Chaperone Spy Protects Outer Membrane Proteins from Folding Stress via Dynamic Complex Formation.

Wei He,Yonghao Pang,Ling Bai,Yuanyuan Yang,Dana Reichmann,Maili Liu,Lichun He,Tianpeng Li,Gangjin Yu,Sebastian Hiller,Shu Quan,Zixiao Xue,Matthew R Chapman

doi:10.1128/mbio.02130-21

Abstract

ABSTRACTGram-negative bacteria have a multicomponent and constitutively active periplasmic chaperone system to ensure the quality control of their outer membrane proteins (OMPs). Recently, OMPs have been identified as a new class of vulnerable targets for antibiotic development, and therefore a comprehensive understanding of OMP quality control network components will be critical for discovering antimicrobials. Here, we demonstrate that the periplasmic chaperone Spy protects certain OMPs against protein-unfolding stress and can functionally compensate for other periplasmic chaperones, namely Skp and FkpA, in the Escherichia coli K-12 MG1655 strain. After extensive in vivo genetic experiments for functional characterization of Spy, we use nuclear magnetic resonance and circular dichroism spectroscopy to elucidate the mechanism by which Spy binds and folds two different OMPs. Along with holding OMP substrates in a dynamic conformational ensemble, Spy binding enables OmpX to form a partially folded β-strand secondary structure. The bound OMP experiences temperature-dependent conformational exchange within the chaperone, pointing to a multitude of local dynamics. Our findings thus deepen the understanding of functional compensation among periplasmic chaperones during OMP biogenesis and will promote the development of innovative antimicrobials against pathogenic Gram-negative bacteria.

Highlights

Gram-negative bacteria have a multicomponent and constitutively active periplasmic chaperone system to ensure the quality control of their outer membrane proteins (OMPs)
By SDS-PAGE analyses of whole-cell lysates from multiple chaperone-deleted strains based on E. coli MG1655, we found that deletion of skp or fkpA alone had little effect on total protein levels, whereas Dskp DfkpA double knockout resulted in decreased protein intensity in the molecular weight range of OmpC, OmpF, and OmpA (35 to 40 kDa) (Fig. S2A)
Our in vivo and in vitro results suggest that Spy, a chaperone proposed to be promiscuous toward many periplasmic proteins [37], contributes to maintaining the proteostasis of certain Outer membrane proteins (OMPs)

Summary

Introduction

Gram-negative bacteria have a multicomponent and constitutively active periplasmic chaperone system to ensure the quality control of their outer membrane proteins (OMPs). We systematically demonstrate that the periplasmic chaperone Spy has a role in maintaining the homeostasis of certain OMPs. Remarkably, Spy utilizes a unique chaperone mechanism to bind OmpX and allows it to form a partially folded b-strand secondary structure in a dynamic exchange of conformations. Spy utilizes a unique chaperone mechanism to bind OmpX and allows it to form a partially folded b-strand secondary structure in a dynamic exchange of conformations This mechanism differs from that of other E. coli periplasmic chaperones such as Skp and SurA, both of which maintain OMPs in disordered conformations. Outer membrane proteins (OMPs) embedded in the OM have a characteristic b-barrel fold and exert various functions, including nutrient transport, cell adhesion, virulence, and multidrug resistance [1, 2]. It is known that spy deletion results in activation of the s E stress response pathway, which monitors OMP biogenesis and can be activated by accumulation of misfolded OMPs upon cell envelope damage or excessive OMP synthesis, implicating that spy deletion may have an influence on OMP homeostasis [21, 22]

Methods

Results

Conclusion