Volume 20, Issue 3, Pages 437-448 (November 2005) Structural Insights into the Roles of Water and the 2′ Hydroxyl of the P Site tRNA in the Peptidyl Transferase Reaction  T. Martin Schmeing, Kevin S. Huang, David E. Kitchen, Scott A. Strobel, Thomas A. Steitz  Molecular Cell  Volume 20, Issue 3, Pages 437-448 (November 2005) DOI: 10.1016/j.molcel.2005.09.006 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Unbiased Fo − Fc Electron Density Maps for Some of the Complexes of the 50S Subunit Bound with Peptidyl Transferase Ligands, All Contoured at 3 σ The figures maintain a consistent coloring scheme, with A site substrates (or A site portions of TSAs) in purple and P site substrates in green. (A) Density for CCPmn (magenta, with orange rRNA) in a map calculated at 2.2 Å resolution. (B) Density for CCApcb (green) and sparsomycin (blue) in a map calculated at 2.4 Å resolution. (C) Density for CCdApcb (green) and sparsomycin (blue) in a map calculated at 2.2 Å resolution. (D) Density for DAN (green and magenta) in a map calculated at 2.3 Å resolution. (E) Density for DAA (green and magenta) in a map calculated at 2.7 Å resolution. (F) Density for RAA (green and magenta) in a map calculated at 2.6 Å resolution. (G–L) Schematic diagrams of the ligands shown in (A)–(F). Molecular Cell 2005 20, 437-448DOI: (10.1016/j.molcel.2005.09.006) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Binding of Substrates to the Peptidyl Transferase Center (A) Aminoacyl-tRNA mimics bound to the A site. CChPmn (purple, with rRNA in brown) from the prereaction complex of 50S subunit, CChPmn, and CCApcb (dark green) (Schmeing et al., 2005) bind to the A loop in a very similar manner, as CCPmn (pink, with orange rRNA) bound alone to the 50S subunit. Only 2541(2506) and 2619–20(2584–5) are in altered positions. (B) Binding of CCApcb to the P site. When bound with sparsomycin (blue), CCApcb (light green, with orange rRNA) is shifted slightly down into the active site relative to when it is bound with CChPmn. (CCApcb in dark green, CChPmn in purple, and rRNA in brown.) (C) Schematic diagrams of the ligands shown in (A). (D) Schematic diagrams of the ligands shown in (B). Molecular Cell 2005 20, 437-448DOI: (10.1016/j.molcel.2005.09.006) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 The Environment of the 2′ Hydroxyl of A76 of the Peptidyl-tRNA (A) An unbiased Fo − Fc electron density map of the TS analog RAP (magenta and green) binding to the 50S subunit calculated at 2.25 Å resolution and contoured at 3 σ. There is density for a solvent atom interacting with the 2′ hydroxyl of A76 of the peptidyl-tRNA. (B) An anomalous difference map from a manganese-soaked twinned P21 crystal, calculated at 3 Å resolution and contoured at 4 σ. There are peaks in the map at known magnesium binding sites, but not near the 2′ hydroxyl of A76 or oxyanion hole. (C) A schematic diagram of RAP, the TSA in (A) and (B). Molecular Cell 2005 20, 437-448DOI: (10.1016/j.molcel.2005.09.006) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Comparison of Ligands Including or Lacking the 2′ Hydroxyl of A76 of the P Site Substrate (A) In complex with sparsomycin, CCdApcb (light green with light blue sparsomycin and orange rRNA) binds in a very similar fashion to CCApcb (dark green with dark blue sparsomycin and brown rRNA). (B) TS analogs RAA (dark green and purple with brown rRNA) and DAA (light green and pink with orange rRNA) bind to the PTC in a near identical manner. (C) An unbiased Fo − Fc electron density map of the active site from crystals soaked with CPmn and CCdApcb, calculated at 2.3 Å resolution and contoured at 3 σ. CCdApcb (green) out competes CPmn and binds to the A site, whereas the P site is empty. (D) Schematic diagrams of the ligands shown in (A). (E) Schematic diagrams of the TSAs shown in (B). (F) Schematic diagrams of CCdApcb and CPmn shown in (C). Molecular Cell 2005 20, 437-448DOI: (10.1016/j.molcel.2005.09.006) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 The Chirality of the Intermediate of the Peptidyl Transferase Reaction and the Identity of the Oxyanion Hole (A) An unbiased Fo − Fc electron density map of the TS analog DCSN (magenta and green) bound to the 50S subunit (rRNA in orange), calculated at 2.3 Å resolution, and contoured at 3 σ. The peptide mimic faces the exit tunnel, whereas the oxyanion mimic (a sulfur atom, in yellow) points away from A2486(2451). (B) An unbiased Fo − Fc electron density map of the complex of DCA (magenta and green) and the 50S subunit after fitting of the TSA, calculated at 2.3 Å resolution, and contoured at 3.5 σ. A peak is visible for a water molecule positioned by the N1 of A2637(2602) and the 2′ hydroxyl of methylU2619(2584), which acts as the oxyanion hole. (C) A schematic diagram of DCSN shown in (A). (D) A schematic diagram of DCA shown in (B). Molecular Cell 2005 20, 437-448DOI: (10.1016/j.molcel.2005.09.006) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 The Reaction Pathway for Peptide Bond Formation (A) The PTC with peptidyl-tRNA bound, represented by CCApcb (green) bound in the uninduced state with ChPmn (not shown). (B) With proper binding of the A site substrate (CChPmn, magenta), the PTC (rRNA in orange) and CCApcb (green) assume a conformation suitable for attack of the amino group of aminoacyl-tRNA on the ester carbon of the peptidyl-tRNA. (C) The attack yields an oxyanion-containing tetrahedral intermediate with S chirality. The oxyanion is stabilized by a water molecule coordinated by A2637(2602) and methylU2619(2584). TSA DCA (green and magenta) is shown. (D) The intermediate breaks down into the products of the reaction, passing the nascent chain (green) from the P site tRNA (green) to the A site aminoacyl-tRNA (magenta). (E–H) Schematic diagrams of the ligands displayed in (A)–(D). A theoretical pathway for the reaction is shown in Movie S1. Molecular Cell 2005 20, 437-448DOI: (10.1016/j.molcel.2005.09.006) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Possible Mechanism of the Peptidyl Transferase Reaction (A) The α-amino group attacks the ester carbon to yield the tetrahedral intermediate, which breaks down to deacylated tRNA and elongated peptidyl-tRNA. (B and C) The zwitterion intermediate may break down through a proton shuttle via the 2′ hydroxyl of A76 in the P site. This pathway might be concerted rather than sequential as shown. (D and E) The proton shuttle could also include the water molecule that interacts with the 2′ and 3′ hydroxyls of A76 in the P site. Modeled hydrogens are shown in white. Molecular Cell 2005 20, 437-448DOI: (10.1016/j.molcel.2005.09.006) Copyright © 2005 Elsevier Inc. Terms and Conditions