title: Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne.
abstract: The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types co-exist in polyubiquitin and are independently regulated in cells. This 'ubiquitin code' determines the fate of the modified protein. Deubiquitinating enzymes of the ovarian tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. Here we reveal how the deubiquitinase Cezanne (also known as OTUD7B) specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with monoubiquitin and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable us to reconstruct the enzymatic cycle in great detail. An intricate mechanism of ubiquitin-assisted conformational changes activates the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the plasticity of deubiquitinases and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Tycho E T
authors.initials: TET
authors.lastname: Mevissen
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Kulathu
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Monique P C
authors.initials: MPC
authors.lastname: Mulder
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Paul P
authors.initials: PP
authors.lastname: Geurink
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Maslen
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Malte
authors.initials: M
authors.lastname: Gersch
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Elliott
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: John E
authors.initials: JE
authors.lastname: Burke
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Bianca D M
authors.initials: BDM
authors.lastname: van Tol
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Masato
authors.initials: M
authors.lastname: Akutsu
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Farid El
authors.initials: FE
authors.lastname: Oualid
authors.affiliation: Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki 305-0801, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Kawasaki
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Stefan M V
authors.initials: SMV
authors.lastname: Freund
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Huib
authors.initials: H
authors.lastname: Ovaa
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: David
authors.initials: D
authors.lastname: Komander
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D000072017:Deubiquitinating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D025821:Polyubiquitin / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D054875:Ubiquitination; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D014452:Ubiquitins; D025821:Polyubiquitin; D010450:Endopeptidases; C476458:OTUD7B protein, human; D000072017:Deubiquitinating Enzymes; D008239:Lysine