Exit tunnel modulation as resistance mechanism of S. aureus erythromycin resistant mutant

The clinical use of the antibiotic erythromycin (ery) is hampered owing to the spread of resistance genes that are mostly mutating rRNA around the ery binding site at the entrance to the protein exit tunnel. Additional effective resistance mechanisms include deletion or insertion mutations in ribosomal protein uL22, which lead to alterations of the exit tunnel shape, located 16 Å away from the drug’s binding site. We determined the cryo-EM structures of the Staphylococcus aureus 70S ribosome, and its ery bound complex with a two amino acid deletion mutation in its ß hairpin loop, which grants the bacteria resistance to ery. The structures reveal that, although the binding of ery is stable, the movement of the flexible shorter uL22 loop towards the tunnel wall creates a wider path for nascent proteins, thus enabling bypass of the barrier formed by the drug. Moreover, upon drug binding, the tunnel widens further.

The high-resolution structures of the ribosome from S. aureus as well as of its complexes with a few clinically useful drugs and new potential inhibitors were determined in our lab 36 and shed light on its explicit drug inhibition properties and selectivity as well as on its specific structural elements to be targeted. Directed evolution can be used for identifying and isolating mutated bacteria that show resistance to antibiotics and is a helpful tool for identifying new resistance mechanisms, which may lead to a better understanding of species-specific resistance mechanisms. It was applied for the isolation a uL22 mutant S. aureus ribosome (SAuL22m) from a wild type strain of S. aureus, which harbors a 2 amino acid deletion in the ß hairpin loop of uL22 26 .
We present here the single particle cryo-EM high resolution structures of the apo and ery bound SAuL22m mutant ribosome, which demonstrates how deletion of R88-A89 in S. aureus ribosomal protein uL22 ß hairpin loop leads to ery resistance although it does not hamper the binding of the antibiotic itself. We also show that upon ery binding additional conformational changes occur that are beyond the expected changes, occur as a result of the deletion per se. In addition, by comparing the current structures with the native SA ribosome and additional uL22 mutants structures from other bacterial species 17,25,26 , we highlight the specific structural changes that occurred in each of the uL22 mutant proteins which nevertheless led to a similar outcome at the cellular level.

Results and Discussion
The single particle cryo-EM structures of the 70S ribosomes from apo uL22 mutant S. aureus (SAuL22m_apo) and of its complex with ery (SAuL22m_ery) were determined at 3.58 Å and 3.2 Å, respectively (Table S1). A focused refinement of both large subunits resulted in a 2.4 Å and 2.3 Å cryo-EM reconstructed maps, respectively. These maps allowed for the unambiguous assignment of ery within SAuL22m_ery complex structure ( Fig. 2A) and for defining the interactions of ery with the rRNA nucleotides at the binding site ( Fig. 2B) including the identification of the nucleotides that bind ery via hydrophobic interactions as well as its hydrogen bonds (Fig. 2C). Ery inhibition assays using SA_WT and SAuL22m ribosomes clearly show that the mutant ribosomes are resistant to ery (Fig. S1).
The cryo-EM maps allowed for the unambiguous tracing of the uL22 hairpin loop of both SAuL22m_apo and its complex structure with ery (Fig. 3A). Upon superposition of the SA50S wild type (PDBID 6HMA, SA50S_ WT) and the SAuL22m_apo structures, we identified conformational changes of uL22 that are due to the deletion mutation that shifts the beta hairpin loop by 6A from its location in the wild type. Consequently, next to the uL22 ß hairpin loop the rRNA nucleotides A1614 (located in H59a tip) is rotated about 45° (Fig. 4A) and H35 is displaced by about ~3.0 Å (Fig. 4B).
In addition, we identified several conformational changes at the macrolide's binding site of nucleotides A2062, A2439, G2505, U2585, G2583 and A2602. A small movement of A-site nucleotides and helix H92 was also detected. Some of these changes are significant, namely, A2439 shifts about 3 Å and U2585 is about 45° rotated (Fig. 4C). Nucleotides G2505 and C2610 are rotated by about 45° and 90° respectively (Fig. 5C).
