Biomedical subjects

Harel Weinstein

Publications and source records attributed to Harel Weinstein.

At least 19 recordsLinked to original sources

PubMed2006

Modeling activated states of GPCRs: the rhodopsin template.

Activation of G Protein-Coupled Receptors (GPCRs) is an allosteric mechanism triggered by ligand binding and resulting in conformational changes transduced by the transmembrane domain. Models of the activated forms of GPCRs have become increasingly necessary for the development of a clear understanding of signal propagation into the cell. Experimental evidence points to a multiplicity of conformations related to the activation of the receptor, rendered important physiologically by the suggestion that different conformations may be responsible for coupling to different signaling pathways. In contrast to the inactive state of rhodopsin (RHO) for which several high quality X-ray structures are available, the structure-related information for the active states of rhodopsin and all other GPCRs is indirect. We have collected and stored such information in a repository we maintain for activation-specific structural data available for rhodopsin-like GPCRs, http://www.physiology.med.cornell.edu/GPCRactivation/gpcrindex.html . Using these data as structural constraints, we have applied Simulated Annealing Molecular Dynamics to construct a number of different active state models of RHO starting from the known inactive structure. The common features of the models indicate that TM3 and TM5 play an important role in activation, in addition to the well-established rearrangement of TM6. Some of the structural changes observed in these models occur in regions that were not involved in the constraints, and have not been previously tested experimentally; they emerge as interesting candidates for further experimental exploration of the conformational space of activated GPCRs. We show that none of the normal modes calculated from the inactive structure has a dominant contribution along the path of conformational rearrangement from inactive to the active forms of RHO in the models. This result may differentiate rhodopsin from other GPCRs, and the reasons for this difference are discussed in the context of the structural properties and the physiological function of the protein.

Models, Molecular↗

PubMed2006

Dynamic models of G-protein coupled receptor dimers: indications of asymmetry in the rhodopsin dimer from molecular dynamics simulations in a POPC bilayer.

Based on the growing evidence that G-protein coupled receptors (GPCRs) form homo- and hetero-oligomers, models of GPCR signaling are now considering macromolecular assemblies rather than monomers, with the homo-dimer regarded as the minimal oligomeric arrangement required for functional coupling to the G-protein. The dynamic mechanisms of such signaling assemblies are unknown. To gain some insight into properties of GPCR dimers that may be relevant to functional mechanisms, we study their current structural prototype, rhodopsin. We have carried out nanosecond time-scale molecular dynamics (MD) simulations of a rhodopsin dimer and compared the results to the monomer simulated in the same type of bilayer membrane model composed of an equilibrated unit cell of hydrated palmitoyl-oleoyl-phosphatidyl choline (POPC). The dynamic representation of the homo-dimer reveals the location of structural changes in several regions of the monomeric subunits. These changes appear to be more pronounced at the dimerization interface that had been shown to be involved in the activation process [Proc Natl Acad Sci USA 102:17495, 2005]. The results are consistent with a model of GPCR activation that involves allosteric modulation through a single GPCR subunit per dimer.

Animals↗

PubMed2006

Thermodynamic basis for promiscuity and selectivity in protein-protein interactions: PDZ domains, a case study.

Like other protein-protein interaction domains, PDZ domains are involved in many key cellular processes. These processes often require that specific multiprotein complexes be assembled, a task that PDZ domains accomplish by binding to specific peptide motifs in target proteins. However, a growing number of experimental studies show that PDZ domains (like other protein-protein interaction domains) can engage in a variety of interactions and bind distinct peptide motifs. Such promiscuity in ligand recognition raises intriguing questions about the molecular and thermodynamic mechanisms that can sustain it. To identify possible sources of promiscuity and selectivity underlying PDZ domain interactions, we performed molecular dynamics simulations of 20 to 25 ns on a set of 12 different PDZ domain complexes (for the proteins PSD-95, Syntenin, Erbin, GRIP, NHERF, Inad, Dishevelled, and Shank). The electrostatic, nonpolar, and configurational entropy binding contributions were evaluated using the MM/PBSA method combined with a quasi-harmonic analysis. The results revealed that PDZ domain interactions are characterized by overwhelmingly favorable nonpolar contributions and almost negligible electrostatic components, a mix that may readily sustain promiscuity. In addition, despite the structural similarity in fold and in recognition modes, the entropic and other dynamical aspects of binding were remarkably variable not only across PDZ domains but also for the same PDZ domain bound to distinct ligands. This variability suggests that entropic and dynamical components can play a role in determining selectivity either of PDZ domain interactions with peptide ligands or of PDZ domain complexes with downstream effectors.

