Reversibly locking a protein fold in an active conformation with a disulfide bond: Integrin αL I domains with high affinity and antagonist activity<i>in vivo</i>

Shimaoka, Motomu; Lu, Chafen; Palframan, Roger; Andrian, Ulrich H. von; McCormack, Alison L.; Takagi, Junichi; Springer, Timothy A.

doi:10.1073/pnas.101130498

Cited by 212 publications

(284 citation statements)

References 47 publications

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“…The present results with the § E I domain agree, in general, with findings on g 2 integrins [6,8,[19][20][21]. Nevertheless, differences have emerged which provide a broader perspective on the role of conformational change in integrin function.…”

Section: Discussionsupporting

confidence: 89%

“…With § L the wild-type adopts a closed conformation having an affinity 10 4 -fold lower than the open version [21] whereas the equilibrium with § M is much nearer to the open form [34]. From our adhesion tests with § E it appears that the locked-open conformation represents an extreme situation that may not be achievable physiologically.…”

Section: Discussionmentioning

confidence: 82%

“…Guided by crystal structures, it has been possible to lock the § I domain of § L, § M and § X into an open, active, conformation and to test for ligand binding activity using the complete integrin holoreceptor or the isolated § I domain. This strategy has greatly increased the affinity of the integrin for its ligand and, in the case of § L g 2, has demonstrated that the § I domain provides the sole contact site for ICAM-1 [19][20][21][22][23]. It has been proposed that this picture provides a paradigm for all § I domain-containing integrins [8].…”

Section: Introductionmentioning

confidence: 99%

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Role of the αI domain in ligand binding by integrin αEβ7

et al. 2003

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The I domain of integrin § E was modeled on the crystal structure of that in CD11b and mutated to produce an open (high affinity) or closed (low affinity) conformation. K562 transfectants expressing mutant § E and wild-type g 7 were tested for adhesion to E-cadherin-Fc. Downward displacement of the C terminus of the § I domain with a disulfide bridge enhanced adhesion and Mn 2+ dependency. Adhesion greatly exceeded that observed using wild type integrin under similar conditions. The closed integrin gave poor adhesion which was greatly improved by PMA-induced clustering. Blocking g 7 function with a g I domain-specific antibody inhibited the wild-type but not the locked open integrin. Isolated open § I domain expressed on K562 cells showed strong Mn 2+ -dependent adhesion to E-cadherin, whereas the wild-type version was ineffective. § E g 7 was shown to bind to monomeric E-cadherin but to only one component of dimeric E-cadherin. Finally, we report that M290, a functionblocking antibody, bound to a conformation-sensitive epitope near the rim of the § I domain MIDAS and recognized wild-type and closed § I domain but not the open conformation. The results broadly support the paradigm for affinity regulation by conformational change that has been established for g 2 integrins. Nevertheless, for § E, the fully open conformation may represent an extreme situation that does not occur physiologically.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 82%

Section: Introductionmentioning

confidence: 99%

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Role of the αI domain in ligand binding by integrin αEβ7

et al. 2003

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“…The binding to ICAM-1 is completely Mg 2ϩ -dependent. The affinity and kinetics of the locked-open I domain for ICAM-1 are comparable with that determined previously for the activated ␣ L ␤ 2 complex [23], indicating that the ␣ L I domain, when locked in an open conformation, is sufficient for maximal affinity-binding to ICAM-1 [15]. When expressed on the cell surface, the locked-open I domain mediates cell adhesion as potently as maximally activated wild-type ␣ L ␤ 2 [13,14].…”

Section: Introductionsupporting

confidence: 63%

“…The ␣ L I domain has been locked in the open or closed conformation with engineered disulfide bonds [11,[13][14][15]. Locking the ␣ L I domain in the open conformation causes a 9000-fold increase in affinity for ICAM-1 compared with the affinity of the inactive wild-type I domain and the locked-closed conformation and can be reversed by disulfide reduction [15].…”

Section: Introductionmentioning

confidence: 99%