Biology subjects

Mooren, O. L.

Publications and source records attributed to Mooren, O. L..

4 recordsLinked to original sources

bioRxiv2026

Biochemical Functions of the Membrane-Binding Domain of CARMIL

Actin assembly at membranes is often associated with proteins with domains that bind and regulate heterodimeric actin capping protein (CP). CP-binding domains can target CP to the membrane and activate CP by promoting dissociation of its stoichiometric inhibitor V-1. The capping protein binding region (CBR) of CARMIL includes a CPI motif and a CSI motif, followed by a membrane binding (MB) domain. The MB domain is necessary for the function of CARMIL in cells, and it is sufficient for targeting GFP to the plasma membrane of cells. Here, we investigated the mechanism and significance of the relationship of the MB domain to CP activity, including capping of actin filament barbed ends and promotion of Arp2/3-nucleated actin assembly. We found that the MB domain is able to bind to lipid-coated beads, bringing the CPI and CSI motifs to the bead, and thus activating CP to promote Arp2/3-based actin assembly. In addition, we discovered that the MB domain can dissociate from the lipid membrane once CP binds; this observation may help account for the long-standing quandary as to how activated CP is released from the membrane and how CP functions to activate Arp2/3-mediated actin assembly near the membrane. We also report that the MB domain released from the membrane enhances the ability of the CPI and CSI domains to activate CP. Thus, the CARMIL MB domain has multiple biochemical functions regulating actin assembly at a membrane. First, it can target CARMIL, CP, and barbed ends to the plasma membrane. Second, the MB domain can leave the membrane, and this promotes the uncapping of capped barbed ends and activates soluble CP, with greater ability than seen with the membrane-attached state.

biochemistry↗

bioRxiv2024

Reconstitution of Arp2/3-Nucleated Actin Assembly with CP, V-1 and CARMIL

Actin polymerization is often associated with membrane proteins containing capping-protein-interacting (CPI) motifs, such as CARMIL, CD2AP, and WASHCAP/Fam21. CPI motifs bind directly to actin capping protein (CP), and this interaction weakens the binding of CP to barbed ends of actin filaments, lessening the ability of CP to functionally cap those ends. The protein V-1 / myotrophin binds to the F-actin binding site on CP and sterically blocks CP from binding barbed ends. CPI-motif proteins also weaken the binding between V-1 and CP, which decreases the inhibitory effects of V-1, thereby freeing CP to cap barbed ends. Here, we address the question of whether CPI-motif proteins on a surface analogous to a membrane lead to net activation or inhibition of actin assembly nucleated by Arp2/3 complex. Using reconstitution with purified components, we discovered that CARMIL at the surface promotes and enhances actin assembly, countering the inhibitory effects of V-1 and thus activating CP. The reconstitution involves the presence of an Arp2/3 activator on the surface, along with Arp2/3 complex, V-1, CP, profilin and actin monomers in solution, recreating key features of cell physiology.

cell biology↗

bioRxiv2023

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein

Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Forster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. CPI-motif peptide association induced conformational changes within CP that propagate in one direction, while V-1 association induced conformational changes in the opposite direction. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wildtype (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI-motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

biochemistry↗

bioRxiv2020

Comparative analysis of CPI-motif regulation of biochemical functions of actin capping protein

The heterodimeric actin capping protein (CP) is regulated by a set of proteins that contain CP-interacting (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and distinct. The CPI motif and surrounding sequences are conserved within a given protein family, when compared to those of other CPI-motif protein families. Using biochemical assays with purified proteins, we compared the ability of CPI-motif-containing peptides from different protein families to a) bind to CP, b) allosterically inhibit barbed-end capping by CP, and c) allosterically inhibit interaction of CP with V-1, another regulator of CP. We found large differences in potency among the different CPI-motif-containing peptides, and the different functional assays showed different orders of potency. These biochemical differences among the CPI-motif peptides presumably reflect interactions between CP and CPI-motif peptides involving amino-acid residues that are conserved but are not part of the strictly defined consensus, as it was originally identified in comparisons of sequences of CPI motifs(1, 2) across all protein families (1, 2). These biochemical differences may be important for conserved distinct functions of CPI-motif protein families in cells with respect to the regulation of CP activity and actin assembly near membranes.

biochemistry↗