Two Distantly Spaced Basic Patches in the Flexible Domain of Huntingtin-Interacting Protein 1 (HIP1) Are Essential for the Binding of Clathrin Light Chain

Abstract

The interaction between HIP family proteins (HIP1 and HIP12/1R) and clathrin is fundamental to endocytosis. We used circular dichroism (CD) to study the stability of a HIP1 sub-fragment (aa468-530) that is splayed open. CD thermal melts show HIP1 468-530 is only stable at low temperatures, but this HIP1 fragment contains a structural unit that does not melt out even at 83°C. We then created HIP1 mutants to probe our hypothesis that a short hydrophobic path in the opened region is the binding site for clathrin light chain. We found that the binding of hub/LCb was sensitive to mutating two distantly separated basic residues (K474 and K494). The basic patches marked by K474 and K494 are conserved in HIP12/1R. The lack of conservation in sla2p (S. cerevisiae), HIP1 from D. melanogaster and HIP1 homolog ZK370.3 from C. elegans implies the binding of HIP1 and HIP1 homologs to clathrin light chain may be different in these organisms.

Unconventional Functions for Clathrin, ESCRTs, and Other Endocytic Regulators in the Cytoskeleton, Cell Cycle, Nucleus, and Beyond: Links to Human Disease

“…( Ybe et al 2007a). There is a second CLC binding determinant (K474) upstream from the DLLRKN region (Ybe et al 2009). The carboxy-terminal region of Hip1 (the crystal structure of the Hip1R region is known; PDB 1R0D) (Brett et al 2006) has a THATCH domain that tethers clathrin-coated vesicles to the cellular cytoskeleton.…”