that will be of increasing importance as additional experimental data is amassed.

Amino Acid Sequence↗

PubMed2003

Batch mode generation of residue-based diagrams of proteins.

SUMMARY: Residue-based diagrams of proteins are graphical representations that can be used in protein information systems. These diagrams make it possible to visually integrate different types of biological information. The approach has been used successfully for membrane proteins. We developed the Residue-based diagram generator to (i) make it possible to generate residue-based diagrams of proteins in a batch mode that is compatible with the needs of information system curators, (ii) allow the generation of residue-based diagrams for families of soluble proteins or domains. AVAILABILITY: Licensed. Royalty free licenses are granted to non-profit institutions for educational and research purposes. http://icb.mssm.edu/crt/RbDg/index.xml

Amino Acid Sequence↗

PubMed2003

Building protein diagrams on the web with the residue-based diagram editor RbDe.

The residue-based diagram editor (RbDe) is web-based software that greatly simplifies the construction of schematic diagrams of proteins. Residue-based diagrams display the sequence of a given protein in the context of its secondary and tertiary structure. Such diagrams are frequently used to summarize mutations or sequence features, in the context of the overall topology of a protein. The initial version of RbDe was designed for transmembrane proteins and has enabled many users to create diagrams of large systems such as G protein-coupled receptors or transporters. We present an extended diagram editor that supports other families of proteins. Users can now import custom-diagram layouts, use them to render members of any protein family and generate high-quality output for publication purposes. RbDe is available free over the web, at http://icb.mssm.edu/crt/RbDe

Amino Acid Sequence↗

PubMed2003

A second site rescue mutation partially restores functional expression to the serotonin transporter mutant V382P.

Transmembrane span 7 (TM7) of the serotonin transporter (SERT) was previously subjected to random mutagenesis, and the mutation V382P was found to abolish transport activity. Val-382 lies next to a threonine residue in the native sequence, creating a TP motif in this mutant. On the basis of molecular modeling studies, which have shown that the presence of a TP motif produces a very large kink in an alpha-helix, it was hypothesized that this motif could be the source of V382P's deleterious effects. We tested this hypothesis by producing second site mutations in the V382P construct that removed the TP motif: T381A-V382P and T381V-V382P. These mutants were tested for the recovery of serotonin transport and binding activities and for expression at the cell surface. The TM7 alpha-helix was modeled computationally, using Biased Monte Carlo simulations to quantify the conformational preferences of the wild type and mutant helices. The double mutation T381A-V382P, which was predicted by modeling to produce a smaller perturbing bend in TM7, was indeed found to allow partial rescue of transport activity. The double mutation T381V-V382P, on the other hand, did not rescue transport activity. Computational analysis of this mutant predicted a markedly different conformational preference from either the V382P or the T381A-V382P mutants. These studies show that changes in the structure of TM7 exert a strong influence on SERT's ability to achieve a mature, properly folded, cell surface conformation.

Amino Acid Motifs↗

PubMed2003

Molecular dynamics simulations of peptides and proteins with a continuum electrostatic model based on screened Coulomb potentials.

