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The structure of the N-terminus of kindlin-1: a domain important for alphaiibbeta3 integrin activation.

Goult BT, Bouaouina M, Harburger DS, Bate N, Patel B, Anthis NJ, Campbell ID, Calderwood DA, Barsukov IL, Roberts GC, Critchley DR - J. Mol. Biol. (2009)

Bottom Line: We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains.Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain.Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Henry Wellcome Building, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.

ABSTRACT
The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the beta-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced alphaIIbbeta3 integrin activation and for the localization of kindlin-1 to focal adhesions.

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Comparison of the domain structure of kindlins and the talin head. (a) Schematic diagram of the domain structure of kindlins and talin. The individual domains—F1, F2, and F3—that make up a canonical FERM domain are shown in green, orange, and blue, respectively; the F0 domain is shown in red. The kindlin PH domain is indicated by a black box. Unstructured regions including the F1–loop region are shown in white. The position of the long C-terminal talin rod is indicated. The horizontal scale in both schematics is the same. (b) The primary sequences of mouse kindlin-1 and talin-1 FERM domains were aligned by T-Coffee. The same color scheme is used as in (a). The sequence of the F1 insert and the kindlin PH domain are not included in the alignment. The six tryptophan residues in kindlin-1 F0 are shown in bold. The overall similarity between talin-1 and kindlin-1 is relatively low (28%), due largely to the inclusion of the kindlin PH domain and the insert in F1. The similarity between the individual F0, F1, F2, and F3 domains is much higher (36–55%).
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fig1: Comparison of the domain structure of kindlins and the talin head. (a) Schematic diagram of the domain structure of kindlins and talin. The individual domains—F1, F2, and F3—that make up a canonical FERM domain are shown in green, orange, and blue, respectively; the F0 domain is shown in red. The kindlin PH domain is indicated by a black box. Unstructured regions including the F1–loop region are shown in white. The position of the long C-terminal talin rod is indicated. The horizontal scale in both schematics is the same. (b) The primary sequences of mouse kindlin-1 and talin-1 FERM domains were aligned by T-Coffee. The same color scheme is used as in (a). The sequence of the F1 insert and the kindlin PH domain are not included in the alignment. The six tryptophan residues in kindlin-1 F0 are shown in bold. The overall similarity between talin-1 and kindlin-1 is relatively low (28%), due largely to the inclusion of the kindlin PH domain and the insert in F1. The similarity between the individual F0, F1, F2, and F3 domains is much higher (36–55%).

Mentions: There are two talin genes in vertebrates,16,17 and both talin-1 and talin-2 (∼ 270 kDa; 2541 amino acids) are composed of an N-terminal head (∼ 50 kDa) and a large, flexible C-terminal rod (∼ 220 kDa).7 The talin head contains a FERM (four-point-one, ezrin, radixin, moesin) domain made up of F1, F2, and F3 domains but is atypical in that F1 contains a large unstructured loop and is preceded by an additional domain (F0) with homology to F1 (Fig. 1a and Goult et al., under revision). The F3 FERM domain has a phosphotyrosine-binding domain fold and selectively binds to the more membrane-proximal of the two NPxY-like motifs in the short cytoplasmic tails of β-integrin subunits.18 The structure of talin F3 bound to residues 739WDTANNPLYDEA750 of the β3 integrin tail shows that the integrin peptide interacts predominantly with the hydrophobic surface on strand S5 of the F3 domain.19 More recently, F3 has also been shown to interact with the membrane-proximal helix of the β3 tail, and integrin residues F727 and F730 bind to a hydrophobic pocket in F3 made up of the flexible loop between β-strands S1 and S2.20 Significantly, mutation of either F727 or F730 in β3 integrin or the interacting residues in F3 (notably L325) markedly reduced activation of αIIbβ3 integrin expressed in Chinese hamster ovary (CHO) cells. This has led to a model in which talin F3 initially binds to the membrane-proximal β3 integrin NPxY motif and subsequently engages the membrane-proximal helix, breaking the clasp between the α- and β-integrin tails, which normally locks the integrin in a low-affinity state. However, the talin F3 domain alone is not sufficient to activate β1 integrins, and the F0 and F1 domains are also required.21


The structure of the N-terminus of kindlin-1: a domain important for alphaiibbeta3 integrin activation.

