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Structure of Rap1b Bound to Talin Reveals a Pathway for Triggering Integrin Activation

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Structure of Rap1b Bound to Talin Reveals a Pathway for Triggering Integrin Activation ARTICLE DOI: 10.1038/s41467-017-01822-8 OPEN Structure of Rap1b bound to talin reveals a pathway for triggering integrin activation Liang Zhu1,2, Jun Yang1, Thomas Bromberger3, Ashley Holly1, Fan Lu1,2, Huan Liu1, Kevin Sun1, Sarah Klapproth3, Jamila Hirbawi1, Tatiana V. Byzova1, Edward F. Plow1, Markus Moser3 & Jun Qin1,2 Activation of transmembrane receptor integrin by talin is essential for inducing cell adhesion. However, the pathway that recruits talin to the membrane, which critically controls talin’s 1234567890 action, remains elusive. Membrane-anchored mammalian small GTPase Rap1 is known to bind talin-F0 domain but the binding was shown to be weak and thus hardly studied. Here we show structurally that talin-F0 binds to human Rap1b like canonical Rap1 effectors despite little sequence homology, and disruption of the binding strongly impairs integrin activation, cell adhesion, and cell spreading. Furthermore, while being weak in conventional binary binding conditions, the Rap1b/talin interaction becomes strong upon attachment of activated Rap1b to vesicular membranes that mimic the agonist-induced microenvironment. These data identify a crucial Rap1-mediated membrane-targeting mechanism for talin to activate integrin. They further broadly caution the analyses of weak protein–protein interactions that may be pivotal for function but neglected in the absence of specific cellular microenvironments. 1 Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA. 2 Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA. 3 Max-Planck-Institute of Biochemistry, Department of Molecular Medicine, 82152 Martinsried, Germany. Liang Zhu, Jun Yang and Thomas Bromberger contributed equally to this work. Correspondence and requests for materials should be addressed to M.M. (email: [email protected]) or to J.Q. (email: [email protected]) NATURE COMMUNICATIONS | 8: 1744 | DOI: 10.1038/s41467-017-01822-8 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01822-8 he adhesion of cells to the extracellular matrix (ECM) is observations strongly suggest a RIAM-independent engagement Tessential for regulating a variety of physiological or between Rap1 and talin to regulate integrin activation. Interest- pathological responses such as platelet aggregation, blood ingly, previous studies reported a direct interaction between Rap1 clotting, stroke, wound-healing, and cancer metastasis. Such and talin in mammals26 and in Dictyostelium27, but the affinity of adhesion critically depends upon integrins, a class of cell surface the interaction was found to be low especially for that in mam- α β 26 – receptors that are ( / ) heterodimers with each subunit con- mals (Kd ~ 0.14 mM) . Since weak protein protein interactions taining a small cytoplasmic tail (CT), a transmembrane segment, (PPIs) are typically assumed to be nonspecific and RIAM was and a large extracellular domain. In unstimulated cells, integrin widely regarded as a key bridge between Rap1 and talin, the adopts an inactive conformation with low affinity for ECM Rap1/talin interaction was hardly studied at the mechanistic level. ligands but, upon agonist stimulation, integrin CT is bound by Through comprehensive structural, biochemical, and cell biolo- intracellular proteins, in particular talin and/or kindlin, which gical analyses, we discovered that talin is actually a distinct trigger global conformational change of the extracellular domain effector of Rap1 that recruits talin to membrane and triggers to acquire high affinity for ECM ligand and initiate firm adhesion. integrin activation. We further found that, while being weak in This process, widely regarded as inside-out signaling or integrin conventional binary binding assays, Rap1/talin interaction activation, has been the central topic of cell adhesion research for became strong when activated Rap1 was anchored to membrane – nearly three decades1 6. Extensive efforts have been focused on that mimics the cellular condition. Our results thus uncover a investigating the mechanism of integrin activation by talin—a mechanism of membrane-targeting of talin by Rap1 and also major cytoskeletal protein comprising an N-terminal head highlight the importance of weak PPIs that may be neglected in domain (talin-H) and a C-terminal rod domain (talin-R). Talin-H the absence of specific cellular microenvironments. contains four subdomains F0, F1, F2, and F3 in which F1–F3 constitutes a so-called FERM (4.1/ezrin/radixin/moesin)-like domain. Talin-R is made up of 13 consecutive helical bundles Results followed by a C-terminal dimerization domain (Fig. 1). Intact Rap1 