Crystal Structure of the Archaeosine Synthase QueF‐Like – Insights into Amidino Transfer and tRNA Recognition by the Tunnel Fold

The 7‐deazaguanosine nucleoside archaeosine (G+) is one of the most remarkable modifications known to occur in tRNA. G+ is found only in Archaea at position‐15 in the dihydrouridine loop (D‐loop) of tRNA, and the presence of the positively charged formamidine group of G+ is believed to be important in stabilization of the tertiary structure of tRNA through electrostatic interactions with the anionic phosphates and by enhancing the hydrogen bonding within the G15‐C48 Levitt base pair. The biosynthesis pathway of G+ is rich in enzymes that belong to the tunneling‐fold (T‐fold) structural superfamily. The T‐fold has emerged as a versatile protein scaffold of diverse catalytic activities. Four members of the T‐fold superfamily have been characterized in the 7‐deazaguanosine pathways and here we report the crystal structure of a fifth enzyme; the recently discovered amidinotransferase QueF‐Like (QueF‐L), responsible for the final step in the biosynthesis of archaeosine in the D‐loop of tRNA in a subset of Crenarchaeota. QueF‐L catalyzes the conversion of the nitrile group of the 7‐cyano‐7‐deazaguanine (preQ 0 ) base of preQ 0 ‐modified tRNA to a formamidino group. The structure, determined in the presence of preQ 0 , reveals a symmetric T‐fold homodecamer of two head‐to‐head facing pentameric subunits, with 10 active sites at the inter‐monomer interfaces. Bound preQ 0 forms a stable covalent thioimide bond with a conserved active site cysteine similar to the intermediate previously observed in the nitrile reductase QueF. Despite distinct catalytic functions, phylogenetic distributions, and only 19% sequence identity, the two enzymes share a common preQ 0 binding pocket, and likely a common mechanism of thioimide formation. However, due to tight twisting of its decamer, QueF‐L lacks the NADPH binding site present in QueF. A large positively charged molecular surface and a docking model suggest simultaneous binding of multiple tRNA molecules and structure‐specific recognition of the D‐loop by a surface groove. The structure sheds light on the mechanism of nitrile amidation, and the evolution of diverse chemistries in a common fold. Support or Funding Information National Institutes of Health (The Stanford Synchrotron Research Lab‐ oratory Structural Molecular Biology Program); Grant number: P41GM103393; Grant sponsor: NSF (D.I.‐R. and M.A.S.); Grant number: CHE‐1309323; Grant sponsor: NIGMS (M.A.S. and D.I.‐R.); Grant number: 1R01GM110588‐01A1.Biosynthesis of G1. (A) The positions of Q and G1 in bacterial and archaeal tRNA, respectively. (B) The de novo biosynthetic pathway to Q and G1. The central boxed portion of the pathway is common in Archaea and Bacteria and occurs outside the context of the tRNA and leads to the formation of the modified base 7‐cyano‐7‐deazaguanine (preQ0). Enzymes that are members of the tunneling‐fold superfamily are GCYH‐IA and IB, QueD, QueF, and QueF‐L.Crystal structure of QueF‐L from P. calidifontis . (A) Ribbon diagram showing a top view (left) and a side view (right) of the QueF‐L homodecamer. The monomers are shown in different colors. PreQ0 molecules bound in the 10 active sites located at the intersubunit interfaces are shown as green Corey–Pauling–Koltun models. Charged side chains lining the tunnel are shown in red (Asp and Glu) and blue (Arg and Lys) stick model. (B) Stereo view of the FOM‐weighted experimental electron density map.Formation of a covalent thioimide intermediate in QueF‐L and induced‐fit binding. (A) Stereo view of the 2Fo‐Fc electron density map (2.75 Å, contour 1.8 σ), superposed on the refined model, in one of the active sites showing bound preQ0 and formation of thioimide bond with active site Cys21. (B) Superposition of the apo structure (subunits in dark and light gray) and the preQ0‐bound structure (subunits in green and pink) around an active site region showing preQ0‐induced conformational changes.Structural comparison of QueF‐L and QueF enzymes. (A) Ribbon diagrams showing the tertiary and quaternary structures of P. calidifontis QueF‐L (left), the homodecamer of unimodular QueF from B. subtilis (middle), and the homodimer of bimodular QueF from V. cholerae (right). (B) Topology diagrams representing the three enzymes. (C) Structure‐based multi‐sequence alignment in the common T‐fold region. Residues shared between the QueF‐L and QueF families are highlighted in red background.The QueF‐L structure lacks space for NADPH binding. (A) View of the NADPH binding site crystallographically observed at the inter‐dimer interface in bimodular QueF (B) Analogous view and representation of the inter‐pentamer interface of unimodular QueF (C) Analogous view and representation of the inter‐pentamer interface of QueF‐L showing the salt‐bridge pairs (Glu18′‐Lys49, Asp53–Arg56′, Asp53′‐Arg56, and Glu18‐Lys49′) that pin together the two pentameric subunits as blue‐red sticksPutative tRNA binding sites. (A) Surface electrostatic potential of QueF‐L. Positive and negative potentials are shown in blue and red, respectively. (B) Energy minimized docking model of a tRNA D‐loop containing preQ0 at position 15 (ball‐and‐stick) onto the crystal structure of QueF‐L (ribbon and surface rendering). The D‐loop phosphate backbone (orange) is recognized by the positively charged groove on the protein surface. The base of G15 is buried deeply in the preQ0 binding pocket.Putative ammonium channel. View of the inter‐pentamer interface near an active site from the preQ0‐bound QueF‐L structure. The putative binding cavity is shown as gray mesh and the enclosing tyrosine side chains are labeled. The asterisk indicates the putative binding site of NH4+ before amidino transfer.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .