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Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins.

ABSTRACT: Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed "Outer Membrane Protein Biogenesis Model" (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway.

SUBMITTER: Costello SM

PROVIDER: S-EPMC4995976 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Gram-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. IMPORTANCE Outer membrane proteins (OMPs) play critical roles in bacterial pathogenicity and provide a new niche for antibiotic development. A comprehensive understanding of the OMP quality control network will strongly impact antimicrobial discovery. Here, 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 β-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. Our study thus deepens the understanding of the complex OMP quality control system and highlights the differences in the mechanisms of ATP-independent chaperones.

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Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins.

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