Kalyan C. Tirupula

Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

Metabotropic glutamate receptors (mGluRs) are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%), the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%), the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86%) and 8/9 (89%) for ArgusLab and 10/14 (71%) and 7/9 (78%) for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by photochemical isomerization of the retinal ligand – despite the extensive differences in sequences between mGluRs and rhodopsin.

Click chemistry on azidoproline: high-affinity dual antagonist for HIV-1 envelope glycoprotein gp120.

Recent advances in the Cu-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and terminal alkynes afford 1,4-disubstituted 1,2,3-triazoles with superior regioselectivity, and almost quantitative transformation under extremely mild conditions. The simple and robust features of this methodology have found application in drug discovery, bioconjugation, and material science. Herein, we report the novel use of stable and chemically accessible azidoproline within an otherwise normally constituted solid-phase-synthesized polypeptide as a platform for side-chain bioconjugation reactions through click chemistry, and its use to introduce triazole conjugates into a dual antagonist of the HIV-1 envelope protein gp120. The procedure enables rapid generation of analogues at an internal side-chain position of the antagonist. This has led to a lead inhibitor for HIV-1 infection with affinity in the nanomolar range. Acquired immunodeficiency syndrome (AIDS), the globally epidemic disease caused by HIV-1, has created an urgent need for new classes of antiviral agents. Viral infection is initiated by the binding of gp120 of HIV-1 to the CD4 antigen on the host T cell surface. The envelope glycoprotein of HIV-1 is a trimer that consists of three gp120 exterior envelope glycoproteins and gp41 transmembrane glycoproteins. The binding of gp120 to CD4 promotes a conformational change in gp120 that increases its affinity for a second host-cell receptor, one of the chemokine receptors, CCR5 and CXCR4. The interaction of gp120 with its receptors is believed to promote further conformational rearrangements in the HIV-1 envelope that drive fusion of the viral and host-cell membranes. Blockage of the interactions between gp120 and cell-surface receptors is an attractive goal for the prevention of HIV-1 infection through the inhibition of membrane fusion and viral entry. The feasibility of therapeutic efficacy with fusion inhibitors has been demonstrated recently. A promising fusion inhibitor lead is a 12-residue peptide (RINNIPWSEAMM, 1) which was discovered initially by phage library screening. Peptide 1 inhibits the interaction between gp120 and both CD4 and 17b, an antibody that recognizes an epitope that overlaps the CCR5 binding site with affinity in the micromolar range. Herein, we report that conjugation at proline 6 of peptide 1 through click chemistry leads to inhibitors with strikingly high affinity for the HIV-1 envelope and which maintain the dual inhibition of CD4 and 17b binding to the viral Env protein. The modification of proline with 4-phenyl-1,4-disubstituted 1,2,3triazole, fabricated through a [3+2] cycloaddition reaction (Scheme 1) leads to a peptide which binds to gp120 with a KD

pH-dependent interaction of rhodopsin with cyanidin-3-glucoside. 1. Structural aspects.

Anthocyanins are a class of natural compounds common in flowers and vegetables. Because of the increasing preference of consumers for food containing natural colorants and the demonstrated beneficial effects of anthocyanins on human health, it is important to decipher the molecular mechanisms of their action. Previous studies indicated that the anthocyanin cyanidin‐3‐glucoside (C3G) modulates the function of the photoreceptor rhodopsin. In this paper, we show using selective excitation 1H NMR spectroscopy that C3G binds to rhodopsin. Ligand resonances broaden upon rhodopsin addition and rhodopsin resonances exhibit chemical shift changes as well as broadening effects in specific resonances, in an activation state‐dependent manner. Furthermore, dark‐adapted and light‐activated states of rhodopsin show preferences for different C3G species. Molecular docking studies of the flavylium cation, quinoidal base, carbinol pseudobase and chalcone forms of C3G to models of the dark, light‐activated and opsin structures of rhodopsin also support this conclusion. The results provide new insights into anthocyanin–protein interactions and may have relevance for the enhancement of night vision by this class of compounds. This work is also the first report of the study of ligand binding to a full‐length membrane receptor in detergent micelles by 1H NMR spectroscopy. Such studies were previously hampered by the presence of detergent micelle resonances, a problem overcome by the selective excitation approach.

Systematic prediction of human membrane receptor interactions

Membrane receptor‐activated signal transduction pathways are integral to cellular functions and disease mechanisms in humans. Identification of the full set of proteins interacting with membrane receptors by high‐throughput experimental means is difficult because methods to directly identify protein interactions are largely not applicable to membrane proteins. Unlike prior approaches that attempted to predict the global human interactome, we used a computational strategy that only focused on discovering the interacting partners of human membrane receptors leading to improved results for these proteins. We predict specific interactions based on statistical integration of biological data containing highly informative direct and indirect evidences together with feedback from experts. The predicted membrane receptor interactome provides a system‐wide view, and generates new biological hypotheses regarding interactions between membrane receptors and other proteins. We have experimentally validated a number of these interactions. The results suggest that a framework of systematically integrating computational predictions, global analyses, biological experimentation and expert feedback is a feasible strategy to study the human membrane receptor interactome.

Characterization of membrane protein non-native states. 1. Extent of unfolding and aggregation of rhodopsin in the presence of chemical denaturants.

