A 1.9 Å Crystal Structure of the HDV Ribozyme Precleavage Suggests both Lewis Acid and General Acid Mechanisms Contribute to Phosphodiester Cleavage

Abstract

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.

HDV Family of Self-Cleaving Ribozymes

“…4.2). 28,[34][35][36] The first pseudoknot is formed between the P1 and P2 helices, and within this pseudoknot a second such structure is formed between the P3 and P1.1 regions. This arrangement …”

“…In the genomic structure of the ribozyme, the P3 helix is extended by invariant U20 and G25 nucleotides that form a reverse wobble pair and stack on another conserved nucleotide, C24. 36 Substitutions to either U20 or G25 that force Watson-Crick base pairing result in ribozymes with markedly decreased activity. 39 Some variability is permitted in the L3 core region, as the number and composition of nucleotides flanking the C24 and G25 residues differs between the genomic and antigenomic HDV ribozymes.…”

“…[41][42][43][44] When this motif is properly positioned in the genomic ribozyme, the N4 of the conserved C75 residue forms two hydrogen bonds, one with a nonbridging oxygen atom of the phosphate backbone linking the C21 and C22 residues that form the 5 0 side of the P1.1 helix, and a second to the 2 0 OH of U20. [34][35][36] These interactions lock the J4/2 region into the active site of the ribozyme. Mutating the genomic C75 or antigenomic C76 residue to a U or G results in a loss of catalytic activity.…”

“…39,40,[44][45][46] The J4/2 strand is further stabilized by the A78 residue of the genomic ribozyme via an A-minor tertiary interaction with the C18 and G29 residues that form the middle base pair of the P3 helix. [34][35][36]47 A purine nucleotide is required 5 0 to A78 to allow for hydrogen bonding between its WatsonCrick face and the 2 0 OH of the C19 nucleotide at the base of P3. [34][35][36] 128…”

“…[34][35][36]47 A purine nucleotide is required 5 0 to A78 to allow for hydrogen bonding between its WatsonCrick face and the 2 0 OH of the C19 nucleotide at the base of P3. [34][35][36] 128…”