Structural characterization of a capping protein interaction motif defines a family of actin filament regulators

Abstract

Capping protein (CP) regulates actin dynamics by binding the barbed ends of actin filaments. Removal of CP may be one means to harness actin polymerization for processes such as cell movement and endocytosis. Here we structurally and biochemically investigated a CP interaction (CPI) motif present in the otherwise unrelated proteins CARMIL and CD2AP. The CPI motif wraps around the stalk of the mushroom-shaped CP at a site distant from the actin-binding interface, which lies on the top of the mushroom cap. We propose that the CPI motif may act as an allosteric modulator, restricting CP to a low-affinity, filament-binding conformation. Structure-based sequence alignments extend the CPI motif–containing family to include CIN85, CKIP-1, CapZIP and a relatively uncharacterized protein, WASHCAP (FAM21). Peptides comprising these CPI motifs are able to inhibit CP and to uncap CP-bound actin filaments.

Molecular Basis for Barbed End Uncapping by CARMIL Homology Domain 3 of Mouse CARMIL-1

“…During preparation of this manuscript, the crystal structure of the CAH3a/b subdomain bound to CP was published (54). Overall, the NMR structure of the CP⅐CAH3a/b complex determined here is consistent with the crystal structure.…”

“…In addition to agreeing generally with the crystal structure, we determined the binding sites on CP for the N- in CAH3a/b affects the loop between strands 6 and 7 of CP␤, which lends support to the idea that this region is required for the local conformational change at the basic patch occurring upon CAH3a/b binding. Interestingly, no dramatic conformational differences were found between free CP (11) and CP bound to the CPI motif (Ser 972 -Cys 1008 ) or the CBR115 fragment (Glu 968 -Ser 1082 ) of CARMIL (54). One possible explanation for this result is that residues Thr 1009 -Val 1026 , which were not observed in the crystal, are involved in driving the conformational change in CP and that this region does not form a stable (i.e.…”

“…One possible explanation for this result is that residues Thr 1009 -Val 1026 , which were not observed in the crystal, are involved in driving the conformational change in CP and that this region does not form a stable (i.e. crystallizable) structure, accounting for the lack of electron density for this region in the crystal (54). Based on the crystal structure, two likely mechanisms for the anti-CP activities of CAH3a/b were proposed: 1) part of the CAH3a subdomain sterically prevents CP from binding the barbed end (the direct competition model), and 2) CAH3a/b binding causes an allosteric change in CP that decreases its affinity for the barbed end (the allosteric model) (54).…”

“…crystallizable) structure, accounting for the lack of electron density for this region in the crystal (54). Based on the crystal structure, two likely mechanisms for the anti-CP activities of CAH3a/b were proposed: 1) part of the CAH3a subdomain sterically prevents CP from binding the barbed end (the direct competition model), and 2) CAH3a/b binding causes an allosteric change in CP that decreases its affinity for the barbed end (the allosteric model) (54). Our data argue that the mechanism of uncapping by CAH3a/b is driven largely by direct interference of CP-barbed end interaction by a portion of the CAH3b subdomain (Gln 1015 -Arg 1025 ), which we name the interference domain.…”

Structure of the Kidney Slit Diaphragm Adapter Protein CD2-Associated Protein as Determined with Electron Microscopy

“…29,30 CD2AP is also directly involved in control of the actin cytoskeleton. The C-terminal segment of CD2AP contains an approximately 20 residue sequence (termed the CARMIL peptide 31,32 ) that binds the actin capping protein (CP). 29,33 CP binds to the barbed ends of actin filaments and thus, caps them, inhibiting the addition and loss of actin monomers at that end.…”