Little is known about the general folding mechanisms of helical membrane proteins. Unfolded, i.e., non-native states, in particular, have not yet been characterized in detail. Here, we establish conditions under which denatured states of the mammalian membrane protein rhodopsin, a prototypic G protein coupled receptor with primary function in vision, can be studied. We investigated the effects of the chemical denaturants sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride (GuHCl), and trifluoroacetic acid (TFA) on rhodopsins secondary structure and propensity for aggregation. Ellipticity at 222 nm decreases in the presence of maximum concentrations of denaturants in the order TFA > GuHCl > urea > SDS + urea > SDS. Interpretation of these changes in ellipticity in terms of helix loss is challenged because the addition of some denaturants leads to aggregation. Through a combination of SDS-PAGE, dependence of ellipticity on protein concentration, and 1D (1)H NMR we show that aggregates form in the presence of GuHCl, TFA, and urea but not in any concentration of SDS, added over a range of 0.05%-30%. Mixed denaturant conditions consisting of 3% SDS and 8 M urea, added in this order, also did not result in aggregation. We conclude that SDS is able to prevent the exposure of large hydrophobic regions present in membrane proteins which otherwise leads to aggregation. Thus, 30% SDS and 3% SDS + 8 M urea are the denaturing conditions of choice to study maximally unfolded rhodopsin without aggregation.

Identification of Motions in Membrane Proteins by Elastic Network Models and Their Experimental Validation

Identifying the functional motions of membrane proteins is difficult because they range from large-scale collective dynamics to local small atomic fluctuations at different timescales that are difficult to measure experimentally due to the hydrophobic nature of these proteins. Elastic Network Models, and in particular their most widely used implementation, the Anisotropic Network Model (ANM), have proven to be useful computational methods in many recent applications to predict membrane protein dynamics. These models are based on the premise that biomolecules possess intrinsic mechanical characteristics uniquely defined by their particular architectures. In the ANM, interactions between residues in close proximity are represented by harmonic potentials with a uniform spring constant. The slow mode shapes generated by the ANM provide valuable information on the global dynamics of biomolecules that are relevant to their function. In its recent extension in the form of ANM-guided molecular dynamics (MD), this coarse-grained approach is augmented with atomic detail. The results from ANM and its extensions can be used to guide experiments and thus speedup the process of quantifying motions in membrane proteins. Testing the predictions can be accomplished through (a) direct observation of motions through studies of structure and biophysical probes, (b) perturbation of the motions by, e.g., cross-linking or site-directed mutagenesis, and (c) by studying the effects of such perturbations on protein function, typically through ligand binding and activity assays. To illustrate the applicability of the combined computational ANM-experimental testing framework to membrane proteins, we describe-alongside the general protocols-here the application of ANM to rhodopsin, a prototypical member of the pharmacologically relevant G-protein coupled receptor family.

A minimal ligand binding pocket within a network of correlated mutations identified by multiple sequence and structural analysis of G protein coupled receptors.

BackgroundG protein coupled receptors (GPCRs) are seven helical transmembrane proteins that function as signal transducers. They bind ligands in their extracellular and transmembrane regions and activate cognate G proteins at their intracellular surface at the other side of the membrane. The relay of allosteric communication between the ligand binding site and the distant G protein binding site is poorly understood. In this study, GREMLIN [1], a recently developed method that identifies networks of co-evolving residues from multiple sequence alignments, was used to identify those that may be involved in communicating the activation signal across the membrane. The GREMLIN-predicted long-range interactions between amino acids were analyzed with respect to the seven GPCR structures that have been crystallized at the time this study was undertaken.ResultsGREMLIN significantly enriches the edges containing residues that are part of the ligand binding pocket, when compared to a control distribution of edges drawn from a random graph. An analysis of these edges reveals a minimal GPCR binding pocket containing four residues (T1183.33, M2075.42, Y2686.51 and A2927.39). Additionally, of the ten residues predicted to have the most long-range interactions (A1173.32, A2726.55, E1133.28, H2115.46, S186EC2, A2927.39, E1223.37, G902.57, G1143.29 and M2075.42), nine are part of the ligand binding pocket.ConclusionsWe demonstrate the use of GREMLIN to reveal a network of statistically correlated and functionally important residues in class A GPCRs. GREMLIN identified that ligand binding pocket residues are extensively correlated with distal residues. An analysis of the GREMLIN edges across multiple structures suggests that there may be a minimal binding pocket common to the seven known GPCRs. Further, the activation of rhodopsin involves these long-range interactions between extracellular and intracellular domain residues mediated by the retinal domain.

Role of Conserved Transmembrane Domain Cysteines in Activation ofMetabotropic Glutamate Receptor Subtype 6

Metabotropic glutamate receptor subtype 6 (mGluR6) is a Class C type G protein coupled receptor (GPCR) uniquely expressed on retinal bipolar cells. mGluR6 plays a key role in dim-light vision but little is known about its structure and function. Here, we characterized the role of the three transmembrane (TM) cysteines in activation through site-directed mutagenesis. Function of the receptors in cells and membranes was assayed using cAMP and G protein activation, respectively. Cysteine mutants in TM helix V displayed slightly elevated or wild-type like activity. In contrast, all mutations involving the cysteine in TM helix VI lacked agonist response. Our results suggest that TM VI plays a key role in Class C activation similar to that observed in rhodopsin-like (Class A) GPCRs.