Effect of N-terminal truncation and solution conditions on chemokine dimer stability: nuclear magnetic resonance structural analysis of macrophage inflammatory protein 1 beta mutants.

Abstract

Chemokines (chemotactic cytokines) are a family of immune system proteins, several of which have been shown to block human immunodeficiency virus (HIV) infection in various cell types. While the solved structures of most chemokines reveal protein dimers, evidence has accumulated for the biological activity of individual chemokine monomers, and a debate has arisen regarding the biological role of the chemokine dimer. Concurrent with this debate, several N-terminal truncations and modifications in the CC subfamily of chemokines have been shown to have functional significance, in many cases antagonizing their respective receptors and in some cases retaining the ability to block HIV entry to the cell. As the dimer interface of CC chemokines is located at their N-terminus, a structural study of N-terminally truncated chemokines will address the effect that this type of mutation has on the dimer-monomer equilibrium. We have studied the structural consequences of N-terminal truncation in macrophage inflammatory protein 1 beta (MIP-1 beta), a CC chemokine that has been shown to block HIV infection. Examination of nuclear magnetic resonance (NMR) spectra of a series of N-terminally truncated MIP-1 beta variants reveals that these proteins possess a range of ability to dimerize. A mutant beginning at amino acid Asp6 [termed MIP(6)] has near wild-type dimer properties, while further truncation results in weakened dimer affinity. The mutant MIP(9) (beginning with amino acid Thr9) has been found to exist solely as a folded monomer. Relaxation measurements yield a rotational correlation time of 8.6 +/- 0.1 ns for wild-type MIP-1 beta and 4.5 +/- 0.1 ns for the MIP(9) mutant, consistent with a wild-type dimer and a fully monomeric MIP(9) variant. The presence of physiological salt concentration drastically changes the monomer-dimer equilibrium for both wild-type and most mutant proteins, heavily favoring the dimeric form of the protein. These results have implications for structure-function analysis of existing chemokine mutants as well as for the larger debate regarding the biological existence and activity of the chemokine dimer.