Computer Simulation↗

PubMed2006

Ab initio computational modeling of loops in G-protein-coupled receptors: lessons from the crystal structure of rhodopsin.

With the help of the crystal structure of rhodopsin an ab initio method has been developed to calculate the three-dimensional structure of the loops that connect the transmembrane helices (TMHs). The goal of this procedure is to calculate the loop structures in other G-protein coupled receptors (GPCRs) for which only model coordinates of the TMHs are available. To mimic this situation a construct of rhodopsin was used that only includes the experimental coordinates of the TMHs while the rest of the structure, including the terminal domains, has been removed. To calculate the structure of the loops a method was designed based on Monte Carlo (MC) simulations which use a temperature annealing protocol, and a scaled collective variables (SCV) technique with proper structural constraints. Because only part of the protein is used in the calculations the usual approach of modeling loops, which consists of finding a single, lowest energy conformation of the system, is abandoned because such a single structure may not be a representative member of the native ensemble. Instead, the method was designed to generate structural ensembles from which the single lowest free energy ensemble is identified as representative of the native folding of the loop. To find the native ensemble a successive series of SCV-MC simulations are carried out to allow the loops to undergo structural changes in a controlled manner. To increase the chances of finding the native funnel for the loop, some of the SCV-MC simulations are carried out at elevated temperatures. The native ensemble can be identified by an MC search starting from any conformation already in the native funnel. The hypothesis is that native structures are trapped in the conformational space because of the high-energy barriers that surround the native funnel. The existence of such ensembles is demonstrated by generating multiple copies of the loops from their crystal structures in rhodopsin and carrying out an extended SCV-MC search. For the extracellular loops e1 and e3, and the intracellular loop i1 that were used in this work, the procedure resulted in dense clusters of structures with Calpha-RMSD approximately 0.5 angstroms. To test the predictive power of the method the crystal structure of each loop was replaced by its extended conformations. For e1 and i1 the procedure identifies native clusters with Calpha-RMSD approximately 0.5 angstroms and good structural overlap of the side chains; for e3, two clusters were found with Calpha-RMSD approximately 1.1 angstroms each, but with poor overlap of the side chains. Further searching led to a single cluster with lower Calpha-RMSD but higher energy than the two previous clusters. This discrepancy was found to be due to the missing elements in the constructs available from experiment for use in the calculations. Because this problem will likely appear whenever parts of the structural information are missing, possible solutions are discussed.

Computer Simulation↗

PubMed2006

A comprehensive structure-based alignment of prokaryotic and eukaryotic neurotransmitter/Na+ symporters (NSS) aids in the use of the LeuT structure to probe NSS structure and function.

The recently elucidated crystal structure of a prokaryotic member of the neurotransmitter/sodium symporter (NSS) family (Yamashita et al., 2005) is a major advance toward understanding structure-function relationships in this important class of transporters. To aid in the generalization of these results, we present here a comprehensive sequence alignment of all known prokaryotic and eukaryotic NSS proteins, based on the crystal structure of the leucine transporter from Aquifex aeolicus (LeuT). Regions of low sequence identity between prokaryotic and eukaryotic transporters were aligned with the aid of a number of bioinformatics tools, and the resulting alignments were validated by comparison with experimental data. In a number of regions, including the transmembrane segments 4, 5, and 9 as well as extracellular loops 2, 3, and 4, our alignment differs from the one proposed previously [Nature (Lond) 437: 215-223, 2005]. Important similarities and differences among the sequences of NSS proteins in regions likely to determine selectivity in substrate binding and mechanisms of transport regulation are discussed in the context of the LeuT structure and the alignment.

Amino Acid Sequence↗

PubMed2006

Functional selectivity and classical concepts of quantitative pharmacology.

