Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD–YeaZ–YjeE complex

Abstract

YgjD from COG0533 is amongst a small group of highly conserved proteins present in all three domains of life. Various roles and biochemical functions (including sialoprotease and endonuclease activities) have been ascribed to YgjD and orthologs, the most recent, however, is involvement in the post transcriptional modification of certain tRNAs by formation of N6-threonyladenosine (t 6 A) at position 37. In bacteria, YgjD is essential and along with YeaZ, YjeE, and YrdC has been shown to be 'necessary and sufficient' for the tRNA modification. To further define interactions and possible roles for some of this set of proteins we have undertaken structural and biochemical studies. We show that formation of the previously reported heterodimer of YgjD-YeaZ involves ordering of the C-terminal region of YeaZ which extends along the surface of YgjD in the crystal structure. ATPcS or AMP is observed in YgjD while no nucleotide is bound on YeaZ. ITC experiments reveal previously unreported binary and ternary complexes which can be nucleotide dependent. The stoichiometry of the YeaZ-YgjD complex is 1:1 with a K D of 0.3 mM. YgjD and YjeE interact only in the presence of ATP, while YjeE binds to YgjD-YeaZ in the presence of ATP or ADP with a K D of 6 mM. YgjD doesn't bind the precursors of t 6 A, threonine, and bicarbonate. These results show a more complex set of interactions than previously thought, which may have a regulatory role. The understanding gained should help in deriving inhibitors of these essential proteins that might have potential as antibacterial drugs.

Structure ofSaccharomyces cerevisiaemitochondrial Qri7 in complex with AMP

“…1 and 2a). Although we could not identify the source of the metal ions, they might be zinc ions, as in the case of the YgjD-ATPS structure (Nichols et al, 2013). Both refined chains A and B consist of residues 31-406.…”

“…The two zinc ions are depicted as grey spheres and the two AMP molecules are shown as orange stick models. interface, Kae1 and YgjD interact with Pcc1 and YeaZ, respectively, in the complexes (Mao et al, 2008;Nichols et al, 2013). On the dimerization surface of Qri7, the side chains of Trp136 form hydrogen bonds to the carbonyl groups of the main chains of Ala135 and Arg104, and Gln87 forms hydrogen bonds to the side chains of Asp127 and Lys130 (Fig.…”

“…2.02 Å for the C atoms of residues 1-321; Hecker et al, 2007), YgjD-ATPS (PDB entry 3zeu, r.m.s.d. 2.25 Å for the C atoms of residues 1-327; Nichols et al, 2013) and YgjD-AMP (PDB entry 3zet, r.m.s.d. 2.33 Å for the C atoms of residues 1-335; Nichols et al, 2013).…”

“…Thus, although the Qri7 homologues of YgjD and Kae1 exhibit threonyl-carbamoyl transferase activity only in the presence of the other subunits, Qri7 forms a homodimer which has the full threonyl-carbamoyl transfer activity (Wan et al, 2013;Deutsch et al, 2012). To date, structures of YgjD in complexes with AMP and with ATPS, of Kae1 in complex with ATP and of the apo form of Qri7 have been determined (Wan et al, 2013;Hecker et al, 2007;Nichols et al, 2013). In the apo structure, Qri7 forms a homodimer related by crystallographic symmetry.…”

Deficiency of BrpB causes major defects in cell division, stress responses and biofilm formation by Streptococcus mutans

“…The results showed no significant differences between the parent strain and the deficient mutant, TW14D (Bitoun et al, 2012a;Wen et al, 2006), during growth in early exponential phase (OD 600 0.3), suggesting that brpB is not directly under the control of BrpA under the conditions studied. In E. coli and Salmonella typhimurium, YjeE was shown to interact with YeaZ (a protease) and YeaZ-YgiD (an atypical DNA-binding protein) complex (Handford et al, 2009;Nichols et al, 2013 Fig. 6.…”