Modularly designed protein fragments combine into thousands of active and structurally diverse enzymes

Lipsh-Sokolik, R.; Khersonsky, O.; Schroder, S. P.; de Boer, C.; Hoch, S. Y.; Davies, G. J.; Overkleeft, H. S.; Fleishman, S. J.

doi:10.1101/2022.09.17.508230

bioRxiv · 10.1101/2022.09.17.508230

Modularly designed protein fragments combine into thousands of active and structurally diverse enzymes

Lipsh-Sokolik, R.·Khersonsky, O.·Schroder, S. P.·de Boer, C.·Hoch, S. Y.·Davies, G. J.·Overkleeft, H. S.·Fleishman, S. J.

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Abstract

Design of structurally diverse enzymes is constrained by long-range interactions that are needed for accurate folding. We introduce an atomistic and machine-learning strategy for Combinatorial Assembly and Design of ENZymes, CADENZ, to design fragments that combine with one another to generate diverse, low-energy structures with stable catalytic constellations. We applied CADENZ to endoxylanases and used activity-based protein profiling to recover thousands of active and structurally diverse enzymes. Functional designs exhibit high active-site preorganization and more stable and compact packing outside the active site. Implementing these lessons into CADENZ led to a tenfold improved hit rate and >10,000 active enzymes. This design-test-learn loop can be applied, in principle, to any modular protein family, yielding huge diversity and general lessons on protein design principles.

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Lipsh-Sokolik, R., Khersonsky, O., Schroder, S. P., de Boer, C., Hoch, S. Y., Davies, G. J., Overkleeft, H. S., Fleishman, S. J.. 2022-09-18. Modularly designed protein fragments combine into thousands of active and structurally diverse enzymes. https://doi.org/10.1101/2022.09.17.508230

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