The concept of intrinsic efficacy has been enshrined in pharmacology for half of a century, yet recent data have revealed that many ligands can differentially activate signaling pathways mediated via a single G protein-coupled receptor in a manner that challenges the traditional definition of intrinsic efficacy. Some terms for this phenomenon include functional selectivity, agonist-directed trafficking, and biased agonism. At the extreme, functionally selective ligands may be both agonists and antagonists at different functions mediated by the same receptor. Data illustrating this phenomenon are presented from serotonin, opioid, dopamine, vasopressin, and adrenergic receptor systems. A variety of mechanisms may influence this apparently ubiquitous phenomenon. It may be initiated by differences in ligand-induced intermediate conformational states, as shown for the beta(2)-adrenergic receptor. Subsequent mechanisms that may play a role include diversity of G proteins, scaffolding and signaling partners, and receptor oligomers. Clearly, expanded research is needed to elucidate the proximal (e.g., how functionally selective ligands cause conformational changes that initiate differential signaling), intermediate (mechanisms that translate conformation changes into differential signaling), and distal mechanisms (differential effects on target tissue or organism). Besides the heuristically interesting nature of functional selectivity, there is a clear impact on drug discovery, because this mechanism raises the possibility of selecting or designing novel ligands that differentially activate only a subset of functions of a single receptor, thereby optimizing therapeutic action. It also may be timely to revise classic concepts in quantitative pharmacology and relevant pharmacological conventions to incorporate these new concepts.

Animals↗

PubMed2006

State-dependent conformations of the translocation pathway in the tyrosine transporter Tyt1, a novel neurotransmitter:sodium symporter from Fusobacterium nucleatum.

The gene of a novel prokaryotic member (Tyt1) of the neurotransmitter:sodium symporter (NSS) family has been cloned from Fusobacterium nucleatum. In contrast to eukaryotic and some prokaryotic NSSs, which contain 12 transmembrane domains (TMs), Tyt1 contains only 11 TMs, a characteristic shared by approximately 70% of prokaryotic NSS homologues. Nonetheless upon heterologous expression in an engineered Escherichia coli host, Tyt1 catalyzes robust Na+-dependent, highly selective l-tyrosine transport. Genetic engineering of Tyt1 variants devoid of cysteines or with individually retained endogenous cysteines at positions 18 or 238, at the cytoplasmic ends of TM1 and TM6, respectively, preserved normal transport activity. Whereas cysteine-less Tyt1 was resistant to the inhibitory effect of sulfhydryl-alkylating reagents, N-ethylmaleimide inhibited transport by Tyt1 variants containing either one or both of the endogenous cysteines, and this inhibition was altered by the substrates sodium and tyrosine, consistent with substrate-induced dynamics in the transport pathway. Our findings support a binding model of Tyt1 function in which an ordered sequence of substrate-induced structural changes reflects distinct conformational states of the transporter. This work identifies Tyt1 as the first functional bacterial NSS member putatively consisting of only 11 TMs and shows that Tyt1 is a suitable model for the study of NSS dynamics with relevance to structure/function relationships of human NSSs, including the dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid transporters.

Amino Acid Sequence↗

PubMed2006

Hallucinogen actions on 5-HT receptors reveal distinct mechanisms of activation and signaling by G protein-coupled receptors.

We review the effect of some key advances in the characterization of molecular mechanisms of signaling by G protein-coupled receptors (GPCRs) on our current understanding of mechanisms of drugs of abuse. These advances are illustrated by results from our ongoing work on the actions of hallucinogens on serotonin (5-HT) receptors. We show how a combined computational and experimental approach can reveal specific modes of receptor activation underlying the difference in properties of hallucinogens compared with nonhallucinogenic congeners. These modes of activation-that can produce distinct ligand-dependent receptor states-are identified in terms of structural motifs (SM) in molecular models of the receptors, which were shown to constitute conserved functional microdomains (FM). The role of several SM/FMs in the activation mechanism of the GPCRs is presented in detail to illustrate how this mechanism can lead to ligand-dependent modes of signaling by the receptors. Novel bioinformatics tools are described that were designed to support the quantitative mathematical modeling of ligand-specific signaling pathways activated by the 5-HT receptors targeted by hallucinogens. The approaches for mathematical modeling of signaling pathways activated by 5-HT receptors are described briefly in the context of ongoing work on detailed biochemical models of 5-HT2A, and combined 5-HT2A/5-HT1A, receptor-mediated activation of the MAPK 1,2 pathway. The continuing need for increasingly more realistic representation of signaling in dynamic compartments within the cell, endowed with spatio-temporal characteristics obtained from experiment, is emphasized. Such developments are essential for attaining a quantitative understanding of how the multiple functions of a cell are coordinated and regulated, and to evaluate the specifics of the perturbations caused by the drugs of abuse that target GPCRs.

