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Protein folding modulates the chemical reactivity of a Gram-positive adhesin

Nature Chemistry volume 13, pages 172–181 (2021)Cite this article

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Abstract

Gram-positive bacteria colonize mucosal tissues, withstanding large mechanical perturbations such as coughing, which generate shear forces that exceed the ability of non-covalent bonds to remain attached. To overcome these challenges, the pathogen Streptococcus pyogenes utilizes the protein Cpa, a pilus tip-end adhesin equipped with a Cys–Gln thioester bond. The reactivity of this bond towards host surface ligands enables covalent anchoring; however, colonization also requires cell migration and spreading over surfaces. The molecular mechanisms underlying these seemingly incompatible requirements remain unknown. Here we demonstrate a magnetic tweezers force spectroscopy assay that resolves the dynamics of the Cpa thioester bond under force. When folded at forces <6 pN, the Cpa thioester bond reacts reversibly with amine ligands, which are common in inflammation sites; however, mechanical unfolding and exposure to forces >6 pN block thioester reformation. We hypothesize that this folding-coupled reactivity switch (termed a smart covalent bond) could allow the adhesin to undergo binding and unbinding to surface ligands under low force and remain covalently attached under mechanical stress.

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Fig. 1: Mechanochemistry of S. pyogenes Cpa adhesin.
Fig. 2: Dynamics of the thioester-intact Cpa polyprotein under force.
Fig. 3: Cpa thioester bond cleavage is negatively force-dependent.
Fig. 4: Protein folding drives thioester bond reformation.
Fig. 5: Cystamine-mediated abrogation of Cpa thioester bond reformation.
Fig. 6: Bacterium mobility strategy model based on the allosteric modulation of the Cpa thioester bond by protein folding.

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All data supporting the results and conclusions are available within this paper and the Supplementary Information. Source data are provided with this paper.

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Acknowledgements

This research was supported by the National Institutes of Health grant no. R35129962 (J.M.F). A.A-C. and R.T-R. express their gratitude to Fundación Ramón Areces (Madrid, Spain) for financial support. We thank C. L. Badilla for assistance in molecular biology procedures, and for reading and reviewing the manuscript. Correspondence and request for materials should be addressed to A.A-C

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Author notes

  1. Daniel J. Echelman

    Present address: Boston Combined Residency Program, Boston Children’s Hospital, Boston, MA, USA

  2. Shubhasis Haldar

    Present address: Ashoka University, Haryana, India

  3. These authors contributed equally: Alvaro Alonso-Caballero, Daniel J. Echelman.

Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, New York, NY, USA

    Alvaro Alonso-Caballero, Daniel J. Echelman, Rafael Tapia-Rojo, Shubhasis Haldar, Edward C. Eckels & Julio M. Fernandez

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  1. Alvaro Alonso-Caballero
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Contributions

D.J.E., A.A-C. and J.M.F designed the research. A.A-C, D.J.E., R.T-R., S.H., E.C.E carried out the experiments. A.A-C, D.J.E., R.T-R. analysed the data. A.A-C., D.J.E., and J.M.F. wrote the manuscript.

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Correspondence to Alvaro Alonso-Caballero.

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Extended data

Extended Data Fig. 1 Cleavage-reformation-cleavage sequence.

Magnetic tweezers force-clamp trajectory of the Cpa polyprotein. After the unfolding of four thioester-intact Cpa domains at 115 pN (circles, ~ 49 nm), the buffer is exchanged and the polyprotein is exposed to a solution containing 100 mM methylamine (+MA). At 21 pN, four steps appear which account for the release of the polypeptide sequence trapped by the thioester bonds (arrows). Then, the force is increased again to 115 pN, revealing the complete extension of the molecule. Immediately after, MA is washed out from the fluid chamber and the polyprotein is allowed to fold and reform the thioester bonds for 100 s at 4.5 pN. A 115 pN pulse reveals three ~ 95 nm steps (empty circles) which correspond with the full extension of Cpa, and one Cpa domain with its thioester bond reformed (circles, ~ 49 nm). Two more quenches at 3 pN are applied to completely recover the thioester-reformed state in all the four domains, as it can be seen in the 115 pN pulse applied approximately after 800 s of experiment (circles). Then, MA is added again and the force quenched to 24 pN to trigger again the cleavage of the thioester bonds of the polyprotein (arrows).

Extended Data Fig. 2 Cystamine permanent blocking of Cpa thioester bond reformation.

Magnetic tweezers force-clamp trajectory of the Cpa polyprotein. After the unfolding of the thioester-intact Cpa domains at 115 pN (circles), the buffer is exchanged and the polyprotein is exposed to a solution containing 100 mM cystamine (+CA). At 115 pN and at 50 pN, no additional extensions are registered as a consequence of thioester bond cleavage, but a drop in force to 25 pN leads to the appearance of four steps which account for the release of the polypeptide sequence trapped by the thioester bonds (empty arrows in the inset). Then, the force is increased again to 115 pN, revealing the complete extension of the molecule. After 100 s at 4 pN and in the presence of CA, a 115 pN pulse reveals three ~95 nm steps (empty circles) which correspond with the full extension of Cpa. CA is then removed from the solution, and several consecutive 100 s force quenches (at 4, 5, and 3 pN) followed by 115 pN pulses are applied. These cycles reveal that, after CA treatment, Cpa is able to fold but not to reform its thioester bond, as it can be observed from the ~95 nm steps observed (empty circles). After the first 300 s of the experiment, one of the Cpa domains stops folding back as a consequence of oxidative damage66. The disturbances observed in the extension during +CA addition (orange block) and washing (gray block) are originated from the movement of buffer volumes in the liquid cell used in the experiments, which transiently alter the measurement.

Extended Data Fig. 3 TCEP rescues Cpa thioester bond reformation.

A Cpa polyprotein previously treated with cystamine shows three ~95 nm steps at 115 pN corresponding with the full extension of each of the domains (empty circles). The addition of 10 mM TCEP and 100 s at 4 pN is enough to trigger thioester bond reformation, as it can be observed in the ~49 nm thioester-intact Cpa steps (circles) registered at 115 pN. The fourth domain not observed at the beginning was probably unfolded and its thioester bond intact, since the difference in the final extension between the first 115 pN pulse and the last is ~140 nm, which matches with the expected final extension decrease from three reformation events. Inset histogram shows the two populations of steps observed after TCEP treatment, thioester-intact Cpa (circles, 48.3 ± 3.5 nm, mean±SD, n=32) and thioester-cleaved Cpa (empty circles, 95.7 ± 6.4 nm mean±SD, n=7). The latter full length steps of Cpa after TCEP treatment could be due to cleavage events induced by remaining cystamine which was not completely washed from the experimental liquid cell. The disturbances observed in the extension during +TCEP addition (green block) are originated from the movement of buffer volumes in the liquid cell used in the experiments, which transiently alter the measurement.

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Alonso-Caballero, A., Echelman, D.J., Tapia-Rojo, R. et al. Protein folding modulates the chemical reactivity of a Gram-positive adhesin. Nat. Chem. 13, 172–181 (2021). https://doi.org/10.1038/s41557-020-00586-x

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