Cryo-EM structure of Shiga toxin 2 in complex with the native ribosomal P-stalk reveals residues involved in the binding interaction

Kulczyk, Arkadiusz W.; Sorzano, Carlos Óscar S.; Grela, Przemysław; Tchórzewski, Marek; Tumer, Nilgun E.; Li, Xiaoping

doi:10.1016/j.jbc.2022.102795

Cited by 13 publications

(14 citation statements)

References 54 publications

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“…Although our model accounts for the effects of M.ECPA8_3172P-PNB-2 on virulence and phage production, one question remains at issue; how does methylation of the small subunit rRNA prevent Stx-mediated ribosome inactivation? This question is especially puzzling since the 1) Stx binding site is located on the P-stalk region, which is on the large subunit ( 13, 56–61 ); 2) Stx acts by removing an adenine base from sarcin-ricin loop (SRL) present in 23S rRNA; 3) the putative positions of the bases modified by M.ECPA8_3172P- PNB-2, are on a solvent exposed surface of 30S subunit (Figure 6b & c), that is on the opposite face of the ribosome from the SRL and the P stalk (data not shown). Hence it seems unlikely that methylation directly inhibits Stx binding to the ribosome.…”

Section: Discussionmentioning

confidence: 99%

The Shiga toxin (Stx)-Phage Encoded Ribosomal RNA Methyltransferase Regulates Stx-producingEscherichia coli(STEC) Virulence by Blocking Stx-Mediated Inactivation of Bacterial Ribosomes

Gong,

Koudelka

2023

Preprint

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Shiga toxin (Stx) produced and released after induction of Stx-encoding prophage resident within Shiga toxin producing E. coli (STEC) causes life-threatening illness. We previously identified that a two-subunit Stx prophage-encoded 16S rRNA methyltransferase, M.ECPA8_3172P-PNB-2, which is both uniquely encoded by and commonly found in Stx2-encoding bacteriophage, regulates both prophage spontaneous induction and STEC virulence. We found here that sequential deletion of these two subunits leads to concomitant, progressive reduction in both prophage spontaneous induction and STEC virulence. This observation indicates that these outcomes are linked. The translation activity of extracts made from a ΔM.ECPA8_3172PΔPNB-2 Stx prophage-containing strain was lower that of extracts made from either the methyltransferase replete STEC strain or from a strain that did not contain a Stx-encoding prophage. We found that the ΔM.ECPA8_3172PΔPNB-2 STEC strain contained significantly fewer ribosomes that did the methyltransferase replete STEC strain. These observations suggested that the M.ECPA8_3172P-PNB-2 methyltransferase may block Stx-mediated ribosome inactivation. Consistent with this idea, we found that translation extracts made from STEC expressing M.ECPA8_3172P-PNB-2 are more resistant to Stx-mediated inactivation than are those made from ΔM.ECPA8_3172PΔPNB-2 STEC. These findings indicate the M.ECPA8_3172P-PNB-2 methylation of 16S rRNA protects the ribosome from Stx-mediated inactivation, thereby allowing more phage and more Stx to be spontaneously produced. Direct 16S rRNA sequencing identified 4 putative M.ECPA8_3172P-PNB-2 methylation sites, all of which map onto the RNA polymerase contacting surface of the 30S ribosome subunit in the expressome, suggesting the M.ECPA8_3172P-PNB-2 may protect the ribosome from inactivation by stabilizing this complex.

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Section: Discussionmentioning

confidence: 99%

The Shiga toxin (Stx)-Phage Encoded Ribosomal RNA Methyltransferase Regulates Stx-producingEscherichia coli(STEC) Virulence by Blocking Stx-Mediated Inactivation of Bacterial Ribosomes

Gong,

Koudelka

2023

Preprint

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“…30,35 Structural changes on the individual B subunits and Stx2A2 were observed by cryo-EM upon binding of the P-stalk pentamer to the Stx2a holotoxin. 37 These results suggested that the different binding mode observed with the Stx2a holotoxin compared to RTA and TCS may be related to the structural differences between the A1 subunit in free form compared to when it is part of the Stx2a holotoxin. 36,37 The Xray structure of the A1 subunit of a Shiga toxin by itself or in complex with a P-stalk peptide has not been solved, and the structural details of the binding of Stx2A1 to the P-stalk have not been determined.…”

