Jose Manuel Perez-Aguilar

Theoretical and Computational Protein Design

From exponentially large numbers of possible sequences, protein design seeks to identify the properties of those that fold to predetermined structures and have targeted structural and functional properties. The interactions that confer structure and function involve intermolecular forces and large numbers of interacting amino acids. As a result, the identification of sequences can be subtle and complex. Sophisticated methods for characterizing sequences consistent with a particular structure have been developed, assisting the design of novel proteins. Developments in such computational protein design are discussed, along with recent accomplishments, ranging from the redesign of existing proteins to the design of new functionalities and nonbiological applications.

Scalable Production of Highly Sensitive Nanosensors Based on Graphene Functionalized with a Designed G Protein-Coupled Receptor

We have developed a novel, all-electronic biosensor for opioids that consists of an engineered μ-opioid receptor protein, with high binding affinity for opioids, chemically bonded to a graphene field-effect transistor to read out ligand binding. A variant of the receptor protein that provided chemical recognition was computationally redesigned to enhance its solubility and stability in an aqueous environment. A shadow mask process was developed to fabricate arrays of hundreds of graphene transistors with average mobility of ∼1500 cm2 V–1 s–1 and yield exceeding 98%. The biosensor exhibits high sensitivity and selectivity for the target naltrexone, an opioid receptor antagonist, with a detection limit of 10 pg/mL.

Computational Protein Design to Reengineer Stromal Cell–Derived Factor-1α Generates an Effective and Translatable Angiogenic Polypeptide Analog

Background— Experimentally, exogenous administration of recombinant stromal cell–derived factor-1&agr; (SDF) enhances neovasculogenesis and cardiac function after myocardial infarction. Smaller analogs of SDF may provide translational advantages including enhanced stability and function, ease of synthesis, lower cost, and potential modulated delivery via engineered biomaterials. In this study, computational protein design was used to create a more efficient evolution of the native SDF protein. Methods and Results— Protein structure modeling was used to engineer an SDF polypeptide analog (engineered SDF analog [ESA]) that splices the N-terminus (activation and binding) and C-terminus (extracellular stabilization) with a diproline segment designed to limit the conformational flexibility of the peptide backbone and retain the relative orientation of these segments observed in the native structure of SDF. Endothelial progenitor cells (EPCs) in ESA gradient, assayed by Boyden chamber, showed significantly increased migration compared with both SDF and control gradients. EPC receptor activation was evaluated by quantification of phosphorylated AKT, and cells treated with ESA yielded significantly greater phosphorylated AKT levels than SDF and control cells. Angiogenic growth factor assays revealed a distinct increase in angiopoietin-1 expression in the ESA- and SDF-treated hearts. In addition, CD-1 mice (n=30) underwent ligation of the left anterior descending coronary artery and peri-infarct intramyocardial injection of ESA, SDF-1&agr;, or saline. At 2 weeks, echocardiography demonstrated a significant gain in ejection fraction, cardiac output, stroke volume, and fractional area change in mice treated with ESA compared with controls. Conclusions— Compared with native SDF, a novel engineered SDF polypeptide analog (ESA) more efficiently induces EPC migration and improves post–myocardial infarction cardiac function and thus offers a more clinically translatable neovasculogenic therapy.

Multiple Hindered Rotators in a Gyroscope-Inspired Tribenzylamine Hemicryptophane

A gyroscope-inspired tribenzylamine hemicryptophane provides a vehicle for exploring the structure and properties of multiple p-phenylene rotators within one molecule. The hemicryptophane was synthesized in three steps in good overall yield using mild conditions. Three rotator-forming linkers were cyclized to form a rigid cyclotriveratrylene (CTV) stator framework, which was then closed with an amine. The gyroscope-like molecule was characterized by (1)H NMR and (13)C NMR spectroscopy, and the structure was solved by X-ray crystallography. The rigidity of the two-component CTV-trismethylamine stator was investigated by (1)H variable-temperature (VT) NMR experiments and molecular dynamics simulations. These techniques identified gyration of the three p-phenylene rotators on the millisecond time scale at -93 °C, with more dynamic but still hindered motion at room temperature (27 °C). The activation energy for the p-phenylene rotation was determined to be ~10 kcal mol(-1). Due to the propeller arrangement of the p-phenylenes, their rotation is hindered but not strongly correlated. The compact size, simple synthetic route, and molecular motions of this gyroscope-inspired tribenzylamine hemicryptophane make it an attractive starting point for controlling the direction and coupling of rotators within molecular systems.

A Computationally Designed Water-Soluble Variant of a G-Protein-Coupled Receptor: The Human Mu Opioid Receptor

G-protein-coupled receptors (GPCRs) play essential roles in various physiological processes, and are widely targeted by pharmaceutical drugs. Despite their importance, studying GPCRs has been problematic due to difficulties in isolating large quantities of these membrane proteins in forms that retain their ligand binding capabilities. Creating water-soluble variants of GPCRs by mutating the exterior, transmembrane residues provides a potential method to overcome these difficulties. Here we present the first study involving the computational design, expression and characterization of water-soluble variant of a human GPCR, the human mu opioid receptor (MUR), which is involved in pain and addiction. An atomistic structure of the transmembrane domain was built using comparative (homology) modeling and known GPCR structures. This structure was highly similar to the subsequently determined structure of the murine receptor and was used to computationally design 53 mutations of exterior residues in the transmembrane region, yielding a variant intended to be soluble in aqueous media. The designed variant expressed in high yield in Escherichia coli and was water soluble. The variant shared structural and functionally related features with the native human MUR, including helical secondary structure and comparable affinity for the antagonist naltrexone (K d  = 65 nM). The roles of cholesterol and disulfide bonds on the stability of the receptor variant were also investigated. This study exemplifies the potential of the computational approach to produce water-soluble variants of GPCRs amenable for structural and functionally related characterization in aqueous solution.

