Massive antibody discovery used to probe structure–function relationships of the essential outer membrane protein LptD

eLife

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[1] Department of Antibody Engineering, Genentech, Inc, South San Francisco, United States;Department of Biochemical and Cellular Pharmacology, Genentech, Inc, South San Francisco, United States;Department of Biomolecular Resources, Genentech, Inc, South San Francisco, United States;Department of Infectious Diseases, Genentech, Inc, South San Francisco, United States;Department of Structural Biology, Genentech, Inc, South San Francisco, United States;

关键词: LptD;    outer membrane;    LPS;    monoclonal antibodies;    E. coli;    K. pneumoniae;    E. coli;    Other;   

DOI  :  10.7554/eLife.46258

来源: publisher

【 摘 要 】

10.7554/eLife.46258.001Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.

【 授权许可】

CC BY