A continuum electrostatics approach for molecular dynamics (MD) simulations of macromolecules is presented and analyzed for its performance on a peptide and a globular protein. The approach incorporates the screened Coulomb potential (SCP) continuum model of electrostatics, which was reported earlier. The model was validated in a broad set of tests some of which were based on Monte Carlo simulations that included single amino acids, peptides, and proteins. The implementation for large-scale MD simulations presented in this article is based on a pairwise potential that makes the electrostatic model suitable for fast analytical calculation of forces. To assess the suitability of the approach, a preliminary validation is conducted, which consists of (i) a 3-ns MD simulation of the immunoglobulin-binding domain of streptococcal protein G, a 56-residue globular protein and (ii) a 3-ns simulation of Dynorphin, a biological peptide of 17 amino acids. In both cases, the results are compared with those obtained from MD simulations using explicit water (EW) molecules in an all-atom representation. The initial structure of Dynorphin was assumed to be an alpha-helix between residues 1 and 9 as suggested from NMR measurements in micelles. The results obtained in the MD simulations show that the helical structure collapses early in the simulation, a behavior observed in the EW simulation and consistent with spectroscopic data that suggest that the peptide may adopt mainly an extended conformation in water. The dynamics of protein G calculated with the SCP implicit solvent model (SCP-ISM) reveals a stable structure that conserves all the elements of secondary structure throughout the entire simulation time. The average structures calculated from the trajectories with the implicit and explicit solvent models had a cRMSD of 1.1 A, whereas each average structure had a cRMSD of about 0.8A with respect to the X-ray structure. The main conformational differences of the average structures with respect to the crystal structure occur in the loop involving residues 8-14. Despite the overall similarity of the simulated dynamics with EW and SCP models, fluctuations of side-chains are larger when the implicit solvent is used, especially in solvent exposed side-chains. The MD simulation of Dynorphin was extended to 40 ns to study its behavior in an aqueous environment. This long simulation showed that the peptide has a tendency to form an alpha-helical structure in water, but the stabilization free energy is too weak, resulting in frequent interconversions between random and helical conformations during the simulation time. The results reported here suggest that the SCP implicit solvent model is adequate to describe electrostatic effects in MD simulation of both peptides and proteins using the same set of parameters. It is suggested that the present approach could form the basis for the development of a reliable and general continuum approach for use in molecular biology, and directions are outlined for attaining this long-term goal.

Amino Acid Sequence↗

PubMed2003

Energetic and structural considerations for the mechanism of protein sliding along DNA in the nonspecific BamHI-DNA complex.

The molecular mechanism by which DNA-binding proteins find their specific binding sites is still unclear. To gain insights into structural and energetic elements of this mechanism, we used the crystal structure of the nonspecific BamHI-DNA complex as a template to study the dominant electrostatic interaction in the nonspecific association of protein with DNA, and the possible sliding pathways that could be sustained by such an interaction. Based on calculations using the nonlinear Poisson-Boltzmann method and Brownian dynamics, a model is proposed for the initial nonspecific binding of BamHI to B-form DNA that differs from that seen in the crystal structure of the nonspecific complex. The model is electrostatically favorable, and the salt dependence as well as other thermodynamic parameters calculated for this model are in good agreement with experimental results. Several residues in BamHI are identified for their important contribution to the energy in the nonspecific binding model, and specific mutagenesis experiments are proposed to test the model on this basis. We show that a favorable sliding pathway of the protein along DNA is helical.

Binding Sites↗

PubMed2003

Molecular basis of partial agonism: orientation of indoleamine ligands in the binding pocket of the human serotonin 5-HT2A receptor determines relative efficacy.

Based on experiment and computational simulation, we present a structural explanation for the differing efficacies of indole agonists at the human serotonin 5-HT2A receptor (5HT2AR). We find that serotonin [5-hydroxytryptamine (5-HT)] forms hydrogen-bonds with Ser3.36 in helix 3 and Ser5.46 in helix 5. Disruption of these hydrogen bonds by methyl-substitution of the cationic primary amine or of the backbone N1-amine, respectively, leads to a reduction in agonist efficacy. Computational simulation predicts that mutation of Ser3.36 to Ala should allow a similar interaction with helix 3 both for agonists that have unmodified cationic amine side chains and for those with substituted amines. Experimentally, this mutation was found to largely eliminate the differences in efficacy caused by cationic amine substitution for a series of indole congeners. Similarly, substitution of the N1-amine, which interacts with Ser5.46, reduced efficacy more markedly at the wild-type (WT) than at the Ser5.46Ala mutant receptor. Computational modeling of binding pocket interactions of ligands with WT and mutant receptor constructs demonstrate how the Ser3.36 and Ser5.46 interactions serve to modify the agonist's favored position in the binding pocket. A striking correlation was found between differences in the position assumed by the indole ring and differences in agonist activity. These data support the hypothesis that the position of the agonist interacting with the receptor is influenced by specific interactions in helices 3 and 5 and determines the degree of receptor activation by agonist through a mechanism that is likely to be shared by other G-protein coupled receptors in this class.

