Abstract
Kidney ankyrin repeat-containing proteins (KANK1/2/3/4) belong to a family of scaffold proteins, playing critical roles in cytoskeleton organization, cell polarity, and migration. Mutations in KANK proteins are implicated in cancers and genetic diseases, such as nephrotic syndrome. KANK proteins can bind various target proteins through different protein regions, including a highly conserved ankyrin repeat domain (ANKRD). However, the molecular basis for target recognition by the ANKRD remains elusive. In this study, we solved a high-resolution crystal structure of the ANKRD of KANK1 in complex with a short sequence of the motor protein kinesin family member 21A (KIF21A), revealing that the highly specific target-binding mode of the ANKRD involves combinatorial use of two interfaces. Mutations in either interface disrupted the KANK1-KIF21A interaction. Cellular immunofluorescence localization analysis indicated that binding-deficient mutations block recruitment of KIF21A to focal adhesions by KANK1. In conclusion, our structural study provides mechanistic explanations for the ANKRD-mediated recognition of KIF21A and for many disease-related mutations identified in human KANK proteins.
Highlights
Kidney ankyrin repeat– containing proteins (KANK1/2/3/4) belong to a family of scaffold proteins, playing critical roles in cytoskeleton organization, cell polarity, and migration
As the main interacting regions of KANK1 and kinesin family member 21A (KIF21A) were reported to be the C-terminal ankyrin repeat domain (ANKRD) (KANK1_ANKRD) and a short helical region (KIF21A_H), respectively (Fig. 1A) [21], these two fragments were expressed in Escherichia coli and purified
Based on isothermal titration calorimetry (ITC) analysis (Figs. 1C and S1), we determined that the 28-residue region contains the necessary and sufficient elements for the binding of KIF21A_H to KANK1_ANKRD
Summary
To obtain insights into the KANK1-mediated target recognition mechanism, we first characterized the binding of KIF21A to KANK1. As extensive trials failed to yield any crystals of the complex, we removed flexible regions on both the N-terminal and C-terminal ends of KANK1_ANKRD without affecting its binding to KIF21A_H (Fig. 1C). By using this KANK1_ANKRD construct (residues 1088 –1338), we successfully obtained high-quality crystals for structural study. The ANKRD contains five ANK repeats (R1–5), capped by an N-terminal helix bundle (␣1–␣5; named R0 hereafter) to form an ␣-solenoid fold The KIF21A_H peptide folds as an extended loop in the N terminus and a short ␣-helix in the C terminus to interact tightly with the concave side of the ANKRD (Fig. 3A).
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