Structural and functional characterization of the CC chemokine macrophage inflammatory protein-1β: Analysis of its propensity for self-association in relation to its interactions with CCR5 and heparin

Jennifer Ann Stowell Laurence, Purdue University

Abstract

Structures of chemokines reveal the presence of distinct patterns of self-association for the two major sub-families, which might be relevant to some of their biological functions. We have undertaken a structural study of chemokine-chemokine interactions and performed biological assays in order to understand what function this phenomenon might serve. Macrophage inflammatory protein 1β, a member of the CC subfamily, was examined by NMR and analytical centrifugation to determine whether mutations made to residues having inter-subunit contacts produced monomeric proteins. It was determined that mutation of Phe13 or Pro8 was sufficient to break up the dimer. Removing residues 1–5 did not cause dissociation, but removal of Asp6 produced a monomer-dimer mixture. Since removal of these residues in closely related chemokines resulted in loss of biological function, we assayed these mutants for their ability to bind and stimulate calcium release through CCR5. Phe13 was shown to be crucial for receptor binding, whereas it appears from studies with truncation mutants that the N-terminal residues promote signaling. Mutational analysis of basic residues using heparin chromatography was done to characterize the heparin binding properties of MIP-1β. The importance of GAG binding in chemokine function was assessed using CCR5 binding and activity assays. Lys45 and Arg46 participate in heparin binding, while Arg46 and Lys48 contribute to CCR5 binding. Mutations made in MIP(9) predict that Lys48 also contributes to heparin affinity and that binding may partially depend on the ability of the protein to dimerize.

Degree

Ph.D.

Advisors

LiWang, Purdue University.

Subject Area

Biochemistry

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