Animals↗

PubMed2006

Trefoil factor family-1 mutations enhance gastric cancer cell invasion through distinct signaling pathways.

BACKGROUND & AIMS: Trefoil factor family-1 (TFF1) is a key gastric tumor-suppressor gene. TFF1 knockout mice develop multiple gastric adenomas and carcinomas, and human gastric cancers typically lack TFF1 expression. Recently, TFF1 mutations have been found in human gastric cancer. The purpose of this study was to determine the functionality of these mutants. METHODS: Recombinant wild-type TFF1 and the gastric cancer-associated TFF1 mutants (A10D and E13K) were produced and tested for their effect on gastric cancer cell proliferation, apoptosis, and invasion. Molecular modeling was used to guide the choice of mutants and to evaluate structure-function relationships. RESULTS: Molecular modeling suggested that A10D and E13K altered the surface charge of the loop 1 region of TFF1 without disturbing protein stability. Recombinant wild-type TFF1 significantly inhibited cell growth; A10D and E13K lost this tumor-suppressive property along with the ability to block etoposide-induced apoptosis. Although wild-type TFF1 promoted cell invasion, A10D and E13K were even more pro-invasive. Invasion induced by both mutants was blocked by inhibiting PI3-kinase or phospholipase-C, but inhibiting Rho-associated kinase (ROCK) blocked only E13K-induced invasion. CONCLUSIONS: The loss of tumor-suppressor activity and gain of invasiveness from single point mutations constitute evidence for a functional role of TFF1 mutations in gastric cancer. These site-directed mutagenesis experiments provide the tools for continued probing of signal transduction mechanisms and structural elements responsible for TFF1 functions.

Apoptosis↗

PubMed2005

Crosstalk in G protein-coupled receptors: changes at the transmembrane homodimer interface determine activation.

Functional crosstalk between G protein-coupled receptors in a homo- or heterodimeric assembly likely involves conformational changes at the dimer interface, but the nature of this interface is not yet established, and the dynamic changes have not yet been identified. We have mapped the homodimer interface in the dopamine D2 receptor over the entire length of the fourth transmembrane segment (TM4) by crosslinking of substituted cysteines. Their susceptibilities to crosslinking are differentially altered by the presence of agonists and inverse agonists. The TM4 dimer interface in the inverse agonist-bound conformation is consistent with the dimer of the inactive form of rhodopsin modeled with constraints from atomic force microscopy. Crosslinking of a different set of cysteines in TM4 was slowed by inverse agonists and accelerated in the presence of agonists; crosslinking of the latter set locks the receptor in an active state. Thus, a conformational change at the TM4 dimer interface is part of the receptor activation mechanism.

Cross-Linking Reagents↗

PubMed2005

A flexible docking procedure for the exploration of peptide binding selectivity to known structures and homology models of PDZ domains.

PDZ domains are important scaffolding modules that typically bind to the C-termini of their interaction partners. Several structures of such complexes have been solved, revealing a conserved binding site in the PDZ domain and an extended conformation of the bound peptide. A compendium of information regarding PDZ complexes demonstrates that dissimilar C-terminal peptides bind to the same PDZ domain, and different PDZ domains can bind the same peptides. A detailed understanding of the PDZ-peptide recognition is needed to elucidate this complexity. To this end, we have designed a family of docking protocols for PDZ domains (termed PDZ-DocScheme) that is based on simulated annealing molecular dynamics and rotamer optimization, and is applicable to the docking of long peptides (20-40 rotatable bonds) to both known PDZ structures and to the more complicated problem of homology models of these domains. The resulting protocol reproduces the structures of PDZ complexes with peptides 4-8 amino acids long within 1-2 A from the experimental structure when the docking is performed to the original structure. If the structure of the target PDZ domain is an apo structure or a homology model, the docking protocol yields structures within 3 A in 9 out of 12 test cases. The automated docking procedure PDZ-DocScheme can serve in the generation of a structural context for validation of PDZ domain specificity from mutagenesis and ligand binding data.

