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

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)29014-29026
Number of pages13
JournalJournal of Biological Chemistry
Volume285
Issue number37
DOIs
Publication statusPublished - Sep 10 2010

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Proteins
Actins
Protozoa
Mutagenesis
Mammals
Molecular interactions
Site-Directed Mutagenesis
Actin Cytoskeleton
Protein Binding
Assays
Magnetic Resonance Spectroscopy
Monomers
Binding Sites
Nuclear magnetic resonance
Association reactions
Imaging techniques
Molecules

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Molecular basis for barbed end uncapping by CARMIL homology domain 3 of mouse CARMIL-1. / Zwolak, Adam; Uruno, Takehito; Piszczek, Grzegorz; Hammer, John A.; Tjandra, Nico.

In: Journal of Biological Chemistry, Vol. 285, No. 37, 10.09.2010, p. 29014-29026.

Research output: Contribution to journalArticle

Zwolak, Adam ; Uruno, Takehito ; Piszczek, Grzegorz ; Hammer, John A. ; Tjandra, Nico. / Molecular basis for barbed end uncapping by CARMIL homology domain 3 of mouse CARMIL-1. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 37. pp. 29014-29026.
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