Promiscuous substrate recognition in folding and assembly activities of the trigger factor chaperone

Abstract

Trigger factor (TF) is a molecular chaperone that binds to bacterial ribosomes where it contacts emerging nascent chains, but TF is also abundant free in the cytosol where its activity is less well characterized. In vitro studies show that TF promotes protein refolding. We find here that ribosome-free TF stably associates with and rescues from misfolding a large repertoire of full-length proteins. We identify over 170 members of this cytosolic Escherichia coli TF substrate proteome, including ribosomal protein S7. We analyzed the biochemical properties of a TF:S7 complex from Thermotoga maritima and determined its crystal structure. Thereby, we obtained an atomic-level picture of a promiscuous chaperone in complex with a physiological substrate protein. The structure of the complex reveals the molecular basis of substrate recognition by TF, indicates how TF could accelerate protein folding, and suggests a role for TF in the biogenesis of protein complexes.

Figure 1. In Vivo TF Function

(A) Proteomic analysis of TF interactions in the E. coli cytosol. SDS PAGE analyses are shown for fractions of TF complexes first purified by metal-affinity chromatography from cytosolic lysates from TF-expressing cells and then separated by size exclusion chromatography. Separations from His₆-tagged wild-type ecTF (center) are compared with those from a tagless control (left) and from a ribosome-binding deficient (RBS⁻) mutant (right), His₆-tagged TF(FRK44-46AAA). Samples were purified with identical protocols. UV-absorbances at 280nm (straight line) and at 254nm (dashed line) are overlaid on the SEC profile of His₆-tagged TF, and gel lanes are from corresponding fractions. Elution volumes of markers are shown (Void, 440kD, 160kD and 50kD). (B) Cell viability dependence on TF. Overnight cultures of wild-type MC4100 and the derivative ΔtigΔdnaKdnaJ mutant cell line transformed with vectors harboring TF variants or the empty control were serially diluted and spotted on LB agar plates at indicated temperatures. (C) TF protection against cellular protein aggregation. The indicated strains of variously transformed cells were grown for approximately 4 h at specified temperatures. Aggregates were isolated as described (Tomoyasu et al., 2001) and visualized on 4–20% SDS–PAGE gels stained with Coomassie blue. White triangles mark the position of S7 as verified by mass spec sequencing of five S7 peptides from an excised gel band.

Figure 2. Characteristics of TF:Substrate Interactions in Vivo and in Vitro

(A) Expression of tmS7 and tmL22 in E. coli with and without tmTF. Gel lanes show proteins in total (T), supernatant (S), and pellet (P) fractions. Lanes correspond to: 1–3 (tmS7), 4–6 (tmS7 + tmTF), 7–9 (tmL22), 10–12 (tmL22 + tmTF). (B) Size-distribution analysis of the T. maritima TF:S7 complex by sedimentation velocity ultracentrifugation and Lamm equation modelling. Open diamonds correspond to tmS7 (Mw 17kD), full diamonds to tmTF (Mw 48kD) and crosses to the TF:S7 complex (Mw 65kD and 130kD).

Figure 3. Structure of TF in Complex with Ribosomal Protein S7

(A) Ribbon diagram of tmTF colored by domains. The N-terminal domain (NTD) is colored blue; the PPIase domain green; the C-terminal domain (CTD) red. NTD and PPIase are connected via a linker colored yellow here and subsequently colored red as part of CTD. Disordered TF residues shown as a blue dotted line correspond to the ribosome binding loop. (B) Ribbon diagram of TF colored by domains as in A except for linker, now red. The yellow ribbon corresponds to tmS7 as it is bound inside the CTD cleft. (C) TF:S7 complex. Two TF molecules (ribbons colored by domains as in b) encapsulate two S7 molecules (yellow molecular surfaces). (D) Surface representation of the TF:S7 complex, TF is colored red and blue, S7 is colored yellow. (E) Ribbon diagram of substrate free tmTF colored by domains as in B. The yellow ribbon represents a symmetry related tmTF with its NTD (solid) bound inside the CTD cleft. (F) Ribbon diagram of substrate free ecTF colored by domains as in B. The yellow ribbon represents a symmetry related ecTF with its NTD (solid) bound inside the CTD cleft.

Figure 4. S7 Interactions with TF

(A) S7 (yellow molecular surface) is encapsulated by NTD and CTD of apposed TFs. TF (colored by domains as in 3B) is rotated by approximately 180° along a vertical axis relative to 3B. (B) Superposition of S7 structures oriented as in A. tmTF bound tmS7 is colored red, ribosome bound Thermus thermophilus S7 (pdbid: 1FJG, chain G) green, isolated T. thermophilus S7 (pdbid: 1RSS) yellow and isolated B. stearothermophilus S7 (pdbid: 1HUS) blue. (C) Ribbon diagram of TF oriented as in A with the molecular surface of S7 colored grey. Included are hydrophilic and polar TF residues that contact S7 with hydrogen bonds or salt bridges. Carbon atoms are colored yellow, nitrogen atoms blue and oxygen atoms red. (D) TF in the ribosome-bound state. Ribosomal RNA is shown as a grey surface, 50S proteins are colored blue, 30S proteins are colored green. TF docks on the 50S subunit by binding to proteins L23 (pink) and L29 (cyan). TF is shown as a ribbon diagram with S7 as a yellow surface. S7 as bound to 30S is also shown as yellow surface.

Figure 5. Surface Representations of T. maritima and E. coli TF

(A) Contact surfaces. The molecular surface of TF (middle) is colored by domains as in 3B but with the imprint of bound S7 colored in yellow. The dark ribbon shows S7. Molecular surfaces of S7 (outside) are rotated to display the imprint of bound TF. Blue residues contact NTD, green contact PPIase, red contact CTD and magenta contact both NTD and CTD. (B) Electrostatic potential of TF and S7. Molecular surfaces are oriented as in A and S7 is drawn as a yellow ribbon. Surfaces are colored in degrees of positive (blue) and negative (red) potential. (C) Hydrophobic patches on the tmTF surface. Vicinal apolar atoms that form continuous hydrophobic surfaces are colored blue. (D) Comparison of the TF:S7 interface with approximately 44,000 structurally defined interfaces between pairs of protein domains catalogued in PYBASE (Davis and Sali, 2005). Properties of the TF:S7 interface are represented by the red and blue spheres (dimer and tetramer, respectively). PYBASE interaction sets are represented by grey spheres. Sc corresponds to Shape Complementarity Value, BSA corresponds to Buried Surface Area and P/NP corresponds to the ratio of polar versus non-polar interfacial residues.

Figure 6. TF and Ribosome Biogenesis

(A) Surfaces of T. thermophilus 30S (tt30S) protein components excluding S7 are shown in red, 16S RNA in blue; S7 is colored grey. (B) Contact imprints on the surface of T. thermophilus S7 (ttS7) oriented as in 5a. Surfaces colored blue uniquely contact tt30S, tmS7 homologs of surfaces colored yellow uniquely contact tmTF, and surfaces colored magenta contact both tt30S and tmTF. (C) Polysome profiles of wild-type cells grown at 30°C and wild-type and Δtig cells grown at 44°C. (D) Average relative peak-height ratios of 70S/30S (light-gray), 70S/50S (dark-blue) and 50S/30S (light-blue) from the indicated strains including TF overexpression (OX) at 44°C are shown. (E) Model of cytosolic TF function. TF could sequester nascent proteins or bind fully synthesized but unstable subunits, like S7, and remain stably associated with these subunits until productive folding or assembly occurs.