5.1 Intermembrane space domains of mitochondrial translocases
In this study, ensemble view of structural properties and dynamics in intermembrane space domains of mitochondrial translocases have been investigated.
We showed that Tim23ims and Tom22ims are disordered in solution possessing transient secondary structural elements. On the other hand, Tim21ims has been shown to exhibit concentration dependent effects and exist in monomer-dimer equilibrium in solution. The monomeric Tim21ims display dynamics in β-strands 2, 3 and 4 and further studies using relaxation dispersion and nuclear spin relaxation (R1rho and Rex) experiments are required to understand the role of these dynamics for either dimerization or various other protein-protein interactions. The expression, purification and solution conditions for Tim50ims have been optimized and further characterization including sequence-specific resonance assignments are currently in progress.
5.2 Presequence-intermembrane space domain interactions
Presequence recognition guides the preprotein translocation in mitochondria.
This study has identified the presequence binding domains of Tim23ims and Tom22ims at single residue level as residues 71-84 and 122-133 respectively. Mutational analysis of the presequence-Tim23 complex showed that both the N and C-terminus of presequence binds to the presequence receptor and R17 in C terminus of presequence is important for binding Tim23ims. Collectively, the Tom22ims-presequence and
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Tim23ims-presequence interaction suggest that a combination of hydrophobic and electrostatic interactions is critical for formation of the presequence-receptor complex.
The affinity Tim23ims-presequence and Tom22ims-preseequnce interactions are in mill-molar range indicative of a lower stability of the complex. Our data also suggests that Tim21ims does not act as presequence receptor.
This study has also established the presence of a presequence binding site in Tim50 164-361. With the availability of assignment, structural insights into the binding site will be obtained and can be compared to full length Tim50ims.
5.3 Interactions between ims domains of the mitochondrial translocases
Biological macromolecular machineries such as TIM23 translocases are functionally complex in nature. The functions of such machineries are encoded by its interacting subunits. Along the same line, the interface of its interacting subunits could primarily be important to modulate the comprehensive function of this biological machinery.
The study of various protein-protein interactions between various subunits of TIM23 translocases is important for the understanding of underlying molecular mechanisms involved in their function. We have used NMR spectroscopy based titrations of various subunits to understand the molecular mechanisms for the functionality of TIM23 at residue specific level (cf. Table 15).
This study envisions the need of disorder in Tim23ims to be multifunctional and establishes it as a hub protein in the intermembrane space of yeast mitochondria.
Most importantly, interactions of disordered Tim23ims are central to this study and that showed active involvement of common hydrophobic linear motifs in mediating multiple interactions with various ligands such as mitochondrial membrane, Tom22ims, Tom40ims, Tim50ims, Tim21ims and presequences. However, we have deduced that the inter-subunit interactions differ in affinity ranging from a micro-molar range affinity in case of Tim23-Tim50 to a milli-molar in case of Tim23ims-Tom22ims. Interestingly, the various low affinity interactions could be collectively involved in the functioning of the TIM23complex.
160 Summary and Outlook
Furthermore, this study has identified the interaction between Tim21-Tim23ims and modeled the structural basis for interaction of Tim21ims-Tim23 mediated by three hydrophobic linear motifs of Tim23 into a single binding site of Tim21ims.
K139 and Y141of Tim21ims are found to be key residues in the binding interface of Tim21ims-Tim23ims have been mutated. NMR titrations of mutant Tim21ims with Tim23 ims would be performed to validate the model of the complex.
Our studies on Tim21ims and Tom22ims showed the absence of any significant interaction between them at the translocation contact site whereas Tim23ims-Tom22ims and Tim23ims –Tom40ims interacts with a lower affinity in the milli-molar range.
Future studies will focus on identification of Tom40ims residues involved in binding to Tim23ims. For this, the labeled Tom40ims peptide would be obtained using a similar strategy as that of Tom22ims using Z2-fusion tag.
Table 15: Comprehensive view of the binding site and affinities of interactions between the intermembrane space domains in yeast mitochondrion deduced during this study. The tabular check board involves unlabeled subunit along the row and labeled subunit along the column. Each checkbox various interactions. Solution condition of Tim50ims has been optimized. Thus, backbone assignment and structural characterization of binary interactions of Tim50ims with Tim23ims, Tom22ims and Tim21ims will form the basis for future studies.
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Additional interactions of Tim23ims with Pam17ims, Tim17ims and the potential role of Mgr2 in modulating the Tim21-Tim23 complex will also be of interest in future.
In conclusion, we showed that disordered Tim23ims acts as the hub protein and demonstrated its role in various interactions involving the other subunits of the TIM23 complex at single residue level. This study provides a basis for the future studies to understand the molecular mechanisms for the function of the TIM23 complex in mitochondrial import.