title: Dynamic interactions between clathrin and locally structured elements in a disordered protein mediate clathrin lattice assembly.
abstract: Assembly of clathrin lattices is mediated by assembly/adaptor proteins that contain domains that bind lipids or membrane-bound cargo proteins and clathrin binding domains (CBDs) that recruit clathrin. Here, we characterize the interaction between clathrin and a large fragment of the CBD of the clathrin assembly protein AP180. Mutational, NMR chemical shift, and analytical ultracentrifugation analyses allowed us to precisely define two clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the clathrin heavy chain (TD). The locations of the two clathrin binding sites are consistent with predictions from sequence alignments of previously identified clathrin binding elements and, by extension, indicate that the complete AP180 CBD contains ∼12 degenerate repeats, each containing a single clathrin binding site. Sequence and circular dichroism analyses have indicated that the AP180 CBD is predominantly unstructured and our NMR analyses confirm that this is largely the case for the AP180 fragment characterized here. Unexpectedly, unlike the many proteins that undergo binding-coupled folding upon interaction with their binding partners, the AP180 fragment is similarly unstructured in its bound and free states. Instead, we find that this fragment exhibits localized β-turn-like structures at the two clathrin binding sites both when free and when bound to clathrin. These observations are incorporated into a model in which weak binding by multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhuo
authors.forename: Udayar
authors.initials: U
authors.lastname: Ilangovan
authors.forename: Virgil
authors.initials: V
authors.lastname: Schirf
authors.forename: Borries
authors.initials: B
authors.lastname: Demeler
authors.forename: Rui
authors.initials: R
authors.lastname: Sousa
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Hinck
authors.forename: Eileen M
authors.initials: EM
authors.lastname: Lafer
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002966:Clathrin / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D017931:DNA Primers; D011994:Recombinant Proteins