By the superposition of the SAuL22m_ery complex structure on the two apo structures above we could identify several conformational changes that occurred upon ery binding; At the binding site, A2062 is 45° rotated, U2585 is 110° rotated, G2505 is 90° and U2506 is about 3.5 Å shifted in order to accomodate G2505 and forms a hydrogen bond with it (Fig. 4C). A small rotation of about 20° of nucleotide G2610 was also identified (Fig. 5C).
Interestingly, upon ery binding, G2505 is about 30° rotated towards C2610, which is 45° rotated, to form a WC base pair (BP) with it, where this movement seems to be at the final step in the mid-way movement shown in SAuL22m_apo relative to SA50S_WT. A similar BP was also identified in the structures of E. coli and T. thermophilus complexes with ery (PDBID 4V7U, EC70S and PDBID 4V7X, respectively); however, in both D. www.nature.com/scientificreports www.nature.com/scientificreports/ radiodurans uL22 mutant apo and ery bound structures (PDBID 4WFN and 4U67, respectively) no such movement has been identified. This movement stabilizes ery binding to the mutant by providing additional hydrophobic interaction (Fig. 5C).  By inspecting the structural changes that occur upon ery binding to the mutant ribosome we could identify a cascade of movements (Fig. 3B). At the ery binding site, nucleotide G2505 rotates almost 90°, forms a new base pair with C2610 and is stabilized by another bond with U2506. U746 moves together with H35 by about ~3.0 Å to form a bond with OP1 of C2611. Then, A1614 rotates by about 45 o where this rotation is stabilized by a bond between N6 from A789 and OP2 of A1614. Due to such cascade of events uL22 ß hairpin loop shifts to the tunnel wall.
By comparison of uL22 ß hairpin loop of the SA50S_WT, SAuL22m_apo and SAuL22m_ery structures, we found that the loop is shifted towards the center of the tunnel in the SAuL22m_apo structure whereas in SAuL22m_ery complex structure it is shifted back closer to its location in SA50S_WT structure (Fig. 4A). Nevertheless, the tip of the beta hairpin loop moves towards the tunnel wall and a new cavity that adds to the tunnel width forms (Figs 5B and S4).
These findings, combined with binding assays that showed no changes in the binding affinity of ery to native SA ribosome compared to the mutant ribosome 26 , are in line with the clear electron density of ery. Thus, it supports the idea that the uL22 mutation does not dramatically affect ery binding site, but provides an alternative mechanism for nascent proteins progression through the NPET.
Previous studies suggested that nascent proteins can bypass the ery in the ribosome by stabilizing A2062 in a conformation that increases the space available for their passage 37 . We add to this proposed stabilization of A2062, the deletion mutation at the tip of the ß hairpin loop that forms an additional free space in the tunnel through which the nascent proteins can bypass the antibiotic (Fig. 5). By comparing the uL22 hairpin region among SA50S_WT, EC70S (PDBID 4V7U) and SAuL22m_apo, a new groove was identified. Upon ery binding, an additional groove form in SAuL22m_ery structure. Thus, we suggest that the uL22m new groove widens the tunnel in which the nascent protein can pass and bypass ery's steric blockage (Fig. S4). This mechanism, which is activated upon drug binding, is a new finding that suggests a rearrangement of the tunnel further to the expected changes due to the deletion mutation. It also supports the necessity to study the complex SAuL22m_ery structure.
Sequence alignment of other, similar, uL22 ery resistant mutant ribosomes from the archaea H. marismortui and the eubacteria D. radiodurans, indicates that the mutations occur in proximity to conserved positions around the tip of the beta hairpin loop (Fig. 5A) and the specific mutated nucleotides in the SAuL22m are highly conserved among bacteria. Our studies explain why changes in this region of uL22 are crucial for the destabilization of its loop and the development of the antibiotic's resistance. Comparative structural studies of the various uL22 mutants' ribosomes reveal different conformations of the loop. The structure of H. marismortui uL22m_del3 with www.nature.com/scientificreports www.nature.com/scientificreports/ no bound antibiotics (PDBID 1YJ9) shows that a three amino acids deletion further downstream of the ß hairpin loop leads to a change of the loop conformation from the tunnel wall which leads to a widening of the tunnel (Fig. 5B). The structure of a three amino acids insertion in D. radiodurans in complex with ery (PDBID 4WFN) displays a widening of the uL22 tip itself and reveals a small movement of the loop towards the tunnel wall (Fig. 5B). The importance of residue 90 of uL22, which is not conserved among bacterial species, for erythromycin resistance was recently reported 38 . This finding further supports our results since the deletion is in proximity to Q90 and changes its location.
