Abstract
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.
Highlights
This study presents the structure of the clathrin heavy chain N-terminal domain (NTD) solved in the presence of cellular and viral clathrin-binding peptides
Site Clathrin box Clathrin box Clathrin box Arrestin box Arrestin box Arrestin box W box W box W box Royle box Royle box Royle box Royle box Reference 25 25 This study This study 13 This study 12 12 14 This study This study 14 14 to clathrin NTD: structures of NTD solved in the presence of β3 adaptin and β-arrestin 2 clathrin-box motifs (CBMs) showed binding only at the clathrin box,[10] only the W box site is occupied in the structure solved in the presence of a peptide derived from the “second” (PWDLW) clathrinbinding motif of amphiphysin,[12] and the structure of NTD in complex with a long splice form arrestin 2 shows binding of 2 different peptide motifs at the clathrin and arrestin box sites
We observe that the CBMs of β2 adaptin, amphiphysin and HDAg-L2 bind to both the clathrin and arrestin box sites
Summary
Clathrin mediates vesicular transport between post-Golgi membranes in eukaryotes and is targeted to specific membranes by interactions with clathrin adaptor proteins.[1,2,3] Individually these interactions are weak,[4] but because clathrin polymerization drives the growth of a network of available binding sites, a wide range of adaptors and accessory factors may be recruited and retained at sites of coated pit formation.[2,5,6,7]. Clathrin:adaptor interactions are typically driven by linear peptide motifs in the unstructured regions of clathrin adaptors that bind the N-terminal β-propeller domain (NTD) of the clathrin heavy chain at several distinct sites (reviewed in reference 8): the “clathrin-box motif” (CBM), consensus sequence LΦxΦ[DE] (where x denotes any amino acid, Φ denotes a bulky hydrophobic residue and [DE] is a glutamate or aspartate), binds in a groove between blades 1 and 2 of the NTD β-propeller[3,9,10]; and the “W box” consensus PWxxW, binds the cleft near the centre of the NTD β-propeller.[11,12] Thirdly, an extended surface loop of the arrestin 2 long isoform (arrestin2L) has been shown to occupy the “arrestin box”, a site lying between blades 4 and 5 of the NTD that binds peptides with consensus [LI][LI]GxL.[13] More recently, a fourth adaptor binding site on the clathrin NTD, between blades 6 and 7, was defined by Willox and Royle[14] on the basis of functional experiments in HeLa cells expressing clathrin heavy chain mutated in the NTD This last study found that even a single functional NTD site was sufficient to sustain transferrin uptake. We provide the first structural characterization of the putative “fourth” adaptor binding site on clathrin NTD
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