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Two basic patches in the flexible region of Huntingtin‐interacting protein 1 (HIP1) drive the binding of clathrin
Author(s) -
Ybe Joel Alcasid,
Clegg Mary,
Illingworth Melissa,
Gonzalez Claire,
Alphonse Ignatius Arun
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.877.7
Subject(s) - clathrin , huntingtin , biophysics , chemistry , cytoskeleton , endocytosis , clathrin adaptor proteins , vesicle , microbiology and biotechnology , biochemistry , biology , gene , membrane , mutant , cell
Huntingtin‐interacting protein 1 (HIP1) is a dumbbell‐shaped molecule that carries out multiple functions in animals. We published two crystal structures of contiguous segments of HIP1 that give us new insights into how HIP proteins participate in clathrin‐mediated endocytosis and Huntington's disease (HD). The objective of the study is to understand if charged patches in the flexible region of HIP1 are involved in mediating the binding of clathrin light chain. A basic patch centered on K494 interrupts the S3 path and acidic residues mark the end of the path. A second basic patch centered on K474 (~50 ? away from K494) is critical for the binding of clathrin light chain b (LCb). These electrostatic features that are also conserved in HIP12/R (related to HIP1, connects clathrin‐coated vesicles to the cytoskeleton) are in a flexible part of the HIP1 coiled‐coil. To assess the impact of replacing K474 and K494 with alanine or glutamic acid, we performed GST‐pulldowns. Circular dichroism (CD) and NMR experiments show the opened region of HIP is stable at low temperatures, but highly labile at 37° C. NMR data indicate that the opened region has "hard wired" structure that is stable even at 83° C. We conclude both K474 and K494 are necessary for the productive binding of clathrin LCb and that this region involved in binding htt, HIPPI and clathrin is flexible.