Goult BT, Bouaouina M, Harburger DS, Bate N, Patel B, Anthis NJ, Campbell ID, Calderwood DA, Barsukov IL, Roberts GC, Critchley DR - J. Mol. Biol. (2009)

Comparison of the domain structure of kindlins and the talin head. (a) Schematic diagram of the domain structure of kindlins and talin. The individual domains—F1, F2, and F3—that make up a canonical FERM domain are shown in green, orange, and blue, respectively; the F0 domain is shown in red. The kindlin PH domain is indicated by a black box. Unstructured regions including the F1–loop region are shown in white. The position of the long C-terminal talin rod is indicated. The horizontal scale in both schematics is the same. (b) The primary sequences of mouse kindlin-1 and talin-1 FERM domains were aligned by T-Coffee. The same color scheme is used as in (a). The sequence of the F1 insert and the kindlin PH domain are not included in the alignment. The six tryptophan residues in kindlin-1 F0 are shown in bold. The overall similarity between talin-1 and kindlin-1 is relatively low (28%), due largely to the inclusion of the kindlin PH domain and the insert in F1. The similarity between the individual F0, F1, F2, and F3 domains is much higher (36–55%).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2963925&req=5

fig1: Comparison of the domain structure of kindlins and the talin head. (a) Schematic diagram of the domain structure of kindlins and talin. The individual domains—F1, F2, and F3—that make up a canonical FERM domain are shown in green, orange, and blue, respectively; the F0 domain is shown in red. The kindlin PH domain is indicated by a black box. Unstructured regions including the F1–loop region are shown in white. The position of the long C-terminal talin rod is indicated. The horizontal scale in both schematics is the same. (b) The primary sequences of mouse kindlin-1 and talin-1 FERM domains were aligned by T-Coffee. The same color scheme is used as in (a). The sequence of the F1 insert and the kindlin PH domain are not included in the alignment. The six tryptophan residues in kindlin-1 F0 are shown in bold. The overall similarity between talin-1 and kindlin-1 is relatively low (28%), due largely to the inclusion of the kindlin PH domain and the insert in F1. The similarity between the individual F0, F1, F2, and F3 domains is much higher (36–55%).
Mentions: There are two talin genes in vertebrates,16,17 and both talin-1 and talin-2 (∼ 270 kDa; 2541 amino acids) are composed of an N-terminal head (∼ 50 kDa) and a large, flexible C-terminal rod (∼ 220 kDa).7 The talin head contains a FERM (four-point-one, ezrin, radixin, moesin) domain made up of F1, F2, and F3 domains but is atypical in that F1 contains a large unstructured loop and is preceded by an additional domain (F0) with homology to F1 (Fig. 1a and Goult et al., under revision). The F3 FERM domain has a phosphotyrosine-binding domain fold and selectively binds to the more membrane-proximal of the two NPxY-like motifs in the short cytoplasmic tails of β-integrin subunits.18 The structure of talin F3 bound to residues 739WDTANNPLYDEA750 of the β3 integrin tail shows that the integrin peptide interacts predominantly with the hydrophobic surface on strand S5 of the F3 domain.19 More recently, F3 has also been shown to interact with the membrane-proximal helix of the β3 tail, and integrin residues F727 and F730 bind to a hydrophobic pocket in F3 made up of the flexible loop between β-strands S1 and S2.20 Significantly, mutation of either F727 or F730 in β3 integrin or the interacting residues in F3 (notably L325) markedly reduced activation of αIIbβ3 integrin expressed in Chinese hamster ovary (CHO) cells. This has led to a model in which talin F3 initially binds to the membrane-proximal β3 integrin NPxY motif and subsequently engages the membrane-proximal helix, breaking the clasp between the α- and β-integrin tails, which normally locks the integrin in a low-affinity state. However, the talin F3 domain alone is not sufficient to activate β1 integrins, and the F0 and F1 domains are also required.21

Bottom Line: We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains.Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain.Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Henry Wellcome Building, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.

ABSTRACT
The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the beta-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced alphaIIbbeta3 integrin activation and for the localization of kindlin-1 to focal adhesions.

Show MeSH
Related in: MedlinePlus