Structural and Biophysical Characterization of the Yersinia Type Three Secretion System ATPase YscN

Structural and Biophysical Characterization of the Yersinia Type Three Secretion System ATPase YscN | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 19 November 2025 V1 Latest version Share on Structural and Biophysical Characterization of the Yersinia Type Three Secretion System ATPase YscN Authors : Samuel Barker A , Porter Ellis K , Andrew Hammer , Sean Johnson 0000-0001-7992-2494 , and Nicholas Dickenson 0000-0003-1572-6077 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176359530.02438941/v1 203 views 121 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Yersinia pestis was responsible for the Black Plague, one of the worst epidemiological disasters in recorded history. Today, Y. pestis , Y. enterocolitica , and Y. pseudotuberculosis remain clinically relevant human pathogens. Each of these pathogenic Yersinia species rely on a Type Three Secretion System (T3SS) for virulence, with the ATPase YscN playing a critical role in T3SS function. T3SS ATPases are responsible for powering apparatus formation and effector protein secretion through ATP hydrolysis. This study provides an extensive enzymatic characterization of recombinant YscN under several conditions, including variable pH and temperature, substrate and protein concentrations, and in the presence of putative inhibitors. Thermal stability data, assessed by circular dichroism, demonstrate that YscN exhibits increased stability in alkaline conditions, coinciding with greatest ATPase activity. Further, we report the first high-resolution crystal structure of YscN and leverage homology data to model an oligomeric active site. Mutational analysis of a predicted active site residue confirms oligomerization as necessary for YscN ATPase activity and corroborates our oligomeric model and enzyme concentration-dependent specific activity. Interestingly, however, AUC analysis reveals that the purified YscN predominantly exists as a monomer, despite oligomerization-dependent active site formation. Thus we propose that transient oligomeric interactions support the observed ATP hydrolysis. Together, these data uncover structural and environmental impacts on YscN activity that may support the highly specialized Yersinia pathogenic lifecycle and leverage its role in virulence in search of pan-effective small molecule T3SS ATPase inhibitors. Introduction Yersinia pestis was responsible for the Black Plague during the 14 th century. 1 It is estimated that this event resulted in the death of 30% to 50% of the European population and stands out as one of the most devastating pandemics in recorded history. 2 Fortunately, global monitoring by service organizations, such as the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO), and the availability of antibiotics significantly reduce the likelihood of such a lethal Y. pestis pandemic occurring again. 1, 3 Despite this, Yersinia infections remain a significant health concern with Y. pestis , Y. enterocolitica , and Y. pseudotuberculosis each infecting humans. 4 Y. enterocolitica , and Y. pseudotuberculosis infections are most common and cause yersiniosis, an acute gastroenteritis . 5 Additionally, worldwide Y. pestis infections resulting in bubonic and pneumonic plage continue to be reported and remain endemic in several countries. 6 Antibiotic-resistant strains of these pathogenic Yersinia species are rapidly emerging, limiting treatment options and prioritizing research to better understand Yersinia virulence mechanisms and motivating the development of new therapeutics against them. 7, 8 As for many Gram-negative pathogenic bacteria, Yersinia rely on a type three secretion system (T3SS) as a primary virulence factor. 9 This system includes the type three secretion apparatus (T3SA), or injectisome, which is a complex, needle-like structure that penetrates a host cell membrane and secretes effector proteins directly into the host cell cytoplasm. 10, 11 These secreted effectors are unique to the pathogens expressing them and support infection through means such as driving phagolysosome maturation, cell and vacuolar membrane disruption, actin remodeling, and many approaches to evade host immune responses. 