VCCI:Chemokine Interactions: Experimental Biochemistry Meets Computational Prediction

Abstract

Inflammation plays a major role in many pathologies including asthma, arthritis, atherosclerosis and traumatic brain injury. As such, controlling inflammation is an important goal. While many biological pathways are involved in inflammation, an appealing target is the chemokine pathway. Chemokines are small immune system proteins that mediate chemotaxis of leukocytes bearing cognate chemokine receptors to the site of infection or inflammation. Many viruses have evolved strategies to counter the chemokine system, including the production of chemokine binding proteins. In particular, poxviruses encode vCCI (viral CC chemokine Inhibitor; also called p35), a protein that binds members of the CC class of chemokines. vCCI has been shown to bind many CC chemokines with high affinity and as such vCCI could be a potent tool as part of an anti‐inflammation strategy.The mechanistic details of how vCCI is able to bind dozens of CC chemokines with nanomolar (or sub‐nanomolar) affinity is still being elucidated. Structures of vCCI alone and in complex have revealed a beta sandwich composed of two beta sheets that binds chemokines using one face of the sandwich in conjunction with a long, highly acidic loop after the second beta strand. Experimental work shows that this highly negatively charged loop in vCCI can act as a lid on the bound chemokine and interacts with positive charges on the chemokine.A particularly puzzling vCCI‐chemokine interaction is in the area at the edge of the negatively charged loop in vCCI, where a conserved tyrosine (Y80) is close to K48 of the chemokine. It was hypothesized that mutating Y80 to Ala in vCCI would allow more room for the chemokine to bind, but experimentally the opposite has been shown to be the case: the Y80A variant in vCCI has a greatly decreased ability to bind chemokines.We report a collaborative effort to understand the role of Y80 and the action of the acidic loop in vCCI to bind chemokines. Molecular dynamics simulations suggest that rather than being a hindrance to the function of the loop, the role of Y80 is actually to prop the loop in the “open” conformation so that the chemokine can bind. When Y80 is replaced with Ala in simulations, the loop closes and blocks the chemokine binding site. This intriguing and unexpected hypothesis from in silico work has been tested experimentally using NMR, isothermal titration calorimetry and other biophysical techniques. We therefore report a collaborative set of experiments that includes computation and experimentation to aid in the understanding of the vCCI:chemokine interactionSupport or Funding InformationSupport provided by UC Merced summer graduate funding.