Model for Tim21-Tim23 complex

Intermolecular NOEs are most commonly used restraints for determination of protein complexes using NMR. The limited solubility of Tim21ims-Tim23ims complex and high Kd (150µM) prevented the use of intermolecular NOEs as restrains. The CBCACONH of Tim23ims in excess of Tim21ims (Tim23: Tim21:: 0.15mM: 1.2mM) was measured. The correlation peaks for residues of Tim23ims involved in binding to Tim21ims, which undergoes intermediate exchange on the NMR timescale between Tim23 free and bound state (that were broadened in ¹H-¹⁵N HSQC), could not encode the transfer of the magnetization from its H^N to C^α and C^β of the previous amino acid and hence were absent in the spectra. However, observable encoded residue showed no significant changes in C^α chemical shift indicating that non-interacting regions of Tim23ims remains disordered in Tim21ims bound state.

Further, ¹H-¹⁵N HetNOE of Tim23ims in complex with 16 fold excess of unlabeled Tim21ims was measured that shows an average value for all residues below

±0.4 indicating that Tim23ims remain dynamic at nanosecond to picosecond time scale in its bound state. On comparing the ¹H-¹⁵N Het NOE values of Tim23ims in bound state (in complex with Tim21ims) with Tim23ims in free state, it was found that C-terminal residues (72-96) showed small yet significant changes .The above results suggest that the Tim23 remains dynamic in the Tim21 bound state with local ordering in the Tim21binding region.

3.5 Structural properties of intermembrane space domains of Tim21-Tim23 127

Figure 50: Tim21 bound Tim23ims is highly dynamic: ¹H-¹⁵N-HetNOE values of Tin23ims in 16 fold excess of Tim21 (grey) and no excess (black) as a function of residue number of Tim23ims. The region at C-terminus, which shows deviations, is highlighted with pink with box.

In order to determine the ensemble view of binding of the above determined three linear motifs namely peptide 1 (residue 2-7, SWLFGD), peptide 2 (residue 67-74 GVEYLDLE) and peptide 3 (residue 90-96, SRWTDD) to a single binding site in Tim21, Rosetta FlexPepDock was used to dock them. Rosetta FlexPepDock can provide the complete flexibility to peptide and the receptor at the binding interface and can be used to refine the interface residues if the approximate binding site is known (Raveh et al. 2011). Indeed, this was the scenario with Tim21-Tim23 where individual binding peptides of Tim23 were known along with binding site in Tim21ims but high resolution binding interface of Tim21-Tim23 complex needs to be defined.

We performed the ab-initio docking and results for the Tim21-peptide 1 complex is based on the top scoring rosetta models whereas for peptide 2 and 3 Tim21 peptide complex, 25000 conformers were obtained and based on rosetta energy score the top 500 models were clustered with a backboneRMSD of 2 Å and binding interface of each cluster was analyzed. The first five clusters for peptide 2 have 17, 11, 8, 5, and 6 conformers whereas peptide 3 has 58, 58, 37, 41 and 29 conformers.

128 Results

The results of docking (Figure 51) revealed that the consensus interactions are formed involving the key residues K139 and Y141 in Tim21with a negatively charged residue of Tim23 (D or E) and the hydrophobic residue (L) or aromatic residue (W) respectively. The Y 141 acts as a main hydrophobic anchor that interacts either to the leucine residue or to the aromatic residue of Tim23ims. Additionally, same peptide can also have the switch of hydrophobic anchor L to Y and W to Y interactions (peptide 1, Figure 51-lower panel).

Furthermore, these peptides are also accommodated at the binding site of Tim21ims with the help of π-π interactions with side chain of F109 if three bulky hydrophobic residues are present for example in peptide1 and 2 that has the WLF and EYLDL respectively.

All the three peptides interact to a single binding site and suggests that the peptides of Tim23ims can dynamically bind in the small hydrophobic cleft of Tim21ims with K139 and Y114 mediating the key interactions. Different clusters of peptide 2 and 3 converge well indicating that the conformer represented in Figure 51B and C are most probable near native solution for these binding motifs in Tim21-Tim23 complex.

However for peptide 1, the switch of L to F to interact with Y141 of Tim21ims was observed. Thus, interaction of peptide 1 with Tim21 cannot be described by single conformation rather can be viewed as the presence of consensus interactions in multiple conformers with each of them representing a member of cluster (Figure 51 A, lower panel). In peptide 3, K139 and Y141 of Tim21 interact with W conferring cation-π and π- π interactions at the interface of Tim21-Tim23 complex.

In conclusion, three linear motifs of Tim23 can be structurally adapted in a shallow single binding site of Tim21ims and residues K139 and Y 141 of Tim21 ims plays an important role at binding interface.

3.5 Structural properties of intermembrane space domains of Tim21-Tim23 129

Figure 51: Model of Tim23ims-Tim21ims complex derived from docking Tim23 peptides to Tim21ims using Rosetta FlexPepDock. Three interacting peptides of Tim23ims based on chemical shift and intermolecular PRE data of Tim21ims and Tim23ims were placed near the binding site in Tim21 (10Å away). Column (A), (B) and (C) represents the results of three different peptides of Tim23 used for docking to monomeric lowest energy NMR structure of Tim21ims. Upper panel represents the peptide sequence and residue number of Tim23ims and the important residues at the docking site is color coded with leucine as blue, aromatic residues as orange and negatively charged residue as yellow. Middle panel shows the surface representation of binding interface of Tim21-Tim23 complex with Tim21ims (light blue) and docked Tim23 peptides. Lower panel shows ribbon representation of binding interface of Tim23 peptides in Tim21ims as elucidated from the lowest energy member of top 5 clusters derived from Rosetta FlexPepDock. The side chains of the key residues of Tim21, K139 and Y141 are shown in red and magenta spheres respectively and important side chains of Tim23ims are shown as sticks and color coded described previously.

130 Results