Conserved hydrophobic residues in the CARP/β‐sheet domain of cyclase‐associated protein are involved in actin monomer regulation

Abstract

Cyclase-associated protein (CAP) is a multi-domain protein that promotes actin filament dynamics. The C-terminal region of CAP contains a CAP and X-linked retinitis pigmentosa 2 protein (CARP) domain (or a β-sheet domain), which binds to actin monomer and is essential for enhancing exchange of actin-bound nucleotides. However, how the CARP domain binds to actin is not clearly understood. Here, we report that conserved hydrophobic residues in the CARP domain play important roles in the function of CAP to regulate actin dynamics. Single mutations of three conserved surface-exposed hydrophobic residues in the CARP domain of CAS-2, a Caenorhabditis elegans CAP, significantly reduce its binding to actin monomers and suppress its nucleotide exchange activity on actin. As a result, these mutants are weaker than wild-type to compete with ADF/cofilin to promote recycling of actin monomers for polymerization. A double mutation (V367A/I373A) eliminates these actin-regulatory functions of CAS-2. These hydrophobic residues and previously identified functional residues are scattered on a concave β-sheet of the CARP domain, suggesting that a wide area of the β-sheet is involved in binding to actin. These observations suggest that the CARP domain of CAP binds to actin in a distinct manner from other known actin-binding proteins.

Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase

“…The CAP1 C-terminal 27 aa are involved in binding monomeric G-actin (66,67). Moreover, recent mutagenesis studies demonstrated a more extended interaction surface, including the C-terminal dimerization domain as well as conserved solvent-exposed hydrophobic residues in the solenoid domain of both units in the dimer (66,68).…”

CAPt’n of Actin Dynamics: Recent Advances in the Molecular, Developmental and Physiological Functions of Cyclase-Associated Protein (CAP)

“…CARP binds ADP-G-actin with high affinity ( Kd 0.02–0.05 μM) and in 1:1 stoichiometry ( Mattila et al, 2004 ; Makkonen et al, 2013 ). This interaction is unique in several ways ( Iwase and Ono, 2017 ; Kotila et al, 2018 ). First, two CARP domains form a homodimer and bind simultaneously two ADP-G-actin, whereby each G-actin interacts with both CARP domains.…”

“…The second CAP homolog CAS-2 has been identified in C. elegans just a few years ago ( Nomura and Ono, 2013 ). In vitro studies unraveled a primary function for CAS-2 in nucleotide exchange on G-actin and, hence, in F-actin assembly, which depends on β-sheets located in its CARP domain and on a C-terminal dimerization motif ( Nomura and Ono, 2013 ; Iwase and Ono, 2016 , 2017 ). CAS-2 has been shown to antagonize F-actin depolymerization and G-actin sequestration by the ADF/Cofilin homolog UNC-60A ( Nomura and Ono, 2013 ).…”

Structural basis of actin monomer re-charging by cyclase-associated protein

“…Despite the fundamental requirement of CAPs for actin cytoskeleton organization and function across the eukaryotic kingdom 36 – 41 , the underlying mechanism by which this protein regulates cytoskeletal dynamics in vivo has remained elusive. Moreover, the CARP domain does not display any structural homology to other known actin-binding domains, and despite extensive mutagenesis 20 , 42 , 43 the mechanism by which CAPs associate with actin monomers and catalyze nucleotide exchange has thus remained a mystery. Here, we determined the crystal structure of CAP1/ADP-G-actin complex.…”