12, 13 These secreted effector proteins must first be recognized by the sorting platform at the base of the T3SS injectisome and partially unfolded prior to passing through the ~2.5 nm inner diameter of the apparatus needle. 14, 15 While the mechanisms supporting effector protein recognition, unfolding, and secretion remain largely unclear and often controversial, it is clear that ATP hydrolysis by ATPases located at the base of all known T3SS injectisomes is essential for proper injectisome assembly, effector protein secretion, and virulence. 16-18 Based on sequence homology and biochemical kinetics experiments, YscN is the Yersinia T3SS ATPase that supports its T3SS function. 11, 19 In fact, Y. pestis expressing an ATPase inactive YscN mutant exhibited an over three million-fold reduction in virulence compared to a strain expressing wild-type YscN. 20 Similar impacts on virulence have been observed in T3SS ATPase inactive mutant strains of Shigella , Salmonella , and enteropathogenic E. coli , highlighting the conserved dependence on T3SS ATPase activity. 18, 21, 22 Native protein-protein interactions between T3SS ATPases and regulatory T3SS proteins, such as MxiN and Spa33 in Shigella, FliI in Salmonella , and YscL in Yersinia, regulate the enzymatic activity of these ATPases, underscoring the importance of the T3SS ATPase in control over T3SS function and virulence. 19, 23-25 In addition, environmental cues specific to the infection niches of each pathogen further influence virulence through mechanisms ranging from apparatus composition and maturation to transcription of key T3SS genes. 26 For example, bile salts found in the human small intestine interact with the protein tip complex of the Shigella T3SS to recruit translocator proteins in preparation of interaction with the colonic epithelium (site of infection). 27-30 Yersinia are highly sensitive to local calcium concentrations, where low calcium environments, such as the host cell cytoplasm, lead to bacteriostasis and the secretion of Yop effector proteins via the T3SS. 31 When Salmonella experience low low-pH environments, such as found in a macrophage Salmonella -containing vacuole (SCV), assembly of the SPI-2 T3SS and secretion of key virulence proteins perturb this immune response. 32 This creates a protected intracellular niche that allows Salmonella to replicate within the SCV, while avoiding immune detection. Eventually, SPI-2 T3SS effectors facilitate disruption of the SCV membrane, promote macrophage lysis, and enable bacterial escape into the extracellular space, spreading the infection. In each of these cases, disruption of the native response pathways markedly reduces virulence. 32-34 Environmental responses such as these appear to have evolved highly specific T3SS-centric mechanisms to support pathogen survival and virulence and mounting evidence suggests that an additional point of regulation may be the conserved T3SS ATPases found in all T3SSs described thus far. Here, we provide a robust analysis of temperature and pH effects on the Yersinia T3SS ATPase, YscN, which reveals a sensitive pH cutoff for ATPase activity, and provides the first high-resolution YscN structure. Interestingly, though T3SS ATPases are almost certainly all associated with T3SS injectisomes as homo-oligomers, 35-37 pure recombinant YscN purifies predominantly as monomers. Structural and functional characterizations suggest that the ATPase activity of monomeric YscN relies on transient oligomer formation to complete an interfacial active site. These observations are consistent with the idea that assembly of oligomeric YscN onto the injectisome is required for YscN activation and T3SS function. This, together with a recent Cryo-EM structure of EscN, the E. coli T3SS ATPase, 35 motivated the construction of a homo-hexameric YscN model that suggests subtle, yet critical, structural differences between YscN and homologous T3SS ATPases. Collectively, these findings catalog diverse environmental impacts on YscN ATPase activity and kinetics, demonstrate the oligomer-dependent ATPase activation of YscN, and provide the first structural characterization of YscN. Further, we assess the impact of recently designed small-molecule T3SS ATPase inhibitors to support the feasibility of pan-effective inhibitor design and, in doing so, highlight the need to