title: Molecular basis for barbed end uncapping by CARMIL homology domain 3 of mouse CARMIL-1.
abstract: Capping protein (CP) is a ubiquitously expressed, 62-kDa heterodimer that binds the barbed end of the actin filament with approximately 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's C terminus. CAH3 binds CP with approximately 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.
authors.affiliation: Laboratory of Molecular Biophysics, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Zwolak
authors.forename: Takehito
authors.initials: T
authors.lastname: Uruno
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Piszczek
authors.forename: John A
authors.initials: JA
authors.lastname: Hammer
authors.forename: Nico
authors.initials: N
authors.lastname: Tjandra
mesh_terms: D051344:Actin Capping Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013499:Surface Properties
chemical_list: D051344:Actin Capping Proteins; C504800:Carmil1 protein, mouse; D002352:Carrier Proteins; D008840:Microfilament Proteins