Allosteric sites can be identified based on the residue–residue interaction energy difference

Allosteric drugs act at a distance to regulate protein functions. They have several advantages over conventional orthosteric drugs, including diverse regulation types and fewer side effects. However, the rational design of allosteric ligands remains a challenge, especially when it comes to the identification allosteric binding sites. As the binding of allosteric ligands may induce changes in the pattern of residue–residue interactions, we calculated the residue–residue interaction energies within the allosteric site based on the molecular mechanics generalized Born surface area energy decomposition scheme. Using a dataset of 17 allosteric proteins with structural data for both the apo and the ligand‐bound state available, we used conformational ensembles generated by molecular dynamics simulations to compute the differences in the residue–residue interaction energies in known allosteric sites from both states. For all the known sites, distinct interaction energy differences (>25%) were observed. We then used CAVITY, a binding site detection program to identify novel putative allosteric sites in the same proteins. This yielded a total of 31 “druggable binding sites,” of which 21 exhibited >25% difference in residue interaction energies, and were hence predicted as novel allosteric sites. Three of the predicted allosteric sites were supported by recent experimental studies. All the predicted sites may serve as novel allosteric sites for allosteric ligand design. Our study provides a computational method for identifying novel allosteric sites for allosteric drug design. Proteins 2015; 83:1375–1384. © 2014 Wiley Periodicals, Inc.     

Allostery mediates ligand binding to WWOX tumor suppressor via a conformational switch

While being devoid of the ability to recognize ligands itself, the WW2 domain is believed to aid ligand binding to the WW1 domain in the context of a WW1–WW2 tandem module of WW domain‐containing oxidoreductase (WWOX) tumor suppressor. In an effort to test the generality of this hypothesis, we have undertaken here a detailed biophysical analysis of the binding of WW domains of WWOX alone and in the context of the WW1–WW2 tandem module to an array of putative proline‐proline‐x–tyrosine (PPXY) ligands. Our data show that while the WW1 domain of WWOX binds to all ligands in a physiologically relevant manner, the WW2 domain does not. Moreover, ligand binding to the WW1 domain in the context of the WW1–WW2 tandem module is two‐to‐three‐fold stronger than when treated alone. We also provide evidence that the WW domains within the WW1–WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. Of particular note is the observation that the physical association of the WW2 domain with WW1 blocks access to ligands. Consequently, ligand binding to the WW1 domain not only results in the displacement of the WW2 lid but also disrupts the physical association of WW domains in the liganded conformation. Taken together, our study underscores a key role of allosteric communication in the ability of the WW2 orphan domain to chaperone physiological action of the WW1 domain within the context of the WW1–WW2 tandem module of WWOX. Copyright © 2015 John Wiley & Sons, Ltd.     

Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B

RING proteins constitute the largest class of E3 ubiquitin ligases. Unlike most RINGs, AO7 (RNF25) binds the E2 ubiquitin-conjugating enzyme, UbcH5B (UBE2D2), with strikingly high affinity. We have defined, by co-crystallization, the distinctive means by which AO7 binds UbcH5B. AO7 contains a structurally unique UbcH5B binding region (U5BR) that is connected by an 11-amino acid linker to its RING domain, forming a clamp surrounding the E2. The U5BR interacts extensively with a region of UbcH5B that is distinct from both the active site and the RING-interacting region, referred to as the backside of the E2. An apparent paradox is that the high-affinity binding of the AO7 clamp to UbcH5B, which is dependent on the U5BR, decreases the rate of ubiquitination. We establish that this is a consequence of blocking the stimulatory, non-covalent, binding of ubiquitin to the backside of UbcH5B. Interestingly, when non-covalent backside ubiquitin binding cannot occur, the AO7 clamp now enhances the rate of ubiquitination. The high-affinity binding of the AO7 clamp to UbcH5B has also allowed for the co-crystallization of previously described and functionally important RING mutants at the RING-E2 interface. We show that mutations having marked effects on function only minimally affect the intermolecular interactions between the AO7 RING and UbcH5B, establishing a high degree of complexity in activation through the RING-E2 interface.     

Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain

The extracellular regions of epithelial Na⁺ channel subunits are highly ordered structures composed of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na⁺ (Na⁺ self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces epithelial Na⁺ channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na⁺ self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na⁺ self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na⁺ self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na⁺ self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.     