Alanine↗

PubMed2002

The local environment at the cytoplasmic end of TM6 of the mu opioid receptor differs from those of rhodopsin and monoamine receptors: introduction of an ionic lock between the cytoplasmic ends of helices 3 and 6 by a L6.30(275)E mutation inactivates the mu opioid receptor and reduces the constitutive activity of its T6.34(279)K mutant.

Activation of rhodopsin and monoamine G protein-coupled receptors (GPCRs) has been proposed to involve in part the disruption of a conserved E6.30-R3.50 ionic interaction between transmembrane segments (TMs) 3 and 6. However, this interaction does not occur in the opioid receptors, which have L275 at 6.30. On the basis of our findings that mutations of T6.34(279) to K and D produced, respectively, a constitutively active and an inactive form of the mu opioid receptor, we previously suggested that the functional role of the 6.30(275) residue could be assumed by T6.34(279), but the interplay between residues at positions 6.30 and 6.34 remained unresolved. In this study, we examined the effects of introducing an E in position 6.30(275) of the wild type (WT) and of the T6.34(279) mutants of the mu opioid receptor to compare the participation of the 6.30 locus in molecular events during activation in this receptor with its role in other GPCRs. The L6.30(275)E and the L6.30(275)E/T6.34(279)D mutants displayed no constitutive activity and could not be activated by the agonist DAMGO or morphine. The L6.30(275)E/T6.34(279)K mutant had some constitutive activity, but much less than the T6.34(279)K mutant, and could be activated by both agonists. The rank order of affinity for the agonist DAMGO is as follows: T6.34(279)K > WT congruent with L6.30(275)E/T6.34(279)K > L6.30(275)E congruent with T6.34(279)D > L6.30(275)E/T6.34(279)D; however, all constructs have a similar affinity for the antagonist [(3)H]diprenorphine. These data are interpreted in the context of interactions with the conserved R3.50(165) in TM3. When L6.30(275) is mutated to E, the favorable E6.30(275)-R3.50(165) interaction stabilizes an inactive state, as in rhodopsin, and hence reduces the activities of T6.34(279) mutants. Thus, the mu opioid receptor is shown to be different from rhodopsin and monoamine GPCRs, of which the WTs with native E6.30 can be activated, and the 6.34D or 6.34K mutants display enhanced constitutive activities. Our molecular modeling results suggest that some specific differences in local geometry at the cytoplasmic ends of TM5 and TM6 may account in part for the observed differences in the molecular mechanisms of receptor activation.

Animals↗

PubMed2002

Conserved helix 7 tyrosine acts as a multistate conformational switch in the 5HT2C receptor. Identification of a novel "locked-on" phenotype and double revertant mutations.

Studies in many rhodopsin-like G-protein-coupled receptors are providing a general scheme of the structural processes underlying receptor activation. Microdomains in several receptors have been identified that appear to function as activation switches. However, evidence is emerging that these receptor proteins exist in multiple conformational states. To study the molecular control of this switching process, we investigated the function of a microdomain involving the conserved helix 7 tyrosine in the serotonin 5HT2C receptor. This tyrosine of the NPXXY motif was substituted for all naturally occurring amino acids. Three distinct constitutively active receptor phenotypes were found: moderate, high, and "locked-on" constitutive activity. In contrast to the activity of the other receptor mutants, the high basal signaling of the locked-on Y7.53N mutant was neither increased by agonists nor decreased by inverse agonists. The Y7.53F mutant was uncoupled. Computational modeling based on the rhodopsin crystal structure suggested that Y7.53 interacts with the conserved aromatic ring at position 7.60 in the recently identified helix 8 domain. This provided a basis for seeking revertant mutations to correct the defective function of the Y7.53F receptor. When the Y7.53F receptor was mutated at position 7.60, the wild-type phenotype was restored. These results suggest that Y7.53 and Y7.60 contribute to a common functional microdomain connecting helices 7 and 8 that influences the switching of the 5HT2C receptor among multiple active and inactive conformations.