recognizes talin-F0 domain in a GTP-dependent manner. talin adopts an inactive conformation where the major integrin- Rap1 is known to contain two highly homologous isoforms called – binding site located in talin-H F3 domain is masked by talin-R Rap1a and Rap1b28 31. In this study, we choose to focus on – (autoinhibition)7 10. Talin may be unmasked via multiple examining the interaction between the platelet-rich Rap1b and – mechanisms to bind integrin β CT7, 8, 11 13 and trigger the talin-F0. Like other small GTPases, Rap1b exists in inactive – receptor activation14 17. (GDP-bound) and active (GTP-bound) states. We first used a While a great deal has been learned about talin, a fundamental highly sensitive NMR technique called heteronuclear single issue still remains unresolved: how is cytoplasmic talin recruited quantum coherence (HSQC)32 to examine the Rap1b/talin-F0 to plasma membrane—a crucial step for talin to bind and activate interaction. Figure 2a and Supplementary Fig. 1a show that integrin? A previous dogma, largely derived from studying pro- Rap1b loaded with GTP analog GMP-PNP induced residue- α β 15 totypic integrin IIb 3 from platelets, is that agonist stimulation specific chemical shift changes of N-talin-F0. Inactive Rap1b activates small GTPase Rap1 on the membrane to recruit an loaded with GDP also binds to talin-F0 but at weaker affinity as effector called RIAM that in turn recruits talin5, 18. However, evidenced by smaller chemical shift changes than those with while both Rap1 and talin are highly abundant19, RIAM is present GMP-PNP (Fig. 2b). Thus, talin-F0 binds favorably to the active at very low levels in platelets with no other homologs detected20. form of Rap1b but, as shown in Fig. 2c, the binding affinity is μ 26 More importantly, mice lacking RIAM are viable and the platelet quite low (Kd ~ 162 M) as previously concluded . On the other α β 20–22 functions including the IIb 3 activation are totally normal . hand, talin-F0 does not bind H-Ras (Supplementary Fig. 1b) that By contrast, Rap1 and talin are both essential for integrin acti- is near 90% homologous in terms of binding interface (overall vation4, 5, 18. For example, ablation of the Rap1 isoform Rap1b similarity is ~76%) to Rap1b (see also Supplementary Fig. 1c), 23 severely impairs αIIbβ3 activation and platelet aggregation — suggesting that despite being weak, the talin-F0/Rap1b interaction defects that were also observed in talin knockout mice24, 25. These is specific. To further investigate the specificity of Rap1b/talin-F0 486 656 786 910 1044 1206 1357 1653 1822 1973 2140 2294 2482 2541 1 86 202 309 402 F0 F1 F2 F3 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 DD Talin-H Talin-R DD PIP2 R2 RIAM-N R4 R8 R13 R6 R9 R11 F0 F1 F2 F3 R5 R7 R12 γ R1 R10 Rap1 PIPKl R3 Integrin-β RIAM-N RIAM-N Fig. 1 Domain organization and a linear structural model of talin. The model was generated from known crystal and NMR structures of talin fragments and shown in cartoon representation. Critical talin-binding partners involved in talin membrane recruitment and activation are indicated in the figure. PIP2 phosphatidylinositol-4,5-bisphosphate, RIAM-N the N terminus of RIAM, PIPKIγ type I phosphatidylinositol phosphate kinase isoform-γ 2 NATURE COMMUNICATIONS | 8: 1744 | DOI: 10.1038/s41467-017-01822-8 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01822-8 ARTICLE a b 105 15 Free N-talin-F0 Free 15N-talin-F0 +Rap1b (GDP) +Rap1b (GMP-PNP) 15N-talin-F0+Rap1b 110 117.1 127.3 116.0 R35 115 117.6 127.6 R33 116.6 120 N (p.p.m.) N (p.p.m.) K15 G11 125 118.1 127.9 117.2 9.10 9.05 8.9 8.8 7.5 7.4 HN (p.p.m.) HN (p.p.m.) HN (p.p.m.) 130 d 15 10.0 9.0 8.0 7.0 Free N-K2F0 15N-K2F0+Rap1b 105 HN (p.p.m.) c 110 15 0.14 Rap1b (GMP-PNP) / N-talin-F0 G42 115 0.12 G11 0.10 K15 R35 120 0.08 N (p.p.m.) 0.06 (HN, N) (p.p.m.) 0.04 125 obs 0.02 Δδ ± μ Kd=162 11 M 0.00 130 0246810 Ratio 10.0 9.0 8.0 7.0 6.0 HN (p.p.m.) Fig. 2 Rap1b/talin-F0 interaction is GTP-dependent and specific but is of weak affinity. a The HSQC spectra of 50 μM 15N-labeled talin-F0 in the absence (black) and presence of 125 μM GMP-PNP loaded Rap1b (red). b The HSQC spectra (four representative residues were shown) of 50 μM 15N-labeled talin- F0 in the absence (black) and presence of 125 μM GDP loaded Rap1b (blue) or GMP-PNP loaded Rap1b (red). Note that Rap1b-GDP induced the same overall pattern of chemical shift changes of 15N-labeled talin-F0 as Rap1b-GMP-PNP does but with less peak shifts and broadenings, indicating a weaker affinity. c The affinity of Rap1b (GMP-PNP)/talin-F0 interaction measured by HSQC titration. d The HSQC spectra of 50 μM 15N-labeled kindlin2-F0 in the absence (black) and presence of 125 μM GMP-PNP loaded Rap1b (red) interaction, we examined the Rap1b binding to F0 domain of determination of the complex structure (Fig.
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