Revisiting the functions of periplasmic chaperones in the quality control of the autotransporter Ag43 using a phenotypically homogeneous Escherichia coli strain.

Antigen 43 is a surface-displayed autotransporter protein that mediates bacterial self-association and pathogenicity. The quality control factors that facilitate Ag43 crossing the periplasm and inserting into the outer membrane remain enigmatic, mostly because Ag43 is phase variable and associated with heterologous phenotypes, which obscures the mutational effects of potential quality control factors. Here, we describe a screening method that allowed us to isolate a subpopulation of Escherichia coli that consistently displays an Ag43-mediated autoaggregation phenotype. Based on this subpopulation, we analyzed how disruptions of known periplasmic chaperones affect Ag43 biogenesis. We found that only the disruption of surA reduced Ag43 levels and abolished the autoaggregation phenotype of cells, but surA disruption did not affect the phase-variable expression of agn43. Using purified proteins, we showed that SurA effectively protected the ß-barrel domain of Ag43 from aggregation. In contrast, the previously reported Ag43 biogenesis factor OsmY showed weak chaperoning effects on Ag43 only in the absence of SurA. Our results shed light on the roles of different periplasmic chaperones in Ag43 biogenesis and provide a methodology applicable to the study of other phase-variable proteins.

Understanding the Stabilization Mechanism of a Thermostable Mutant of Hygromycin B Phosphotransferase by Protein Sector-Guided Dynamic Analysis.

Point mutations can exert beneficial effects on proteins, including stabilization. The stabilizing effects of mutations are typically attributed to changes in free energy and residue interactions. However, these explanations lack detail and physical insights, which hinder the mechanistic study of protein stabilization and prevent accurate computational prediction of stabilizing mutations. Here, we investigate the physical mechanism underlying the enhanced thermostability of a Hygromycin B phosphotransferase mutant, Hph5. We find that the unpredictable mutation A118V induces rotation of F199, allowing it to establish an aromatic-aromatic interaction with W235. In contrast, the predictable mutation T246A acts through static hydrophobic interactions within the protein core. These discoveries were accelerated by a residue-coevolution-based theory, which links mutational effects to stability-associated local structures, providing valuable guidance for mechanistic exploration. The established workflow will benefit the development of accurate stability prediction programs and can be used to mine a protein stability database for undiscovered physical mechanisms.

Temperature-Dependent Mechanical Properties of a Metal-Organic Framework: Creep Behavior of a Zeolitic Imidazolate Framework-8 Single Crystal.

Zeolite Imidazole Framework-8 (ZIF-8) with a robust structure and high thermal stability is a strong candidate to act as the catalyst matrix for various chemical applications, especially for those at higher temperatures, like hydrogenation. In this study, the time-dependent plasticity of a ZIF-8 single crystal was explored by a dynamic indentation technique to explore its mechanical stability at higher temperatures. The thermal dynamic parameters for the creep behaviors, like activation volume and activation energy, were determined, and possible mechanisms for the creep of ZIF-8 were then discussed. A small activation volume implies the localization of the thermo-activated events, while high activation energy, high stress exponent n, and weak dependence of the creep rate on temperature all favor pore collapse over volumetric diffusion as the creep mechanism.

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

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 ß-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 ß-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.