The Tim8-Tim13 complex has multiple substrate binding sites and binds cooperatively to Tim23

Abstract

The Tim8-Tim13 complex, located in the mitochondrial intermembrane space, functions in the TIM22 import pathway that mediates the import of the mitochondrial carriers Tim23, Tim22, and Tim17 into the mitochondrial inner membrane. The Tim8-Tim13 complex assembles as a hexamer and binds to the substrate Tim23 to chaperone the hydrophobic Tim23 across the aqueous intermembrane space. However, both structural features of the Tim8-Tim13 complex and the binding interaction to Tim23 remain poorly defined. The crystal structure of the yeast Tim8-Tim13 complex, reported here at 2.6 A resolution, reveals that the architecture of the Tim8-Tim13 complex is similar to those of other chaperones such as Tim9-Tim10, prefoldin, and Skp, in which long helices extend from a central body like tentacles from a jellyfish. Surface plasmon resonance was applied to investigate interactions between the Tim8-Tim13 complex and Tim23. The Tim8-Tim13 complex contained approximately six binding sites and showed a complex binding interaction indicative of positive cooperativity rather than a simple bimolecular interaction. By combining results from the structural and binding studies, we provide a molecular model of the Tim8-Tim13 complex binding to Tim23. The regions where the tentacle helices attach to the body of the Tim8-Tim13 complex contain six hydrophobic pockets that likely interact with specific sequences of Tim23 and possibly other substrates. Smaller hydrophobic patches on the tentacles themselves likely interact nonspecifically with the substrate's transmembrane helices, shielding it from the aqueous intermembrane space. The central region of Tim23, which enters the intermembrane space first, may serve to nucleate the binding of the Tim8-Tim13 complex, thereby initiating the chaperoned translocation of Tim23 to the mitochondrial inner membrane.

Fig. 1. Comparison of Tim8–Tim13 and Tim9–Tim10 structures

(a) Ribbon diagrams of the Tim8–Tim13 (TIM 8·13) complex and the Tim9–Tim10 (TIM 9·10) complex (PDB ID 2bsk) illustrate similarities in the oligomeric assembly. In this view, the “tentacle” helices are pointed toward the viewer. The largest differences appear in the N-terminal helix of Tim8 and Tim10 (blue and cyan helices lining the central pore). Red sticks indicate the location of conserved intrachain disulfide bonds. (b) Superposition of the Tim8–Tim13 and Tim9–Tim10 complexes. (C) Structure based sequence alignment of Tim8, Tim9, Tim10, and Tim13. The secondary structure elements (top) are mapped to the four sequences. This figure was prepared using the programs JalView and PyMol .

Fig. 2. The Tim8–Tim13 complex and Tim13 monomer are folded

(a) Schematic of the recombinant Tim8–Tim13 and Tim13 constructs that were purified from E. coli. The black box represents the location of the His-tag and the black triangle represents the ribosomal binding site. (b) Schematic showing the location of the peptides derived from Tim23. The transmembrane domains (designated TM1–TM4) are marked by white boxes. (c) Recombinant Tim8–Tim13 complex, Tim13, and a mitochondrial lysate (mito) were separated by BN-PAGE. After transfer to a PVDF membrane, the membrane was blotted with a polyclonal antibody against Tim13. (d and e) CD spectral analysis shows that the recombinant Tim8–Tim13 complex and Tim13 contain mostly alpha-helical structure.

Fig. 3. Tim13 bound to pep^91–103 in a bimolecular interaction

For SPR analysis, the Tim13 monomer was coupled to the Ni²⁺-coated sensor surface at 1500 RU. Pep^91–103 [0–500 µM] was analyzed for binding to the Tim13 monomer (described in Materials and Methods) and the saturation curve is shown.

Fig. 4. Pep^91–103 bound cooperatively to the Tim8–Tim13 complex

(a) Sensorgram showing the binding of pep^91–103 to the Tim8–Tim13 complex. The complex was coupled to the Ni²⁺-coated sensor chip and binding of the Tim23 pep^91–103 [0–630 µM] was investigated (described in Materials and Methods). A representative SPR sensorgram in the ligand series is shown. (b) The kinetics of binding were too rapid for curve fitting, so the equilibrium sensorgram values from ‘a’ (RU at equilibrium vs. pep^91–103 concentration) were used to plot a saturation curve. Two representative experiments are marked by triangle and square symbols. (c) Because the analysis from ‘b’ did not show the expected 1:1 binding model, a Hill representation of the pep^91–103 — Tim8–Tim13 complex association was graphed. A function of the bound pep^91–103 [log B/(B_max - B)] was plotted against the logarithmic concentration of the amount of injected pep^91–103. The slope provides an estimate of the Hill coefficient (n), and the intercept with the x-axis provides an estimate of the concentration of free pep^91–103 required to occupy half of the binding sites.

Fig. 5. The Tim8–Tim13 complex displayed different binding properties with different peptides derived from regions of Tim23

(a) Peptides derived from Tim23 (see Fig. 2b) were analyzed for binding to the coupled Tim8–Tim13 complex. The saturation curve was plotted. (b) Binding between pep^77–88 from ‘a’ was analyzed by the Hill equation as described in Fig. 4c.

Fig. 6. The addition of pep^91–103 increased the binding affinity of Tim8–Tim13 for pep^136–148

(a) Pep^91–103 was maintained at a constant concentration of 63 µM and pep^136–148 was added over a range of 0–400 µM in the buffer (designated pep^136–148 + 63 µM pep^91–103). Binding to the Tim8– Tim13 complex was measured. The binding curve for pep^136–148 alone was included for a reference. (b) The concentration of pep^91–103 and pep^136–148 were varied inversely such that the total concentration was fixed at 500 µM in the SPR analysis with Tim8–Tim13. The binding of pep^91–103 alone was included for a reference. (c) The data from ‘b’ was plotted using the Hill equation as in Fig. 4c.

Fig. 7. Model showing on the Tim8–Tim13 complex binds to Tim23

(a) Surface hydrophobicity diagrams of the Tim8–Tim13 complex. The hydrophobic residues are shown in pink and hydrophilic residues shown in green. N and C-terminal residues disordered in the Tim8–Tim13 structure are modeled here as α-helices. (b) Model proposing how Tim8–Tim13 binds to the Tim23 substrate. Tim23 folding is based on weak homology to AAC2. Magenta color segments correspond to peptide sequences shown to bind Tim8–Tim13 and dark magenta segments mark conserved residues that are predicted to bind to the substrate. Evolutionarily conserved residues in Tim13 Ile37, Glu40, Ala42, Ala44, Asn45, Ala46 and Leu49 are represented as sticks. See Discussion for details.