Structural basis of the leukocyte integrin Mac-1 I-domain interactions with the platelet glycoprotein Ib

Cell-surface receptor interactions between leukocyte integrin macrophage-1 antigen (Mac-1, also known as CR3, alpha Mb2, CD11b/CD18) and platelet glycoprotein Ib alpha (GPIb alpha) are critical to vascular inflammation. To define the key residues at the binding interface, we used nuclear magnetic resonance (NMR) to assign the spectra of the mouse Mac-1 I-domain and mapped the residues contacting the mouse GPIb alpha N-terminal domain (GPIb alpha N) to the locality of the integrin metal ion-dependant adhesion site (MIDAS) surface. We next determined the crystal structures of the mouse GPIb alpha N and Mac-1 I-domain to 2 angstrom and 2.5 angstrom resolution, respectively. The mouse Mac-1 I-domain crystal structure reveals an active conformation that is stabilized by a crystal contact from the alpha 7-helix with a glutamate side chain completing the octahedral coordination sphere of the MIDAS Mg2+ ion. The amino acid sequence of the alpha 7-helix and disposition of the glutamic acid matches the C-terminal capping region alpha-helix of GPIb alpha effectively acting as a ligand mimetic. Using these crystal structures in combination with NMR measurements and docking analysis, we developed a model whereby an acidic residue from the GPIb alpha leucine-rich repeat (LRR) capping alpha-helix coordinates directly to the Mac-1 MIDAS Mg2+ ion. The Mac-1: GPIb alpha N complex involves additional interactions consolidated by an elongated pocket flanking the GPIb alpha N LRR capping alpha-helix. The GPIb alpha N alpha-helix has an HxxxE motif, which is equivalent by homology to RxxxD from the human GPIb alpha N. Subsequent mutagenesis of residues at this interface, coupled with surface plasmon resonance studies, confirmed the importance of GPIb alpha N residues H218, E222, and the Mac-1 MIDAS residue T209 to formation of the complex.