title: Identification of primary and secondary UBA footprints on the surface of ubiquitin in cell-mimicking crowded solution.
abstract: Despite significant advancements in our understanding of ubiquitin-mediated signaling, the influence of the intracellular environment on the formation of transient ubiquitin-partner complexes remains poorly explored. In our work, we introduce macromolecular crowding as a first level of complexity toward the imitation of a cellular environment in the study of such interactions. Using NMR spectroscopy, we find that the stereospecific complex of ubiquitin and the ubiquitin-associated domain (UBA) is minimally perturbed by the crowding agent Ficoll. However, in addition to the primary canonical recognition patch on ubiquitin, secondary patches are identified, indicating that in cell-mimicking crowded solution, UBA contacts ubiquitin at multiple sites.
authors.affiliation: Department of Biotechnology, University of Verona, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Munari
authors.affiliation: Department of Biotechnology, University of Verona, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bortot
authors.affiliation: Department of Biotechnology, University of Verona, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Zanzoni
authors.affiliation: Department of Biotechnology, University of Verona, Italy.
authors.forename: Mariapina
authors.identifier: http://orcid.org/0000-0002-8699-0847
authors.initials: M
authors.lastname: D'Onofrio
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, USA.
authors.forename: David
authors.initials: D
authors.lastname: Fushman
authors.affiliation: Department of Biotechnology, University of Verona, Italy.
authors.forename: Michael
authors.identifier: http://orcid.org/0000-0001-9331-3169
authors.initials: M
authors.lastname: Assfalg
mesh_terms: D000465:Algorithms; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D005362:Ficoll / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D012996:Solutions / Q000737:chemistry; D013237:Stereoisomerism; D013499:Surface Properties; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D012996:Solutions; D025801:Ubiquitin; D005362:Ficoll
keywords: NMR spectroscopy; biomolecular recognition; macromolecular crowding; paramagnetic relaxation enhancement; ubiquitin; ubiquitin-associated domain