(A) CARMIL domain organization: PH, pleckstrin-homology domain; L, linker; N-cap (N), LRR, leucine-rich repeat domain; C, C-cap; HD, helical dimerization domain; CBR, Capping Protein binding domain, consisting of CPI, Capping Protein interaction domain, and CSI, CARMIL-specific interaction sequence; MBD, membrane binding domain; PRD, proline-rich domain. Alignment between the Capping Protein Interaction (CPI) motif consensus sequence, and the CPI regions of H. sapiens (H.s.) CARMIL1 (UniProtKB Q5VZK9.1), CARMIL2 (UniProtKB Q6F5E8.2), CARMIL3 (UniProtKB Q8ND23.2), CKIP1 UniProtKB Q53GL0.2), CD2AP (CBI NP_036252.1), WASHCAP (Fam21) (UniProtKB Q9Y4E1.3), CapZIP (CBI NP_443094.3), CIN85 (UniProtKB Q96B97.2), and the tail sequences of Twinfilin homologs from D. melanogaster (D.m), C. lectularius (C.l.), S. cerevisiae (S.c.), O. Taurus (O.t.), S. litura (S.l.), D. rerio (D.r.), H. sapiens (H.s.), and M. musculus (M.m.). Twinfilin isoforms (D.m. Twf1 UniProtKB NP_650338, C.l. Twf1 UniProtKB XP_014258437.1, S.c. Twf1 GenBank GAX68393.1, O.t. Twf1 XP_022917989.1, S.l. Twf1 XP_022816377.1, D.r. Twf1 AAH67638.1, H.s. Twf1 UniProtKB NO_001229326.1, and M.m. Twf1 GenBank AAH15081.1). Amino acid color coding illustrates side chain chemistry similarities. The asterisk marks the residue we mutated in mTwf1 in panel. (C) The alignments were generated using the MAFFT algorithm in the DNASTAR Lasergene Suite/MegAlign Pro application (MegAlign Pro. Version 15.0. DNASTAR. Madison, WI.).(B) Fluorescence anisotropy measurement of 60 nM HiLyte488-labeled mTwf1 tail peptide mixed with increasing concentrations of the indicated CP construct. (C) Fluorescence anisotropy measurement of 40 nM TAMRA-labeled mTwf1 tail peptide incubated with 1 µM CP and different concentrations of wild type and mutant mTwf1 tail peptides. (D) Fluorescence anisotropy measurement of 60 nM HiLyte488-labeled mTwf1 tail peptide incubated in the presence of 240 nM CP and increasing concentrations of the indicated CARMIL fragment (CBR, CSI, or CPI). CSI failed to compete with HiLyte 488-mTwf1 tail peptide at the concentrations tested. Anisotropy values for each condition were averaged from three independent experiments.