Structure and function of the N-terminal extension of the formin INF2

Abstract

In INF2—a formin linked to inherited renal and neurological disease in humans—the DID is preceded by a short N-terminal extension of unknown structure and function. INF2 activation is achieved by Ca2+-dependent association of calmodulin (CaM). Here, we show that the N-terminal extension of INF2 is organized into two α-helices, the first of which is necessary to maintain the perinuclear F-actin ring and normal cytosolic F-actin content. Biochemical assays indicated that this helix interacts directly with CaM and contains the sole CaM-binding site (CaMBS) detected in INF2. The residues W11, L14 and L18 of INF2, arranged as a 1-4-8 motif, were identified as the most important residues for the binding, W11 being the most critical of the three. This motif is conserved in vertebrate INF2 and in the human population. NMR and biochemical analyses revealed that CaM interacts directly through its C-terminal lobe with the INF2 CaMBS. Unlike control cells, INF2 KO cells lacked the perinuclear F-actin ring, had little cytosolic F-actin content, did not respond to increased Ca2+ concentrations by making more F-actin, and maintained the transcriptional cofactor MRTF predominantly in the cytoplasm. Whereas expression of intact INF2 restored all these defects, INF2 with inactivated CaMBS did not. Our study reveals the structure of the N-terminal extension, its interaction with Ca2+/CaM, and its function in INF2 activation.

INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death

“…As previously reported [12], compared with control cells, INF2 KO cells had a scarce cytosolic F-actin content and lacked the ring of F-actin that surrounds the nucleus (Fig. S2G).…”

“…Similar to CaM, centrins bound to the 2-340 INF2 fragment and to the sequence encompassing residues 2-21 that house the CaM-binding site, but they did not bind to the 19-340 fragment or the 2-340 INF2 fragment with the triple W11L14L18A mutation (Fig. 6B), which disrupts CaM binding [12]. Mutation of the W11 residue to Ala or the double mutation of residues L14 and L18 to Ala abolished centrin binding (Fig.…”

“…Ca 2+ /CaM binds to INF2 and initiates its actin polymerization activity [13]. The EF-hands in the C-terminal lobe of CaM specifically interacts with a unique CaM-binding site in the first of the two α-helices situated in the short N-terminal extension adjacent to the DID [12]. Human centrins 1-3 constitute a family of Ca 2+ sensors that, similar to CaM, encompass N-terminal and the C-terminal EF-hands [45].…”

“…Human centrins 1-3 constitute a family of Ca 2+ sensors that, similar to CaM, encompass N-terminal and the C-terminal EF-hands [45]. Notably, centrin-2 binds targets with helical sequences featuring a WxxLxxxL (where x is a spacer amino acid) motif, which coincides with the INF2 Ca 2+ /CaM-binding motif [12,46]. To determine whether centrins bind to INF2, we performed pull-down assays utilizing GST fusions of amino acids 2-340 INF2 and extracts of HEK293T cells expressing GFP fused to individual centrins.…”

“…Specific Rho GTPase binding in the GTP-loaded active state disrupts the inhibitory DID-DAD interaction in mDia1-3 and other formins, liberating the FH2 domain [9]. However, INF2 activation involves calmodulin (CaM) binding to a helical peptide motif in the N-terminal extension preceding the DID [12,13]. In addition to cytoplasmic F-actin formation, INF2 is implicated in nuclear F-actin assembly [14], microtubule binding and stabilization [15,16], vesicular transport [17,18], mitochondrial fission [19,20], and cell extrusion [21].…”