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| Subject Categories: Structural Biology | Membranes & Transport |
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| The EMBO Journal
(2002) 21, 6072–6082, doi: 10.1093/emboj/cdf594 |
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Clathrin light and heavy chain interface:  -helix binding superhelix loops via critical tryptophans |
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| Chih-Ying Chen1, Michael L. Reese2, Peter K. Hwang3, Nobuyuki Ota3, David Agard3 and Frances M. Brodsky1 |
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1 The G.W.Hooper Foundation, Department of Microbiology and Immunology and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0552, USA 2 Graduate Group in Biophysics, University of California, San Francisco, CA 94143-0552, USA 3 Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0552, USA To whom correspondence should be addressed Frances M. Brodsky, fmarbro@itsa.ucsf.edu Received 12 August 2002; Revised 19 September 2002; Accepted 19 September 2002. |
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| Abstract |
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| Clathrin light chain subunits (LCa and LCb) contribute to regulation of coated vesicle formation to sort proteins during receptor-mediated endocytosis and organelle biogenesis. LC binding to clathrin heavy chain (HC) was characterized by genetic and structural approaches. The core interactions were mapped to HC residues 1267−1522 (out of 1675) and LCb residues 90−157 (out of 228), using yeast two-hybrid assays. The C-termini of both subunits also displayed interactions extending beyond the core domains. Mutations to helix breakers within the LCb core disrupted HC association. Further suppressor mutagenesis uncovered compensatory mutations in HC (K1415E or K1326E) capable of rescuing the binding defects of LCb mutations W127R or W105R plus W138R, thereby pinpointing contacts between HC and LCb. Mutant HC K1415E also rescued loss of binding by LCa W130R, indicating that both LCs interact similarly with HC. Based on circular dichroism data, mapping and mutagenesis, LCa and LCb were represented as -helices, aligned along the HC and, using molecular dynamics, a structural model of their interaction was generated with novel implications for LC control of clathrin assembly. |
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Keywords: cation− interaction, clathrin, coat assembly, molecular dynamics, reverse two-hybrid screen |
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