Models, Molecular↗

PubMed2005

A pincer-like configuration of TM2 in the human dopamine transporter is responsible for indirect effects on cocaine binding.

The second transmembrane segment (TM2) of DAT and other neurotransmitter transporters has been proposed to play a role in oligomerization as well as in cocaine binding. In an attempt to determine whether TM2 contributes to the binding site and/or transport pathway of DAT, we mutated to cysteine, one at a time, 25 residues in TM2 - from Phe98 to Gln122 - in an appropriate DAT background construct. Four of the mutants, F98C, G110C, P112C, and E117C, did not express at the cell surface, and G121C was inactive, despite its presence on the cell surface. Of the 21 mutants that expressed, none of the substituted cysteines reacted with MTSEA biotin-CAP, and none of the 20 functional mutants was sensitive to MTSEA or MTSET. Thus, TM2 does not appear to be water-accessible, based both on the lack of functional effects of charged MTS derivatives, and on the biochemical determination of lack of reaction with a biotinylated MTS derivative. This leads to the conclusion that TM2 does not contribute directly to the substrate-binding site or the transport pathway, and suggests that the observed effect of mutations in this region on cocaine binding is indirect. Three mutants, M106C, V107C and I108C, were crosslinked by treatment with HgCl(2). This crosslinking was inhibited by the presence of the cocaine analogue MFZ 2-12, likely due to a conformational rearrangement in TM2 upon inhibitor binding. However, the lack of crosslinking of cysteines substituted for Leu99, Leu113 and Leu120 - three of the residues that along with Met106 form a leucine heptad repeat in TM2 - makes it unlikely that this leucine repeat plays a role in symmetrical TM2 dimerization. Importantly, a high-resolution structure of LeuT, a sodium-dependent leucine transporter that is sufficiently homologous to DAT to suggest a high degree of structural similarity, became available while this manuscript was under review. We have taken advantage of this structure to explore further and interpret our experimental results in a rigorous structural context.

Analysis of Variance↗

PubMed2005

Crooked tail (Cd) model of human folate-responsive neural tube defects is mutated in Wnt coreceptor lipoprotein receptor-related protein 6.

A cranial neural tube defect in Crooked tail (Cd) mice is prevented with prenatal dietary folic acid Cd positional cloning reveals a missense mutation of a highly conserved amino acid in the low density lipoprotein receptor-related protein 6 (Lrp6), a coreceptor required for Wnt canonical signaling. Molecular modeling predicts that Lrp6(Cd) alters a hinge region of the second YWTD beta-propeller domain. Mutant LRP6 binds to Wnt and Dickkopf1 (Dkk1) but not Mesd1, and Dkk1 cannot antagonize Wnt in Cd/Cd cells, resulting in hyperactivity. NIH 3T3 cells transfected with a mutant Lrp6 plasmid resist Dkk1 antagonism much like Cd/+ cells, confirming the significance of the mutation. The Lrp6 mutation in Cd mice provides evidence for a functional connection between Wnt signaling and folate rescue of neural tube defects.

Amino Acid Sequence↗

PubMed2005

Modeling membrane proteins based on low-resolution electron microscopy maps: a template for the TM domains of the oxalate transporter OxlT.

The availability of both EM and high-resolution crystallographic data for several membrane proteins (MPs) permits a detailed evaluation of the ability of molecular modeling techniques to complement EM data in the development of models of MPs. A protocol for this purpose is presented, consisting of (1) identifying transmembrane (TM) domains from sequence; (2) assigning buried and lipid-exposed faces of the TM domains; and (3) assembling the TM domains into a bundle, based on geometric restraints obtained from the EM data. The protocol is validated by predicting the structures of several 7- and 12-TM MPs to within 3-5 A r.m.s.d. from their crystal structures. The protocol is applied to generate a model of the oxalate transporter OxlT, for which a high-resolution structure is not yet available.

Algorithms↗

PubMed2005

Molecular determinants for the complex binding specificity of the PDZ domain in PICK1.

