title: Structural requirements for the ubiquitin-associated domain of the mRNA export factor Mex67 to bind its specific targets, the transcription elongation THO complex component Hpr1 and nucleoporin FXFG repeats.
abstract: The ubiquitin-associated (UBA) domain of the principal Saccharomyces cerevisiae mRNA nuclear export factor, Mex67, can bind both nuclear pore protein (nucleoporin) FG repeats and Hpr1, a component of the TREX.THO complex that functions to link transcription and export. Using fluorescence resonance energy transfer-based assays, we show here that Hpr1 and the FG repeats interact with overlapping binding sites on the Mex67 UBA domain. We present the solution structure of the Mex67 UBA domain (UBA-Mex67) complexed with a FXFG nucleoporin peptide and define residues engaged in the interaction and those involved in the FXFG-induced conformational change. We show by NMR titration that the binding of Hpr1 produces analogous changes in chemical shifts in similar regions of the UBA domain. Together the data presented here indicate that both Hpr1 and FXFG nucleoporins may bind in a similar way to the UBA-Mex67 domain. However, whereas binding of Hpr1 allows UBA-Mex67 to interact with tetra-ubiquitin, the complex between UBA-Mex67 and FXFG is unable to bind mono- or tetra-ubiquitin, suggesting that both substrate binding and also the nature of the substrate may influence the affinity of the UBA-Mex67 domain for ubiquitin.
authors.affiliation: Institut Jacques Monod, Université Paris VII, CNRS, 2 Place Jussieu, Tour 43, 75251 Paris Cedex 05, France.
authors.forename: Maria
authors.initials: M
authors.lastname: Hobeika
authors.forename: Christoph
authors.initials: C
authors.lastname: Brockmann
authors.forename: Florian
authors.initials: F
authors.lastname: Gruessing
authors.forename: David
authors.initials: D
authors.lastname: Neuhaus
authors.forename: Gilles
authors.initials: G
authors.lastname: Divita
authors.forename: Murray
authors.initials: M
authors.lastname: Stewart
authors.forename: Catherine
authors.initials: C
authors.lastname: Dargemont
mesh_terms: D001692:Biological Transport; D018360:Crystallography, X-Ray; D005453:Fluorescence; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D012996:Solutions; D013329:Structure-Activity Relationship
chemical_list: C063494:HPR1 protein, S cerevisiae; C106898:MEX67 protein, S cerevisiae; C062748:NSP1 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D010446:Peptide Fragments; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions