Recognition of the Regulatory Nascent Chain TnaC by the Ribosome Leonardo G. Trabuco, Christopher B. Harrison, Eduard Schreiner, Klaus Schulten  Structure  Volume 18, Issue 5, Pages 627-637 (May 2010) DOI: 10.1016/j.str.2010.02.011 Copyright © 2010 Elsevier Ltd Terms and Conditions

Structure 2010 18, 627-637DOI: (10.1016/j.str.2010.02.011) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 1 Structure of TnaC in the Exit Tunnel Shown are selected residues of the 23S rRNA, the portions of ribosomal proteins L4 and L22 forming the exit tunnel's constriction site, and ten models of the nascent chain found to be consistent with cryo-EM data (Seidelt et al., 2009). 23S rRNA residues displayed in black represent bacterial sequence signatures (Roberts et al., 2008). Movie S1 shows a 360° view of this figure. See also Figure S1. Structure 2010 18, 627-637DOI: (10.1016/j.str.2010.02.011) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 2 Nonredundant Sequence Alignments Shown are TnaC (A) and the regions of ribosomal proteins L4 (B) and L22 (C) comprising the exit tunnel's constriction site. The sequence alignments and corresponding structures are colored by sequence similarity using the BLOSUM 30 matrix and a red-white-blue color scale, with most conserved residues shown in blue. For TnaC (A), complete sequences are shown, that is, there exists a stop codon at the end of each presented sequence. See also Table S1. Structure 2010 18, 627-637DOI: (10.1016/j.str.2010.02.011) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 3 Recognition of Key TnaC Residues by the Exit Tunnel (A) Interaction energies between W12 (TnaC) and R92 (L22) calculated using the MD force field from the ten independent simulations starting from the different TnaC models shown in Figure 1 (labeled model 0–9). Several trajectory frames were selected as indicative for cation-π interactions (circles; see Figure S2A), for which the interaction energies were also determined using QM calculations (diamonds; full results given in Table S2). The same procedure was followed for all positively charged residues in the vicinity of W12 (Figure S2B). (B) Snapshot from one of the simulation trajectories showing a cation-π interaction between W12 (TnaC) and R92 (L22) as well as a salt bridge between D16 (TnaC) and K90 (L22) (see Figure 4). See also Figure S2 and Table S2. Structure 2010 18, 627-637DOI: (10.1016/j.str.2010.02.011) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 4 Salt Bridges Formed between D16 (TnaC) and the Exit Tunnel in all Simulations The plots show the distance between the center of mass of the oxygen atoms in the acidic side chain and the center of mass of the nitrogen atoms in the basic side chain. For each interaction pair, only simulations in which a stable salt bridge was observed are shown. The ten independent simulations starting from different TnaC models shown in Figure 1 are labeled model 0–9. See also Figure S3. Structure 2010 18, 627-637DOI: (10.1016/j.str.2010.02.011) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 5 Frequency of Contacts between TnaC Residues I19 and V20 and Ribosomal Residue Contacts were defined based on a cutoff distance of 3.5 Å between heavy atoms of side chains, and the last 40 ns of each of the ten independent simulations were considered in the analysis. See also Table S3 and Figure S4. Structure 2010 18, 627-637DOI: (10.1016/j.str.2010.02.011) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 6 Path of the TnaC Peptide inside the Exit Tunnel (A) Atomic model of ribosomal proteins L4 and L22 obtained from the 70S·TnaC cryo-EM map (Seidelt et al., 2009), along with the density corresponding to TnaC. For comparison, the swung conformation of the tip of L22 is shown in red (PDB 1OND) (Berisio et al., 2003). (B) Conformations explored by TnaC during the MD simulations according to clustering analysis of the Cα atoms (see Experimental Procedures). Two possible paths for a polyalanine nascent chain had been previously proposed, namely case-over and case-under R92 from L22 (Ishida and Hayward, 2008). Structure 2010 18, 627-637DOI: (10.1016/j.str.2010.02.011) Copyright © 2010 Elsevier Ltd Terms and Conditions