The Q163C/Q309C mutant of α_MI-domain is an active variant suitable for NMR characterization

by Hoa Nguyen, Tianwei Jing, Xu Wang

Integrin α_Mβ₂ (Mac-1, CD11b/CD18, CR3) is an important adhesion receptor expressed on monocytes. Mac-1 is responsible for mediating cell migration, phagocytosis, degranulation as well as cell-cell fusion. It is also the most promiscuous integrin in terms of ligand specificity with over 100 ligands, most of which use the α_MI-domain as their binding site. Despite the importance of α_MI-domain in defining ligand interactions of Mac-1, structural studies of α_MI-domain’s interactions with ligands are lacking. In particular, solution NMR studies of α_MI-domain’s interaction with ligands have not been possible because the most commonly used active α_MI-domain mutants (I316G and ΔK315) are not sufficiently stable and soluble to be used in solution NMR. The goal of this study is to identify an α_MI-domain active mutant that’s amenable to NMR characterization. By screening known activating mutations of α_MI-domain, we determined that the Q163C/Q309C mutant, which converts the α_MI-domain into its active form through the formation of an intramolecular disulfide bond, can be produced with a high yield and is more stable than other active mutants. In addition, the Q163C/Q309C mutant has better NMR spectral quality than other active mutants and its affinity for ligands is comparable to other active mutants. Analysis of the Co²⁺-induced pseudocontact shifts in the Q163C/Q309C mutant showed the structure of the mutant is consistent with the active conformation. Finally, we show that the minor fraction of the Q163C/Q309C mutant without the disulfide bond can be removed through the use of carboxymethyl sepharose chromatography. We think the availability of this mutant for NMR study will significantly enhance structural characterizations of α_MI-domain-ligand interactions.