Abstract
Capping protein (CP) controls the polymerization of actin filaments by capping their barbed ends. In lamellipodia, CP dissociates from the actin cytoskeleton rapidly, suggesting the possible existence of an uncapping factor, for which the protein CARMIL (capping protein, Arp2/3 and myosin-I linker) is a candidate. CARMIL binds to CP via two motifs. One, the CP interaction (CPI) motif, is found in a number of unrelated proteins; the other motif is unique to CARMILs, the CARMIL-specific interaction motif. A 115-aa CARMIL fragment of CARMIL with both motifs, termed the CP-binding region (CBR), binds to CP with high affinity, inhibits capping, and causes uncapping. We wanted to understand the structural basis for this function. We used a collection of mutants affecting the actin-binding surface of CP to test the possibility of a steric-blocking model, which remained open because a region of CBR was not resolved in the CBR/CP co-crystal structure. The CP actin-binding mutants bound CBR normally. In addition, a CBR mutant with all residues of the unresolved region changed showed nearly normal binding to CP. Having ruled out a steric blocking model, we tested an allosteric model with molecular dynamics. We found that CBR binding induces changes in the conformation of the actin-binding surface of CP. In addition, ∼30-aa truncations on the actin-binding surface of CP decreased the affinity of CBR for CP. Thus, CARMIL promotes uncapping by binding to a freely accessible site on CP bound to a filament barbed end and inducing a change in the conformation of the actin-binding surface of CP.
Highlights
CARMIL inhibits the actin capping action of heterodimeric capping protein
We used a collection of mutants affecting the actin-binding surface of Capping protein (CP) to test the possibility of a steric-blocking model, which remained open because a region of CP-binding region (CBR) was not resolved in the CBR/CP co-crystal structure
The F-actin cap conformation has a more curved conformation, whereas the CBR cap is slightly flatter. Based on these structures and the principal component analysis (PCA) analysis, we suggest that CARMIL CBR keeps CP in a conformation close to the unbound form of CP, inhibiting F-actin binding by inducing the cap to have an unfavorable actin-binding configuration
Summary
CARMIL inhibits the actin capping action of heterodimeric capping protein. Results: Results argue against a steric blocking model and provide evidence for an allosteric mechanism. Conclusion: The conformations of the actin- and CARMIL-binding sites on capping protein are linked. CARMIL promotes uncapping by binding to a freely accessible site on CP bound to a filament barbed end and inducing a change in the conformation of the actin-binding surface of CP. CP binds to the actin filament network very near the membrane, and it dissociates from the network after a short time (14, 15) This rate of dissociation is much higher than what would be expected from in vitro rate constants, suggesting that another factor plays a role, perhaps by removing CP from barbed ends or by severing filaments. All-atom molecular dynamics simulations provide evidence for an allosteric model in which the conformation of the binding sites on CP for CARMIL and actin are linked
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