Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 20, Issue 22, Pages -Publisher
MDPI
DOI: 10.3390/ijms20225647
Keywords
cyclase-associated proteins; oligomerization; coiled coils; actin severing; actin depolymerization; alpha-barrels
Funding
- National Institute of General Medical Sciences of the NIH [R01 GM114666]
- National Institutes of Health [P41 GM128577]
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Actin-depolymerizing factor (ADF)/cofilins accelerate actin turnover by severing aged actin filaments and promoting the dissociation of actin subunits. In the cell, ADF/cofilins are assisted by other proteins, among which cyclase-associated proteins 1 and 2 (CAP1,2) are particularly important. The N-terminal half of CAP has been shown to promote actin filament dynamics by enhancing ADF-/cofilin-mediated actin severing, while the central and C-terminal domains are involved in recharging the depolymerized ADP-G-actin/cofilin complexes with ATP and profilin. We analyzed the ability of the N-terminal fragments of human CAP1 and CAP2 to assist human isoforms of muscle (CFL2) and non-muscle (CFL1) cofilins in accelerating actin dynamics. By conducting bulk actin depolymerization assays and monitoring single-filament severing by total internal reflection fluorescence (TIRF) microscopy, we found that the N-terminal domains of both isoforms enhanced cofilin-mediated severing and depolymerization at similar rates. According to our analytical sedimentation and native mass spectrometry data, the N-terminal recombinant fragments of both human CAP isoforms form tetramers. Replacement of the original oligomerization domain of CAPs with artificial coiled-coil sequences of known oligomerization patterns showed that the activity of the proteins is directly proportional to the stoichiometry of their oligomerization; i.e., tetramers and trimers are more potent than dimers, which are more effective than monomers. Along with higher binding affinities of the higher-order oligomers to actin, this observation suggests that the mechanism of actin severing and depolymerization involves simultaneous or consequent and coordinated binding of more than one N-CAP domain to F-actin/cofilin complexes.
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