title: Multiple Weak Linear Motifs Enhance Recruitment and Processivity in SPOP-Mediated Substrate Ubiquitination.
abstract: Primary sequence motifs, with millimolar affinities for binding partners, are abundant in disordered protein regions. In multivalent interactions, such weak linear motifs can cooperate to recruit binding partners via avidity effects. If linear motifs recruit modifying enzymes, optimal placement of weak motifs may regulate access to modification sites. Weak motifs may thus exert physiological relevance stronger than that suggested by their affinities, but molecular mechanisms of their function are still poorly understood. Herein, we use the N-terminal disordered region of the Hedgehog transcriptional regulator Gli3 (Gli3(1-90)) to determine the role of weak motifs encoded in its primary sequence for the recruitment of its ubiquitin ligase CRL3(SPOP) and the subsequent effect on ubiquitination efficiency. The substrate adaptor SPOP binds linear motifs through its MATH (meprin and TRAF homology) domain and forms higher-order oligomers through its oligomerization domains, rendering SPOP multivalent for its substrates. Gli3 has multiple weak SPOP binding motifs. We map three such motifs in Gli3(1-90), the weakest of which has a millimolar dissociation constant. Multivalency of ligase and substrate for each other facilitates enhanced ligase recruitment and stimulates Gli3(1-90) ubiquitination in in vitro ubiquitination assays. We speculate that the weak motifs enable processivity through avidity effects and by providing steric access to lysine residues that are otherwise not prioritized for polyubiquitination. Weak motifs may generally be employed in multivalent systems to act as gatekeepers regulating post-translational modification.
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Wendy K
authors.initials: WK
authors.lastname: Pierce
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Christy R
authors.initials: CR
authors.lastname: Grace
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Jihun
authors.initials: J
authors.lastname: Lee
authors.affiliation: Molecular Interactions Analysis Shared Resource, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Nourse
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Melissa R
authors.initials: MR
authors.lastname: Marzahn
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA; Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Sciences Center, Memphis, TN 38105, USA.
authors.forename: Edmond R
authors.initials: ER
authors.lastname: Watson
authors.affiliation: St. Jude Proteomics Facility, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: High
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA; St. Jude Proteomics Facility, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA; Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Junmin
authors.initials: J
authors.lastname: Peng
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA; Howard Hughes Medical Institute, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Brenda A
authors.initials: BA
authors.lastname: Schulman
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA. Electronic address: tanja.mittag@stjude.org.
authors.forename: Tanja
authors.initials: T
authors.lastname: Mittag
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D006364:Hedgehogs; D011485:Protein Binding; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D014157:Transcription Factors; D044767:Ubiquitin-Protein Ligases
keywords: NMR spectroscopy; Speckle-type POZ protein; cancer; degron; multivalency