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

doi:10.1073/pnas.0601164103

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

*The CBR Institute for Biomedical Research and Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115; and
^†Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139

Contributed by Timothy A. Springer, February 13, 2006

Abstract

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 α_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 α7-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.

Footnotes

^‡To whom correspondence should be addressed. E-mail: springeroffice{at}cbr.med.harvard.edu
Author contributions: M.J. and T.A.S. designed research; M.J., G.S., N.S.A., and C.V.C. performed research; Y.-S.K. and D.K.W. contributed new reagents/analytic tools; M.J., M.S., and T.A.S. analyzed data; and M.J. and T.A.S. wrote the paper.
Conflict of interest statement: No conflicts declared.
Abbreviations:

Abbreviations:

I domain,

inserted domain;

MIDAS,

metal ion-dependent adhesion site;

ICAM-1,

intercellular adhesion molecule-1;

IA,

intermediate affinity;

HA,

high affinity;

SFI,

specific fluorescence intensity;

ASFI,

adjusted SFI;

LFA-1,

lymphocyte function-associated antigen 1;

PMA,

phorbol ester 12-tetradecanoylphorbol-13 acetate;

MFI,

mean fluorescence intensity.