Cellular and viral peptides bind multiple sites on the N-terminal domain of clathrin.
Short peptide motifs in unstructured regions of clathrin-adaptor proteins recruit clathrin to membranes to facilitate post-Golgi membrane transport. Three consensus clathrin-binding peptide sequences have been identified and structural studies show that each binds distinct sites on the clathrin heavy chain N-terminal domain (NTD). A fourth binding site for adaptors on NTD has been functionally identified but not structurally characterised. We have solved high resolution structures of NTD bound to peptide motifs from the cellular clathrin adaptors β2 adaptin and amphiphysin plus a putative viral clathrin adaptor, hepatitis D virus large antigen (HDAg-L). Surprisingly, with each peptide we observe simultaneous peptide binding at multiple sites on NTD and viral peptides binding to the same sites as cellular peptides. Peptides containing clathrin-box motifs (CBMs) with the consensus sequence LΦxΦ[DE] bind at the 'arrestin box' on NTD, between β-propeller blades 4 and 5, which had previously been thought to bind a distinct consensus sequence. Further, we structurally define the fourth peptide binding site on NTD, which we term the Royle box. In vitro binding assays show that clathrin is more readily captured by cellular CBMs than by HDAg-L, and site-directed mutagenesis confirms that multiple binding sites on NTD contribute to efficient capture by CBM peptides.
Department of Pathology, University of Cambridge, Cambridge, UK.
Julia
J
Muenzner
Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA.
Linton M
LM
Traub
Cambridge Institute for Medical Research, Department of Clinical Biochemistry, University of Cambridge, Cambridge, UK.
Bernard T
BT
Kelly
Department of Pathology, University of Cambridge, Cambridge, UK.
Stephen C
SC
Graham
D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology*; D033922:Clathrin Heavy Chains / Q000378:metabolism*; D035923:Hepatitis delta Antigens / Q000378:metabolism; D006801:Humans; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology*
D033942:Adaptor Proteins, Vesicular Transport; D035923:Hepatitis delta Antigens; D009419:Nerve Tissue Proteins; D010455:Peptides; C442244:hepatitis delta virus large antigen; D033922:Clathrin Heavy Chains; C075637:amphiphysin
amphiphysin; arrestin; assembly polypeptide 2 (AP2); clathrin-mediated endocytosis; endocytosis; hepatitis D virus