Adaptation of Aminoacyl-tRNA Synthetase Catalytic Core to Carrier Protein Aminoacylation  Marko Mocibob, Nives Ivic, Marija Luic, Ivana Weygand-Durasevic  Structure  Volume 21, Issue 4, Pages 614-626 (April 2013) DOI: 10.1016/j.str.2013.02.017 Copyright © 2013 Elsevier Ltd Terms and Conditions

Structure 2013 21, 614-626DOI: (10.1016/j.str.2013.02.017) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 The Structure of B. japonicum Gly:CP Ligase 1 in Complex with Cognate CP (A and B) Structure comparison of Bj Gly:CP ligase 1 complexed with Bj CP (A) and the model of mMbSerRS complexed with tRNA (B). The two subunits of the enzymes are colored in gray and yellow. Both enzymes contain Zn2+ in the active site, shown as a cyan sphere. The N-terminal domain (pink), important for recognition of tRNA, does not exist in Bj Gly:CP ligase 1. Macromolecular substrates, CP and tRNA, are shown in teal. One tRNA molecule binds across both subunits of mMbSerRS, while CP binds to a distinct CP-binding helix (light pink) of the ligase. The CP-binding helix is a part of loop-helix motif (light pink) characteristic for aa:CP ligases. In mMbSerRS, the loop-helix is replaced by the serine ordering loop (light pink) that is formed upon the serine binding. The thin ribbon in panel A corresponds to the poorly structured part of the CP untraceable in the electron density maps. See also Figures S1 and S2. Structure 2013 21, 614-626DOI: (10.1016/j.str.2013.02.017) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Protein:Protein Interaction between Bj Gly:CP Ligase 1 and Cognate CP (A) CP (teal) binds solely to the CP-binding helix (light pink) of the ligase (gray). (B) Putative hydrogen bonds formed between CP and Bj Gly:CP ligase 1 are located on solvent-exposed part of the interface. (C) Hydrophobic interactions involved in CP-ligase interaction. Zinc ion is shown as a cyan sphere. Residues coordinating the zinc ion, AMP, 4′-phosphopantetheine, and residues involved in CP:ligase interaction are shown as sticks. Putative hydrogen bonds are marked as black dashes. Roman numerals designate four characteristic CP helices. Structure 2013 21, 614-626DOI: (10.1016/j.str.2013.02.017) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 Active Site of the B. japonicum Gly:CP Ligase Complex (A) Active site of Bj Gly:CP ligase 1 complexed with glycyl-adenylate analog (GlyAMS). The prosthetic arm (Ppant) is positioned toward the carbonyl moiety of GlyAMS. (B) Proposed mechanism for the transfer of activated glycine to Ppant. Prosthetic group and GlyAMS are displayed as yellow sticks and the residues involved in the reaction are shown as gray sticks. Putative hydrogen bonds are marked as black dashes. The red sphere represents a water molecule, while the cyan and green spheres correspond to zinc and magnesium ions, respectively. See also Figure S3. Structure 2013 21, 614-626DOI: (10.1016/j.str.2013.02.017) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 Pull-Down Assay of Bj Gly:CP Ligase 1 (A–D) At Ala:CP ligase (B), hybrid Gly:CP ligase (C), and Bj Gly:CP ligase with deleted LH motif (D). The ligases were incubated with various cognate, heterologous, and holo- and apo-carrier proteins, as indicated on the picture. The mixture of His-tagged CP and the ligase (input; left side of each panel) was loaded on Ni-NTA agarose, and proteins retained on the resin were analyzed by SDS-PAGE (pull-down; right side of each panel). Bold arrows (lig.→) mark the position of the ligases, hollow arrows (CP, open right arrow) designate the position of the CPs on the gel. The numbers in the middle of the gels designate the molecular weight of the MW standards. See also Figures S4 and S5. Structure 2013 21, 614-626DOI: (10.1016/j.str.2013.02.017) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 5 Influence of apoCPs on CP Aminoacylation By Bj Gly:CP ligase 1 (left panel) and At Ala:CP ligase (right panel). The concentration of the holoCP was varied, while the concentration of the apoCP was kept constant, as indicated on the graphs, in excess of the holoCP concentration to facilitate the competition between apo- and holo-CP forms. See also Figure S6. Structure 2013 21, 614-626DOI: (10.1016/j.str.2013.02.017) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 6 Structural Analysis of aa:CP Ligases’ Specificity toward CP (A) Superimposed complex of Bj Gly:CP ligase 1 (gray) and Bj CP (teal) on the complex of hybrid Gly:CP ligase (violet) and At CP (yellow). The centers of mass for the CPs are shown as black spheres. (B and C) Interface between hybrid Gly:CP ligase (violet) and At CP (yellow). Residues involved in polar (B) and hydrophobic (C) interactions are shown as sticks. Putative hydrogen bonds are marked as black dashes. (D–G)Surface charge representations of Bj CP (D) and At CP (E) oriented to display part of the CP surface interacting with CP binding helices (gray, Bj Gly:CP ligase 1; violet, At helix incorporated in hybrid Gly:CP ligase). Surface charge of Bj (F) and At (G) aa:CP ligase CP-binding helices. The upper parts of the helices correspond to their N termini. See also Figures S7 and S8. Structure 2013 21, 614-626DOI: (10.1016/j.str.2013.02.017) Copyright © 2013 Elsevier Ltd Terms and Conditions