October 4, 2023
120 (41) e2309607120
Small proteins have high prevalence, vast diversity, and primarily regulatory functions in biological processes across all domains of life. However, their mechanisms of action remain largely elusive. In this study, we investigate the mechanism of the small protein, MgrB. It interacts with the sensor kinase PhoQ, rearranges its conformation, represses its kinase activity, and regulates bacterial response to environmental changes. In particular, for antimicrobial peptides, MgrB is required for bacteria to have a selective response to this host-exclusive stimulus. Our findings underline the importance of a small protein in bacterial fitness and drug resistance and provide a molecular basis for engineering peptide-based regulators.
A large number of small membrane proteins have been uncovered in bacteria, but their mechanism of action has remained mostly elusive. Here, we investigate the mechanism of a physiologically important small protein, MgrB, which represses the activity of the sensor kinase PhoQ and is widely distributed among enterobacteria. The PhoQ/PhoP two-component system is a master regulator of the bacterial virulence program and interacts with MgrB to modulate bacterial virulence, fitness, and drug resistance. A combination of cross-linking approaches with functional assays and protein dynamic simulations revealed structural rearrangements due to interactions between MgrB and PhoQ near the membrane/periplasm interface and along the transmembrane helices. These interactions induce the movement of the PhoQ catalytic domain and the repression of its activity. Without MgrB, PhoQ appears to be much less sensitive to antimicrobial peptides, including the commonly used C18G. In the presence of MgrB, C18G promotes MgrB to dissociate from PhoQ, thus activating PhoQ via derepression. Our findings reveal the inhibitory mechanism of the small protein MgrB and uncover its importance in antimicrobial peptide sensing.
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Small proteins are defined by their short sequences (usually < 50 aa) and their direct synthesis via ribosomal translation. An increasing number of small proteins have been identified in individual species in all domains of life and complex biological communities, suggesting a high prevalence and vast diversity in biological systems (reviewed in refs. 1–4). Many of these small proteins (~30%) are located in the cell membrane or have been predicted to be located there (e.g., refs. 5 and 6). Although only a few detailed studies of small membrane proteins have been conducted, results show that small proteins may target larger proteins and the lipid bilayer, regulating protein function, abundance, and changing membrane properties, demonstrating their predominant role as regulators in the lipid bilayer (reviewed in ref. 7).
The membrane located PhoQ sensor kinase, a master regulator of a bacterial virulence program, detects host-associated stimuli (review in ref. 8), such as cationic antimicrobial peptides (CAMPs) (9), mild acidic pH (10, 11), increased osmolarity (12), biliary unsaturated long-chain fatty acids (13), and magnesium limitation (14). Three small membrane proteins have been identified to control PhoQ activity via direct protein–protein interactions. In E. coli, MgrB (47aa) and SafA (65aa) modulate PhoQ activity, with MgrB acting as an inhibitor and SafA as an activator (15, 16). Deletion of mgrB or safA has physiological consequences, resulting in a cell division block under magnesium starvation or lowered acid resistance, respectively (17, 18). In Salmonella enterica serovar Typhimurium, a species-specific small protein, UgtS (34 aa), was reported recently and shown to control the timing of virulence gene expression by preventing premature PhoQ activation when inside macrophages (19).
The PhoQ sensor kinase is found as a dimer on the bacterial inner membrane. It consists of a periplasmic, a transmembrane (TM), a HAMP, a DHp, and a catalytic ATP-binding domain. In the periplasmic domain, several molecular interactions are essential for the functional state of the PhoQ molecule. These include the central hydrogen bonding network (T48 D179 K186) in the PAS-fold (20–23), the intermonomeric salt bridge (D179-R50') (24), the membrane-facing acidic cluster (148 EDDDDAE), and the juxta-membrane region at the periplasm/inner-membrane interface (9, 23, 25). Mutations altering these molecular interactions change the conformation of the periplasmic domain and the sensitivity of PhoQ to magnesium, low pH, and/or CAMPs. Additionally, different stimuli may result in distinct conformational states of the periplasmic domain, at least in an in vitro setting. For example, the PhoQ periplasmic domain showed divergent NMR spectra in low pH compared to that in low magnesium conditions (10). Besides the periplasmic domain, the PhoQ transmembrane domain was shown to detect environmental osmotic upshifts, where the transmembrane four-helix bundle adopts a more compact and kinase-active conformation (12).
