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. 2003 May 15;372(Pt 1):121–127. doi: 10.1042/BJ20021273

Engineered allosteric mutants of the integrin alphaMbeta2 I domain: structural and functional studies.

Clare J McCleverty 1, Robert C Liddington 1
PMCID: PMC1223386  PMID: 12611591

Abstract

The alpha-I domain, found in the alpha-subunit of the leucocyte integrins such as alphaMbeta2 and alphaLbeta2, switches between the open and closed tertiary conformations, reflecting the high- and low-affinity ligand-binding states of the integrin that are required for regulated cell adhesion and migration. In the present study we show, by using point mutations and engineered disulphide bonds, that ligand affinity can be reduced or increased allosterically by altering the equilibrium between the closed and open states. We determined equilibrium constants for the binding of two ligands, fibrinogen and intercellular cell-adhesion molecule 1, to the alphaM-I domain by surface plasmon resonance, and determined crystal structures of a low-affinity mutant. Locking the domain in the open conformation increases affinity by a factor of no greater than 10, consistent with a closely balanced equilibrium between the two conformations in the absence of ligand. This behaviour contrasts with that of the unliganded alphaL-I domain, for which the equilibrium lies strongly in favour of the closed conformation. These results suggest significant differences in the way the parent integrins regulate I domain conformation and hence ligand affinity.