PICK1 (protein interacting with C kinase 1) contains a single PDZ domain known to mediate interaction with the C termini of several receptors, transporters, ion channels, and kinases. In contrast to most PDZ domains, the PICK1 PDZ domain interacts with binding sequences classifiable as type I (terminating in (S/T)XPhi; X, any residue) as well as type II (PhiXPhi; Phi, any hydrophobic residue). To enable direct assessment of the affinity of the PICK1 PDZ domain for its binding partners we developed a purification scheme for PICK1 and a novel quantitative binding assay based on fluorescence polarization. Our results showed that the PICK1 PDZ domain binds the type II sequence presented by the human dopamine transporter (-WLKV) with an almost 15-fold and >100-fold higher affinity than the type I sequences presented by protein kinase Calpha (-QSAV) and the beta(2)-adrenergic receptor (-DSLL), respectively. Mutational analysis of Lys(83) in the alphaB1 position of the PDZ domain suggested that this residue mimics the function of hydrophobic residues present in this position in regular type II PDZ domains. The PICK1 PDZ domain was moreover found to prefer small hydrophobic residues in the C-terminal P(0) position of the ligand. Molecular modeling predicted a rank order of (Val > Ile > Leu) that was verified experimentally with up to a approximately 16-fold difference in binding affinity between a valine and a leucine in P(0). The results define the structural basis for the unusual binding pattern of the PICK1 PDZ domain by substantiating the critical role of the alphaB1 position (Lys(83)) and of discrete side chain differences in position P(0) of the ligands.

Animals↗

PubMed2005

The study of G-protein coupled receptor oligomerization with computational modeling and bioinformatics.

To achieve a structural context for the analysis of G-protein coupled receptor (GPCR) oligomers, molecular modeling must be used to predict the corresponding interaction interfaces. The task is complicated by the paucity of detailed structural data at atomic resolution, and the large number of possible modes in which the bundles of seven transmembrane (TM) segments of the interacting GPCR monomers can be packed together into dimers and/or higher-order oligomers. Approaches and tools offered by bioinformatics can be used to reduce the complexity of this task and, combined with computational modeling, can serve to yield testable predictions for the structural properties of oligomers. Most of the bioinformatics methods take advantage of the evolutionary relation that exists among GPCRs, as expressed in their sequences and measurable in the common elements of their structural and functional features. These common elements are responsible for the presence of detectable patterns of motifs and correlated mutations evident from the alignment of the sequences of these complex biological systems. The decoding of these patterns in terms of structural and functional determinants can provide indications about the most likely interfaces of dimerization/oligomerization of GPCRs. We review here the main approaches from bioinformatics, enhanced by computational molecular modeling, that have been used to predict likely interfaces of dimerization/oligomerization of GPCRs, and compare results from their application to rhodopsin-like GPCRs. A compilation of the most frequently predicted GPCR oligomerization interfaces points to specific regions of TMs 4-6.

Computational Biology↗

PubMed2005

The structure and dynamics of GPCR oligomers: a new focus in models of cell-signaling mechanisms and drug design.

G protein-coupled receptors (GPCRs) represent approximately half of the potential pharmaceutical targets for current drugs, and thus the way in which these receptors assemble into dimeric/oligomeric structures is of vital interest in practical as well as conceptual aspects of current drug discovery efforts. The significance of such structures is based on the recent realization that ligand-dependent signaling by GPCRs is not necessarily transduced to the G protein by receptor monomers, but possibly by GPCR dimers or even oligomers that function as dynamic macromolecular assemblies. In addition, recent evidence that GPCR hetero-oligomerization can produce signaling units with unexpected combinations of pharmacological properties suggests entirely new methods for developing successful drugs. The dynamic mechanisms of these signaling assemblies remain to be elucidated. The development of increasingly accurate dynamic molecular models of GPCR dimers is expected to produce a more complete structural context for understanding the molecular mechanisms of GPCR function, and to aid in drug discovery.

Animals↗

PubMed2004

PDZBase: a protein-protein interaction database for PDZ-domains.

SUMMARY: PDZBase is a database that aims to contain all known PDZ-domain-mediated protein-protein interactions. Currently, PDZBase contains approximately 300 such interactions, which have been manually extracted from > 200 articles. The database can be queried through both sequence motif and keyword-based searches, and the sequences of interacting proteins can be visually inspected through alignments (for the comparison of several interactions), or as residue-based diagrams including schematic secondary structure information (for individual complexes).

Database Management Systems↗