The glycine rich motifs in Mic10’s might not be the only site driving the oligomerization

3.4.1 TM2 domain homo-oligomerization is highly likely by bioinformatics predictions

Since both Mic10’s TM domains have a high content of glycine rich motifs (GxxxG), we wonder about the propensity of these TM domain to interact individually.These motifs are well known to promote helix-helix interaction (228-230). Anderson et al. observed high propensity of the helix-helix interaction in peptides containing ZxxxZ motif (Z = small amino acid residues like G, S or A). Interestingly, including this definition into Mic10 an extended packing-packing region is observed in both TM domains (Fig. 4.14B). On the one hand, TM1

11 10 9 8 7

shows a AxGxGxGxxxS motif, thus three ZxxxZ motifs (Fig. 4.14B) or one GxxxGxxxZ motif (Fig. 4.14B, red). On the other hand, TM2 has a GxGxGxGxGxAxG motif depicted in four ZxxxZ motifs (Fig. 4.14B) or two ZxxxZxxxZ motifs (Fig. 4.14B, red for GxxxGxxxZ and black bold for GxxxGxxxG). To investigate the strength of the interaction between the TM domains, both TM domains were submitted to preddimer a bioinformatic tool, which predicts the probability of two transmembrane α-helical domains to dimerize (231). The three combinations TM1-TM1, TM2-TM2 and TM1-TM2 were submitted. Interestingly, different dimer structures were predicted by preddimer：whereas for TM1 a higher dimerization score is obtained for a parallel TM1-TM1 homo-dimer, referred to “parallel-dimer” (score of 4.8, fig. 4.14A, TM1); for TM2 the highest score is obtained for two helices interacting with each other at a specific location of the TM2 domain, the RGY region, referred to “cross-dimer”(score of 6.8, fig. 4.14B, TM2).. In the cross-dimer situation, where the score obtained is the highest, the dimer seems to be stabilized by a cation-π interaction between the arginine and tyrosine side-chains. Interestingly, the score obtained for dimerization between the two TM domains (TM1-TM2) drops to 3.0 suggesting that Mic10 homo-oligomerization is more likely to occur between the interfaces of the “parallel-dimer” (TM1-TM1) and “cross-dimer” (TM2-TM2) rather than the interface of the hetero-dimer (TM1 – TM2). Judging from the number of the ZxxxZ motifs, it is not surprising that the “cross-dimer” (TM2-TM2) interaction obtains the higher score since it contains more GxxxG motifs. Additionally, the presence of L, V and I residues neighboring the glycines of the GxxxG motifs in both domains, TM1 and TM2, could further stabilize the helix-helix interaction. Such residues have been reported to increase the stability for helix-helix packing (230).

Overall, the bioinformatics predictions are consistent with the available biochemical studies, suggesting that oligomerization is in fact a prominent behavior for Mic10. However, the predictions that TM2 domain has a higher propensity to dimerize opposes to the biochemical data. Previous biochemical studies showed that TM1 domain is responsible for the oligomerization of Mic10. Mutations at residues G50 and G52, found facing in opposite direction of the TM1, to alanine induce the complete loss of Mic10 homo-oligomerization (194). However, so far no atomic data reported have determined the helix-helix packing interface or explained the relationship between the ZxxxZ motifs present in Mic10 regarding oligomerization and membrane curvature. Additional biochemical data targeting the RGY

region to disrupt the predicted cation-π interaction, which seems to be involved in stabilizing the “cross-dimer”, would be necessary to further shed light into the possible role of the TM2 domain in the Mic10 oligomerization process. Considering that the predicted length of the TM domains are of 19/25 residues for the TM1 domain and 19 residues for the TM2 domain, and that the total number of residues is of 96, Mic10 has more than half of the protein exposed to the mitochondrial interspace. This suggests the possibility that the soluble domains are involved in the oligomer formation as well.

Figure 4.14. Predicted helical-helical interaction from the different Mic10 transmembrane domains.

A, shows the TM1 helical structure prediction including the position of the two glycine (Gly50 and Gly52, in pink) responsible for homo-oligomerization. B, shows the amino acid sequence of Mic10 with the (Z)xxx(G) motifs in green, (G)xxx(G)xxx(Z) motifs in red and the (G)xxx(G)xxx(G) motifs in black bold. From C to D, the predicted parallel-dimer (TM1-TM1, in C), cross-dimer (TM2-TM2, in D) and the hetero-dimer (TM1-TM2, in E) by preddimer from SIB tools are shown. The TMs are color coded on the amino acid sequence with TM1 in purple and TM2 in cyan 


3.4.2 Soluble domains might have a role in the stabilization of oligomers

In order to disrupt the homo-oligomerization of Mic10 and thus decrease spectral complexity for solid-state NMR studies, a double mutant G50A and G52A which was kindly provided by our collaborators, Professor Dr. Meinecke at the University of Göttingen, was expressed. However, yield of the expression was too low to pursue NMR studies. Moreover, after the affinity chromatography column, the eluted double mutant Mic10 protein, when analyzed by SDS-PAGE exhibited high molecular bands as observed for Mic10His.

