Folding of the b-propeller domain of the integrin aL subunit is independent of the I domain and dependent on the b2 subunit

We have studied the folding during biosynthesis of the lymphocyte function-associated antigen 1 (LFA-1) aL subunit using mAb to epitopes that map to seven different regions within the amino acid sequence. The N-terminal portion of aL is predicted to contain a b-propeller domain, consisting of seven b-sheets, and an I domain that is predicted to be inserted between b-sheet 2 and b-sheet 3 of the b-propeller. The I domain of aL folds before association with the b2 subunit, as shown by immunoprecipitation of the unassociated aL subunit by mAbs specific for four different sequence elements within the I domain. By contrast, the b-propeller domain is not folded in unassociated aL after a chase of as long as 12 h after synthesis, but does fold upon association with b2. This is shown with mAbs to regions of aL, that precede and follow the I domain in the primary structure. A mAb that maps near the junction of the C terminus of the I domain with the b-propeller domain suggests that this region is partially folded before subunit association. The results show that the I domain and b-propeller domains fold independently of one another, and suggest that the b-propeller domain bears an interface for association with the b subunit. Lymphocyte function-associated antigen 1 (LFA-1) is a member of the leukocyte integrin or b2 integrin subfamily that is expressed on all leukocytes and is important in adhesive interactions in immune and inflammatory responses (1). LFA-1 consists of aL (CD11a) and b2 (CD18) subunits that are noncovalently associated in a 1:1 complex (2). LFA-1 binds three cell surface ligands that are members of the Ig superfamily, intercellular adhesion molecule (ICAM)-1, ICAM-2, and ICAM-3 (1). Interactions with ligands can be transiently up-regulated by cellular activation, apparently both by increased avidity or affinity and changes in association with the cytoskeleton (3–5). Regions important for ligand binding by integrins have been mapped to theN-terminal portions of integrin a and b subunits (6). One region corresponds to seven repeats of about 60 aa each that have weak sequence homology to one another. The homologies include FG (phenylalanyl-glycyl) andGAP (glycylalanyl-prolyl) consensus sequences (7, 8). Seven FG-GAP repeats are found in all integrin a subunits, and the last three or four contain putative Ca21-binding motifs (9). The FGGAP repeats have been found to be important for ligand binding particularly for integrins that lack inserted (I) domains (6). Within the third FG-GAP repeat, in 7 of the 16 known integrins in mammals including LFA-1, an I domain is inserted (Fig. 1). In the integrins that contain I domains, the I domain appears to be important for or has been directly implicated in ligand binding (10–19). The three-dimensional structures of the I domains of the leukocyte integrins Mac-1 and LFA-1 were recently solved by x-ray crystallography (20, 21). The I domain folds to a doubly wound ayb structure with a Mg21 binding site in its crevice. A specific binding site on LFA-1 for ICAM-1 was mapped to residues of the I domain that are on the same face, and surround the Mg21 binding site (19). This is consistent with the idea that a glutamic acid residue in ICAM-1 that is crucial for binding to LFA-1 (22) could coordinate with the Mg21. Although previously the FG-GAP repeats have been conceptualized as independent domains, it was recently suggested that they fold cooperatively into a single domain known as a b-propeller (23). In the b-propeller fold, four, six, seven, or eight b-sheets are arranged radially around a central pseudosymmetry axis (24). Each sheet contains four anti-parallel b-strands that form the legs of a W. These sheets are twisted, like the blades of a propeller. The predicted integrin b-propeller contains seven sheets, and hence is a seven-bladed b-propeller (Fig. 1). The sequence threads through the propeller in a circular fashion, so that the N and C termini of the b-propeller sequence are adjacent in the structure, and b-sheet W7 is in between b-sheets W6 and W1. The FG-GAP repeats are predicted to be offset with respect to the Ws, so that the seventh W contains b-strands contributed by the most Nterminal and C-terminal FG-GAP repeats (Fig. 1). The b-propeller is a tightly folded domain, with extensive hydrophobic contacts between each neighboring W (24). The upper surface of the integrin b-propeller is predicted to bind ligand (23). This is consistent with results from mutagenesis studies and with findings that multiple FG-GAP repeats contribute to ligand binding, since all FG-GAP repeats contribute two loops to this surface (reviewed in ref. 23). The Ca21-binding motifs are predicted to be in close proximity to one another in the loops between b-strands 1 and 2 on the lower surface of the b-propeller. Because removal of Ca21 activates ligand binding by many integrins and destabilizes association between the a and b subunits in several integrins (4, 5, 25, 26), including LFA-1 (27, 28), the lower surface of the b-propeller may be involved in interactions with the b subunit that regulate ligand The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y943162-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: LFA, lymphocyte function-associated antigen; ICAM, intercellular adhesion molecule. *To whom reprint requests should be addressed. FIG. 1. Folding topology of the predicted LFA-1 a subunit b-propeller domain. b-Strands are arrows, predicted disulfide bonds are horizontal lines, putative Ca21 ions bound to 1–2 loops are filled circles, and boundaries between FG-GAP repeats are marked with vertical dashes. The Ws are shown upright, with the 1–2 and 3–4 loops on the bottom, and 2–3 loops and 4–1 loops connecting adjacent Ws on the top.