The Structure of a CXCR4:Chemokine Complex

CXCR4 was the first chemokine receptor to be identified as an HIV coreceptor in 1996 (1). Along with the importance of CXCR4 in development, it was also discovered as a key chemokine receptor in the metastasis of breast (2) and numerous other cancers (3). These were the main reasons that motivated us to pursue structural studies of CXCR4 with synthetic inhibitors and chemokines. However, my laboratory took a rather circuitous route to this goal, and I did a lot of reinventing myself as a scientist along the way. As background, I got my Ph.D. in chemistry/membrane biophysics at the California Institute of Technology, and then in 1989 accepted a postdoctoral position to do “protein design” with Bill DeGrado at E. I. Du Pont de Nemours (we called it Du Pont University back then, given the amazing freedom we had to do truly basic research). Du Pont (primarily a chemical company) formed a Joint Venture and became Du Pont Merck Pharmaceuticals; and when I was transitioning to a full time employee in 1992, we were tasked with coming up with new therapeutic targets for the expanded pharmaceutical side of the business. I was hired as part of the macromolecular NMR group headed by Peter Domaille, and thus my target choices were biased by some of the exciting work emerging from the structural biology community. This included the first structure of a chemokine, interleukin-8 (IL-8, now CXCL8), which was published in 1990 by Angela Gronenborn’s NMR group at NIH (4). I remember being intrigued by the dimeric structure and thinking (as they described in their paper) that the dimeric architecture of two alpha helices on top of a beta sheet platform might provide a perfect binding site for the IL-8 receptor, as it was reminiscent of the human class I histocompatibility antigen HLAA2 binding pocket for antigenic peptides. In 1989, two separate groups had cloned the gene for the related CC chemokine, monocyte chemotactic protein-1 (MCP-1, also called MCAF, now CCL2) (5, 6), and although the MCP-1 receptor (CCR2) had not yet been cloned, it looked like this system might be a good target for inflammation. It was consequently adopted as a focus of the Du Pont Merck inflammatory disease group, with the goal of inhibiting the receptor. Inspired by the IL-8 structure and the expectation that MCP-1 would also be a tractable target for NMR, Peter Domaille and I began working on its structure around 1992. We were hoping to obtain the structure of the first CC chemokine, but not surprisingly, the powerhouse NIH group beat us by a long shot and solved MIP-1β in 1994 (7), as did Nick Skelton and Tom Schall at Genentech, who solved the structure of RANTES in 1995 (8). Nevertheless, we persisted, and although I left Du Pont Merck for a faculty position at the University of California Berkeley in 1994, we published the structure of MCP-1 in 1996 (9). At Berkeley, I continued working on MCP-1 in collaboration with a group at Roche led by Kurt Jarnagin. A major question that arose from the prevalence of dimeric chemokine structures that had been solved was whether they bound receptors as dimers (the prevailing hypothesis) or as monomers. By identifying a mutant that was incapable of dimerizing but was as potent as WT MCP-1 in migration and receptor binding assays, we demonstrated that it bound CCR2 as a monomer (10). This conclusion was consistent with a prior study by Ian ClarkLewis who had shown that IL-8 was also a functional monomer (11). We also did a fairly comprehensive mutagenesis study of the residues involved in binding and signaling and came up with a model, which was published in 1999 (Figure 1A) (12). Although we never properly docked MCP-1 to the rhodopsin-based model of the receptor, we were qualitatively on the right track of what the structure might look like. However, it was just a model based on mutagenesis data, and I really wanted to determine highresolution structures of intact receptors with chemokines and/or small molecule antagonists. Because membrane receptors are so challenging, there was no way I was going to even consider working on intact chemokine receptor structures until/unless I got tenure at Berkeley, and fortunately that occurred in 2000. In 2002, I managed to hire a talented postdoc, Samantha Allen, from University of Bristol. She had a background in protein folding studies of bacteriorhodopsin, was interested in moving onto studies of eukaryotic membrane receptors and had the bravery (or perhaps naivety) to join me in the pursuit of chemokine receptor structures. Not having a track record in the expression, biochemistry or structural biology of membrane receptors, it was very difficult to get funding. Fortunately, Richard Horuk managed to convince his company, Berlex, to provide matching funds for a UC Discovery grant to pursue CCR1. It was not a lot of money, but that money along with fellowships that Samantha managed to garner, enabled us