Jufang Shan

The Substrate-Driven Transition to an Inward-Facing Conformation in the Functional Mechanism of the Dopamine Transporter

Background The dopamine transporter (DAT), a member of the neurotransmitter:Na+ symporter (NSS) family, terminates dopaminergic neurotransmission and is a major molecular target for psychostimulants such as cocaine and amphetamine, and for the treatment of attention deficit disorder and depression. The crystal structures of the prokaryotic NSS homolog of DAT, the leucine transporter LeuT, have provided critical structural insights about the occluded and outward-facing conformations visited during the substrate transport, but only limited clues regarding mechanism. To understand the transport mechanism in DAT we have used a homology model based on the LeuT structure in a computational protocol validated previously for LeuT, in which steered molecular dynamics (SMD) simulations guide the substrate along a pathway leading from the extracellular end to the intracellular (cytoplasmic) end. Methodology/Principal Findings Key findings are (1) a second substrate binding site in the extracellular vestibule, and (2) models of the conformational states identified as occluded, doubly occupied, and inward-facing. The transition between these states involve a spatially ordered sequence of interactions between the two substrate-binding sites, followed by rearrangements in structural elements located between the primary binding site and the cytoplasmic end. These rearrangements are facilitated by identified conserved hinge regions and a reorganization of interaction networks that had been identified as gates. Conclusions/Significance Computational simulations supported by information available from experiments in DAT and other NSS transporters have produced a detailed mechanistic proposal for the dynamic changes associated with substrate transport in DAT. This allosteric mechanism is triggered by the binding of substrate in the S2 site in the presence of the substrate in the S1 site. Specific structural elements involved in this mechanism, and their roles in the conformational transitions illuminated here describe, a specific substrate-driven allosteric mechanism that is directly amenable to experiment as shown previously for LeuT.

Quantitative Modeling of Membrane Deformations by Multihelical Membrane Proteins: Application to G-Protein Coupled Receptors

The interpretation of experimental observations of the dependence of membrane protein function on the properties of the lipid membrane environment calls for a consideration of the energy cost of protein-bilayer interactions, including the protein-bilayer hydrophobic mismatch. We present a novel (to our knowledge) multiscale computational approach for quantifying the hydrophobic mismatch-driven remodeling of membrane bilayers by multihelical membrane proteins. The method accounts for both the membrane remodeling energy and the energy contribution from any partial (incomplete) alleviation of the hydrophobic mismatch by membrane remodeling. Overcoming previous limitations, it allows for radially asymmetric bilayer deformations produced by multihelical proteins, and takes into account the irregular membrane-protein boundaries. The approach is illustrated by application to two G-protein coupled receptors: rhodopsin in bilayers of different thickness, and the serotonin 5-HT(2A) receptor bound to pharmacologically different ligands. Analysis of the results identifies the residual exposure that is not alleviated by bilayer adaptation, and its quantification at specific transmembrane segments is shown to predict favorable contact interfaces in oligomeric arrays. In addition, our results suggest how distinct ligand-induced conformations of G-protein coupled receptors may elicit different functional responses through differential effects on the membrane environment.

Ligand-dependent conformations and dynamics of the serotonin 5-HT(2A) receptor determine its activation and membrane-driven oligomerization properties.

From computational simulations of a serotonin 2A receptor (5-HT2AR) model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD) simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011), we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i)-the involvement of cholesterol in the activation of the 5-HT2AR, and (ii)-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization.

GPCR-OKB

SUMMARY Rapid expansion of available data about G Protein Coupled Receptor (GPCR) dimers/oligomers over the past few years requires an effective system to organize this information electronically. Based on an ontology derived from a community dialog involving colleagues using experimental and computational methodologies, we developed the GPCR-Oligomerization Knowledge Base (GPCR-OKB). GPCR-OKB is a system that supports browsing and searching for GPCR oligomer data. Such data were manually derived from the literature. While focused on GPCR oligomers, GPCR-OKB is seamlessly connected to GPCRDB, facilitating the correlation of information about GPCR protomers and oligomers. AVAILABILITY AND IMPLEMENTATION The GPCR-OKB web application is freely available at http://www.gpcr-okb.org

A Functional Selectivity Mechanism at the Serotonin-2A GPCR Involves Ligand-Dependent Conformations of Intracellular Loop 2

With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT2AR) in the absence of ligand and bound to four distinct serotonergic agonists. The 5-HT2AR is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT2AR agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT2AR interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. The findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT2AR activation.

PROBING THE STRUCTURAL DETERMINANTS FOR THE FUNCTION OF INTRACELLULAR LOOP 2 IN STRUCTURALLY COGNATE G-PROTEIN COUPLED RECEPTORS

Intracellular loop 2 (IL2) in G-protein-coupled receptors (GPCRs) is functionally important, e.g., in binding to G-protein and β-arrestin. Differences in secondary structure of IL2 in the crystal structures of the very similar β(1)- and β(2)-adrenergic receptors (β(1)AR and β(2)AR, respectively), i.e., an α-helix and an L-shaped strand, respectively, emphasize the need to understand the structural basis for IL2 functionality. We studied the properties of IL2 in the context of experimental data using a Monte Carlo-based ab initio method. The procedure was validated first by verifying that the IL2 structures in β(1)AR and β(2)AR crystals were correctly reproduced, even after conformational ensemble searches at >1200 K where most secondary structure had been lost. We found that IL2 in β(1)AR and β(2)AR sampled each others conformation but adopted different energetically preferred conformations, consistent with the crystal structures. The results indicate a persistent contextual preference for the structure of IL2, which was conserved when the IL2 sequences were interchanged between the receptors. We conclude that the protein environment, more than the IL2 sequence, regulates the IL2 structures. We extended the approach to the molecular model of 5-HT(2A)R for which no crystal structure is available and found that IL2 is predominantly helical, similar to IL2 in β(1)AR. Because the P3.57A mutation in IL2 had been shown to decrease β-arrestin binding and internalization, we predicted the effects of the mutation and found that it decreased the propensity of IL2 to form helix, identifying the helical IL2 as a component of the GPCR active form.

Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation.

The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.

Calculation of pKa in proteins with the microenvironment modulated-screened coulomb potential†

The MM‐SCP has been applied to predict pKa values of titratable residues in wild type and mutants of staphylococcal nuclease (SNase). The calculations were based on crystal structures made available by the Garcia‐Moreno Laboratory. In the mutants, mostly deeply buried hydrophobic residues were replaced with ionizable residues, and thus their pKa values could be measured and calculated using various methods. The data set used here consisted of a set of WT SNase for which His pKa at several ionic strengths had been measured, a set of mutants for which measured pKa were available and a set of 11 mutants for which the measured pKa were not known at the time of calculation. For this latter set, blind predictions were submitted to the protein pKa cooperative, 2009 workshop at Telluride, where the results of the blind predictions were discussed (the RMSD of the submitted set was 1.10 pH units). The calculations on the structures with known pKa indicated that in addition to weaknesses of the method, structural issues were observed that led to larger errors (>1) in pKa predictions. For example, different crystallographic conditions or steric clashes can lead to differences in the local environment around the titratable residue, which can produce large differences in the calculated pKa. To gain further insight into the reliability of the MM‐SCP, pKa of an extended set of 54 proteins belonging to several structural classes were carried out. Here some initial results from this study are reported to help place the SNase results in the appropriate context. Proteins 2011.