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Selected References

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  1. Alonso José Luis, Essafi Makram, Xiong Jian Ping, Stehle Thilo, Arnaout M. Amin. Does the integrin alphaA domain act as a ligand for its betaA domain? Curr Biol. 2002 May 14;12(10):R340–R342. doi: 10.1016/s0960-9822(02)00852-7. [DOI] [PubMed] [Google Scholar]
  2. Baldwin E. T., Sarver R. W., Bryant G. L., Jr, Curry K. A., Fairbanks M. B., Finzel B. C., Garlick R. L., Heinrikson R. L., Horton N. C., Kelley L. L. Cation binding to the integrin CD11b I domain and activation model assessment. Structure. 1998 Jul 15;6(7):923–935. doi: 10.1016/s0969-2126(98)00093-8. [DOI] [PubMed] [Google Scholar]
  3. Brünger A. T., Adams P. D., Clore G. M., DeLano W. L., Gros P., Grosse-Kunstleve R. W., Jiang J. S., Kuszewski J., Nilges M., Pannu N. S. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905–921. doi: 10.1107/s0907444998003254. [DOI] [PubMed] [Google Scholar]
  4. Edwards C. P., Champe M., Gonzalez T., Wessinger M. E., Spencer S. A., Presta L. G., Berman P. W., Bodary S. C. Identification of amino acids in the CD11a I-domain important for binding of the leukocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1) J Biol Chem. 1995 May 26;270(21):12635–12640. doi: 10.1074/jbc.270.21.12635. [DOI] [PubMed] [Google Scholar]
  5. Emsley J., Knight C. G., Farndale R. W., Barnes M. J., Liddington R. C. Structural basis of collagen recognition by integrin alpha2beta1. Cell. 2000 Mar 31;101(1):47–56. doi: 10.1016/S0092-8674(00)80622-4. [DOI] [PubMed] [Google Scholar]
  6. García-Alvarez Begoña, de Pereda José M., Calderwood David A., Ulmer Tobias S., Critchley David, Campbell Iain D., Ginsberg Mark H., Liddington Robert C. Structural determinants of integrin recognition by talin. Mol Cell. 2003 Jan;11(1):49–58. doi: 10.1016/s1097-2765(02)00823-7. [DOI] [PubMed] [Google Scholar]
  7. Huang C., Springer T. A. A binding interface on the I domain of lymphocyte function-associated antigen-1 (LFA-1) required for specific interaction with intercellular adhesion molecule 1 (ICAM-1). J Biol Chem. 1995 Aug 11;270(32):19008–19016. doi: 10.1074/jbc.270.32.19008. [DOI] [PubMed] [Google Scholar]
  8. Hughes P. E., Diaz-Gonzalez F., Leong L., Wu C., McDonald J. A., Shattil S. J., Ginsberg M. H. Breaking the integrin hinge. A defined structural constraint regulates integrin signaling. J Biol Chem. 1996 Mar 22;271(12):6571–6574. doi: 10.1074/jbc.271.12.6571. [DOI] [PubMed] [Google Scholar]
  9. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  10. Kamata T., Liddington R. C., Takada Y. Interaction between collagen and the alpha(2) I-domain of integrin alpha(2)beta(1). Critical role of conserved residues in the metal ion-dependent adhesion site (MIDAS) region. J Biol Chem. 1999 Nov 5;274(45):32108–32111. doi: 10.1074/jbc.274.45.32108. [DOI] [PubMed] [Google Scholar]
  11. Kamata T., Puzon W., Takada Y. Identification of putative ligand binding sites within I domain of integrin alpha 2 beta 1 (VLA-2, CD49b/CD29) J Biol Chem. 1994 Apr 1;269(13):9659–9663. [PubMed] [Google Scholar]
  12. Kamata T., Takada Y. Direct binding of collagen to the I domain of integrin alpha 2 beta 1 (VLA-2, CD49b/CD29) in a divalent cation-independent manner. J Biol Chem. 1994 Oct 21;269(42):26006–26010. [PubMed] [Google Scholar]
  13. Kern A., Briesewitz R., Bank I., Marcantonio E. E. The role of the I domain in ligand binding of the human integrin alpha 1 beta 1. J Biol Chem. 1994 Sep 9;269(36):22811–22816. [PubMed] [Google Scholar]
  14. Lee J. O., Bankston L. A., Arnaout M. A., Liddington R. C. Two conformations of the integrin A-domain (I-domain): a pathway for activation? Structure. 1995 Dec 15;3(12):1333–1340. doi: 10.1016/s0969-2126(01)00271-4. [DOI] [PubMed] [Google Scholar]
  15. Lee J. O., Rieu P., Arnaout M. A., Liddington R. Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell. 1995 Feb 24;80(4):631–638. doi: 10.1016/0092-8674(95)90517-0. [DOI] [PubMed] [Google Scholar]
  16. Li R., Rieu P., Griffith D. L., Scott D., Arnaout M. A. Two functional states of the CD11b A-domain: correlations with key features of two Mn2+-complexed crystal structures. J Cell Biol. 1998 Dec 14;143(6):1523–1534. doi: 10.1083/jcb.143.6.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liddington R. C., Ginsberg M. H. Integrin activation takes shape. J Cell Biol. 2002 Sep 3;158(5):833–839. doi: 10.1083/jcb.200206011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lupher M. L., Jr, Harris E. A., Beals C. R., Sui L. M., Liddington R. C., Staunton D. E. Cellular activation of leukocyte function-associated antigen-1 and its affinity are regulated at the I domain allosteric site. J Immunol. 2001 Aug 1;167(3):1431–1439. doi: 10.4049/jimmunol.167.3.1431. [DOI] [PubMed] [Google Scholar]