Computational Design of Membrane Proteins

Membrane proteins are involved in a wide variety of cellular processes, and are typically part of the first interaction a cell has with extracellular molecules. As a result, these proteins comprise a majority of known drug targets. Membrane proteins are among the most difficult proteins to obtain and characterize, and a structure-based understanding of their properties can be difficult to elucidate. Notwithstanding, the design of membrane proteins can provide stringent tests of our understanding of these crucial biological systems, as well as introduce novel or targeted functionalities. Computational design methods have been particularly helpful in addressing these issues, and this review discusses recent studies that tailor membrane proteins to display specific structures or functions and examines how redesigned membrane proteins are being used to facilitate structural and functional studies.

NMR structure and dynamics of a designed water-soluble transmembrane domain of nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828-1833, 2004; Ma et al., PNAS, 105: 16537-42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ~83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T(2) dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.

Opioid binding sites in human serum albumin.

BACKGROUND Human serum albumin (HSA) is an important carrier for opioids. However, the locations of the binding sites remain unclear. In the present study, we have characterized opioid-HSA interactions using multiple biochemical and biophysical techniques to reveal: (a) the location of the binding site(s); (b) whether naloxone shares the binding site with morphine; and (c) whether opioid agonists share their binding site(s) with general anesthetics. METHODS Elution chromatography to determine the global interactions and tryptophan intrinsic fluorescence to determine the localized interactions of opioids with HSA were used. Competition studies using isothermal titration calorimetry were used to determine the overlap of binding site(s) among opioid agonists, antagonists, and general anesthetics. An automatic docking calculation was used to predict the possible binding sites and to assess findings of the solution studies. RESULTS For elution chromatography with immobilized HSA, the retention times of naloxone, morphine, and fentanyl were prolonged but shorter than that of propofol. The inhibition of tryptophan fluorescence by naloxone was not affected by morphine or fentanyl. The calorimetric heat profiles of propofol and halothane interaction with HSA were changed significantly, but not equally by morphine, naloxone, or fentanyl. Consistent with direct binding studies, docking results demonstrated that opioids share sites with general anesthetics; a distinct binding site for naloxone was revealed near the sole tryptophan in HSA that is not shared with morphine. CONCLUSIONS The interaction of opioids with HSA is weak in comparison with propofol. Naloxone has a distinct binding site in HSA not shared with opioid agonists. Opioids share binding sites with general anesthetics in HSA.

Molecular recognition of ketamine by a subset of olfactory G protein–coupled receptors

The anesthetic ketamine activates G protein–coupled receptors involved in smell. Wake up and smell the ketamine! Ketamine antagonizes ion channels known as N-methyl-d-aspartate (NMDA) receptors to mediate its effects as an anesthetic; however, ketamine also has other properties, which suggests that it has other targets. Ho et al. found that ketamine activated four mouse olfactory receptors, which are a subfamily of G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs). Structural analysis revealed residues that formed a ketamine-binding site in these olfactory receptors, and mutation of nonresponsive olfactory receptors to engineer the ketamine-binding site rendered them responsive to ketamine. Together, these data suggest that members of this family of GPCRs are functional ketamine receptors and provide a means to identify other potential targets of this pleiotropic drug. Ketamine elicits various neuropharmacological effects, including sedation, analgesia, general anesthesia, and antidepressant activity. Through an in vitro screen, we identified four mouse olfactory receptors (ORs) that responded to ketamine. In addition to their presence in the olfactory epithelium, these G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) are distributed throughout the central nervous system. To better understand the molecular basis of the interactions between ketamine and ORs, we used sequence comparison and molecular modeling to design mutations that (i) increased, reduced, or abolished ketamine responsiveness in responding receptors, and (ii) rendered nonresponding receptors responsive to ketamine. We showed that olfactory sensory neurons (OSNs) that expressed distinct ORs responded to ketamine in vivo, suggesting that ORs may serve as functional targets for ketamine. The ability to both abolish and introduce responsiveness to ketamine in GPCRs enabled us to identify and confirm distinct interaction loci in the binding site, which suggested a signature ketamine-binding pocket that may guide exploration of additional receptors for this general anesthetic drug.

Human μ Opioid Receptor Models with Evaluation of the Accuracy Using the Crystal Structure of the Murine μ Opioid Receptor.

Models of the human μ opioid receptor were constructed using available G-protein-coupled receptor (GPCR) structures using homology (comparative) modeling techniques. The recent publication of a high-resolution crystal structure of a construct based on the murine μ opioid receptor offers a unique opportunity to evaluate the reliability of the homology models and test the relevance of introducing more templates (known structures) to increase the accuracy of the comparative models. In the first model two templates were used: the β2 adrenergic and bovine rhodopsin receptors. For the second model, four templates were utilized: the β2 adrenergic, bovine rhodopsin, β1 adrenergic, and A2A adenosine receptors. Including additional templates improved the accuracy of structural motifs and other features of the model when the same sequence alignment was used. The predicted structures were especially relevant in the case of important receptor regions such as the DRY motif, which has been associated with receptor activation. Additionally, this study showed that receptor sequence similarity is crucial in homology modeling, as indicated in the case of the highly diverse EC2 loop. This study demonstrates the reliability of the homology modeling technique in the case of the μ opioid receptor, a member of the rhodopsin-like family class of GPCRs. The addition of more templates improved the accuracy of the model. The findings regarding the modeling has significant implication to other GPCRs where the crystal structure is still unknown and suggest that homology modeling techniques can provide high quality structural models for interpreting experimental findings and formulating structurally based hypotheses regarding the activity of these important receptors.