Structural and functional differences between Krit1A and Krit1B isoforms: a framework for understanding CCM pathogenesis

Krit1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). This gene encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs, three ankyrin repeats, and a FERM domain. Previously, we isolated Krit1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormal expressed. Combining the homology modelling method of protein-structure prediction with the yeast two-hybrid method of in vivo protein-protein interaction assay and cellular biology analyses, we identified structural and functional differences between Krit1A and Krit1B isoforms. We found that the 15th exon encodes for the distal -sheet of the F3/PTB-like subdomain of Krit1A FERM domain, indicating that the Krit1B isoform is devoid of a functional PTB binding pocket. As a consequence, Krit1B is not able to bind Rap1A, while the FERM domain of Krit1A is even sufficient for this function. Indeed, we showed that Rap1A binding requires both the F1 and F3 lobes of the FERM domain. Importantly, we also demonstrated that Krit1A, but not Krit1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and showed that a functional PTB subdomain enables the nucleocytoplasmic shuttling of Krit1A, while its absence confers a restricted cytoplasmic localization to Krit1B. The new insights into the structural and functional differences between Krit1A and Krit1B isoforms constitute an useful framework for understanding the molecular mechanisms underlying the CCM pathogenesis.