Crystal Structure of CCM3, a Cerebral Cavernous Malformation Protein Critical for Vascular Integrity

Abstract

CCM3 mutations are associated with cerebral cavernous malformation (CCM), a disease affecting 0.1-0.5% of the human population. CCM3 (PDCD10, TFAR15) is thought to form a CCM complex with CCM1 and CCM2; however, the molecular basis for these interactions is not known. We have determined the 2.5 Å crystal structure of CCM3. This structure shows an all ␣-helical protein containing two domains, an N-terminal dimerization domain with a fold not previously observed, and a C-terminal focal adhesion targeting (FAT)-homology domain. We show that CCM3 binds CCM2 via this FAThomology domain and that mutation of a highly conserved FAK-like hydrophobic pocket (HP1) abrogates CCM3-CCM2 interaction. This CCM3 FAT-homology domain also interacts with paxillin LD motifs using the same surface, and partial CCM3 co-localization with paxillin in cells is lost on HP1 mutation. Disease-related CCM3 truncations affect the FAT-homology domain suggesting a role for the FAT-homology domain in the etiology of CCM. Cerebral cavernous malformation (CCM)4 is a common vascular lesion that affects the central nervous system vasculature with a prevalence of 0.1-0.5% in the human population (1, 2) (OMIM 116860, 603284, 603285). CCMs manifest as thinwalled, dilated blood vessels lined by a monolayer of endothelial cells that lack tight junctions. The clinical effects of these lesions include seizures, headaches, and stroke in midlife and are often associated with focal hemorrhage (1, 2). These lesions can occur sporadically or as a familial form attributable to mutations in three different genes: CCM1, CCM2, and CCM3.A majority of mutations in CCM genes result in truncations of their protein products, CCM1 (Krev/Rap1 Interacting Trapped 1; KRIT1) (3, 4), CCM2 (malcavernin, MGC4607, osmosensing scaffold for mitogen-activated protein kinase kinase kinase-3; OSM) (5, 6), and CCM3 (programmed cell death 10; PDCD10, TF-1 cell apoptosis-related protein 15; TFAR15) (7,8). These mutations are inherited in an autosomal dominant fashion (9) with acquisition of CCM lesions hypothesized to be due to a two-hit mechanism (10 -12). Expression of the CCM proteins is required for both development and maintenance of endothelial cells in the vasculature; they are required for normal vasculogenesis (13) and global deletion of CCM1 renders mice nonviable (14), a result also seen in global or endothelial-specific deletion of CCM2 (15) and CCM3 (16). Overall, the clinical and in vivo data point to an essential role for the CCM proteins in endothelial cells that makes them critical for vasculogenesis and survival.CCM1, CCM2, and CCM3 interact with one another and play roles in multiple signaling pathways with CCM2 acting as a hub that directly interacts with both CCM3 (17) and CCM1 (18,19). The structural characteristics of the CCM proteins have not been directly assessed but have been inferred by molecular modeling and homology studies that suggest three regions in CCM1 (NPXY-rich, ankyrin repeat, and FERM domains) (20) and a predicted phosphotyrosine binding (PTB) dom...

Structure-Function Analysis of Core STRIPAK Proteins

“…Finally, although our data point to the STRIPAK complex as the major interactor for epitope-tagged or endogenous CCM3 protein in HEK293 cells (4), HeLa cells, C2C12 myoblasts, and myotubes and in bovine endothelial aortic cells (data not shown) CCM3 is also capable of interacting with CCM2 (48) and paxillin (30). Because these interactions are apparently mediated via the same surface as the striatin binding site on CCM3, we propose here that they may be mutually exclusive.…”

“…On this basis, an interaction of CCM3 with paxillin was validated previously (30) and shown to require four lysine residues that establish interactions with paxillin (Fig. 3B); the same residues were also implicated in mediating the interaction between CCM3 and CCM2, as CCM2 shares a stretch of homology to paxillin (30). The striatin peptide responsible for association with CCM3 exhibits an amino acid composition similar to the paxillin and CCM2-derived peptide (Fig.…”

“…This region of CCM3 forms a globular domain consisting of four ␣-helices exhibiting structural resemblance to the FAT domain, which mediates the interaction between focal adhesion kinase and paxillin. On this basis, an interaction of CCM3 with paxillin was validated previously (30) and shown to require four lysine residues that establish interactions with paxillin (Fig. 3B); the same residues were also implicated in mediating the interaction between CCM3 and CCM2, as CCM2 shares a stretch of homology to paxillin (30).…”

“…B, structural modeling of the CCM3 FAT domain with a peptide derived from paxillin. The CCM3 crystal structure (30) revealed that the region that we have mapped as interacting with STRN3 also folds as a focal adhesion targeting domain (yellow) similar to focal adhesion kinase (FAK, cyan). Interaction with peptides derived from paxillin (shown in red) are mediated via four lysine residues (highlighted).…”

Germinal center kinases in immune regulation

“…54,77,78 The N-terminal domain of PDCD10 mediates homodimerization while the C-terminal domain structurally resembles the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). 79 Constitutive expression of PDCD10 in peripheral blood T-cells has been correlated with cutaneous T-cell lymphoma. 80 The homodimers of PDCD10 and GCK-III may undergo dissociation and form a PDCD10-GCK-III heterodimer, which stabilizes and activates GCK-III.…”