Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module.
Well-ordered crystals of a genomic hepatitis delta virus (HDV) ribozyme, a large, globular RNA, were obtained employing a new crystallization method. A high-affinity binding site for the spliceosomal protein U1A was engineered into a segment of the catalytic RNA that is dispensable for catalysis. Because molecular surfaces of proteins are more chemically varied than those of RNA, the presence of the protein moiety was expected to facilitate crystallization and improve crystal order. The HDV ribozyme-U1A complex crystallized readily, and its structure was solved using standard techniques for heavy-atom derivatization of protein crystals. Over 1200 A(2) of the solvent-accessible surface area of the complex are involved in crystal contacts. As protein-protein interactions comprise 85% of this buried area, these crystals appear to be held together predominantly by the protein component of the complex. Our crystallization method should be useful for the structure determination of other biochemically important RNAs for which protein partners do not exist or are experimentally intractable. The refined model of the complex (R-free=27.9% for all reflections between 20.0 and 2.3 A) reveals an RNA with a deep active site cleft. Well-ordered metal ions are not observed crystallographically in this cavity. Biochemical results of previous workers had suggested an important role in catalysis for cytosine 75. The pyrimidine base of this residue is buried at the bottom of the active site in an environment that could raise its pK(a) value. We propose that this highly conserved cytosine may be the general base that catalyzes the transesterification.
Ferré-D'Amaré
A R
AR
Department of Molecular Biophysics, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA.
Doudna
J A
JA
D001665:Binding Sites; D003460:Crystallization; D003596:Cytosine / Q000737:chemistry; D003698:Hepatitis Delta Virus / Q000201:enzymology*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D016337:RNA, Catalytic / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry*
D016337:RNA, Catalytic; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; D003596:Cytosine