Chemokine:Receptor Structure and Interactions

Chemokine receptors belong to the G Protein-Coupled Receptor (GPCR) family of membrane proteins. They are incredibly versatile allosteric machines that undergo conformational changes upon binding ligands in order to transmit information from the outside to the inside of cells. By virtue of their role in cell migration and activation, chemokine receptors are involved in an extraordinary number of diseases which has motivated major initiatives in the pharmaceutical industry to develop drugs that block chemokine receptors for many disease indications. Structural information is therefore highly desirable for optimizing drug leads. Additionally, there is a great deal of interest in understanding the conformational changes induced by ligands, how they elicit changes in interactions with downstream signaling partners, and the consequences on signaling output. Our laboratory is therefore interested in determining structures of receptors, and understanding ligand induced conformational changes by crystallography, EPR and other biophysical methods. We are also interested in investigating the ligand dependent and independent interactions of receptors with intracellular proteins using Bioluminescence Energy Transfer (BRET) and mass spectrometry-based proteomic approaches.

Receptor Structure

Structure determination of any membrane protein is difficult and GPCRs are no exception. The major challenges include expression of sufficient quantities of receptor, purification and reconstitution of receptors in artificial membranes with preservation of function, and crystallization which is thwarted by the inherently dynamic nature of these proteins. Nevertheless, there has been an enormous breakthrough in the determination of GPCRs besides Rhodopsin since 2007 (see the GPCR network for an update). These include five structures of the chemokine receptor CXCR4 in complex with a small molecule and peptide ligands (see Figure 1).

Figure 1. View of the binding pocket of CXCR4 with small molecule antagonist, IT1t

Fortunately, we have been awarded a UO1 grant as part of the Protein Structure Initiative, PSI:Biology to determine structures of additional receptor complexes in collaboration with Stevens Lab and the GPCR network at Scripps. In addition, we will use EPR and other biophysical methods to provide complementary and functionally important dynamic information related to receptor activation. Finally, we use the structures to guide mutagenesis/protein engineering coupled with cell-based assays of receptor function to further interrogate the relationship between structure and function. The experimental is tightly integrated with computational modeling approaches with the Abagyan lab, in order to minimize the trial-and-error in experimental construct design, and to rationalize the processes of ligand binding and activation in a three-dimensional context.

Receptor Interactions with Intracellular Proteins

In addition to in vitro studies of receptor structure and activation dynamics, we are interested in how chemokine receptors function through interaction with intracellular proteins, identifying what are the agonist-dependent interactions, how they change with time post-activation, how they control receptor trafficking and the structural determinants of these interactions. We use BRET to study the interactions with known intracellular partners. Mass-spectrometry based proteomics is being used to identify unknown interacting partners. These studies are coupled with cell based assays of function (cell migration, internalization, immunoblot, immunofluorescence etc). We are also interested in how these interactions change in normal versus diseased (e.g. cancer ) cells. The figure below shows constitutive internalization of a chemokine receptor and a mutant in which the internalization is significantly retarded.