Molecular Recognition of Leucine-Aspartate Repeat (LD) Motifs by the Focal Adhesion Targeting Homology Domain of Cerebral Cavernous Malformation 3 (CCM3)

Abstract

Cerebral cavernous malformation (CCM) is a disease that affects between 0.1 and 0.5% of the human population, with mutations in CCM3 accounting for ϳ15% of the autosomal dominant form of the disease. We recently reported that CCM3 contains an N-terminal dimerization domain (CCM3D) and a C-terminal focal adhesion targeting (FAT) homology domain. Intermolecular protein-protein interactions of CCM3 are mediated by a highly conserved surface on the FAT homology domain and are affected by CCM3 truncations in the human disease. Here we report the crystal structures of CCM3 in complex with three different leucine-aspartate repeat (LD) motifs (LD1, LD2, and LD4) from the scaffolding protein paxillin, at 2.8, 2.7, and 2.5 Å resolution. We show that CCM3 binds LD motifs using the highly conserved hydrophobic patch 1 (HP1) and that this binding is similar to the binding of focal adhesion kinase and Pyk2 FAT domains to paxillin LD motifs. We further show by surface plasmon resonance that CCM3 binds paxillin LD motifs with affinities in the micromolar range, similar to FAK family FAT domains. Finally, we show that endogenous CCM3 and paxillin co-localize in mouse cerebral pericytes. These studies provide a molecular-level framework to investigate the protein-protein interactions of CCM3.Mutations in three genes, CCM1, CCM2, and CCM3, are implicated in the inherited autosomal dominant form of cerebral cavernous malformation (CCM) 2 (1, 2). CCM is a dysplasia that affects the central nervous system, resulting in thin-walled, dilated blood vessels. Complications resulting from CCM include hemorrhagic stroke, seizure, epilepsy, and other focal neurological outcomes (3)(4)(5). Approximately 15% of inherited CCM cases are associated with truncations or mutations in CCM3, the protein product of which is cerebral cavernous malformation 3 (CCM3, also called PDCD10 (programmed cell death 10), or TFAR15 (TF-1 cell apoptosis-related protein 15) (6, 7). The function of this protein is not fully described, and its protein binding partners are still being determined; however, both global and endothelial-specific deletions of CCM3 are incompatible with life (8). This protein seems to have a scaffolding function and has been shown to directly interact with CCM2 (9 -11), germinal center kinase III family members STK24, STK25, and MST4 (10, 12-17), the STRIPAK complex (18), and paxillin (11) and can associate the cytoplasmic tail of VEGFR2 (8). The atomic-level mechanisms of CCM3 interactions with its binding partners have not been described.Paxillin is a scaffolding protein important for localization of many proteins to the intracellular side of cell adhesions (19 -21). Importantly, the localization of protein kinases, phosphatases, and GTPase regulating proteins (guanine nucleotide exchange factors, GTPase-activating proteins, and effectors) to the actin cytoskeleton and the spatial-temporal dynamics of cellular adhesion are impacted by paxillin (22-24). Examples of proteins that paxillin recruits include FAK, Pyk2, PTP-PEST, CrkII, DOCK180,...

Structural Basis for Paxillin Binding and Focal Adhesion Targeting of β-Parvin

“…Furthermore, the affinities of LD motifs for other paxillin binding proteins, e.g. focal adhesion kinase (1-9 M) (26, 44 -46), CCM3 (17-39 M) (26), and Pyk2 (45 M) (44) are similar to ␤-parvin CH2, indicating that ␤-parvin is a paxillin binding protein.…”

“…CCM3 (25,26), focal adhesion kinase, vinculin, and GIT1 (21). Recombinant ␤-parvin protein was expressed and purified, and binding to paxillin GST-LD fusion proteins assessed.…”

“…1A) (20), which act as protein-protein interaction modules. Specific LD motifs are responsible for binding focal adhesion kinase (21-23), proline-rich tyrosine kinase (Pyk2) (24), vinculin (21), GIT1/GIT2 (21), and CCM3 (cerebral cavernous malformation 3) (25,26).…”

How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions

“…2C) (Brown et al, 1996). FAT binds two helical LD motifs, one between helices H1 and H4 (site 1/4), the other one between helices H2 and H3 (site 2/3) (Bertolucci et al, 2005;Gao et al, 2004;Hoellerer et al, 2003), whereas FAT-homology domains (FAHs) of other FAlocalising proteins (CCM3, GIT1/2 and vinculin) bind only one LD motif (Alam et al, 2014;Brown et al, 1996;Li et al, 2011;Schmalzigaug et al, 2007;Turner et al, 1990;Zhang et al, 2008). FAT binds also two CD4 endocytosis motifs, in a structurally similar way to LD motifs, allowing CD4 to recruit FAK for T cell receptor signalling (Garron et al, 2008).…”

Pericytes of the neurovascular unit: key functions and signaling pathways

“…Disrupted TGFβ signaling at the BBB is reported in studies using iCcm1 ΔEC/EC mice, human brain tissue of CCM subjects, and cultured human brain vascular cells 99,103 and contributes to CCM by inducing structural instability of capillaries. Human CCM3 also binds to paxillin, a scaffolding protein, and CCM3 and paxillin co-localize in mouse cerebral pericytes 105 .…”