Biochemical properties of the HtrA homolog from bacterium Stenotrophomonas maltophilia

Zarzecka, Urszula; Modrak-Wójcik, Anna; Bayassi, Martyna; Szewczyk, Maciej; Giełdoń, Artur; Lesner, Adam; Koper, Tomasz; Bzowska, Agnieszka; Sanguinetti, Maurizio; Backert, Steffen; Lipińska, Barbara; Skórko-Glonek, Joanna

doi:10.1016/j.ijbiomac.2017.11.086

Cited by 11 publications

(34 citation statements)

References 56 publications

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“…43 As shown in Table 1, the melting point for HtrA Cj was approximately 78° C, and was higher than T m of HtrA Ec and HtrA Sm , but lower by about 10°C compared to HtrA Hp . 41,42 The T m values seem to be in the agreement with the temperatures at which the enzymes exhibited their highest proteolytic activity (Table 1).…”

Section: Thermal Stability Of the Htra Cj Proteinsupporting

confidence: 64%

“…The melting point for HtrA Cj was approximately 78°C, and was higher than T m HtrA Ec and HtrA Sm , but lower about 10°C than HtrA Hp . 41,42 The environmental conditions of E. coli and C. jejuni are similar, as well as showing maximum proteolytic activity at a temperature of 50-55°C, and similar T m can be explained by their environmental adaptation. In contrast to H. pylori, which lives in extremely adverse stomach conditions and it is necessary that secreted fractions of HtrA Hp function properly, so Tm over 85°C has great importance in pathogenesis.…”

Section: Discussionmentioning

confidence: 92%

“…The start and end sites of the peptides were evaluated and the frequency of digestion in a given place is marked with heatmap-colored triangles. The crosses indicate similarity of published cleavage sites obtained with HtrA Hp and HtrA Ec (according to 41,42 ). S197A substitution mutant of HtrA Cj in E. coli, and after purification of the protein, the structure was investigated by Cryo-EM.…”

Section: Cryo-em Of Htra Cj and Structural Descriptionmentioning

confidence: 84%

“…Interestingly, the molecules of proteolytically active HtrA Cj migrated as smaller particles, probably hexamers ( Figure 6e) as judged from their elution volume (almost identical to that of the HtrA Ec hexamers). 41,42 Pre-incubation with β-casein, however, did not change the elution pattern of the wild-type HtrA Cj , so we can expect that the active enzyme completely degraded its substrate and returned to the resting state.…”

Section: Htra Cj Oligomerization Statusmentioning

confidence: 93%

“…As controls, HtrA Ec and HtrA Hp proteins were included, whose oligomer states have been previously established. 41,42 Proteolytically inactive S/A mutants of each of these proteins were purified in presence of urea and subsequently refolded to remove co-purifying peptides. As expected, in the absence of substrate, the refolded S/A mutant of HtrA Ec forms hexamers ( Figure 6a) and that of HtrA Hp is mainly present as trimers with a small fraction of hexamers (Figure 6b), while large complexes are formed in presence of β-casein.…”

Section: Htra Cj Oligomerization Statusmentioning

confidence: 99%

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Functional analysis and cryo-electron microscopy of Campylobacter jejuni serine protease HtrA

et al. 2020

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Campylobacter jejuni is a predominant zoonotic pathogen causing gastroenteritis and other diseases in humans. An important bacterial virulence factor is the secreted serine protease HtrA (HtrA Cj), which targets tight and adherens junctional proteins in the gut epithelium. Here we have investigated the function and structure of HtrA Cj using biochemical assays and cryo-electron microscopy. Mass spectrometry analysis identified differences and similarities in the cleavage site specificity for HtrA Cj by comparison to the HtrA counterparts from Helicobacter pylori and Escherichia coli. We defined the architecture of HtrA Cj at 5.8 Å resolution as a dodecamer, built of four trimers. The contacts between the trimers are quite loose, a fact that explains the flexibility and mobility of the dodecameric assembly. This flexibility has also been studied through molecular dynamics simulation, which revealed opening of the dodecamer to expose the proteolytically active site of the protease. Moreover, we examined the rearrangements at the level of oligomerization in the presence or absence of substrate using size exclusion chromatography, which revealed hexamers, dodecamers and larger oligomeric forms, as well as remarkable stability of higher oligomeric forms (> 12-mers) compared to previously tested homologs from other bacteria. Extremely dynamic decay of the higher oligomeric forms into lower forms was observed after full cleavage of the substrate by the proteolytically active variant of HtrA Cj. Together, this is the first report on the in-depth functional and structural analysis of HtrA Cj , which may allow the construction of therapeutically relevant HtrA Cj inhibitors in the near future.

