title: Patterns of structural dynamics in RACK1 protein retained throughout evolution: a hydrogen-deuterium exchange study of three orthologs.
abstract: RACK1 is a member of the WD repeat family of proteins and is involved in multiple fundamental cellular processes. An intriguing feature of RACK1 is its ability to interact with at least 80 different protein partners. Thus, the structural features enabling such interactomic flexibility are of great interest. Several previous studies of the crystal structures of RACK1 orthologs described its detailed architecture and confirmed predictions that RACK1 adopts a seven-bladed β-propeller fold. However, this did not explain its ability to bind to multiple partners. We performed hydrogen-deuterium (H-D) exchange mass spectrometry on three orthologs of RACK1 (human, yeast, and plant) to obtain insights into the dynamic properties of RACK1 in solution. All three variants retained similar patterns of deuterium uptake, with some pronounced differences that can be attributed to RACK1's divergent biological functions. In all cases, the most rigid structural elements were confined to B-C turns and, to some extent, strands B and C, while the remaining regions retained much flexibility. We also compared the average rate constants for H-D exchange in different regions of RACK1 and found that amide protons in some regions exchanged at least 1000-fold faster than in others. We conclude that its evolutionarily retained structural architecture might have allowed RACK1 to accommodate multiple molecular partners. This was exemplified by our additional analysis of yeast RACK1 dimer, which showed stabilization, as well as destabilization, of several interface regions upon dimer formation.
authors.affiliation: Institute of Biochemistry and Biophysics Department, Polish Academy of Science, 02-106, Warsaw, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Tarnowski
authors.forename: Kinga
authors.initials: K
authors.lastname: Fituch
authors.forename: Roman H
authors.initials: RH
authors.lastname: Szczepanowski
authors.forename: Michal
authors.initials: M
authors.lastname: Dadlez
authors.forename: Magdalena
authors.initials: M
authors.lastname: Kaus-Drobek
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry*; D029681:Arabidopsis Proteins / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry*; D011487:Protein Conformation; D055503:Protein Multimerization; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D016415:Sequence Alignment
chemical_list: C111825:ASC1 protein, S cerevisiae; D048868:Adaptor Proteins, Signal Transducing; D029681:Arabidopsis Proteins; D009363:Neoplasm Proteins; C515163:RACK1 protein, Arabidopsis; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins
keywords: WD repeats; cell signaling; hydrogen deuterium exchange; mass spectrometry; protein dynamics; receptor for activated C kinase; scaffolding protein