This comprehensive and high resolution study provides five main insights into the quaternary structure of the TIM23 complex. First, after importing each of 54 different Tim23p monocysteine mutants into intact and energized mitochondria, specific regions of a Tim23p molecule were shown to be adjacent to Tim17p, Tim50p, and at least one other Tim23p protein in a fully assembled and functional TIM23 complex (see below). Second, the structural proximity and hence arrangement of proteins within the TIM23 complex is dependent on the Δψ. Third, the Δψ-dependent conformational rearrangements are reversible, suggesting that they represent a true physiological response to the energized state of the IM. Fourth, different protein–protein proximities in the TIM23 complex are detected when a substrate is trapped inside the translocase, and these changes reflect Δψ-dependent changes. Fifth, these combined results show that an assembled TIM23 complex exists in a minimum of three states (energized, de-energized, and energized with substrate) within the IM that differ both structurally and functionally. The TIM23 complex is therefore a dynamic multicomponent assembly whose functional state is dictated, and presumably regulated, by changes in quaternary structure in response to cellular conditions.
Tim23p TMS1 is adjacent to Tim17p (A and A) because nine residues of TMS1 (from Tyr105 to Phe114) cross-linked to Tim17p. Interestingly, these sites are found on two oppositely oriented faces of the TMS1 helix when mapped onto a helical projection (D). Although rotational degeneracy of a transmembrane helix in the TIM23 complex could explain such differential cross-linking, the specificity with which cross-linked adducts were formed on the two faces strongly suggests that TMS1 of Tim23p contacts two separate Tim17p molecules on opposite sides. Given the topological placement of native Cys residues within Tim17p (A), its two Cys residues in the N-terminal part of TMS4 are the most likely cross-linking partners for Tim23p TMS1 monocysteine mutants.
Figure 8. Summary of Tim23p cross-linking results. Schematic representation of imported [35S]Tim23p monocysteine mutants (red) and (A) Tim17p (yellow), (B) endogenous Tim23p (green), and (C) Tim50p (cyan) in the TIM23 complex. Stars represent approximate sites (more ...)
Among the TIM23 complex structural relationships characterized, the juxtaposition of Tim23p TMS1 and Tim17p was the most sensitive both to changes in Δψ (A) and to the presence of substrate (). It is not clear whether the Δψ- and substrate-dependent changes in Tim23p TMS1-Tim17p cross-linking have the same molecular basis (i.e., whether the collapse of the Δψ or the addition of substrate elicit the same conformational changes in the TIM23 complex). But whatever the origin, this dynamic Δψ- and substrate-dependent proximity may very well be related to the recently identified role of Tim17p in regulating the substrate-dependent voltage gating of Tim23p (Meier et al., 2005
; Martinez-Caballero et al., 2007
The Tim23p IMS region is proximal to other Tim23p subunits in the TIM23 complex (B and B). The wide range of sites in the [35
S]Tim23p mutants that cross-link to native Tim23p presumably through its single Cys in the IMS indicates a high degree of Tim23p conformational flexibility in the IMS. A decrease in the Δψ leads to a modest reduction in cross-linking between Tim23p IMS sites and other Tim23p subunits (B), whereas the presence of a translocation intermediate significantly reduces cross-linking between Tim23p sites in the C-terminal half of the IMS region and other Tim23p subunits (B). These results are consistent with previous work, indicating that Tim23p dimerizes via a putative leucine zipper structure in the C-terminal half of its IMS domain in a manner dependent on the functional state of the channel: dimer formation requires the Δψ, and matrix targeting presequences stimulate dimer dissociation (Bauer et al., 1996
). Finally, the strong effect of antimycin A on Tim23p IMS cross-linking (B) could result in part from the recently described coupling between Tim21p of the TIM23 complex and respiratory complexes III-IV (van der Laan et al., 2006
). Alternatively, the effect could arise from the binding of antimycin A to a site other than complex III (e.g., Tzung et al., 2001
The extreme C-terminal end of the IMS region of Tim23p is proximal to Tim50p (C and C), consistent with previous work showing an interaction between the IMS region of Tim50p and the C-terminal half of the Tim23p IMS domain (Geissler et al., 2002
; Yamamoto et al., 2002
; Mokranjac et al., 2003
). In addition, we unexpectedly found cross-links between Tim50p and TMS1 of Tim23p (C and C). Given that the putative cross-linking site on Tim50p (Cys268 in the mature protein) is in the C-terminal IMS domain, this suggests that either this region of Tim50p protrudes into the bilayer and/or that Tim23p has the conformational freedom to move TMS1 into the aqueous IMS to some degree. In any case, a helical projection of the sites that cross-link Tim23p TMS1 to Tim50p (D) suggests that, in contrast to well-defined sites that cross-link Tim23p TMS1 to Tim17p (D), there is significant rotational freedom between TMS1 and Tim50p such that cross-linking occurs over most of the helical circumference.
The cross-linking observed between Tim23p and Tim50p may also provide some insight into the functional role of Tim50p. The reactive Cys in Tim50p falls within a region of the IMS domain that is homologous to NIF/CDCc domains of CTD phosphatases (residues 165-344; Geissler et al., 2002
; Mokranjac et al., 2003
). Although the possible role of this domain in Tim50p function is not known, the potential specific interaction between Tim23p TMS1 and this region may provide clues to its function. Our unexpected observation that cross-linking between the IMS region of Tim23p and Tim50p increased when the Δψ collapsed suggests that Tim23p-Tim50p proximity and/or affinity increases upon loss of the Δψ. When coupled with the recent observation that the IMS region of Tim50p maintains the permeability barrier across the IM by promoting the closed state of the Tim23p channel (Meinecke et al., 2006
), a regulatory mechanism is suggested. Specifically, the closer interaction between Tim23p and Tim50p that we detected upon lowering the Δψ may have evolved to maintain the IM permeability barrier and prevent unregulated collapse of IM gradients.
Our comprehensive cross-linking study has therefore provided unprecedented resolution in characterizing the proximity between TIM23 complex subunits, thereby significantly extending our understanding of the TIM23 complex quaternary structure. Although studies describing protein interactions based on coimmunoprecipitation cannot distinguish between direct contact and association through an intermediary subunit, a covalent bond that directly cross-links two macromolecules shows unambiguously that residues from each protein were adjacent. Moreover, the formation of covalent adducts usually identifies protein surfaces that interact for functional or structural reasons. But even though a lack of cross-linking cannot be taken as evidence for lack of proximity, the cross-linking approach used here maps the proximity of specific helical faces to other proteins at high resolution.
Several models could account for the multiple Δψ- and substrate-induced conformational changes in quaternary structure characterized here. Two possibilities are depicted in D. Partial de-insertion of TMS1 from the membrane (case 1) would require that the energetic penalties of moving nonpolar residues into the aqueous phase and disrupting any associative energies with other TMSs in the membrane be offset by some compensatory binding energy (e.g., binding to Tim50p). Alternatively, loss of membrane potential and/or substrate addition may, for example, induce helix tilting and expansion of an aqueous crevice in this region (case 2). Future work will determine to what extent either model, or some combination thereof, is correct. But the conformational changes characterized by this study have provided insight into the Δψ- and substrate-dependent dynamics of the TIM23 complex and hence will direct further investigations.