Dual recruitment of two CCM2 molecules to KRIT1 suppresses KLF4 expression

Regulated expression of Kruppel like factor (KLF) transcription factors is essential for normal maintenance of endothelial cells, but loss of either K-Rev interaction trapped 1 (KRIT1) or cerebral cavernous malformations 2 (CCM2) proteins results in significant over-expression of KLF4 protein, causing the cerebrovascular disorder, cerebral cavernous malformations. Here, combining knockdown and reconstitution in an endothelial cell line, with co-immunoprecipitation, biophysical analysis of purified proteins, and co-crystallography, we find that to restrain KLF4 expression, two CCM2 proteins must cluster on a single KRIT1, with the PTB domain of each CCM2 protein binding either the second or third NPxF motif within KRIT1. This clustering of two PTB domains to a single peptide reveals a previously unobserved mechanism for PTB domain recruitment to partner proteins. Overall, our data support a model where clustering of two CCM2 molecules to one KRIT1 is required for normal regulation of expression of KLF4 transcription factor.