Dependency map of proteins in the small ribosomal subunit

Abstract

The assembly of the ribosome has recently become an interesting target for antibiotics in several bacteria. In this work, we extended an analytical procedure to determine native state fluctuations and contact breaking to investigate the protein stability dependence in the 30S small ribosomal subunit of Thermus thermophilus. We determined the causal influence of the presence and absence of proteins in the 30S complex on the binding free energies of other proteins. The predicted dependencies are in overall agreement with the experimentally determined assembly map for another organism, Escherichia coli. We found that the causal influences result from two distinct mechanisms: one is pure internal energy change, the other originates from the entropy change. We discuss the implications on how to target the ribosomal assembly most effectively by suggesting six proteins as targets for mutations or other hindering of their binding. Our results show that by blocking one out of this set of proteins, the association of other proteins is eventually reduced, thus reducing the translation efficiency even more. We could additionally determine the binding dependency of THX--a peptide not present in the ribosome of E. coli--and suggest its assembly path.

Figure 1. The 30S Subunit In Vitro Experimental Assembly Map for E. coli

The primary, secondary and tertiary binding proteins are shown in black, orange, and blue, respectively. Arrows indicate facilitating effect of binding of one protein on another. Adapted from the review of Culver [6] based on the work of the Nomura Laboratory [4,5].

Figure 2. The Influence Network of the Presence of Single Proteins on the Binding Stability of the Other Proteins

The three clusters and the four unaffected proteins are visible. The colors of the arrows indicate Δ_r (the larger the number, the stronger the influence). The arrows point from the removed protein to the affected protein, e.g., removal of S4 alters the binding strength of S5 with the “influence strength” of 3. The gray arrows indicate the interaction that is due to very small relative energy change and involves the “suspicious” protein S12 (see text for details).

Figure 3. Color-Coded Contribution to Destabilization, Determined by Eigenvector Analysis of the Two-Protein Removal Experiment

Red diamonds indicate contributions from the smallest and blue diamonds from the largest eigenvalue, respectively. The symbol × indicates influences that were already found in the one protein removal experiment of the previous section. The green × were discussed in the previous section. The respective energy differences of those two interdependencies are rather small and enter into the magnitude of entries of the eigenvectors only slightly. Entries for were marked if the value deviated more than 10% from their most likely value, while for this threshold was set to 1%, reflecting the respective order of magnitude of λ_1/20.

Figure 4. The Additional Destabilization from the Two-Protein Removal Case Mapped onto the E. coli Map (Blue Diamonds in Figure 3)

The green proteins are not in contact with any other protein. The peptide THX was placed close to the proteins that influence its binding stability. All interactions are non-local, as the respective proteins are not in proximity.

Figure 5. The Angle α_s between the Respective Eigenvectors for an Interaction Strength κ of Protein S12 and for the Full Parameter Set

The broken vertical lines indicate the average values used in the first two validation experiments in the section Sensitivity to Parameters. The full parameter set refers to the one in the Results section under Influence Map, Second-order stabilization revealed by two-protein removal. Inset: Illustration of the deviation in the eigenvector contributions for the two different average interaction values (shown for the worst case of S7).