Among the known PhoQ-regulating small proteins, MgrB is the most widely distributed PhoQ repressor in enterobacteria. In Salmonella, the deletion of mgrB was shown to affect its infection of macrophages and epithelial cells (26). In Klebsiella pneumonia, the disruption of the mgrB gene is the most prevalent among other chromosomal mutations in colistin-resistant strains (27). The expression of mgrB is PhoPQ dependent. Thus, the inhibitory function of MgrB provides negative feedback to the two-component system, resulting in a partial adaptation of E. coli to environmental changes (15, 28). MgrB is a bitopic inner membrane protein with a short cytosolic N terminus and a periplasmic C-terminal region (Fig. 1 A). Two conserved cysteines (C28 and C39) in the periplasmic region were shown to be essential for MgrB function and suggested to form a disulfide bond (15). This disulfide bond was proposed to allow MgrB to act as a sensor for the redox state of the bacterial periplasm and create an entry point for redox sensing by the PhoQ/PhoP system (29). Besides the two cysteines, eight functionally essential residues were identified spread across the entire molecule (30). However, the mechanism of MgrB inhibiting PhoQ remained largely unclear.
Fig. 1.
Image 1
Identification of the interacting residues between MgrB and PhoQ in the transmembrane helices. (A) The sequence of MgrB from E. coli. Residues predicted to be in the transmembrane helix are underlined. (B) The tertiary structure of the top-ranked PhoQ/MgrB complex predicted by AlphaFold2. The two PhoQ protomers are colored in wheat and teal. MgrB is colored in magenta. (C) The structural models of MgrB predicted by AlphaFold2 are shown in the ribbon. The model of MgrB alone (Left) is colored in the rainbow spectrum with the N terminus in blue and the C terminus in red. The positions involved in cross-linking (20, 22, and 24) are in purple and shown with side chains in ball-and-stick. The predicted structure of MgrB in complex (Right) is colored in magenta. All structural figures were prepared using PyMOL unless otherwise stated. (D, E Top, F Top) Western blot analysis of E. coli membrane extracts after disulfide cross-linking. The indicated PhoQ and FLAG-tagged MgrB variants were expressed in E. coli, followed by Cu-phenanthroline catalyzed disulfide cross-linking (details in Materials and Methods). The total protein stain of the PVDF membranes serves as loading control. Residues from PhoQ TM1 and TM2 are colored in wheat and teal, respectively. Magenta indicates the residues in the MgrB TM helix. Data are representative of at least three independent experiments. (D bottom, F bottom) The functional assay of PhoQ Cys-variants with or without MgrB. A GFP reporter plasmid carrying the gfp gene fused with a PhoP-regulated promoter P mgtLA was introduced to the E. coli strain. The expression of phoQ and mgrB variants was induced, and cells were grown to the early log phase. The function of PhoQ variants was then monitored by measuring the GFP fluorescence of the cell culture. Each data point is shown with the calculated average and SD from three independent biological replicates.
In this study, we combined cross-linking approaches with functional assays and protein dynamic simulations to identify interactions between PhoQ and MgrB, probe MgrB-induced PhoQ conformational changes, and examine the impact of MgrB on PhoQ sensing environmental stimuli. We propose that the binding of MgrB i) changes the conformation of the linker region between PhoQ periplasmic and transmembrane domains, ii) rearranges the transmembrane four-helix bundle, and iii) initiates the translocation of the catalytic domain. MgrB interferes with PhoQ signaling, resulting in an overall lowered activity, but still allows PhoQ activation under low pH, osmotic upshift, and magnesium limitation. We further reveal that MgrB mediates PhoQ sensing antimicrobial peptides, enabling bacteria to have a selective response to this exclusively host-associated stimulus.
We used AlphaFold2 multimer (31–33) to predict the structures of the PhoQ/MgrB complex as well as the individual component of the complex (Fig. 1 B and C and SI Appendix, Fig. S1). The overall prediction of structural elements agrees with previous experimental evidence. For example, the two conserved periplasmic cysteine residues (C28 and C39) in MgrB were positioned in close proximity in the models, structurally supporting the presence of a disulfide bond. In the PhoQ predictions, the PAS fold in the periplasmic domain, the HAMP, the DHp, and the cytosolic catalytic-ATP binding domains resembled the experimental results of these structural motifs (24, 34). Notably, the region connecting the periplasmic and TM domains and that connecting the HAMP and DHp domains showed a lower predicted local distance difference test (pLDDT) score (colored in yellow to green in SI Appendix, Fig. S1), indicating that these linker regions might have a higher capability of adopting different conformations. Unlike the asymmetric crystal structure of the PhoQ sensor domain (24), the AlphaFold2 predicted PhoQ dimer was symmetric, which likely represented only one of many conformations that PhoQ could adopt. In all five PhoQ/MgrB complex models (Fig. 1 B and SI Appendix, Fig. S1) the