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Section: Thermal Stability Of the Htra Cj Proteinsupporting

confidence: 64%

Section: Discussionmentioning

confidence: 92%

Section: Cryo-em Of Htra Cj and Structural Descriptionmentioning

confidence: 84%

Section: Htra Cj Oligomerization Statusmentioning

confidence: 93%

Section: Htra Cj Oligomerization Statusmentioning

confidence: 99%

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Functional analysis and cryo-electron microscopy of Campylobacter jejuni serine protease HtrA

et al. 2020

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Application of biophysical methods for improved protein production and characterization: A case study on an high‐temperature requirement A‐family bacterial protease

et al. 2022

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The high-temperature requirement A (HtrA) serine protease family presents an attractive target class for antibacterial therapeutics development. These proteins possess dual protease and chaperone functions and contain numerous binding sites and regulatory loops, displaying diverse oligomerization patterns dependent on substrate type and occupancy. HtrA proteins that are natively purified coelute with contaminating peptides and activating species, shifting oligomerization and protein structure to differently activated populations.Here, a redesigned HtrA production results in cleaner preparations with high yields by overexpressing and purifying target protein from inclusion bodies under denaturing conditions, followed by a high-throughput screen for optimal refolding buffer composition using function-agnostic biophysical techniques that do not rely on target-specific measurements. We use Borrelia burgdorferi HtrA to demonstrate the effectiveness of our function-agnostic approach, while characterization with both new and established biophysical methods shows the retention of proteolytic and chaperone activity of the refolded protein. This systematic workflow and toolset will translate to the production of HtrA-family proteins in higher quantities of pure and monodisperse composition than the current literature standard, with applicability to a broad array of protein purification strategies.

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Trimer stability of Helicobacter pylori HtrA is regulated by a natural mutation in the protease domain

Zarzecka

Tegtmeyer

Sticht

et al. 2023

Med Microbiol Immunol

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The human pathogen Helicobacter pylori is a major risk factor for gastric disease development. Serine protease HtrA is an important bacterial virulence factor that cleaves the cell junction proteins occludin, claudin-8 and E-cadherin, which causes gastric tissue damage. Using casein zymography, we discovered that HtrA trimer stability varies in clinical H. pylori strains. Subsequent sequence analyses revealed that HtrA trimer stability correlated with the presence of leucine or serine residue at position 171. The importance of these amino acids in determining trimer stability was confirmed by leucine-to-serine swapping experiments using isogenic H. pylori mutant strains as well as recombinant HtrA proteins. In addition, this sequence position displays a high sequence variability among various bacterial species, but generally exhibits a preference for hydrophilic amino acids. This natural L/S171 polymorphism in H. pylori may affect the protease activity of HtrA during infection, which could be of clinical importance and may determine gastric disease development.

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Biochemical properties of the HtrA homolog from bacterium Stenotrophomonas maltophilia

Cited by 11 publications

References 56 publications

Functional analysis and cryo-electron microscopy of Campylobacter jejuni serine protease HtrA

Functional analysis and cryo-electron microscopy of Campylobacter jejuni serine protease HtrA

Application of biophysical methods for improved protein production and characterization: A case study on an high‐temperature requirement A‐family bacterial protease

Trimer stability of Helicobacter pylori HtrA is regulated by a natural mutation in the protease domain

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