Amino Acid Motifs↗

PubMed2002

Structural mimicry of proline kinks: tertiary packing interactions support local structural distortions.

Proline residues in the helical segments of soluble and transmembrane proteins have received special attention from both a structural and functional perspective. A feature of these helices is the structural distortion termed "proline-kink", which has been associated with the presence of the proline residue. However, a recent report on the yeast heat-shock transcription factor of Kluyveromyces lactis (HSF_KL) suggests that these proline-associated deformations can be achieved in the absence of proline residues, thus raising the question of the mechanisms responsible for the structural mimicry of proline-related features. In this study, the specific interactions responsible for the distortion were characterized by comparative analysis of the atomic details of the packing interactions that surround the evolutionarily conserved proline-kink in the alpha2 helix of HSF_KL and a set of 39 structurally related proteins that lacked the distortion. The mechanistic details inferred from this analysis were confirmed with molecular dynamics simulations. The study shows that the packing interactions between the alpha2 and alpha1 helices in HSF_KL are responsible for the stabilization of the conserved kink, whether a proline residue that divides the helix into segments is present or not. The proline-kink can facilitate the formation of tertiary packing interactions that would otherwise not be possible. However, it is the ability to establish differential packing interactions for the helix segments, rather than the structural properties of the proline-kink itself, that emerges as the key factor for the characteristic distortion.

Amino Acid Sequence↗

PubMed2002

Structural models for dimerization of G-protein coupled receptors: the opioid receptor homodimers.

Among the most exciting functional features of G-protein coupled receptors (GPCRs) that are coming into focus lately are those relating to the role and structural characteristics of their oligomerization (mostly homo- and heterodimers). The structural underpinnings of these novel functional insights are still not clear, as current experimental techniques have not yet succeeded in identifying the dimerization interfaces between GPCR monomers. Two computational approaches have recently been designed in our lab to provide reasonable three-dimensional (3D) molecular models of the transmembrane (TM) regions of GPCR dimers based on a combination of the structural information of receptor monomers and analyses of correlated mutations in receptor families. The modeling of GPCR heterodimers has been described recently. We present here a related approach for modeling of GPCR homodimers that identifies the interfaces in the most likely configurations of the complexes. The approach is illustrated for the three cloned opioid receptor subtypes (OPRD, OPRM, and OPRK).

Computer Simulation↗

PubMed2002

Three-dimensional representations of G protein-coupled receptor structures and mechanisms.

Explore the source record for details and available documents.

Amino Acid Sequence↗

PubMed2002

Prediction of heterodimerization interfaces of G-protein coupled receptors with a new subtractive correlated mutation method.

Recent studies employing differential epitope tagging, selective immunoprecipitation of receptor complexes and fluorescence or bioluminescence resonance energy transfer techniques provide direct evidence for heterodimerization between both closely and distantly related members of the G-protein coupled receptor (GPCR) family. Since heterodimerization appears to play a role in modulating agonist affinity, efficacy and/or trafficking properties, the molecular models of GPCRs required to understand receptor function must consider these oligomerization hypotheses. To advance knowledge in this field, we present here a computational approach based on correlated mutation analysis and the structural information contained in three-dimensional molecular models of the transmembrane regions of GPCRs built using the rhodopsin crystal structure as a template. The new subtractive correlated mutation method reveals likely heterodimerization interfaces amongst the different alternatives for the positioning of two tightly packed bundles of seven transmembrane domains next to each other in contact heterodimers of GPCRs. Predictions are applied to GPCRs in the class of opioid receptors. However, in the absence of a known structure of any GPCR dimer, the features of the method and predictions are also illustrated and analyzed for a dimeric complex of known structure.

Mutation↗