Structures of RNA ligase RtcB in complexes with divalent cations and GTP

Abstract

Pyrococcus horikoshii (Pho) RtcB exemplifies a family of binuclear transition metal- and GTP-dependent RNA ligases that join 3'-phosphate and 5'-OH ends via RtcB-(histidinyl-N)–GMP and RNA3'pp5'G intermediates. We find that guanylylation of PhoRtcB is optimal with manganese and less effective with cobalt and nickel. Zinc and copper are inactive and potently inhibit manganese-dependent guanylylation. We report crystal structures of PhoRtcB in complexes with GTP and permissive (Mn, Co, Ni) or inhibitory (Zn, Cu) metals. Zinc and copper occupy the M1 and M2 sites adjacent to the GTP phosphates, as do manganese, cobalt and nickel. The identity/positions of enzymic ligands for M1 (His234, His329, Cys98) and M2 (Cys98, Asp95, His203) are the same for permissive and inhibitory metals. The differences pertain to: (i) the coordination geometries and phosphate contacts of the metals; and (ii) the orientation of the His404 nucleophile with respect to the GTP α-phosphate and pyrophosphate leaving group. M2 metal coordination geometry correlates with metal cofactor activity, whereby inhibitory Zn2 and Cu2 assume a tetrahedral configuration and contact only the GTP γ-phosphate, whereas Mn2, Co2, and Ni2 coordination complexes are pentahedral and contact the β- and γ-phosphates. The His404-Nε–Pα–O(α-β) angle is closer to apical in Mn (179˚), Co (171˚), and Ni (169˚) structures than in Zn (160˚) and Cu (155˚) structures. The octahedral Mn1 geometry in our RtcB•GTP•Mn2+ structure, in which Mn1 contacts α-, β-, and γ-phosphates, transitions to a tetrahedral configuration after formation of RtcB•(His404)–GMP•Mn2+ and departure of pyrophosphate.

Structural and mechanistic insights into activation of the human RNA ligase RTCB by Archease

“…Co 2+ ions promote guanylylation in P. horikoshii and human RTCB, while E. coli RTCB cannot use Co 2+ for activation. 15,21,31,32 The described trace levels of guanylylation and complete lack of ligation with Co 2+ in our study is at odds with the previous results for human RTCB, which report the strongest activity in the presence of Co 2+ . 21 We recognize that the possibility of more than one type of metal ion in the active site under physiological conditions and in the context of the holo-complex will require further exploration.…”

“…The lack of guanylylation in the presence of Zn 2+ and the competing effect with manganese were previously observed for E. coli and P. horikoshii RtcB. 15,31,32 The general argument is that zinc as a softer Lewis acid binds stronger than manganese due to the conserved cysteine in the active site while it does not support guanylylation chemistry. With regards to Co 2+ , the various studies come to different conclusions.…”

“…A previous report showed inhibitory effects on guanylylation by adding equimolar amounts of Zn 2+ compared to Mn 2+ on PhRtcB. 31,32 Thus, we tested equimolar mixtures of Mn 2+ and Zn 2+ as well as Mn 2+ and Co 2+ in RTCB activation assays. In the former case, there was no detectable guanylylation of RTCB (Figure 5C, lane 7) while the latter displays diminished guanylylation (Figure 5C, lane 8).…”

“…While the importance of divalent metal ions for RTCB-catalyzed ligation is generally accepted, their precise role and identity is debated. 15,21,31,32 In order to determine the catalytic functionality in the presence of different divalent metal ions, we prepared metal-free RTCB and Archease by using the metal chelator ethylenediaminetetraacetic acid (EDTA) (see Materials and Methods for details). Without addition of any metal ions to the EDTA-treated proteins, Archease did not promote guanylylation of RTCB (Figure 5C, lane 1).…”

Fungi of the order Mucorales express a “sealing-only” tRNA ligase

“…Most Metazoa and some protozoa use a “direct ligation” mechanism to join the tRNA exons, which is carried out by RTCB enzymes. RCTB uses a catalytic histidine residue to transfer GMP onto the 3’ end of the 5’ exon, generating an activated exon that can then be ligated to the 3’ exon (Chakravarty et al 2012; Englert et al 2012; Desai et al 2013; Jacewicz et al 2022). RTCB has been implicated in tRNA splicing in humans, Caenorhabditis elegans , and Drosophila melanogaster , and homologs of RTCB are encoded in most metazoan genomes (Popow et al 2011; Kosmaczewski et al 2014; Lu et al 2014; Nandy et al 2017).…”