3 RESULTS
4.3 Visualization of the ribosome-bound insertase complex
In order to understand and accurately depict how YidC inserts proteins in a co- translational manner into the inner membrane of E. coli, it is essential to solve the structure of YidC bound to substrate specific RNCs at high resolution. The structural information for Ec-YidC was then limited to an X-ray structure of the large periplasmic domain (Oliver and Paetzel, 2008; Ravaud et al., 2008), a 10 Å 2D projection map of the membrane-integrated Ec-YidC dimer (Lotz et al., 2008) and a low-resolution (14,4 Å) cryo- EM reconstruction of an Ec-YidC:RNC complex (Kohler et al., 2009). None of those structures, though, had enough performance to answer for instance the question about the functional oligomeric state or to give further insights how YidC acts at a molecular level. To address these questions, we performed an advanced structural analysis of YidC- ribosome complexes using high-resolution cryo-EM with the C-terminally elongated YidC- chimera YidC-Rb. Due to the extension of Ec-YidC with the C-terminal ribosome binding domain, that drastically increases the ribosome affinity in vitro (Fig. 3.8), we were able to isolate stable YidC-Rb:RNC complexes (2.13). Those complexes were visualized using high-performance cryo-EM and the reconstruction of the complex could be refined to 8,6 Å (2.14). This improved resolution now allows a more detailed interpretation of the structural and functional features of the YidC-insertase complex. The TnaC-stalled ribosomes carried nascent polypeptide chains encompassing the first two TM segments of MscL, a
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native YidC substrate in E. coli (Fig. 3.10). The path and position of the nascent chain within and at the ribosome can be traced from the peptidyl transferase center (PTC) through the ribosomal exit tunnel into an extra density at the tunnel exit representing YidC-Rb (Fig. 3.13). Although our overall resolution was in the subnanometer range, the alpha-helical secondary structures were barely resolved in the YidC density, most probably due to some flexibility in this region (Fig. 3.12). Interestingly, the presence of the C-terminal extension increases the affinity of YidC to the ribosome without changing its overall ribosome interaction mode. The spatial position and topology of the YidC-Rb chimera in this study agreed well with the position of Ec-YidC observed in the cryo-EM structure from an earlier study of ribosome-bound E. coli YidC (Kohler et al., 2009). Moreover, we observed a similar interaction pattern between our C-terminally extended YidC-Rb and the ribosome. We could resolve in molecular detail the contact sites of YidC- Rb to the ribosome: The helix H59 of the 23S rRNA showed the strongest contact to YidC- Rb followed by close interaction with the two ribosomal proteins L24 and L29. Surprisingly, we observed a relatively weak connecting density with L23, the main contact site for SRP and TF, and also for a proposed contact derived from the lower resolution structure of the Ec-YidC-RNC with a FOc nascent chain (Kohler et al., 2009). However, the ribosomal proteins L24 and L29, together with L23, also surround the ribosomal tunnel exit and provide binding sites for diverse factors involved in co-translational processing, folding, targeting and membrane insertion of nascent chains (Fig. 1.3). Since an enhanced interaction interface of YidC-Rb to the ribosomal protein L29 was observed (Fig. 3.11 C and D), compared to the Kohler et al. (2009) structure, the YidC-Rb protein as well as the Ec-YidC were tested for their ability to interact directly with isolated ribosomal proteins in in vitro pull down assays (2.15). Indeed, L29 only co-eluted with YidC-Rb (Fig. 3.16, lower panels), whereas L24 co-eluted both with Ec-YidC and in similar amounts as with YidC-Rb (Fig. 3.16, upper panels). Thus, L24 seems to be a major contact site for the YidC- insertase core domain, while the enhanced affinity of YidC-Rb to the ribosome is at least partially caused by the interaction between the C-terminal R. baltica YidC tail domain with the ribosomal protein L29, suggesting that the C-tail of YidC-Rb faces towards the L29 moiety of the ribosome.
Recently, Wickles et al. (2014) reconstituted YidC-Rb with RNCs exposing the first TM helix of FOc and subjected the purified complex to cryo-EM and single particle analysis to a resolution of ~ 8 Å. In contrast to our first cryo-EM structure of ribosome-bound YidC-Rb (Seitl et al., 2014), it was now possible to separate the weaker electron density of the detergent micelle from that of the YidC-Rb protein moiety (Fig. 4.1 B). Furthermore, Wickles et al. (2014) calculated a structural model of E. coli YidC via the intramolecular
125 co-variation analysis that could be docked in a distinct orientation into the cryo-EM structure of the YidC-Rb:RNC complex (Fig. 4.1 B and C). Shortly after, the X-ray structure of Ec-YidC was published and confirmed that the built Ec-YidC model (Wickles et al., 2014) appeared in good agreement with the experimentally solved molecular structure (Kumazaki et al., 2014-b). Interestingly, in both YidC-Rb:RNC densities an additional density which is aligned with the ribosomal exit tunnel, neighboring TM3, was found that could be attributed to the TM helix of the nascent MscL- or FOc- chain, respectively (Fig. 4.1 A and C). This hypothesis is supported by data from several independent studies which show that YidC substrates, and also nascent FOc chains (Wickles et al., 2014), can be crosslinked to TM3 (Klenner et al., 2008; Yu et al., 2008; Neugebauer et al., 2012; Klenner and Kuhn, 2012). Strikingly, at the same relative position nascent chains have been observed inside the SecY channel (Frauenfeld et al., 2011; Wickles et al., 2014). Independently of the nascent chain substrate used in our experiments, YidC-Rb revealed an almost identical interaction pattern on the ribosomal exit site (Fig. 4.1) for both cryo-EM reconstructions (Seitl et al., 2014; Wickles et al., 2014).
