Reversibly locking a protein fold in an active conformation with a disulfide bond: integrin alphaL I domains with high affinity and antagonist activity in vivo.
The integrin alphaLbeta2 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the alphaM and alpha2 subunits has been crystallized in both open and closed conformations; however, the alphaL I domain has been crystallized in only the closed conformation. We hypothesized that the alphaL domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the alphaL I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on Mg(2+). Orders of affinity were ICAM-1 > ICAM-2 > ICAM-3. The k(on), k(off), and K(D) values for the locked open I domain were within 1.5-fold of values previously determined for the alphaLbeta2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized alphaLbeta2-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.
Shimaoka
M
M
The Center for Blood Research and Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
Lu
C
C
Palframan
R T
RT
von Andrian
U H
UH
McCormack
A
A
Takagi
J
J
Springer
T A
TA
D015703:Antigens, CD / Q000378:metabolism*; D000943:Antigens, Differentiation; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D004220:Disulfides; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D020014:K562 Cells; D007700:Kinetics; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding
D015703:Antigens, CD; D000943:Antigens, Differentiation; D015815:Cell Adhesion Molecules; D004220:Disulfides; C065289:ICAM2 protein, human; C483616:ICAM3 protein, human; D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1