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). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs 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 abnormally expressed. Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms. We found that the 15th exon encodes for the distal beta-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. 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 proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner. As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.
Francalanci
Floriana
F
Molecular Biotechnology Centre, Department of Genetics, Biology and Biochemistry, Via Nizza 52, 10126 Torino, Italy.
Avolio
Maria
M
De Luca
Elisa
E
Longo
Dario
D
Menchise
Valeria
V
Guazzi
Paolo
P
Sgrò
Francesco
F
Marino
Marco
M
Goitre
Luca
L
Balzac
Fiorella
F
Trabalzini
Lorenza
L
Retta
Saverio Francesco
SF
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D020785:Central Nervous System Vascular Malformations / Q000235:genetics / Q000503:physiopathology*; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D006367:HeLa Cells; D020786:Hemangioma, Cavernous, Central Nervous System / Q000235:genetics / Q000503:physiopathology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D000075927:KRIT1 Protein; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020827:rap1 GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C512603:ICAP-1 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D000075927:KRIT1 Protein; C491535:Krit1 protein, mouse; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C480498:osmosensing scaffold for MEKK3 protein, mouse; D020827:rap1 GTP-Binding Proteins