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De novo design of mini-protein binders broadly neutralizing Clostridioides difficile toxin B variants

Affiliations
  • 1Research Center for Industries of the Future, Westlake University, Hangzhou, Zhejiang, 310024, China.
  • 2Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, 310024, China.
  • 3Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, 310024, China.
  • 4Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, 310024, China.
  • 5Center for Infectious Disease Research, School of Medicine, Westlake University, Hangzhou, Zhejiang, 310024, China.
  • 6Center for General Practice Medicine, Department of Infectious Diseases, Zhejiang Provincial People’s Hospital, Hangzhou Medical College Affiliated People’s Hospital, Hangzhou, Zhejiang, 310014, China.
  • 7Research Center for Industries of the Future, Westlake University, Hangzhou, Zhejiang, 310024, China. taoliang@westlake.edu.cn.
  • 8Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, 310024, China. taoliang@westlake.edu.cn.
  • 9Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, 310024, China. taoliang@westlake.edu.cn.
  • 10Center for Infectious Disease Research, School of Medicine, Westlake University, Hangzhou, Zhejiang, 310024, China. taoliang@westlake.edu.cn.
  • 11Research Center for Industries of the Future, Westlake University, Hangzhou, Zhejiang, 310024, China. lupeilong@westlake.edu.cn.
  • 12Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, 310024, China. lupeilong@westlake.edu.cn.
  • 13Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, 310024, China. lupeilong@westlake.edu.cn.
  • 14Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, 310024, China. lupeilong@westlake.edu.cn.

Published on:

October 2, 2024

Abstract

Clostridioides difficile toxin B (TcdB) is the key virulence factor accounting for C. difficile infection-associated symptoms. Effectively neutralizing different TcdB variants with a universal solution poses a significant challenge. Here we present the de novo design and characterization of pan-specific mini-protein binders against major TcdB subtypes. Our design successfully binds to the first receptor binding interface (RBI-1) of the varied TcdB subtypes, exhibiting affinities ranging from 20 pM to 10 nM. The cryo-electron microscopy (cryo-EM) structures of the mini protein binder in complex with TcdB1 and TcdB4 are consistent with the computational design models. The engineered and evolved variants of the mini-protein binder and chondroitin sulfate proteoglycan 4 (CSPG4), another natural receptor that binds to the second RBI (RBI-2) of TcdB, better neutralize major TcdB variants both in cells and in vivo, as demonstrated by the colon-loop assay using female mice. Our findings provide valuable starting points for the development of therapeutics targeting C. difficile infections (CDI).