Molecular basis for barbed end uncapping by CARMIL homology domain 3 of mouse CARMIL-1

Adam Zwolak, Takehito Uruno, Grzegorz Piszczek, John A. Hammer, Nico Tjandra

研究成果: ジャーナルへの寄稿学術誌査読

22 被引用数 (Scopus)

抄録

Capping protein (CP) is a ubiquitously expressed, 62-kDa heterodimer that binds the barbed end of the actin filament with ∼0.1 nM affinity to prevent further monomer addition. CARMIL is a multidomain protein, present from protozoa to mammals, that binds CP and is important for normal actin dynamics in vivo. The CARMIL CP binding site resides in its CAH3 domain (CARMIL homology domain 3) located at or near the protein'sC terminus. CAH3 binds CP with ∼1 nM affinity, resulting in a complex with weak capping activity (30-200 nM). Solution assays and single-molecule imaging show that CAH3 binds CP already present on the barbed end, causing a 300-fold increase in the dissociation rate of CP from the end (i.e. uncapping). Here we used nuclear magnetic resonance (NMR) to define the molecular interaction between the minimal CAH3 domain (CAH3a/b) of mouse CARMIL-1 and CP. Specifically, we show that the highly basic CAH3a subdomain is required for the high affinity interaction of CAH3 with a complementary "acidic groove" on CP opposite its actin-binding surface. This CAH3a-CP interaction orients the CAH3b subdomain, which we show is also required for potent anti-CP activity, directly adjacent to the basic patch of CP, shown previously to be required for CP association to and high affinity interaction with the barbed end. The importance of specific residue interactions between CP and CAH3a/b was confirmed by site-directed mutagenesis of both proteins. Together, these results offer a mechanistic explanation for the barbed end uncapping activity of CARMIL, and they identify the basic patch on CP as a crucial regulatory site.

本文言語英語
ページ(範囲)29014-29026
ページ数13
ジャーナルJournal of Biological Chemistry
285
37
DOI
出版ステータス出版済み - 9月 10 2010
外部発表はい

!!!All Science Journal Classification (ASJC) codes

  • 生化学
  • 分子生物学
  • 細胞生物学

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