A 1.9 A crystal structure of the HDV ribozyme precleavage suggests both Lewis acid and general acid mechanisms contribute to phosphodiester cleavage.
The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are self-cleaving RNAs found throughout all kingdoms of life. These RNAs fold into a double-nested pseudoknot structure and cleave RNA, yielding 2',3'-cyclic phosphate and 5'-hydroxyl termini. The active site nucleotide C75 has a pK(a) shifted >2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg(2+) ion that helps activate the 2'-hydroxyl for nucleophilic attack has been characterized biochemically; however, this ion has not been visualized in any previous structures. To create a snapshot of the ribozyme in a state poised for catalysis, we have crystallized and determined the structure of the HDV ribozyme bound to an inhibitor RNA containing a deoxynucleotide at the cleavage site. This structure includes the wild-type C75 nucleotide and Mg(2+) ions, both of which are required for maximal ribozyme activity. This structure suggests that the position of C75 does not change during the cleavage reaction. A partially hydrated Mg(2+) ion is also found within the active site where it interacts with a newly resolved G.U reverse wobble. Although the inhibitor exhibits crystallographic disorder, we modeled the ribozyme-substrate complex using the conformation of the inhibitor strand observed in the hammerhead ribozyme. This model suggests that the pro-R(P) oxygen of the scissile phosphate and the 2'-hydroxyl nucleophile are inner-sphere ligands to the active site Mg(2+) ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.
Chen
Jui-Hui
JH
Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47906, USA.
Yajima
Rieko
R
Chadalavada
Durga M
DM
Chase
Elaine
E
Bevilacqua
Philip C
PC
Golden
Barbara L
BL
D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003698:Hepatitis Delta Virus / Q000201:enzymology*; D006868:Hydrolysis; D008274:Magnesium; D055601:Organic Chemistry Phenomena; D010755:Organophosphates / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism
D010755:Organophosphates; D016337:RNA, Catalytic; D008274:Magnesium