Student Theses and Dissertations

Carlos Rico

Date of Award

2017

Document Type

Thesis

RU Laboratory

Sakmar Laboratory

Abstract

The G protein-coupled receptor (GPCR) C-C chemokine receptor 5 (CCR5) is the major co-receptor for HIV-1. CCR5 binds to the viral glycoprotein gp120 allowing HIV particles to infect T cells. Currently, maraviroc is the only Food and Drug Administration (FDA) approved entry inhibitor for HIV-1 but resistance to maraviroc has been reported indicating the need for novel entry inhibitors. Recently, four peptides derived from RANTES (Regulated on Activation, Normal T cell Expressed and Secreted), the endogenous ligand of CCR5, were shown to induce different CCR5 signaling pathways and efficiently block viral entry. The peptide analogues are 5P12, which blocks HIV-1 entry but does not induce signaling or receptor internalization; 6P4, which is a non-biased ligand; PSC, which is a super agonist relative to RANTES; and 5P14, which does not activate G-protein mediated signaling yet induces receptor internalization. Yet, we lack mechanistic knowledge about how the analogues bind to CCR5. To address this issue, we have employed fluorescence cross-correlation spectroscopy (FCCS) to determine equilibrium dissociation and inhibition constants for the RANTES analogues binding to CCR5. We developed a tandem affinity purification protocol to purify full-length, monomeric CCR5 from receptor truncations and oligomers. We fluorescently labeled CCR5 using the SNAP tag, a functional tag derived from DNA alkyl transferase, with Alexa-488. We show similar cell surface expression for CCR5-SNAP and wild-type CCR5 using flow cytometry with different epitope recognizing antibodies. We demonstrate that CCR5-SNAP activates G-proteins and internalizes similarly as wild type CCR5 in response to chemokine stimulus. We perform saturation and competition binding with the Alexa-647 labeled RANTES analogues and show that they bind with picomolar to nanomolar affinities. Global fitting on the binding isotherms shows that the RANTES analogues bind to 38% of CCR5-SNAP and recognize two different species that are non-interconvertible. Competition binding with gp120 complexed to soluble CD4 shows that the RANTES analogues are efficient at blocking Env binding. We also show that the native chemokines are incapable of displacing the RANTES analogues from CCR5-SNAP. We speculate that CCR5-SNAP is modified differentially with post-translational modifications that affect receptor affinity for the analogues. Our studies were performed with CCR5-SNAP in detergent solution that is not suitable to investigate the role G-protein on CCR5-SNAP ligand binding. To address this issue, we developed a novel zebrafish derived apolipoprotein AI (ZapN1) for the assembly of nanoscale apolipoprotein bound bilayers (NABBs). We optimized expression and purification of ZapN1 from E. coli and the assembly of NABBs with different lipids and detergent conditions. We performed FCCS ligand binding with CCR5-SNAP in NABBs to show that the receptor can recognize a conformationally sensitive antibody. Our studies illustrate the advantage of single molecule ligand binding assays to study receptor species that are averaged in ensemble measurements.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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