title: Nuclear Magnetic Resonance Structural Mapping Reveals Promiscuous Interactions between Clathrin-Box Motif Sequences and the N-Terminal Domain of the Clathrin Heavy Chain.
abstract: The recruitment and organization of clathrin at endocytic sites first to form coated pits and then clathrin-coated vesicles depend on interactions between the clathrin N-terminal domain (TD) and multiple clathrin binding sequences on the cargo adaptor and accessory proteins that are concentrated at such sites. Up to four distinct protein binding sites have been proposed to be present on the clathrin TD, with each site proposed to interact with a distinct clathrin binding motif. However, an understanding of how such interactions contribute to clathrin coat assembly must take into account observations that any three of these four sites on clathrin TD can be mutationally ablated without causing loss of clathrin-mediated endocytosis. To take an unbiased approach to mapping binding sites for clathrin-box motifs on clathrin TD, we used isothermal titration calorimetry (ITC) and nuclear magnetic resonance spectroscopy. Our ITC experiments revealed that a canonical clathrin-box motif peptide from the AP-2 adaptor binds to clathrin TD with a stoichiometry of 3:1. Assignment of 90% of the total visible amide resonances in the TROSY-HSQC spectrum of (13)C-, (2)H-, and (15)N-labeled TD40 allowed us to map these three binding sites by analyzing the chemical shift changes as clathrin-box motif peptides were titrated into clathrin TD. We found that three different clathrin-box motif peptides can each simultaneously bind not only to the previously characterized clathrin-box site but also to the W-box site and the β-arrestin splice loop site on a single TD. The promiscuity of these binding sites can help explain why their mutation does not lead to larger effects on clathrin function and suggests a mechanism by which clathrin may be transferred between different proteins during the course of an endocytic event.
authors.affiliation: Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhuo
authors.affiliation: Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States.
authors.forename: Kristin E
authors.initials: KE
authors.lastname: Cano
authors.affiliation: Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States.
authors.forename: Liping
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States.
authors.forename: Udayar
authors.initials: U
authors.lastname: Ilangovan
authors.affiliation: Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Hinck
authors.affiliation: Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States.
authors.forename: Rui
authors.initials: R
authors.lastname: Sousa
authors.affiliation: Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States.
authors.forename: Eileen M
authors.initials: EM
authors.lastname: Lafer
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D019390:Arrestins / Q000737:chemistry* / Q000378:metabolism; D002153:Calorimetry, Indirect; D002417:Cattle; D033922:Clathrin Heavy Chains / Q000737:chemistry* / Q000378:metabolism; D004705:Endocytosis; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D010455:Peptides; D000071557:beta-Arrestins; D033922:Clathrin Heavy Chains
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