Structure of H/ACA RNP protein Nhp2p reveals cis/trans isomerization of a conserved proline at the RNA and Nop10 binding interface

J Mol Biol. 2011 Sep 2;411(5):927-42. doi: 10.1016/j.jmb.2011.06.022. Epub 2011 Jun 25.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Blotting, Western
  • Immunoprecipitation
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Proteins / metabolism*
  • Proline / chemistry*
  • Protein Binding
  • Protein Conformation
  • RNA, Fungal / metabolism*
  • RNA, Small Nucleolar / metabolism*
  • RNA-Binding Proteins / metabolism*
  • Ribonucleoproteins, Small Nuclear / chemistry*
  • Ribonucleoproteins, Small Nuclear / genetics
  • Ribonucleoproteins, Small Nuclear / metabolism*
  • Ribonucleoproteins, Small Nucleolar / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Homology, Amino Acid
  • Stereoisomerism

Substances

  • NOP10 protein, S cerevisiae
  • Nuclear Proteins
  • RNA, Fungal
  • RNA, Small Nucleolar
  • RNA-Binding Proteins
  • Ribonucleoproteins, Small Nuclear
  • Ribonucleoproteins, Small Nucleolar
  • Saccharomyces cerevisiae Proteins
  • NHP2 protein, S cerevisiae
  • Proline

Associated data

  • PDB/2LBW
  • PDB/2LBX