To alleviate the NMR spectral complexity, we opted for reducing the length of the protein. Since our focus is in the TM domains (residue ~33 to 82) of Mic10, we wonder if by

MSYYHHHHHH DYDIPTTENL YFQGAMGILMSEQAQTQQPA KSTPSKDSNK NGSSVSTILD TKWDIVLSNM LVKTAMGF50GV 52GVFTSVLFFK RRAFPVWLGIGFGVGRGYAE

GDAIFRSSAG LRSSK TM1

Gly52

Gly50 TM1 - TM1 TM2 - TM2 TM1 -TM2

A B

C D E

removing the soluble domains one could increase spectral resolution. To evaluate this possibility, an enzymatic treatment was performed in the refolded Mic10His to cleave out the accessible regions of the protein. Using this approach, we expected to cleave out the extracellular domains of Mic10His while keeping the TM domains since they should be protected from the proteases by the membrane. We tested two distinct endoproteases, trypsin and chymotrypsin.
 baseline distortions, which in here it indicates truncation of the FID.

Figure 4.15 shows the (H)NH and (H)NHj spectra of Mic10His recorded in DMPC lipids after trypsin treatment. The trypsin treatment does not improve spectral quality (Fig.

4.15A, DMPC cleaved). The (H)NHj shows that there is still flexible regions on the sample (Fig. 4.15B). Although the TM domains where not accessible for assignment proposes, we wonder if there have been any changes on the flexible domains compare to the micelles prior to trypsin treatment. Slightly differences on the glycine region from LDAO to DMPC cleaved sample (above 110 ppm nitrogen peak) can be observed (Appendix 4.VII). Other peaks seemed to be affected as well, however it is still to be seen if the (H)NHj spectra of a non cleaved Mic10His in DMPC shows the same differences.

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Figure 4.16. Enzymatic treatments disrupt Mic10His oligomers.

White and red stars show the lines where Mic10His was found by mass spectrometry analysis (Appendix 4.VIII). From left to right of the gel is Chymotrypsin (1), Trypsin (2), leader marker (3), Mic10-His (4) as control for the next lines where we treated Mic10His with Trypsin for different times of 48 and 36 hours (5 and 6) at room temperature. The same treatment was performed with a buffer containing 10 mM arginine and 10 mM glutamic acid (9 and 10). The same treatment conditions were used for Chymotrypsin treatment without additives (lines 7 and 8) and with additives (lines 11 and 12). The cleavage sites are represented over Mic10His amino acid sequence. A total of 15 and 11 cleavage sites are found for Chymotrypsin and Trypsin over the full Mic10His sequence, respectively.


The efficiency of the cleavage and the accessible regions of the enzyme tested was followed by SDS-PAGE (Fig. 4.16). Figure 4.16 shows the SDS-PAGE gels stained using silver nitrate (232). In both enzymatic treatments, enzyme was added at 0.75 U/mg and the reaction was followed by SDS-PAGE at different time points. Previous data reported the role of arginine as stabilizer for membrane proteins (233), thus Mic10 was refolded in two conditions: one with and one without the addition of 20 mM arginine in 0.5% DPC. The SDS-PAGE gel shows that the oligomers of Mic10His were largely digested by the treatment of both enzymes (Fig. 4.16). Surprisingly, in contrast to the sample treated with chymotrypsin, the trypsin treated sample showed a major band above ~12 kDa. To confirm the presence of Mic10His in this band, the bands present on the gel were cut out and analyzed by mass spectrometry. Even though the sequence coverage was low (~30%, see appendix VIII), Mic10His sequence was identified from residue W34 to R87, which is comprised in the TM domains (residue 34 to 62) including residues G50 and G52 shown to be important for Mic10 oligomerization (Barbot et al.). None of the mass spectrometry data showed the presence of

(kDa)M Mic10

the N-terminal including the His tag or the C-terminal of Mic10His (Appendix 4.VIII). This indicates that the flexible regions are likely to be cleaved by the enzymatic treatment.

Although further investigation is needed, these results suggest that Trypsin cleavage can be used in the presence of DPC to reduce the presence of oligomers and obtain a ~12 kDa domain.


To conclude, the combination of bioinformatics, ssNMR in more native environments and enzymatic treatments suggest a strong protein-protein interaction behind the stable non homogenous homo-oligomers formed by the full-length protein. The enzymatic treatment raises additional questions regarding the oligomerization of Mic10. Is the oligomerization in part modulated by the soluble domains of Mic10His as well? Our data suggest that it might be a more complex mechanism behind the formation of Mic10 homo-oligomers and that including other proteins of the MICOS complex may be important for the further progress in structurally defining the Mic10 protein.

3.5 BOTH TRANSMEMBRANE DOMAINS OF MIC10 SHOW HELICAL PROPENSITY IN