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

Integrin alpha(M)beta(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 alpha I-M-domain as their binding site. Despite the importance of alpha I-M-domain in defining ligand interactions of Mac-1, structural studies of alpha I-M-domain's interactions with ligands are lacking. In particular, solution NMR studies of alpha I-M-domain's interaction with ligands have not been possible because the most commonly used active alpha I-M-domain mutants (I316G and Delta K315) are not sufficiently stable and soluble to be used in solution NMR. The goal of this study is to identify an alpha I-M-domain active mutant that's amenable to NMR characterization. By screening known activating mutations of alpha I-M-domain, we determined that the Q163C/Q309C mutant, which converts the alpha I-M-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 alpha I-M-domain-ligand interactions.

Automated summary

Integrin alpha(M)beta(2) (Mac-1, CD11b/CD18, CR3) is an important adhesion receptor expressed on monocytes. It is responsible for mediating various cellular processes such as migration, phagocytosis, degranulation, and cell-cell fusion. Despite the lack of structural studies, the alpha I-M-domain plays a crucial role in ligand interactions of Mac-1. This study successfully identified a stable and soluble mutant, Q163C/Q309C, which can be used for NMR characterization and enhance the understanding of alpha I-M-domain-ligand interactions.