A recent study reported that a Vibiro export monitoring polypeptide (VemP) acts as a cis-regulatory polypeptide and interacts with R92 and R95 of uL22 in order to stall the ribosome 13 . Upon superposition of VemP of E. coli (PDBID 5NWY) on SA50S_WT, SAuL22m_apo and SAuL22m_ery we observed that while in S. aureus uL22 residue 95 is alanine instead of arginine the overall structure remains the same in SA50S_WT. However, in mutant SAuL22m_apo there is a vast opening in proximity to the ß hairpin loop and the new groove lead to a more spacious tunnel for peptides movement through the tunnel. Upon ery binding to the mutant, in SAuL22m_ ery structure, additional grooves are formed, which leads to a wider tunnel (Fig. 6). We suggest that this change affects resistance to ery while potentially preserving the stalling function of the cis-regulatory polypeptides, which are important to the normal function of the bacteria. A recent computational study 39 suggests that erythromycin slows or stalls synthesis of ErmCL compared to H-NS due to stronger interactions with particular residue positions along the nascent protein. uL22 various mutations may change the rate of stalling of specific protein synthesis while ery is bound at the NPET and changes the electrostatic and dispersion interactions with nascent proteins.
Our results confirm that diverse mutations in the rProtein uL22 ß hairpin loop occur in various bacterial species and, by applying slightly different mechanisms, they facilitate nascent protein progression in the exit

Materials and Methods
Ribosome purification. The bacteria were isolated and grown as described 26,36  eM Sample preparation. Both samples were flash frozen using Vitrobot TM VI (FEI) using the following conditions: For the SAuL22m_apo grids a ribosome concentration of 1 mg/ml was used on QUANTIFOIL ® R 1.2/1.3. while for SAuL22M_ery grids, a ribosome concentration of 0.3 mg/ml was used on QUANTIFOIL ® R 2/2 grids with continues carbon support.
Data collection, processing and refinement. The cryo-EM data for both SAuL22m_apo and SAuL22m_ ery structures were collected at the ESRF CM01 beamline 41 using FEI Titan Krios (FEI) with K2 Summit (Gatan) direct electron detector, and Quantum LS imaging filter (Gatan) at a magnification of x130K, at defocus range of −0.5-1.0 nm and Pixel size of 1.067 A. The cryo-EM data for SAuL22m_apo structure were collected at 6.367 e − / pix/s and 5.227 e − /A2/s. 40 frames per micrograph of 8 sec total length, 0.2 sec per frame and a total dose of 40 e − / A2. 3542 movies were collected. From these micrographs 529,786 particles of SAuL22m_apo were selected for the 2D classification, from them 145,897 particles were selected for 3D classification and 124,731 particles were used for the 3D refinement which gave a 3.58 Å resolution map for the whole 70 S ribosome and a resolution of 3.2 Å resolution map for the 50 S ribosomal LSU (Fig. S2, Table S1).
The cryo-EM data for SAuL22m_ery complex structure were collected at 5.231 e − /pix/s and 4.288 e − /A2/s. 28 frames per micrograph of 7 sec with total length of 0.25 sec per frame and total dose of 30 e − /A2. 4161 movies were collected. From these micrographs 734,247 particles of SAuL22m_ery were selected for the 2D classification, from them 426,250 particles were selected for 3D classification and 378,309 particles were used for the 3D Figure 6. VemP interaction with uL22. A view of VemP (green) with a surface representation of uL22 from E. coli (green), SA50S_WT (blue) both shows a similar structure while SAuL22m_apo (coral) and SAuL22m_ery complex (grey) reveals a wider path made possible by the shortening of the ß hairpin loop and the additional grove upon ery binding to SAuL22m_ery. Ery position is shown in red.