Directed evolution to probe protein allostery and integrin I domains of 200,000-fold higher affinity.

Understanding allostery may serve to both elucidate mechanisms of protein regulation and provide a basis for engineering active mutants. Herein we describe directed evolution applied to the integrin alpha(L) inserted domain for studying allostery by using a yeast surface display system. Many hot spots for activation are identified, and some single mutants exhibit remarkable increases of 10,000-fold in affinity for a physiological ligand, intercellular adhesion molecule-1. The location of activating mutations traces out an allosteric interface in the interior of the inserted domain that connects the ligand binding site to the alpha7-helix, which communicates allostery to neighboring domains in intact integrins. The combination of two activating mutations (F265S/F292G) leads to an increase of 200,000-fold in affinity to intercellular adhesion molecule-1. The F265S/F292G mutant is potent in antagonizing lymphocyte function-associated antigen 1-dependent lymphocyte adhesion, aggregation, and transmigration.

[1]  K D Wittrup,et al.  Yeast surface display for directed evolution of protein expression, affinity, and stability. , 2000, Methods in enzymology.

[2]  Timothy A. Springer,et al.  Reversibly locking a protein fold in an active conformation with a disulfide bond: Integrin αL I domains with high affinity and antagonist activity in vivo , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Junichi Takagi,et al.  Structures of the αL I Domain and Its Complex with ICAM-1 Reveal a Shape-Shifting Pathway for Integrin Regulation , 2003, Cell.

[4]  M. Arnaout,et al.  Does the Integrin αA Domain Act as a Ligand for its βA Domain? , 2002, Current Biology.

[5]  Michael Loran Dustin,et al.  A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. , 1986, Journal of immunology.

[6]  P. Nollert,et al.  Lipidic cubic phases as matrices for membrane protein crystallization. , 2004, Methods.

[7]  D. Leahy,et al.  Crystal structure of the I-domain from the CD11a/CD18 (LFA-1, alpha L beta 2) integrin. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[8]  M. Shimaoka,et al.  The Binding Sites for Competitive Antagonistic, Allosteric Antagonistic, and Agonistic Antibodies to the I Domain of Integrin LFA-11 , 2004, The Journal of Immunology.

[9]  M. Weetall,et al.  A homogeneous fluorometric assay for measuring cell adhesion to immobilized ligand using V-well microtiter plates. , 2001, Analytical biochemistry.

[10]  A. Fersht.  Enzyme structure and mechanism , 1977 .

[11]  D E Wemmer,et al.  Two-state allosteric behavior in a single-domain signaling protein. , 2001, Science.

[12]  A. McMichael,et al.  A human lymphocyte‐associated antigen involved in cell‐mediated lympholysis , 1983, European journal of immunology.

[13]  R. Liddington,et al.  Engineered allosteric mutants of the integrin alphaMbeta2 I domain: structural and functional studies. , 2003, The Biochemical journal.

[14]  D. L. Griffith,et al.  Two Functional States of the CD11b A-Domain: Correlations with Key Features of Two Mn2+-complexed Crystal Structures , 1998, The Journal of cell biology.

[15]  David W. Colby,et al.  Engineering antibody affinity by yeast surface display. , 2004, Methods in enzymology.

[16]  C. Carman,et al.  A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them , 2004, The Journal of cell biology.

[17]  J. R. Huth,et al.  NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Thornton,et al.  Influence of proline residues on protein conformation. , 1991, Journal of molecular biology.

[19]  Richard W. Farndale,et al.  Structural Basis of Collagen Recognition by Integrin α2β1 , 2000, Cell.

[20]  T. Springer,et al.  The requirement for lymphocyte function-associated antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester , 1986, The Journal of experimental medicine.

[21]  Ioan Andricioaei,et al.  Conversion between three conformational states of integrin I domains with a C-terminal pull spring studied with molecular dynamics. , 2004, Structure.