Characterization of the SAM domain of the PKD-related protein ANKS6 and its interaction with ANKS3.

Catherine N Leettola,Duilio Cascio,Mary Jane Knight,James U Bowie,Sigrid Hoffman

doi:10.1186/1472-6807-14-17

Abstract

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder leading to end-stage renal failure in humans. In the PKD/Mhm(cy/+) rat model of ADPKD, the point mutation R823W in the sterile alpha motif (SAM) domain of the protein ANKS6 is responsible for disease. SAM domains are known protein-protein interaction domains, capable of binding each other to form polymers and heterodimers. Despite its physiological importance, little is known about the function of ANKS6 and how the R823W point mutation leads to PKD. Recent work has revealed that ANKS6 interacts with a related protein called ANKS3. Both ANKS6 and ANKS3 have a similar domain structure, with ankyrin repeats at the N-terminus and a SAM domain at the C-terminus.ResultsThe SAM domain of ANKS3 is identified as a direct binding partner of the ANKS6 SAM domain. We find that ANKS3-SAM polymerizes and ANKS6-SAM can bind to one end of the polymer. We present crystal structures of both the ANKS3-SAM polymer and the ANKS3-SAM/ANKS6-SAM complex, revealing the molecular details of their association. We also learn how the R823W mutation disrupts ANKS6 function by dramatically destabilizing the SAM domain such that the interaction with ANKS3-SAM is lost.ConclusionsANKS3 is a direct interacting partner of ANKS6. By structurally and biochemically characterizing the interaction between the ANKS3 and ANKS6 SAM domains, our work provides a basis for future investigation of how the interaction between these proteins mediates kidney function.

Highlights

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder leading to end-stage renal failure in humans
The R823W mutation of Pkd/Mhm(cy/+) rats perturbs the structure of Ankyrin repeat and sterile alpha motif (SAM)-domain containing protein 6 (ANKS6)-SAM Using our native gel binding assay, we discovered that the Cy mutation (R54W in our numbering of the construct, R823W in the full-length protein) in ANKS6-SAM destroys the interaction with Ankyrin repeat and SAM-domain containing protein 3 (ANKS3)-SAM (Figure 4B)
We have provided a molecular explanation for the defect in the R823W mutation: it disrupts the tertiary structure of the ANKS6-SAM domain and prevents the ML-surface of ANKS6-SAM from adopting a conformation capable of binding ANKS3-SAM

Summary

Introduction

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder leading to end-stage renal failure in humans. In the PKD/Mhm(cy/+) rat model of ADPKD, the point mutation R823W in the sterile alpha motif (SAM) domain of the protein ANKS6 is responsible for disease. Recent work has revealed that ANKS6 interacts with a related protein called ANKS3 Both ANKS6 and ANKS3 have a similar domain structure, with ankyrin repeats at the N-terminus and a SAM domain at the C-terminus. The Cy mutation was found to be a missense mutation in the ANKS6 gene, encoding ankyrin repeat and SAM-domain containing protein 6 (ANKS6) [11]. Human ANKS6 is an 871 amino acid protein containing 11 ankyrin repeats at its N-terminus and a SAM domain near its C-terminus (Figure 1A). The R823W point mutation acts in a dominant-negative fashion, as evidenced by the PKD phenotype of transgenic rats over-expressing mutated ANKS6(p.R823W) [12]

Methods

Results

Discussion

Conclusion