title: Structural model and functional significance of pH-dependent talin-actin binding for focal adhesion remodeling.
abstract: Actin filament binding by the focal adhesion (FA)-associated protein talin stabilizes cell-substrate adhesions and is thought to be rate-limiting in cell migration. Although F-actin binding by talin is known to be pH-sensitive in vitro, with lower affinity at higher pH, the functional significance of this pH dependence is unknown. Because increased intracellular pH (pH(i)) promotes cell migration and is a hallmark of metastatic carcinomas, we asked whether it increases FA remodeling through lower-affinity talin-actin binding. Talin contains several actin binding sites, but we found that only the COOH-terminal USH-I/LWEQ module showed pH-dependent actin binding, with lower affinity and decreased maximal binding at higher pH. Molecular dynamics simulations and NMR of this module revealed a structural mechanism for pH-dependent actin binding. A cluster of titratable amino acids with upshifted pK(a) values, including His-2418, was identified at one end of the five-helix bundle distal from the actin binding site. Protonation of His-2418 induces changes in the conformation and dynamics of the remote actin binding site. Structural analyses of a mutant talin-H2418F at pH 6.0 and 8.0 suggested changes different from the WT protein, and we confirmed that actin binding by talin-H2418F was relatively pH-insensitive. In motile fibroblasts, increasing pH(i) decreased FA lifetime and increased the migratory rate. However, expression of talin-H2418F increased lifetime 2-fold and decreased the migratory rate. These data identify a molecular mechanism for pH-sensitive actin binding by talin and suggest that FA turnover is pH-dependent and in part mediated by pH-dependent affinity of talin for binding actin.
authors.affiliation: Department of Cell and Tissue Biology, University of California, San Francisco, CA 94143, USA.
authors.forename: J
authors.initials: J
authors.lastname: Srivastava
authors.forename: G
authors.initials: G
authors.lastname: Barreiro
authors.forename: S
authors.initials: S
authors.lastname: Groscurth
authors.forename: A R
authors.initials: AR
authors.lastname: Gingras
authors.forename: B T
authors.initials: BT
authors.lastname: Goult
authors.forename: D R
authors.initials: DR
authors.lastname: Critchley
authors.forename: M J S
authors.initials: MJ
authors.lastname: Kelly
authors.forename: M P
authors.initials: MP
authors.lastname: Jacobson
authors.forename: D L
authors.initials: DL
authors.lastname: Barber
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D003198:Computer Simulation; D022001:Focal Adhesions / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016608:Talin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D016608:Talin; C496077:Tln1 protein, mouse
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