Abstract
Adhesin P1 (aka AgI/II) plays a pivotal role in mediating Streptococcus mutans attachment in the oral cavity, as well as in regulating biofilm development and maturation. P1’s naturally occurring truncation product, Antigen II (AgII), adopts both soluble, monomeric and insoluble, amyloidogenic forms within the bacterial life cycle. Monomers are involved in important quaternary interactions that promote cell adhesion and the functional amyloid form promotes detachment of mature biofilms. The heterologous, 51-kD C123 construct comprises most of AgII and was previously characterized by X-ray crystallography. C123 contains three structurally homologous domains, C1, C2, and C3. NMR samples made using the original C123 construct, or its C3 domain, yielded moderately resolved NMR spectra. Using Alphafold, we re-analyzed the P1 sequence to better identify domain boundaries for C123, and in particular the C3 domain. We then generated a more tractable construct for NMR studies of the monomeric form, including quaternary interactions with other proteins. The addition of seven amino acids at the C-terminus greatly improved the spectral dispersion for C3 relative to the prior construct. Here we report the backbone NMR resonance assignments for the new construct and characterize some of its quaternary interactions. These data are in good agreement with the structure predicted by Alphafold, which contains additional β-sheet secondary structure compared to the C3 domain in the C123 crystal structure for a construct lacking the seven C-terminal amino acids. Its quaternary interactions with known protein partners are in good agreement with prior competitive binding assays. This construct can be used for further NMR studies, including protein-protein interaction studies and assessing the impact of environmental conditions on C3 structure and dynamics within C123 as it transitions from monomer to amyloid form.
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Data availability
The chemical shift values for P1 Adhesin C3 domain were deposited in the Biological Magnetic Resonance Data Bank (BMRB) under accession code 52097.
Abbreviations
- CSI:
-
Chemical shift index
- Δδ:
-
Chemical shift perturbation
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Acknowledgements
We thank James Collins and James Rocca for technical assistance with NMR experiments.
Funding
This work was supported by NIH/NIDCR R01 DE021789 to L.J.B. and J.R.L. A portion of this work was performed in the McKnight Brain Institute at the National High Magnetic Field Laboratory’s AMRIS Facility, which is supported by National Science Foundation Cooperative Agreements DMR-1644779 and DMR-2128556, the State of Florida, and an NIH award, S10 OD028753, for magnetic resonance instrumentation.
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EQP performed protein expression and purification, collected the NMR data, analyzed the data and wrote the first draft of the manuscript. MLCN assisted with plasmid design, NMR data collection, and analysis. GR assisted with data analysis and preparation of figures. All authors commented on previous versions of the manuscript and reviewed the final submitted manuscript. Funding acquisition and supervision were done by LJB and JRL.
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Peng, EQ., Caldas Nogueira, M.L., Rivière, G. et al. Backbone NMR resonance assignments for the C terminal domain of the Streptococcus mutans adhesin P1. Biomol NMR Assign 17, 293–299 (2023). https://doi.org/10.1007/s12104-023-10158-y
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DOI: https://doi.org/10.1007/s12104-023-10158-y