carefully consider the structure and activation mechanism of each T3SS ATPase target. Materials and Methods Materials - Escherichia coli strains and DNA ligase were from Novagen (Madison, WI). Restriction enzymes, the pTYB21 protein expression plasmid, Q5 DNA polymerase, and chitin resin were from New England Biolabs (Ipswich, MA). DNA primers and double stranded gBlocks were from Integrated DNA Technologies (Coralville, IA). The Superdex 200 size exclusion and HiTrapQ FF columns were purchased from GE Healthcare (Pittsburgh, PA). Dithiothreitol (DTT), isopropyl ß-D-1-thiogalactopyranoside (IPTG), 4- (2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), and ampicillin were from Gold Biotechnology (St. Louis, MO). Tryptone, and yeast extract were from Genesee Scientific (San Diego, CA) and ATP was from Sigma-Aldrich (St. Louis, MO). The malachite green inorganic phosphate detection kit was from BioAssay Systems (Hayward, CA). The small molecule inhibitors 1870, 3812, 4000, 8573, and 8771 were from Enamine (Monmouth Jct., NJ), 4967 was from ChemBridge (San Diego, CA), and 1691, 2357, 5765, and 6573 were from ChemDiv (San Diego, CA). All other solutions and chemicals were of reagent grade. The UniProtKB accession numbers for YscN and Spa47 are P40290 and P0A1C1, respectively. Cloning - The wild-type yscN gene was purchased as a double stranded gBlock from IDT (Coralville, IA) and was ligated into the commercial pTYB21 expression plasmid encoding an N-terminal chitin-binding domain (CBD) and self-cleaving intein linker. Subsequently, the ligation product was transformed into E. coli NovaBlue competent cells via heat shock. A single ampicillin resistant colony was selected for outgrowth, the yscN /pTYB21 plasmid isolated by miniprep, and the sequence verified by whole plasmid sequencing (Azenta Life Sciences). The YscN ∆1-92 , YscN R359A , and YscN ∆1-92, R359A mutant constructs were created by inverse PCR using non-overlapping 5’ phosphorylated primers. The PCR products were ligated and transformed into NovaBlue competent cells prior to colony selection, plasmid isolation, and sequence verification. Each of the sequence verified constructs were subsequently transformed into NiCo21(DE3) competent E. coli cells for protein expression. A spa47 /pTYB21 expression construct that was cloned and validated previously was used for Spa47 expression. 22 Protein Expression and Purification - NiCo21(DE3) E. coli carrying the pTYB21 expression constructs encoding YscN WT , YscN R359A , YscN Δ1-92 , YscN Δ1-92, R359A , or Spa47 WT were used to inoculate small overnight cultures in Luria Broth (LB, Miller) media supplemented with 0.1 mg/mL ampicillin. These saturated cultures were then used to inoculate larger cultures in Terrific Broth (TB) media containing 0.1 mg/mL ampicillin. The TB cultures were grown at 37 C and 200 rpm to an A 600 ~0.8. Cultures were subsequently cooled to 17 C and induced with 1 mM isopropyl β -D-1-thiogalactopyranoside (IPTG) for ~20 h (17 C, 200 rpm). All subsequent steps were performed at 4 C unless otherwise noted. The NiCo21(DE3) cells were harvested by centrifugation, and the cell pellets were resuspended in chitin binding buffer (20 mM Tris, 500 mM NaCl, 0.48 µg/mL AEBSF, pH 7.9) and lysed via sonication. The lysate was clarified by centrifugation and soluble proteins in the supernatant exposed to chitin affinity resin for capture of the CBD-intein-YscN or CBD-intein-Spa47 complexes. Chitin elution buffer (20 mM Tris, 500 mM NaCl, 50 mM DTT, pH 7.9) was then added to the column to facilitate self-cleavage of the intein domain and release of the expressed protein of interest. In all subsequent purification steps, solutions containing YscN Δ1-92 or YscN Δ1-92, R359A crystallization constructs were supplemented with 5% glycerol (v/v). Chitin column elution fractions were evaluated via SDS PAGE and appropriate fractions pooled and diluted to 20 mM Tris, 100 mM NaCl, 10 mM DTT, pH 7.9. The protein was further purified using a Q-sepharose anion exchange column for positive (YscN WT and YscN R359A ) or negative (YscN ∆1-92 , YscN ∆1-92, R359A , and Spa47) selection. Elution fractions of YscN WT and YscN R359A or flow through of YscN ∆1-92 , YscN ∆1-92, R359A , and Spa47 were subsequently concentrated and subjected to a final gel filtration