Chaperonin recognition of protein dynamics drives drug resistance

Liu, J.; Qi, Z.; Rodrigues, J. V.; Zhang, Z.; Xu, Q.; Dubey, A.; Wang, H. K.; Cui, D.; Li, Z.; Aly, F.; Hekstra, D. R.; Shakhnovich, E. I.

doi:10.64898/2026.06.03.729952

bioRxiv · 10.64898/2026.06.03.729952

Chaperonin recognition of protein dynamics drives drug resistance

Liu, J.·Qi, Z.·Rodrigues, J. V.·Zhang, Z.·Xu, Q.·Dubey, A.·Wang, H. K.·Cui, D.·Li, Z.·Aly, F.·Hekstra, D. R.·Shakhnovich, E. I.

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Abstract

The emergence of drug resistance is typically driven by mutations that alter drug-target affinity, yet the role of host cellular machinery regulating these processes remains unclear. Here, we reveal that the chaperonin GroEL/S promotes drug resistance through recognition of protein dynamics. Using directed evolution of E. coli DHFR under antibiotic stress and varying GroEL/S expression, we identify a well-folded resistance variant whose fitness, despite tight inhibitor binding, is critically potentiated by GroEL/S engagement. X-ray crystallography, NMR, molecular dynamics and kinetic modeling reveal that millisecond-timescale flipping of the M20 loop generates steric accessibility and a kinetic window for chaperonin interaction that forcibly displaces tightly bound inhibitors, thereby overriding thermodynamic equilibrium of inhibitor binding to restore the active enzyme pool and preserve metabolic flux. Our findings not only reveal a novel paradigm of "dynamic recognition" where both conformational kinetics and distribution govern chaperonin recognition but also establish chaperonins as "deligandases" that actively modulate in vivo drug binding, suggesting that chaperone surveillance of the cellular proteome extends beyond quality control to govern native protein function. This mechanism defines a previously unrecognized route for the rapid development of drug resistance, with implications for understanding therapeutics of microbial infections and human malignancies.

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Liu, J., Qi, Z., Rodrigues, J. V., Zhang, Z., Xu, Q., Dubey, A., Wang, H. K., Cui, D., Li, Z., Aly, F., Hekstra, D. R., Shakhnovich, E. I.. 2026-06-07. Chaperonin recognition of protein dynamics drives drug resistance. https://doi.org/10.64898/2026.06.03.729952

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