The goal of this work is to understand the structural and biochemical basis for the recruitment of lymphocytes by human Lymphotactin, a chemokine that defies the traditional paradigm by interconverting between two entirely different tertiary structures. Inflammation in the vertebrate immune system is orchestrated by approximately 50 chemokines that specifically activate members of a group of 20 G protein-coupled receptors. These secreted signaling proteins also bind extracellular matrix glycosaminoglycans (GAG) in order to direct migration along a gradient of chemokine concentration. Lymphotactin (Ltn/XCL1), the prototype and single member of the C class of chemokines, acts through its cognate receptor XCR1 to selectively recruit T and NK cells. Functional roles in tumor regression and tissue transplant rejection highlight two disease states that may respond to treatment with either Ltn mimetics or antagonists. Ltn contains only one of the two disulfides conserved in all other chemokines, and is further distinguished by a unique disordered C-terminal extension that is essential for activity. A consequence of these divergent sequence features is that Ltn simultaneously adopts two distinct tertiary structures in physiological solution conditions, only one of which resembles the canonical chemokine fold. Experiments in Specific Aim 1 will use NMR spectroscopy and mutagenesis to determine the novel non-chemokine structure of Ltn, measure the dynamics of interconversion, and test the hypothesis that its conformational equilibrium derives from the lack of a conserved disulfide bridge. The goal of Aim 2 is to alter the conformational equilibrium and measure binding affinities for each species by surface plasmon resonance, in order to test the hypothesis that the structural interconversion creates a high affinity GAG binding site essential for in vivo activity.
In Specific Aim 3, conserved residues in the essential C-terminus will be probed for their role in GPCR activation, and comparisons of activity in vitro and in vivo for conformationally-restricted Ltn variants will be compared in order to test the hypothesis that only the chemokine-like structure is competent to bind and activate the receptor. These structural and biochemical studies are designed to open new avenues for in vivo modulation of lymphocyte trafficking directed by Lymphotactin.
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