Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

Abstract

Dictyostelium Formin C (ForC) is involved in the regulation of local actin cytoskeleton reorganization (e.g. during cellular adhesion or migration). ForC contains formin homology 2 and 3 (FH2 and -3) domains and an N-terminal putative GTPase-binding domain (GBD) but lacks a canonical FH1 region. To better understand the role of the GBD, its structure, dynamics, lipid-binding properties, and cellular functions were analyzed by NMR and CD spectroscopy and by in vivo fluorescence microscopy. Moreover, the program CS-Rosetta was tested for the structure prediction based on chemical shift data only. The ForC GBD adopts an ubiquitinlike ␣/␤-roll fold with an unusually long loop between ␤-strands 1 and 2. Based on the lipid-binding data, the presence of DPC micelles induces the formation of ␣-helical secondary structure and a rearrangement of the tertiary structure. Lipid-binding studies with a mutant protein and a peptide suggest that the ␤1-␤2 loop is not relevant for these conformational changes. Whereas small amounts of negatively charged phosphoinositides (1,2-dioctanoyl-sn-glycero-3-(phosphoinositol 4,5-bisphosphate) and 1,2-dihexanoyl-sn-glycero-3-(phosphoinositol 3,4,5-trisphosphate)) lower the micelle concentration necessary to induce the observed spectral changes, other negatively charged phospholipids (1,2-dihexanoyl-sn-glycero-3-(phospho-L-serine) and 1,2-dihexanoyl-snglycero-3-phospho-(1 -rac-glycerol)) had no such effect. Interestingly, bicelles and micelles composed of diacylphosphocholines had no effect on the GBD structure. Our data suggest a model in which part of the large positively charged surface area of the GBD mediates localization to specific membrane patches, thereby regulating interactions with signaling proteins. Our cellular localization studies show that both the GBD and the FH3 domain are required for ForC targeting to cell-cell contacts and early phagocytic cups and macropinosomes.Formins are widely expressed multidomain proteins that regulate the spatial and temporal organization of the actin and microtubule cytoskeleton, thereby affecting important cellular functions, such as cytokinesis and cell-cell adhesion (1-3). The defining element is the ϳ400-amino acid-encompassing "formin homology 2" (FH2) 2 domain that can tightly associate with the barbed end of elongating actin filaments (4). In nearly all formins, the FH2 domain is preceded by a prolinerich "formin homology 1" (FH1) region that varies in length and the number of binding sites for profilin and that is used to recruit profilin-actin complexes for filament elongation. The influence of the FH2 domain on the rate of filament elongation depends on the length and composition of the FH1 region and the connecting linker (2, 4, 5). Most formins contain additional regulatory domains. The N-terminal "formin homology 3" (FH3) domain mediates interactions with autoinhibitory regulatory elements in the C terminus as well as with other formin regulators. The FH3 domain can be further divided into functional subdomains, such as an armadill...

NMR- and Circular Dichroism-monitored Lipid Binding Studies Suggest a General Role for the FATC Domain as Membrane Anchor of Phosphatidylinositol 3-Kinase-related Kinases (PIKK)

“…In the absence of micelles, the presence of the FATC domain in a mostly unstructured, flexible form may further result in an additional viscous drag. A similar effect has been observed for the N-terminal domain of formin C, which contains a large unstructured loop (56). The 15 N T 1 and T 2 and { 1 H}-15 N NOE data for only the FATC part of the free and micelle-immersed fusion protein are shown in Fig.…”

ADiaphanous-related formin links Ras signaling directly to actin assembly in macropinocytosis and phagocytosis

“…Of note, the tertiary structure of the ForC-GBD was shown to adapt an ubiquitin-like fold (66), which is, when combined with positively charged patches at the interaction surface, a hallmark of RBDs (45). In higher eukaryotes, the closest homologs of ForG are members of the FHOD family (28), which are actin-bundling proteins mainly found in stress fibers (67).…”

Characterization of residue‐dependent differences in the peripheral membrane association of the FATC domain of the kinase ‘target of rapamycin’ by NMR and CD spectroscopy

“…To mimic the restriction of mobility in the full-length protein, the 26-mer peptide could further be circularized by oxidation of terminal cysteines. Whereas the full N-terminal domain and the mutant form lacking the loop showed strong spectral changes in the presence of pure DPC micelles and even more if containing negatively charged lipids, neither the reduced nor the oxidized 26-mer peptide showed any spectral changes in CD or NMR spectra upon addition of 50 mM DPC [49]. In contrast to the y1fatc protein analyzed in this study, the Formin C N-terminal domain showed only very minor spectral changes with bicelles, presumably because its interaction with membrane mimetics may be more sensitive to the packing and accessibility of the hydrophobic acyl chains and not only to the curvature [49].…”

“…Whereas the full N-terminal domain and the mutant form lacking the loop showed strong spectral changes in the presence of pure DPC micelles and even more if containing negatively charged lipids, neither the reduced nor the oxidized 26-mer peptide showed any spectral changes in CD or NMR spectra upon addition of 50 mM DPC [49]. In contrast to the y1fatc protein analyzed in this study, the Formin C N-terminal domain showed only very minor spectral changes with bicelles, presumably because its interaction with membrane mimetics may be more sensitive to the packing and accessibility of the hydrophobic acyl chains and not only to the curvature [49]. Another example for a protein region that shows no interactions with DPC micelles is the N-terminal natively unstructured region of the mycobacterial kinase G (PknG, residues 1-76, Uniprot ID P65728) (unpublished data).…”