Wely B. Floriano

Prediction of structure and function of G protein-coupled receptors

G protein-coupled receptors (GPCRs) mediate our sense of vision, smell, taste, and pain. They are also involved in cell recognition and communication processes, and hence have emerged as a prominent superfamily for drug targets. Unfortunately, the atomic-level structure is available for only one GPCR (bovine rhodopsin), making it difficult to use structure-based methods to design drugs and mutation experiments. We have recently developed first principles methods (MembStruk and HierDock) for predicting structure of GPCRs, and for predicting the ligand binding sites and relative binding affinities. Comparing to the one case with structural data, bovine rhodopsin, we find good accuracy in both the structure of the protein and of the bound ligand. We report here the application of MembStruk and HierDock to β1-adrenergic receptor, endothelial differential gene 6, mouse and rat I7 olfactory receptors, and human sweet receptor. We find that the predicted structure of β1-adrenergic receptor leads to a binding site for epinephrine that agrees well with the mutation experiments. Similarly the predicted binding sites and affinities for endothelial differential gene 6, mouse and rat I7 olfactory receptors, and human sweet receptor are consistent with the available experimental data. These predicted structures and binding sites allow the design of mutation experiments to validate and improve the structure and function prediction methods. As these structures are validated they can be used as targets for the design of new receptor-selective antagonists or agonists for GPCRs.

Predictions of CCR1 Chemokine Receptor Structure and BX 471 Antagonist Binding Followed by Experimental Validation

A major challenge in the application of structure-based drug design methods to proteins belonging to the superfamily of G protein-coupled receptors (GPCRs) is the paucity of structural information (1). The 19 chemokine receptors, belonging to the Class A family of GPCRs, are important drug targets not only for autoimmune diseases like multiple sclerosis but also for the blockade of human immunodeficiency virus type 1 entry (2). Using the MembStruk computational method (3), we predicted the three-dimensional structure of the human CCR1 receptor. In addition, we predicted the binding site of the small molecule CCR1 antagonist BX 471, which is currently in Phase II clinical trials (4). Based on the predicted antagonist binding site we designed 17 point mutants of CCR1 to validate the predictions. Subsequent competitive ligand binding and chemotaxis experiments with these mutants gave an excellent correlation to these predictions. In particular, we find that Tyr-113 and Tyr-114 on transmembrane domain 3 and Ile-259 on transmembrane 6 contribute significantly to the binding of BX 471. Finally, we used the predicted and validated structure of CCR1 in a virtual screening validation of the Maybridge data base, seeded with selective CCR1 antagonists. The screen identified 63% of CCR1 antagonists in the top 5% of the hits. Our results indicate that rational drug design for GPCR targets is a feasible approach.

Test of the Binding Threshold Hypothesis for olfactory receptors: explanation of the differential binding of ketones to the mouse and human orthologs of olfactory receptor 912-93.

We tested the Binding Threshold Hypothesis (BTH) for activation of olfactory receptors (ORs): To activate an OR, the odorant must bind to the OR with binding energy above some threshold value. The olfactory receptor (OR) 912‐93 is known experimentally to be activated by ketones in mouse, but is inactive to ketones in human, despite an amino acid sequence identity of ∼66%. To investigate the origins of this difference, we used the MembStruk first‐principles method to predict the tertiary structure of the mouse OR 912‐93 (mOR912‐93), and the HierDock first‐principles method to predict the binding site for ketones to this receptor. We found that the strong binding of ketones to mOR912‐93 is dominated by a hydrogen bond of the ketone carbonyl group to Ser105. All ketones predicted to have a binding energy stronger than EBindThresh = 26 kcal/mol were observed experimentally to activate this OR, while the two ketones predicted to bind more weakly do not. In addition, we predict that 2‐undecanone and 2‐dodecanone both bind sufficiently strongly to activate mOR912‐93. A similar binding site for ketones was predicted in hOR912‐93, but the binding is much weaker because the human ortholog has a Gly at the position of Ser105. We predict that mutating this Gly to Ser in human should lead to activation of hOR912‐93 by these ketones. Experimental substantiations of the above predictions would provide further tests of the validity of the BTH, our predicted 3D structures, and our predicted binding sites for these ORs.

Interaction of E. coli Outer-Membrane Protein A with Sugars on the Receptors of the Brain Microvascular Endothelial Cells

Esherichia coli, the most common gram‐negative bacteria, can penetrate the brain microvascular endothelial cells (BMECs) during the neonatal period to cause meningitis with significant morbidity and mortality. Experimental studies have shown that outer‐membrane protein A (OmpA) of E. coli plays a key role in the initial steps of the invasion process by binding to specific sugar moieties present on the glycoproteins of BMEC. These experiments also show that polymers of chitobiose (GlcNAcβ1‐4GlcNAc) block the invasion, while epitopes substituted with the L‐fucosyl group do not. We used HierDock computational technique that consists of a hierarchy of coarse grain docking method with molecular dynamics (MD) to predict the binding sites and energies of interactions of GlcNAcβ1‐4GlcNAc and other sugars with OmpA. The results suggest two important binding sites for the interaction of carbohydrate epitopes of BMEC glycoproteins to OmpA. We identify one site as the binding pocket for chitobiose (GlcNAcβ1‐4GlcNAc) in OmpA, while the second region (including loops 1 and 2) may be important for recognition of specific sugars. We find that the site involving loops 1 and 2 has relative binding energies that correlate well with experimental observations. This theoretical study elucidates the interaction sites of chitobiose with OmpA and the binding site predictions made in this article are testable either by mutation studies or invasion assays. These results can be further extended in suggesting possible peptide antagonists and drug design for therapeutic strategies. Proteins 2003;50:213–221.

Strategies for Multiscale Modeling and Simulation of Organic Materials: Polymers and Biopolymers

Abstract Advances in theory and methods are making it practical to consider fully first principles (de novo) predictions of structures, properties and processes for organic materials. However, despite the progress there remains an enormous challenge in bridging the vast range of distances and time scales between de novo atomistic simulations and the quantitative continuum models for the macroscopic systems essential in industrial design and operations. Recent advances relevant to such developments include: quantum chemistry including continuum solvation and force field embedding, de novo force fields to describe phase transitions, molecular dynamics (MD) including continuum solvent, non equilibrium MD for rheology and thermal conductivity and mesoscale simulations. To provide some flavor for the opportunities we will illustrate some of the progress and challenges by summarizing some recent developments in methods and their applications to polymers and biopolymers. Four different topics will be covered: (1) hierarchical modeling approach applied to modeling olfactory receptors, (2) stabilization of leucine zipper coils by introduction of trifluoroleucine, (3) modeling response of polymers sensors for electronic nose, and (4) diffusion of gases in amorphous polymers.