  19. Michishita M., Videm V., Arnaout M. A. A novel divalent cation-binding site in the A domain of the beta 2 integrin CR3 (CD11b/CD18) is essential for ligand binding. Cell. 1993 Mar 26;72(6):857–867. doi: 10.1016/0092-8674(93)90575-b. [DOI] [PubMed] [Google Scholar]
  20. Mould A. P., Askari J. A., Aota S. i., Yamada K. M., Irie A., Takada Y., Mardon H. J., Humphries M. J. Defining the topology of integrin alpha5beta1-fibronectin interactions using inhibitory anti-alpha5 and anti-beta1 monoclonal antibodies. Evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the alpha5 subunit. J Biol Chem. 1997 Jul 11;272(28):17283–17292. doi: 10.1074/jbc.272.28.17283. [DOI] [PubMed] [Google Scholar]
  21. Oxvig C., Lu C., Springer T. A. Conformational changes in tertiary structure near the ligand binding site of an integrin I domain. Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2215–2220. doi: 10.1073/pnas.96.5.2215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schwartz Martin A., Ginsberg Mark H. Networks and crosstalk: integrin signalling spreads. Nat Cell Biol. 2002 Apr;4(4):E65–E68. doi: 10.1038/ncb0402-e65. [DOI] [PubMed] [Google Scholar]
  23. Shimaoka M., Lu C., Palframan R. T., von Andrian U. H., McCormack A., Takagi J., Springer T. A. 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. Proc Natl Acad Sci U S A. 2001 May 15;98(11):6009–6014. doi: 10.1073/pnas.101130498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Smith C., Estavillo D., Emsley J., Bankston L. A., Liddington R. C., Cruz M. A. Mapping the collagen-binding site in the I domain of the glycoprotein Ia/IIa (integrin alpha(2)beta(1)). J Biol Chem. 2000 Feb 11;275(6):4205–4209. doi: 10.1074/jbc.275.6.4205. [DOI] [PubMed] [Google Scholar]
  25. Takagi J., Erickson H. P., Springer T. A. C-terminal opening mimics 'inside-out' activation of integrin alpha5beta1. Nat Struct Biol. 2001 May;8(5):412–416. doi: 10.1038/87569. [DOI] [PubMed] [Google Scholar]
  26. Takagi Junichi, Petre Benjamin M., Walz Thomas, Springer Timothy A. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell. 2002 Sep 6;110(5):599–511. doi: 10.1016/s0092-8674(02)00935-2. [DOI] [PubMed] [Google Scholar]
  27. Tuckwell D., Calderwood D. A., Green L. J., Humphries M. J. Integrin alpha 2 I-domain is a binding site for collagens. J Cell Sci. 1995 Apr;108(Pt 4):1629–1637. doi: 10.1242/jcs.108.4.1629. [DOI] [PubMed] [Google Scholar]
  28. Vinogradova Olga, Velyvis Algirdas, Velyviene Asta, Hu Bin, Haas Thomas, Plow Edward, Qin Jun. A structural mechanism of integrin alpha(IIb)beta(3) "inside-out" activation as regulated by its cytoplasmic face. Cell. 2002 Sep 6;110(5):587–597. doi: 10.1016/s0092-8674(02)00906-6. [DOI] [PubMed] [Google Scholar]
  29. Xiong J. P., Li R., Essafi M., Stehle T., Arnaout M. A. An isoleucine-based allosteric switch controls affinity and shape shifting in integrin CD11b A-domain. J Biol Chem. 2000 Dec 8;275(49):38762–38767. doi: 10.1074/jbc.C000563200. [DOI] [PubMed] [Google Scholar]
  30. Xiong J. P., Stehle T., Diefenbach B., Zhang R., Dunker R., Scott D. L., Joachimiak A., Goodman S. L., Arnaout M. A. Crystal structure of the extracellular segment of integrin alpha Vbeta3. Science. 2001 Sep 6;294(5541):339–345. doi: 10.1126/science.1064535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zhang L., Plow E. F. A discrete site modulates activation of I domains. Application to integrin alphaMbeta2. J Biol Chem. 1996 Nov 22;271(47):29953–29957. doi: 10.1074/jbc.271.47.29953. [DOI] [PubMed] [Google Scholar]
  32. Zhang L., Plow E. F. Amino acid sequences within the alpha subunit of integrin alpha M beta 2 (Mac-1) critical for specific recognition of C3bi. Biochemistry. 1999 Jun 22;38(25):8064–8071. doi: 10.1021/bi990141h. [DOI] [PubMed] [Google Scholar]
  33. Zhang L., Plow E. F. Identification and reconstruction of the binding site within alphaMbeta2 for a specific and high affinity ligand, NIF. J Biol Chem. 1997 Jul 11;272(28):17558–17564. doi: 10.1074/jbc.272.28.17558. [DOI] [PubMed] [Google Scholar]

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