Saturation Mutagenesis of the Antithrombin Reactive Center Loop P14 Residue Supports a Three-step Mechanism of Heparin Allosteric Activation Involving Intermediate and Fully Activated States

Past studies have suggested that a key feature of the mechanism of heparin allosteric activation of the anticoagulant serpin, antithrombin, is the release of the reactive center loop P14 residue from a native state stabilizing interaction with the hydrophobic core. However, more recent studies have indicated that this structural change plays a secondary role in the activation mechanism. To clarify this role, we expressed and characterized 15 antithrombin P14 variants. The variants exhibited basal reactivities with factors Xa and IXa, heparin affinities and thermal stabilities that were dramatically altered from wild type, consistent with the P14 mutations perturbing native state stability and shifting an allosteric equilibrium between native and activated states. Rapid kinetic studies confirmed that limiting rate constants for heparin allosteric activation of the mutants were altered in conjunction with the observed shifts of the allosteric equilibrium. However, correlations of the P14 mutations'' effects on parameters reflecting the allosteric activation state of the serpin were inconsistent with a two-state model of allosteric activation and suggested multiple activated states. Together, these findings support a minimal three-state model of allosteric activation in which the P14 mutations perturb equilibria involving distinct native, intermediate, and fully activated states wherein the P14 residue retains an interaction with the hydrophobic core in the intermediate state but is released from the core in the fully activated state, and the bulk of allosteric activation has occurred in the intermediate.     

Oligomerization and nanocluster organization render specificity

Nanoclusters are anchored to membranes, either within them or in the cytoplasm latched onto the cytoskeleton, whose reorganization can regulate their activity. Nanoclusters have been viewed in terms of cooperativity and activation; here we perceive nanocluster organization from a conformational standpoint. This leads us to suggest that while single molecules encode activity, nanoclusters induce specificity, and that this is their main evolutionary aim. Distinct, isoform‐specific nanocluster organization can drive the preferred effector (and ligand) interactions and thereby designate signalling pathways. The absence of detailed structural information across the nanocluster, due to size and dynamics, hinders an in‐depth grasp of its mechanistic features; however, available data already capture some of the principles and their functional ‘raison d'être’. Collectively, clustering lends stability and reduces the likelihood of proteolytic cleavage; it also increases the effective local concentration and enables efficient cooperative activation. However, clustering does not determine the ability of the single molecule to function. Drugs targeting nanoclusters can attenuate activity by hampering cooperativity; however, this may not perturb activation and signalling, which originate from the molecules themselves, and as such, are likely to endure. What then is the major role of nanoclustering? Assuming that single molecules evolved first, with a subsequent increase in cellular complexity and emergence of highly similar isoform variants, evolution faced the threat of signalling promiscuity. We reason that this potential risk was thwarted by oligomerization and clustering; clustering confers higher specificity, and a concomitant extra layer of cellular control. In our Ras example, signalling will be more accurate as a dimer than as a monomer, where its isomer specificity could be compromised.     

3-(Pyridin-2-yl-ethynyl)benzamide metabotropic glutamate receptor 5 negative allosteric modulators: hit to lead studies

A series of 3-(pyridin-2-yl-ethynyl)benzamide negative allosteric modulators of the metabotropic glutamate receptor 5 (mGluR5 NAMs) have been prepared. Starting from HTS hit 1 (IC₅₀: 926 nM), potent mGluR5 NAMs showing excellent potencies (IC₅₀s <50 nM) and good physicochemical profiles were prepared by monitoring LipE values. One compound 26 showed excellent mGluR5 binding (K_i: 21 nM) and antagonism (IC₅₀: 8 nM), an excellent rat PK profile (CL: 12 mL/min/kg, %F: 85) and showed oral activity in a mouse 4-Plate Behavioral model of anxiety (MED: 30 mpk) and a mouse Stress Induced Hyperthermia model of anxiety (MED 17.8 mpk).     

NMR reveals novel mechanisms of protein activity regulation

NMR spectroscopy is one of the most powerful tools for the characterization of biomolecular systems. A unique aspect of NMR is its capacity to provide an integrated insight into both the structure and intrinsic dynamics of biomolecules. In addition, NMR can provide site-resolved information about the conformation entropy of binding, as well as about energetically excited conformational states. Recent advances have enabled the application of NMR for the characterization of supramolecular systems. A summary of mechanisms underpinning protein activity regulation revealed by the application of NMR spectroscopy in a number of biological systems studied in the lab is provided.