Structures of RNA ligase RtcB in complexes with divalent cations and GTP.
Publication Type:Journal Article
<p>(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•Mn structure, in which Mn1 contacts α-, β-, and γ-phosphates, transitions to a tetrahedral configuration after formation of RtcB•(His404)-GMP•Mn and departure of pyrophosphate.</p>