Structural Basis for the Disruption of the Cerebral Cavernous Malformations 2 (CCM2) Interaction with Krev Interaction Trapped 1 (KRIT1) by Disease-associated Mutations

Abstract

Background: Mutations in Krev interaction trapped 1 (KRIT1) and cerebral cavernous malformations 2 (CCM2) are associated with CCM disease. Results: The CCM2-KRIT1 interaction is characterized structurally and biochemically. Conclusion: CCM2 preferentially binds the third NPX(Y/F) motif of KRIT1, and disease-associated mutations destabilize this interaction. Significance: These data may inform future studies into the biology of CCM disease.

CCM2–CCM3 interaction stabilizes their protein expression and permits endothelial network formation

“…To understand the functional significance of the CCM2–CCM3 interaction, we first sought a detailed characterization of the CCM2–CCM3 interface. CCM2 contains an N-terminal PTB domain ( Liquori et al, 2003 ; Fisher et al, 2015 ), followed by a linker region and an HHD ( Fisher et al, 2013 ; Fig. 1 A ), but the portion of CCM2 that binds to CCM3 has not previously been mapped.…”

“…CCM3 also binds an LD-like motif within the striatin family of proteins ( Kean et al, 2011 ), but how CCM2 and CCM3 interact has not been well described. Sequence alignments of CCM2 with both paxillin and striatin family LD and LD-like motifs revealed a putative CCM2 LD–like motif ( Kean et al, 2011 ), located between the N-terminal phosphotyrosine-binding (PTB) domain ( Liquori et al, 2003 ; Fisher et al, 2015 ) and the C-terminal harmonin homology domain (HHD; Fisher et al, 2013 ), but this has not been investigated further.…”

Novel CCM2 missense variants abrogating the CCM1–CCM2 interaction cause cerebral cavernous malformations

“…The CCM2 p.L198R missense variant located in the PTB domain and reported in a CCM family has been shown to be deleterious by aborting the interaction between CCM1 and CCM2 11 12. Additional missense point variants have been shown to destabilise CCM2 PTB domain and weaken its interaction with CCM1 13. In a molecular diagnostic context, the interpretation of missense CCM variants is challenging since frequency in databases and in silico predictive tools are insufficient to consider them as pathogenic or not.…”

Structural analysis of the KRIT1 ankyrin repeat and FERM domains reveals a conformationally stable ARD–FERM interface

“…These protein products are generally considered scaffolding proteins and can directly interact with one another (Draheim et al, 2015; Fisher and Boggon, 2014; Fisher et al, 2015a). Structural studies have been conducted for CCM2 and CCM3 that discovered novel domains in both proteins (Fisher et al, 2013; Li et al, 2010), and the structural basis for CCM2 and CCM3 interactions with binding partners (Draheim et al, 2015; Fisher et al, 2015b; Li et al, 2011; Xu et al, 2013; Zhang et al, 2013).…”

“…Structural studies have been conducted for CCM2 and CCM3 that discovered novel domains in both proteins (Fisher et al, 2013; Li et al, 2010), and the structural basis for CCM2 and CCM3 interactions with binding partners (Draheim et al, 2015; Fisher et al, 2015b; Li et al, 2011; Xu et al, 2013; Zhang et al, 2013). Similarly, structural studies have been conducted for KRIT1, and have facilitated the discovery of a Nudix fold domain at the KRIT1 N-terminus (Liu et al, 2013), a novel PTB-binding site which interacts with the protein ICAP1 (integrin cytoplasmic associated protein 1) (Liu and Boggon, 2013; Liu et al, 2013), the mode of interactions with CCM2 (Fisher et al, 2015a) and (SNX17) sorting nexin 17 (Stiegler et al, 2014), and the distinct and unusual manner by which the C-terminal FERM (band 4-point-1, ezrin, radixin, moesin) domain binds to the small GTPase Rap1 (Ras related protein Rap1) and the cell adhesion molecule HEG1 (Heart of Glass 1) (Gingras et al, 2012; Gingras et al, 2013; Li et al, 2012). These structural studies have therefore provided insights into the molecular basis for the CCM complex proteins and their interactions (Fisher and Boggon, 2014), however gaps still remain in our structural understanding of KRIT1.…”