Luo B.-H., Strokovich K., Walz T., Springer T. requires a change in orientation between domains 1 and 2 of VCAM for binding to 41. Mapping of species-specific differences onto 41 and 47 shows that their ligand-binding sites are highly conserved. Skewing away from these conserved regions of the epitopes recognized by current therapeutic function-blocking antibodies has resulted in previously unanticipated mechanisms of action. Keywords: Adhesion, Cell Surface, Crystal Structure, Immunology, Integrins, Structure Introduction Adhesion receptors of the immune system are required for all cell-cell interactions in innate and adaptive immune responses, as well as for leukocyte emigration from the bloodstream and migration Pirozadil within tissues (1). A new class of therapeutics termed selective adhesion molecule inhibitors target these receptors (2). A leading example is usually natalizumab, directed to the integrin 4 subunit that is present in both integrins 41 and 47 (1, 3, 4). Natalizumab is more effective than any other drug in preventing relapse in multiple Pirozadil sclerosis, where it has been used in over 100,000 patients (5, 6). Natalizumab also is efficacious in an inflammatory bowel disease, Crohn’s disease (2, 7). However, little is known about the mechanism of adhesion blockade by natalizumab. Integrin 41 binds to the immunoglobulin supergene family (IgSF)3 molecule, vascular cell adhesion molecule (VCAM) (1). VCAM is usually induced on venular endothelium in inflammation, including in brain in experimental autoimmune encephalitis. Antibody to 41 blocks lymphocyte emigration into brain, 41-dependent costimulation of immune responses, and experimental autoimmune encephalitis (8, 9). Integrin 47 binds to the IgSF member mucosal addressin cell adhesion molecule (MAdCAM), Pirozadil which is usually expressed on vascular cells in mucosal tissues (1). 41 and not 47 is critical for Pirozadil experimental autoimmune encephalitis (10). Conversely, the efficacy of natalizumab in Crohn’s disease appears to reflect blockade of 47-dependent interactions (2). Vedolizumab, an antibody specific for 47, shows promise in Crohn’s disease (7). We have previously reported the crystal structure of the integrin 47 headpiece bound to a small molecule antagonist, and to Fab of the mouse precursor of vedolizumab, Act-1 Fab (11). Because the complementarity-determining regions (CDRs) of Act-1 and vedolizumab are identical, we use these names interchangeably here. Vedolizumab bound around the 7 side of a long, wide ligand-binding groove at an extensive interface between the 4-subunit -propeller domain name and the 7-subunit I domain name. Here, we report the crystal structure of natalizumab Fab bound to the integrin 4-subunit. Natalizumab binds on the opposite, 4 side of the ligand-binding groove. Because natalizumab did not appear to block binding of domain name 1 of VCAM to the groove, we examined its mechanism of inhibition, which turns out GP9 to be noncompetitive. Apparently, natalizumab Fab imposes a change in orientation between D1 and D2 of VCAM bound to 41. Species-specific differences outside but near ligand-binding pockets appear to be targeted by many mouse anti-human therapeutic antibodies. Greater complexity exists in the mechanism of action of selective adhesion molecule inhibitors than was originally envisioned. EXPERIMENTAL PROCEDURES Protein Preparation and Purification An 47 headpiece fragment made up of 4 residues 1C587 and 7 residues 1C493, and C-terminal TEV cleavage sites, ACID-BASE coiled-coil, and purification tags secreted by CHO-Lec3.2.8.1 was prepared and purified as described (11). Pharmaceutical natalizumab (1 mg/ml) was digested with papain (0.01 mg/ml).