Section: ■ Introductionmentioning

confidence: 89%

Structure–Function Analysis of the A1 Subunit of Shiga Toxin 2 with Peptides That Target the P-Stalk Binding Site and Inhibit Activity

Li,

Rudolph,

Chen

et al. 2024

Biochemistry

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Shiga toxin 2a (Stx2a) is the virulence factor of Escherichia coli (STEC), which is associated with hemolytic uremic syndrome, the leading cause of pediatric kidney failure. The A1 subunit of Stx2a (Stx2A1) binds to the conserved C-terminal domain (CTD) of the ribosomal P-stalk proteins to remove an adenine from the sarcin−ricin loop (SRL) in the 28S rRNA, inhibiting protein synthesis. There are no antidotes against Stx2a or any other ribosome-inactivating protein (RIP). The structural and functional details of the binding of Stx2A1 to the P-stalk CTD are not known. Here, we carry out a deletion analysis of the conserved P-stalk CTD and show that the last eight amino acids (P8) of the P-stalk proteins are the minimal sequence required for optimal affinity and maximal inhibitory activity against Stx2A1. We determined the first X-ray crystal structure of Stx2A1 alone and in complex with P8 and identified the exact binding site. The C-terminal aspartic acid of the Pstalk CTD serves as an anchor, forming key contacts with the conserved arginine residues at the P-stalk binding pocket of Stx2A1. Although the ricin A subunit (RTA) binds to the P-stalk CTD, the last aspartic acid is more critical for the interaction with Stx2A1, indicating that RIPs differ in their requirements for the P-stalk. These results demonstrate that the catalytic activity of Stx2A1 is inhibited by blocking its interactions with the P-stalk, providing evidence that P-stalk binding is an essential first step in the recruitment of Stx2A1 to the SRL for depurination.

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“…The initial model of the trimeric Lm complex was obtained using a combination of Phenix 1.18 29 , COOT 0.9 30 , UCSF Chimera 25 , and Colab-Fold with AlphaFold2 and the sequence search module MMseq2 31 . In brief, atomic coordinates of previously determined X-ray structures of α5 (PDB ID: 2Y38 [https://doi.org/10.2210/pdb2Y38/pdb] 5 , β1 (PDB ID: 4AQS [https://doi.org/10.2210/pdb4AQS/pdb] 6 and γ1 (PDB ID: 4AQT [https://doi.org/10.2210/pdb4AQT/pdb]) 6 were docked into the Coulomb density map in UCSF Chimera.…”

Section: Model Buildingmentioning

confidence: 99%

“…The calculations confirmed the correctness of the assignment. The model of the Lm polymer node was then iteratively refined using a combination of COOT 0.9 30 and Phenix 1.18 29 with imposed NCS, secondary structure and Ramachandran restrains. We systematically improved the quality of the model by addressing specific issues, such us interatomic clashes, violations of Ramachandran and other geometry restrains including errors in side chain rotamers.…”

Section: Model Buildingmentioning

confidence: 99%

Cryo-EM reveals the molecular basis of laminin polymerization and LN-lamininopathies

et al. 2023

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Laminin polymerization is the major step in basement membranes assembly. Its failures cause laminin N-terminal domain lamininopathies including Pierson syndrome. We have employed cryo-electron microscopy to determine a 3.7 Å structure of the trimeric laminin polymer node containing α1, β1 and γ1 subunits. The structure reveals the molecular basis of calcium-dependent formation of laminin lattice, and provides insights into polymerization defects manifesting in human disease.

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Cryo-EM structure of Shiga toxin 2 in complex with the native ribosomal P-stalk reveals residues involved in the binding interaction

Cited by 13 publications

References 54 publications

The Shiga toxin (Stx)-Phage Encoded Ribosomal RNA Methyltransferase Regulates Stx-producingEscherichia coli(STEC) Virulence by Blocking Stx-Mediated Inactivation of Bacterial Ribosomes

The Shiga toxin (Stx)-Phage Encoded Ribosomal RNA Methyltransferase Regulates Stx-producingEscherichia coli(STEC) Virulence by Blocking Stx-Mediated Inactivation of Bacterial Ribosomes

Structure–Function Analysis of the A1 Subunit of Shiga Toxin 2 with Peptides That Target the P-Stalk Binding Site and Inhibit Activity

Cryo-EM reveals the molecular basis of laminin polymerization and LN-lamininopathies

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