purification step using a Superdex 200 16/600 size exclusion column and isocratic elution in 20 mM Tris, 100 mM NaCl, 5 mM DTT, pH 8.0. Protein purity was assessed by SDS-PAGE. Kinetic Characterization of YscN - YscN ATPase activity was monitored using a malachite green colorimetric assay. ATPase reactions were initiated with the addition of indicated concentrations of ATP and 10 mM MgCl 2 to a 20 mM Tris, 100 mM NaCl, 5 mM DTT solution containing YscN. Reaction samples were taken at indicated time points, quenched with the malachite green detection agent solution, and each kinetic assay was analyzed in triplicate to allow evaluation of statistical significance. Malachite green absorbance was measured at 620 nm with a BioTek Synergy H4 hybrid reader thirty minutes after quenching the reaction and P i concentrations were quantified against a standard curve. Reaction conditions, including temperature, pH, substrate concentration, enzyme concentration, and the presence of small molecule inhibitors were examined in this study and the specific conditions of each are given in the figures presenting the resulting data sets. The general reaction conditions to which these modifications were made are 20 mM Tris, 100 mM NaCl, 5 mM DTT, pH 8.0, and 28 C with 10 mM MgCl 2 , 600 µM ATP, and 1.75 µM YscN. For temperature dependence assays, activity was quantified in the general assay condition described above, but the reactions were performed in a heat block at the indicated temperatures. For pH dependence assays, YscN was buffer-exchanged into 20 mM Tris, 100 mM NaCl, 5 mM DTT at the indicated pH values using spin desalting columns just prior to filtering and re-assessing protein concentration and initiating ATPase reactions. Substrate and enzyme dependence assays were performed in the general assay conditions described above with varied substrate or YscN concentrations, as indicated. To test recently described Spa47 ATPase inhibitors on YscN, the inhibitors were first dissolved in DMSO to a concentration of 200 mM and then added to the YscN general reaction condition (lacking ATP and MgCl 2 ) to a final concentration of 250 µM inhibitor and 2.5% DMSO and incubated for 30 minutes before initiating the reactions. Because YscN purifies as a monomer and the Spa47 inhibitors had been previously tested against purified Spa47 oligomers, each inhibitor was also tested against monomeric Spa47 with the following modifications from YscN conditions (based upon previously published studies 17 ): the Spa47 activity buffer was 10 mM Tris, 100 mM NaCl, 5 mM DTT, pH 7.9 and the assay was performed at 22 C with a Spa47 protein concentration of 0.15 µM. SigmaPlot was used to fit kinetic data sets to the Michaelis-Menton equation: v 0 = (V max, app [S])/(K M, app +[S]) Where v 0 is the initial reaction velocity, V max, app is the maximal reaction velocity under the tested conditions, [S] is the concentration of ATP, and K M, app is the Michaelis constant of the enzyme under the tested conditions. Far-Ultraviolet (far-UV) Circular Dichroism (CD) - Far-UV CD spectra and secondary structure thermal stability profiles were collected for YscN WT using a JASCO model J-1500 spectropolarimeter equipped with a temperature-controlled sample chamber. CD spectra were collected from 190 to 260 nm at 28 C using 0.1 cm quartz cuvettes, 1 nm data sampling, a 50 nm/min scan rate, and a 1 s data integration time. Secondary structure thermal stability profiles were collected in the same 0.1 cm quartz cuvettes by monitoring the CD signal at the 208 nm spectra minima, while the solution temperature was increased from 10 C to 90 C at a rate of 0.5 C/min. Measurements were performed at protein concentrations of 0.4-0.8 mg/mL and CD signals were concentration normalized by converting ellipticity units to mean residue molar ellipticity. Spectral analysis was performed using the BeStSel CD spectral analysis server 38 to quantify the protein secondary structure content. Thermal unfolding transition temperatures ( T m ) were determined by fitting the unfolding data to a 4 parameter sigmoidal function and identifying the corresponding local maxima of the function derivative. T onset values representing the temperature corresponding to 40% of the maximal transition slope were also identified using the derivatives of the sigmoidal fits. Cystallization, Data Collection, and Structure Determination - Crystallization of YscN ∆1-92 and YscN ∆1-92, R359A was achieved using standard hanging drop vapor diffusion techniques. Initial crystallization conditions were identified from the commercial Hampton Research (Aliso Viejo, CA) MCSG-1 screen (condition F11) and Additive Screen kits. Crystals used in this study resulted from conditions spanning 19-32% PEG 3350 and 0.16-0.24 M (NH 4 )SO 4 in 0.1 M HEPES, pH 7.5. 