The Q163C/Q309C mutant of α(M)I-domain is an active variant suitable for NMR characterization

Integrin α(M)β(2) (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(M)I-domain as their binding site. Despite the importance of α(M)I-domain in defining ligand interactions of Mac-1, structural studies of α(M)I-domain’s interactions with ligands are lacking. In particular, solution NMR studies of α(M)I-domain’s interaction with ligands have not been possible because the most commonly used active α(M)I-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(M)I-domain active mutant that’s amenable to NMR characterization. By screening known activating mutations of α(M)I-domain, we determined that the Q163C/Q309C mutant, which converts the α(M)I-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 Co(2+)-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(M)I-domain-ligand interactions.