Systematic Cys mutagenesis of FlgI, the flagellar P-ring component of Escherichia coli

The bacterial flagellar motor is embedded in the cytoplasmic membrane, and penetrates the peptidoglycan layer and the outer membrane. A ring structure of the basal body called the P ring, which is located in the peptidoglycan layer, is thought to be required for smooth rotation and to function as a bushing. In this work, we characterized 32 cysteine-substituted Escherichia coli P-ring protein FlgI variants which were designed to substitute every 10th residue in the 346 aa mature form of FlgI. Immunoblot analysis against FlgI protein revealed that the cellular amounts of five FlgI variants were significantly decreased. Swarm assays showed that almost all of the variants had nearly wild-type function, but five variants significantly reduced the motility of the cells, and one of them in particular, FlgI G21C, completely disrupted FlgI function. The five residues that impaired motility of the cells were localized in the N terminus of FlgI. To demonstrate which residue(s) of FlgI is exposed to solvent on the surface of the protein, we examined cysteine modification by using the thiol-specific reagent methoxypolyethylene glycol 5000 maleimide, and classified the FlgI Cys variants into three groups: well-, moderately and less-labelled. Interestingly, the well- and moderately labelled residues of FlgI never overlapped with the residues known to be important for protein amount or motility. From these results and multiple alignments of amino acid sequences of various FlgI proteins, the highly conserved region in the N terminus, residues 1–120, of FlgI is speculated to play important roles in the stabilization of FlgI structure and the formation of the P ring by interacting with FlgI molecules and/or other flagellar components

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

タンパク質の抗体ラベリング技術を改良し、構造解析をアシスト --電子顕微鏡やX線結晶解析による構造決定を加速化--. 京都大学プレスリリース. 2021-04-20.Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

細胞膜の中ではたらく特殊なタンパク質分解酵素の構造を解明 --細菌感染症の新たな治療法の開発へ期待--. 京都大学プレスリリース. 2022-08-25.Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments

Roles of the membrane-reentrant β-hairpin-like loop of RseP protease in selective substrate cleavage.

Molecular mechanisms underlying substrate recognition and cleavage by Escherichia coli RseP, which belongs to S2P family of intramembrane-cleaving proteases, remain unclear. We examined the function of a conserved region looped into the membrane domain of RseP to form a β-hairpin-like structure near its active site in substrate recognition and cleavage. We observed that mutations disturbing the possible β-strand conformation of the loop impaired RseP proteolytic activity and that some of these mutations resulted in the differential cleavage of different substrates. Co-immunoprecipitation and crosslinking experiments suggest that the loop directly interacts with the transmembrane segments of substrates. Helix-destabilising mutations in the transmembrane segments of substrates suppressed the effect of loop mutations in an allele-specific manner. These results suggest that the loop promotes substrate cleavage by selectively recognising the transmembrane segments of substrates in an extended conformation and by presenting them to the proteolytic active site, which contributes to substrate discrimination

Role of the Intramolecular Disulfide Bond in FlgI, the Flagellar P-Ring Component of Escherichia coli

The P ring of the bacterial flagellar motor consists of multiple copies of FlgI, a periplasmic protein. The intramolecular disulfide bond in FlgI has previously been reported to be essential for P-ring assembly in Escherichia coli, because the P ring was not assembled in a dsbB strain that was defective for disulfide bond formation in periplasmic proteins. We, however, found that the two Cys residues of FlgI are not conserved in other bacterial species. We then assessed the role of this intramolecular disulfide bond in FlgI. A Cys-eliminated FlgI derivative formed a P ring that complemented the flagellation defect of our ΔflgI strain when it was overproduced, suggesting that disulfide bond formation in FlgI is not absolutely required for P-ring assembly. The levels of the mature forms of the FlgI derivatives were significantly lower than that of wild-type FlgI, although the precursor protein levels were unchanged. Moreover, the FlgI derivatives were more susceptible to degradation than wild-type FlgI. Overproduction of FlgI suppressed the motility defect of ΔdsbB cells. Additionally, the low level of FlgI observed in the ΔdsbB strain increased in the presence of l-cystine, an oxidative agent. We propose that intramolecular disulfide bond formation facilitates the rapid folding of the FlgI monomer to protect against degradation in the periplasmic space, thereby allowing its efficient self-assembly into the P ring

The Escherichia coli S2P intramembrane protease RseP regulates ferric citrate uptake by cleaving the sigma factor regulator FecR

Escherichia coli RseP, a member of the S2P family of intramembrane proteases, is involved in the activation of the σE extracytoplasmic stress response and elimination of signal peptides from the cytoplasmic membrane. However, whether RseP has additional cellular functions is unclear. In this study, we used mass spectrometry-based quantitative proteomic analysis to search for new substrates that might reveal unknown physiological roles for RseP. Our data showed that the levels of several Fec system proteins encoded by the fecABCDE operon (fec operon) were significantly decreased in an RseP-deficient strain. The Fec system is responsible for the uptake of ferric citrate, and the transcription of the fec operon is controlled by FecI, an alternative sigma factor, and its regulator FecR, a single-pass transmembrane protein. Assays with a fec operon expression reporter demonstrated that the proteolytic activity of RseP is essential for the ferric citrate-dependent upregulation of the fec operon. Analysis using the FecR protein and FecR-derived model proteins showed that FecR undergoes sequential processing at the membrane and that RseP participates in the last step of this sequential processing to generate the N-terminal cytoplasmic fragment of FecR that participates in the transcription of the fec operon with FecI. A shortened FecR construct was not dependent on RseP for activation, confirming this cleavage step is the essential and sufficient role of RseP. Our study unveiled that E. coli RseP performs the intramembrane proteolysis of FecR, a novel physiological role that is essential for regulating iron uptake by the ferric citrate transport system