title: Structure of H/ACA RNP protein Nhp2p reveals cis/trans isomerization of a conserved proline at the RNA and Nop10 binding interface.
abstract: H/ACA small nucleolar and Cajal body ribonucleoproteins (RNPs) function in site-specific pseudouridylation of eukaryotic rRNA and snRNA, rRNA processing, and vertebrate telomerase biogenesis. Nhp2, one of four essential protein components of eukaryotic H/ACA RNPs, forms a core trimer with the pseudouridylase Cbf5 and Nop10 that binds to H/ACA RNAs specifically. Crystal structures of archaeal H/ACA RNPs have revealed how the protein components interact with each other and with the H/ACA RNA. However, in place of Nhp2p, archaeal H/ACA RNPs contain L7Ae, which binds specifically to an RNA K-loop motif absent from eukaryotic H/ACA RNPs, while Nhp2 binds a broader range of RNA structures. We report solution NMR studies of Saccharomyces cerevisiae Nhp2 (Nhp2p), which reveal that Nhp2p exhibits two major conformations in solution due to cis/trans isomerization of the evolutionarily conserved Pro83. The equivalent proline is in the cis conformation in all reported structures of L7Ae and other homologous proteins. Nhp2p has the expected α-β-α fold, but the solution structures of the major conformation of Nhp2p with trans Pro83 and of Nhp2p-S82W with cis Pro83 reveal that Pro83 cis/trans isomerization affects the positions of numerous residues at the Nop10 and RNA binding interface. An S82W substitution, which stabilizes the cis conformation, also stabilizes the association of Nhp2p with H/ACA snoRNPs expressed in vivo. We propose that Pro83 plays a key role in the assembly of the eukaryotic H/ACA RNP, with the cis conformation locking in a stable Cbf5-Nop10-Nhp2 ternary complex and positioning the protein backbone to interact with the H/ACA RNA.
authors.affiliation: Department of Chemistry and Biochemistry, and the Molecular Biology Institute, PO Box 951569,University of California, Los Angeles, CA 90095-1569, USA.
authors.forename: Bon-Kyung
authors.initials: BK
authors.lastname: Koo
authors.forename: Chin-Ju
authors.initials: CJ
authors.lastname: Park
authors.forename: Cesar F
authors.initials: CF
authors.lastname: Fernandez
authors.forename: Nicholas
authors.initials: N
authors.lastname: Chim
authors.forename: Yi
authors.initials: Y
authors.lastname: Ding
authors.forename: Guillaume
authors.initials: G
authors.lastname: Chanfreau
authors.forename: Juli
authors.initials: J
authors.lastname: Feigon
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D015153:Blotting, Western; D047468:Immunoprecipitation; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D012331:RNA, Fungal / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism
chemical_list: C117016:NOP10 protein, S cerevisiae; D009687:Nuclear Proteins; D012331:RNA, Fungal; D020537:RNA, Small Nucleolar; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; D029701:Saccharomyces cerevisiae Proteins; C069628:NHP2 protein, S cerevisiae; D011392:Proline