Crystallographic structure of the nuclease domain of 3'hExo, a DEDDh family member, bound to rAMP

Abstract

A human 3'-5'-exoribonuclease (3'hExo) has recently been identified and shown to be responsible for histone mRNA degradation. Functionally, 3'hExo and a stem-loop binding protein (SLBP) target opposite faces of a unique highly conserved stem-loop RNA scaffold towards the 3' end of histone mRNA, which is composed of a 6 bp stem and a 4 nt loop, followed by an ACCCA sequence. Its Caenorhabditis elegans homologue, ERI-1, has been shown to degrade small interfering RNA in vitro and to function as a negative regulator of RNA interference in neuronal cells. We have determined the structure of the nuclease domain (Nuc) of 3'hExo complexed with rAMP in the presence of Mg2+ at 1.6 A resolution. The Nuc domain adopts an alpha/beta globular fold, with four acidic residues coordinating a binuclear metal cluster within the active site, whose topology is related to DEDDh exonuclease family members, despite a very low level of primary sequence identity. The two magnesium cations in the Nuc active site are coordinated to D134, E136, D234 and D298, and together with H293, which can potentially act as a general base, provide a platform for hydrolytic cleavage of bound RNA in the 3' --> 5' direction. The bound rAMP is positioned within a deep active-site pocket, with its purine ring close-packed with the hydrophobic F185 and L189 side-chains and its sugar 2'-OH and 3'-OH groups hydrogen bonded to backbone atoms of Nuc. There are striking similarities between the active sites of Nuc and epsilon186, an Escherichia coli DNA polymerase III proofreading domain, providing a common hydrolytic cleavage mechanism for RNA degradation and DNA editing, respectively.

Figure 1

The structure of rAMP-bound Nuc. (A) β-Strands are labeled from β1 to β6, while α-helices are labeled α1 to α8 on proceeding from the N to C terminus within the sequence. The bound rAMP is presented in a stick representation and is colored by atom type. The side-chains of C139 and C186 are shown. All the Figures were drawn by PyMol (DeLano, W.L. The PyMOL Molecular Graphics System on http://www.pymol.org). (B) Potential electrostatics surface of Nuc is colored from blue (basic) to red (acidic), while bound rAMP is shown in yellow. (C) Ribbon diagram of Nuc with B-factor colored from blue (low) to red (high). Nuc in (B) and (C) are in the same orientation.

Figure 2

Structural comparison of the folded topologies of Nuc, ERI-1 and ε186. (A) Ternary structure comparison of Nuc (orange) and ε186 (blue). Nuc is shown in the same orientation as in Figure 1(A), left panel. (B) Primary and secondary structural alignment of Nuc, ERI-1 and ε186. The exonuclease domain of 3′hExo (123 to 322 of NP699163, Nuc), ERI-1 (144 to 346 of AAK39278) and ε186 are aligned on the basis of sequence conservation (Nuc versus ERI-1, sequence identity 39%) and following secondary structure similarity (Nuc versus ε186, sequence identity 13%). Besides five critical (DEDDh) residues, which are colored purple, the identical residues throughout the three sequences are colored red, while the semi-identical residues are colored yellow.

Figure 3

Stereo views looking into the binding cavity of rAMP-bound Nuc. Overview (A) and close-up view (B) of the rAMP binding pocket within Nuc, with hydrogen bonds defining the divalent cation coordination sites (in A) and defining intermolecular contacts (in B) indicated by broken lines. The 2F_o−F_c map contoured at 1.0 is shown in B.

Figure 4

Active sites and proposed hydrolysis mechanism. The active sites of (A) Nuc and (B) ε186. Two conformations of dTMP bound to ε186 are indicated by different occupancy (in B). (C) The proposed hydrolysis mechanism of Nuc. The coordination between DEDDh residues, rAMP and divalent ions is indicated by broken lines. The remaining residues are omitted for clarity.