Fig. 4.1 Comparison of two YidC-Rb structures bound to RNCs carrying MscL (8.6 Å) – and
FOc (8 Å) nascent chains, respectively. YidC-Rb revealed an almost identical interaction pattern
on the ribosomal exit site for both cryo-EM reconstructions and both nascent chains enter the insertase core within particular proximity of TM3. (A) Density blot (bottom view) of YidC-Rb shows the entering of the nascent MscL chain (green) in the center of the YidC-Rb density (red) and the proposed TM domain arrangement (1-6; red), relative to the indicated ribosomal proteins L23, L24, L29 and the rRNA helix H59 (blue). The assumed position of the C-terminal ribosome-binding
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domain (CTD) was assigned to an additional density near L29. Cytoplasmic loops C1 (47aa) and C2 (13aa) are schematically (dotted lines) included in the figure for orientation. (B) Close up view from within the membrane region of YidC-Rb bound to FOc-RNC, highlighting the predicted
contacts between the C1 and C2 loops of YidC and the ribosome, indicated by magenta spheres. The detergent micelle is shown in blue and the FOc TM helix in pink. (C) Periplasmic view of the
active ribosome-bound YidC model, with the YidC contour outlined in red. The polypeptide exit tunnel is indicated with an asterisk. Structure (A) was adapted from Seitl et al., 2014 and structure (B/C) from Wickles et al., 2014.
Although the C-terminally extended domain of YidC-Rb was excluded in the built Ec-YidC model (Fig. 4.1 B and C), we suggest that the additional density, located between the ribosomal proteins L23 and L29, accounts for the C-terminal ribosome-binding domain (CTD) of the marine Rhodopirellula YidC homolog (Fig. 4.1 A). As discussed above, YidC- Rb shows a much higher affinity to L29 than Ec-YidC in pull down assays (Fig. 3.16). Therefore, we assume that the C-tail of YidC-Rb faces the ribosomal rRNA helix H59, which is located next to TM6, up to the opposite site of the exit tunnel. There it contacts the ribosomal protein L29 next to TM1, stabilizing the whole insertase-ribosome complex. This model also explains why we observed in both YidC-Rb structures only weak contacts to the ribosomal protein L23, compared to the Ec-YidC:RNC structure (Kohler et al., 2009). It is possible that the large CTD, positioned by the high affinity to L29, shields the L23 majority and therefore prevents to a large extent the interaction with the YidC- insertase core. As additional contacts to the ribosome, Wickles et al. (2014) suggested residues in the positively charged C1 (Y370; Y377, contacts to rRNA helix H59) and C2 (D488, contact to L23) loops of YidC (Fig. 4.1 B). Indeed, mutations in these residues compromised the growth of YidC-depleted E. coli cells, emphasizing their functional significance. However, as discussed in chapter 4.2, Geng et al. (2015) probed the influence of the C1 and C2 loop regions in direct ribosome-binding efficiency and showed that only C2 contributes to YidC:RNC assembly, while the C1 loop is involved in other vital functions. Due to the local proximity of the C1 region to the nascent chain portal site in the YidC-Rb density (Fig. 4.1 A), it is feasible that the C1 loop directly contacts the emerging polypeptide chain and assists the correct folding of inserting membrane proteins into the lipid bilayer.
Another striking finding of the cryo-EM YidC-Rb:RNC structures was that only a single monomer of YidC was bound to the translating ribosome (Fig. 4.1; Seitl et al., 2014; Wickles et al., 2014). Until then, results from the early studies on YidC extracted from bacterial membranes suggested that the protein is present in both monomeric and dimeric forms (van der Laan et al., 2001; Heuberger et al., 2002). Also crystallographic analysis showed that YidC forms symmetric dimers in the membrane (Lotz et al., 2008). Additionally, the low resolution cryo-EM structure on a YidC:RNC-FOc complex also
127 suggested that two copies of YidC bind to the ribosomal tunnel exit (Kohler et al., 2009). However, this view changed during the last years due to the YidC-Rb:RNC structures and also by the recently published X-ray structures of both Gram-positive and Gram-negative YidC proteins, clearly showing YidC to be in a monomeric state (Kumazaki et al., 2014-a & 2014-b). Kedrov et al. (2013) probed the oligomeric state of Ec-YidC in its free form and bound at the ribosome via fluorescence cross-correlation spectroscopy (FCCS). In this study it was shown that a single membrane-embedded YidC copy is sufficient to bind a substrate-translating ribosome and YidC only oligomerizes on the ribosome when YidC was applied in excess, non-physiological concentrations, compared to the amount of applied RNCs. Since a 10-fold excess of YidC was used to form YidC:RNC complexes in the previously cryo-EM reconstitution (Kohler et al., 2009), the corresponding structure likely represented a concentration-dependent oligomer of YidC. Taken together our observation of a single YidC-Rb protein bound to a 70S E. coli ribosome is in agreement with the recent literature, showing clearly that a single YidC copy is fully active as monomer, thus being the minimal and probably true-to-life functional unit for YidC- dependent, co-translational insertion of membrane proteins.