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 engineere d into a segment of the catalytic RNA that is dispensable for catalysis. Be cause molecular surfaces of proteins are more chemically varied than those of RNA, the presence of the protein moiety was expected to facilitate cryst allization and improve crystal order. The HDV ribozyme-U1A complex crystall ized readily, and its structure was solved using standard techniques for he avy-atom derivatization of protein crystals. Over 1200 Angstrom(2) Of the s olvent-accessible surface area of the complex are involved in crystal conta cts. As protein-protein interactions comprise 85 % of this buried area, the se crystals appear to be held together predominantly by the protein compone nt of the complex. Our crystallization method should be useful for the stru cture 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 An gstrom) reveals an RNA with a deep active site cleft. Well-ordered metal io ns are not observed crystallographically in this cavity. Biochemical result s of previous workers had suggested an important role in catalysis for cyto sine 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. (C) 2000 Academic Press.