1 Visualizing Molecular Interactions

Visualizing Molecular Interactions: Computational Investigations of Structure-Function Relationships and Dynamics in Biomolecular Systems Kristina E. Furse Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 Email: [email protected] Computer modeling has emerged as a powerful complement The de novo receptor models also yielded some interesting and to experimental and theoretical investigations. Visualization of testable hypotheses for the structural basis of !-AR subtype experimental data in the context of a three-dimensional, ligand selectivity. atomic-scale model can not only help to explain unexpected Four years after we completed our study, the first high- results but often raises new questions, driving future research. resolution X-ray structure of !-AR was released.3 Many of our Models of sufficient quality can be set in motion in molecular structural predictions were validated by the X-ray structure, dynamics (MD) simulations to move beyond a static picture including an unusual configuration of three active site serines and provide insight into the dynamics of important biological and the unique spatial arrangement of two extracellular processes. disulfide bonds that introduced a previously unknown loop The power of computer modeling and simulation is residue, Phe-193, into the binding site (Figure 1). Our de novo nowhere more evident than in the study of small-molecule models were included in a virtual screening study by an interactions with proteins and DNA. Detailed knowledge of independent lab.4 They found that our model outperformed these molecular-scale interactions is vital to rational drug rhodopsin-based homology models as well as other de novo design, understanding and harnessing the catalytic power of models. In fact, ours was the only model to rival the !-AR X- enzymes, and unraveling the mysteries of molecular ray structure itself in virtual screening of agonists and recognition, which is at the heart of innumerable physiological antagonists. This demonstrates the potential power of processes from signal transduction to DNA repair. Computer constraint-based modeling approaches for structure prediction, models and simulations have been used in concert with given a methodical approach and sufficient high quality experiment and theory to gain insight into these processes. experimental data to refine the model. The following describes three distinct biological problems requiring distinct computational approaches: (1) theoretical modeling of a structurally underdetermined member of the G- protein coupled receptor (GPCR) superfamily, (2) mechanistic investigations of the pharmacologically important cyclooxygenase (COX) enzymes, and (3) simulation of time- resolved fluorescence spectroscopy experiments to understand water dynamics at the DNA interface. GPCR: De novo model building. G-protein coupled receptors (GPCR) are prime therapeutic targets since they play a key role in signal transduction in many cells and are implicated in control or regulation of a wide array of biological functions. There is great interest in exploiting the details of GPCR structure for rational drug design, however like many integral membrane proteins they are challenging molecules to study experimentally and high-resolution structural characterization is extremely difficult. As a result, great importance has been placed on indirect structural Figure 1. Extracellular view of the complex of photolabile antagonist evidence obtained from a variety of biophysical techniques, as aminoflisopolol with the de novo !2-AR model. The ligand is well as detailed sequence analysis and molecular modeling displayed in white with key active site residues shown in magenta. studies. De novo modeling is a flexible technique that utilizes Y199 in TM5 is the site of covalent attachment in photoaffinity this indirect structural evidence as constraint data to guide the labeling experiments. Other key contacts include a charge reinforced construction of theoretical three-dimensional (3D) models of a hydrogen bond between the protonated amine and D113 in TM3 and a target without using a related protein of known structure as a hydrogen bond between the ligand sidechain hydroxyl and N293 in template (the latter describes “homology modeling"). TM6, both indicated by dashed yellow lines. N312 in TM7, along with F289 and H296 in TM6, interact with the isopropyl N- In this work, we constructed 3D models for agonist and substituent. For this complex, the second extracellular loop is antagonist complexes with !-adrenergic receptors (!-AR) included, with the two disulfide bonds highlighted in yellow and loop using both standard homology modeling methods (template: residue F193 shown interacting with the fluorenone ring of the ligand. rhodopsin crystal structure),1 as well as de novo modeling Adapted from Figure 6 in Ref 2. techniques.2 While the de novo and homology-derived receptor models were generally quite similar, there were some COX Enzymes: Mechanistic studies. The two localized structural differences that impacted the putative cyclooxygenase enzymes, COX-1 and COX-2, are responsible ligand-binding site significantly. The de novo receptor models for the committed step in prostaglandin biosynthesis, and are appeared to provide much better agreement with experimental the targets of the non-steroidal anti-inflammatory drugs data, particularly for receptor complexes with agonist ligands. aspirin, ibuprofen and the COX-2 selective inhibitors, 1 CelebrexTM, VioxxTM and BextraTM. The enzymes are both COX-1 and COX-2 with the substrate-derived remarkable in that they catalyze two dioxygenations and two pentadienyl radical intermediate bound in the active site.7 The cyclizations of the native substrate, arachidonic acid, with near enzymes’ influence on the conformation of the pentadienyl absolute regio- and stereoselectivity. In stark contrast, the non- radical was investigated, along with the accessible space enzymatic (solution) oxidation of AA or its esters yields many above and below the radical plane, and the width of several different products. Several theories have been advanced to channels to the active site that could function as access routes explain the nature of enzymatic control over this series of for molecular oxygen. Additional simulations demonstrated reactions. the extent of molecular oxygen mobility within the active site (Figure 2). The results suggested that spin localization (Scenario 3) is unlikely to play a role in enzymatic control of this reaction. Instead, a combination of oxygen channeling, steric shielding and selective radical trapping appear to be responsible. This work adds a dynamic perspective to the strong foundation of static structural data (X-ray structures) available for these enzymes. Figure 2. Traces of oxygen position from explicit O2 simulations. Each panel is a sliced solid surface view of the cyclooxygenase active site of COX-1/AA-13-yl complexes in the snapshot used as a starting point for 2 different trajectories (rows 1-2). The AA-13-yl radical is shown in red, with C11 highlighted in yellow, and the surfaces of active site residues Arg-120 (top) and Ser-530 (bottom) highlighted in cyan. The position of O2 in the active site over time is indicated by a line that changes color from blue to red from 0-500 ps. While the Figure 3. Fluorescent probe Hoechst 33258 bound to the minor surface views were sliced with a vertical clipping plane (cut surface is groove of DNA. The solvent accessible surface of the DNA is white) to show the interior space, no clipping planes were used on the displayed along with water molecules in the first solvation shell of the oxygen traces in order to show them in their entirety. Adapted from probe. Figure 7 in Ref 7. DNA: Simulating solvation dynamics. Together, We identified four possible scenarios to explain how the spectroscopy combined with computational studies that relate enzyme guides the first addition of oxygen to the pentadienyl directly to the experimental measurements have the potential radical intermediate almost exclusively at the pro-R face of to provide unprecedented insight into the dynamics of C11: (1) molecular oxygen is directed to the substrate via a important biological processes. It is well known that hydration designated oxygen channel in the enzyme; (2) the substrate is is essential to the stability and function of biological bound in the active site in a configuration that sterically molecules. Recent time-resolved fluorescence experiments shields alternative positive spin-bearing positions along the 5,6 have shown that the time scales for collective reorganization at pentadienyl radical; (3) the enzyme localizes spin at the pro- the interface of proteins and DNA with water are more than an R face of C11 by forcing the radical intermediate to adopt a order of magnitude slower than in bulk aqueous solution. The higher energy, non-planar conformation; (4) simple molecular interpretation of this change in the collective competition kinetics governs the product distribution—oxygen response is somewhat controversial—some attribute the adds to all possible positions along the substrate radical but slower reorganization to dramatically retarded water motion, only oxygenation at the pro-R face of C11 yields an while others describe rapid water dynamics at the interface intermediate that can be efficiently trapped (via cyclization to combined with a slower biomolecular response. form the endoperoxide ring). In order to gain further insight into

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