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A Sterile Alpha Motif Domain Network Involved in Kidney Development

  • Author(s): Leettola, Catherine
  • Advisor(s): Bowie, James U
  • et al.
Abstract

Cystic kidney diseases including polycystic kidney disease (PKD) and nephronophthisis (NPHP) are the most common genetic disorders leading to end-stage renal failure in humans. Animal models and human cases of PKD and NPHP have implicated the sterile alpha motif (SAM) domain containing proteins bicaudal C homolog 1 (BICC1) and ankyrin repeat and SAM-domain containing protein 6 (ANKS6) as being involved in these conditions and important for renal development. SAM domains are known protein-protein interaction domains that are capable of binding each other to form polymers and heterodimers. Using a negGFP native gel assay, we have identified the SAM domain of the previously uncharacterized protein ankyrin repeat and SAM-domain containing protein 3 (ANKS3) as a direct binding partner of the BICC1 and ANKS6 SAM domains. We found the ANKS3 SAM domain to polymerize with moderate affinity and determined the ANKS6 SAM domain can bind to a single end of this polymer. Crystal structures of the ANKS3 SAM domain polymer and the ANKS3 SAM-ANKS6 SAM heterodimer are presented to reveal typical ML-EH SAM domain interaction interfaces with a pronounced charge complementarity. A crystal structure of the BICC1 SAM domain, which forms a helical polymer with moderate affinity, is also presented. The SAM domains of BICC1 and ANKS3 exhibit high sequence and structural homology, allowing these SAM domains to bind each other using both of their conserved ML and EH interaction surfaces. The measured binding affinities of the two possible interfaces between the BICC1 and ANKS3 SAM domains are nearly identical, suggesting these SAM domains may associate to form the first observed alternating SAM domain co-polymer. The BICC1 and ANKS6 SAM domains are also shown to bind each other as a heterodimer with strong affinity. The R823W and I817N point mutations in the SAM domain of ANKS6 are responsible for cystic kidney disease in the PKD/Mhm(cy/+) rat and a newly identified mouse model, respectively. We show that these point mutations dramatically destabilize the ANKS6 SAM domain and are responsible for a loss of interaction with the SAM domains of ANKS3 and BICC1, suggesting these lost interactions are part of a possible disease mechanism. The network of interactions between the SAM domains of BICC1, ANKS3, and ANKS6 may allow regulation of polymer formation and the creation of diverse cellular scaffolds that are suggested to be important for renal development. This work provides a structural and biochemical foundation for the continued investigation of how the interactions between the BICC1, ANKS3, and ANKS6 SAM domains mediate cellular development.

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