10mg/mL YscN ∆1-92 was plated in 2:1.5 protein:well drop volume ratios and supplemented with 1 part 0.1 M spermidine. 10 mg/mL YscN ∆1-92, R359A was plated in 2:2 protein:well drop volume ratios. Crystals were grown at room temperature and appeared within one week. YscN ∆1-92 crystals were cryo-preserved in 29% PEG 3350, 10% glycerol, 0.18 M (NH 4 )SO 4 , and 0.1 M HEPES, pH 7.5. YscN ∆1-92, R359A crystals were cryo-preserved in 30% PEG 3350, 5% Glycerol, 0.2 M (NH 4 )SO 4 , and 0.1 M HEPES, pH 7.5. YscN ∆1-92 crystal X-ray intensity data were collected to 2.0 Å resolution on a home source diffractometer system equipped with a RIGAKU MICROMAX-007 HF rotating anode X-ray generator and RIGAKU RAXIS IV++ detector. YscN ∆1-92, R359A crystal X-ray intensity data were collected to 2.5 Å resolution on BEAMLINE BL9-2 at the Stanford Synchrotron Radiation Lightsource. Diffraction data were processed using HKL2000. 39 The YscN ∆1-92 structure was solved in the P1 space group, with two molecules in the asymmetric unit, by molecular replacement using a Spa47 ∆1-79 (PDB: 5SWJ) search model. The YscN ∆1-92, R359A structure was also solved in the P1 space group, with two molecules in the asymmetric unit, using the YscN ∆1-92 structure from this study as a search model for molecular replacement. Model building was performed in Coot and refinement, carried out to convergence, was performed in the PHENIX suite. 40, 41 A full summary of the collection, data reduction, and refinement statistics is included in Table 2. The PyMOL molecular graphics system was used to generate and render molecular models. 42 Analytical Ultracentrifugation - Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC) experiments were conducted using an Optima XL-I (Beckman Coulter, Fullerton, CA) analytical ultracentrifuge equipped with an absorbance optical detection system. 16, 24, and 32 µM YscN WT and YscN Δ1-92 and buffer references were loaded into separate Beckman Charcoal-Epon two-sector cells with quartz windows and 12-mm path lengths. Samples were centrifuged at 20 C and 40,000 rpm in a 50 Ti eight-hole rotor until complete sedimentation was achieved. The buffer density (1.00310 g/mL), buffer viscosity (0.01018 Poise), and protein partial specific volumes for YscN WT and YscN Δ1-92 (0.74059 mL/g and 0.73637 mL/g, respectively) were calculated using Sednterp. 43 Sedimentation data for three independent sedimentation profiles were analyzed using a continuous c(s) distribution with SEDFIT. 44 Results YscN ATPase activity is strongly influenced by temperature and pH – Like most pathogens, Yersinia spp. experience changing environmental conditions during the course of an infection and it is likely that these conditions provide important cues as to how virulence associated pathways respond. After recombinantly expressing and purifying the Yersinia T3SS ATPase YscN, we identified robust activity conditions by first examining temperature and pH influences on its ATPase activity (Figure 1). Figure 1. Temperature and pH impacts on YscN catalyzed ATP hydrolysis. (A) The effect of temperature on YscN ATPase activity was examined (at pH 8.0). A strong positive correlation between activity and temperature is observed between 20 C and 28 C. Activity levels off to 32 C and is followed by a sharp decline as temperature increases to 36 C. (B) YscN ATPase activity is strongly influenced by pH (tested at 28 C), with no activity observed at pH 6.75 and a strong enhancement observed as the pH increases to 8.0-8.5. Activity declines as the pH further increases to 9.25. Each data point in both (A) and (B) represents the mean rate (specific activity) ± standard deviation determined from the initial velocity values calculated from multiple-timepoint activity profiles of three independent biological replicates. Based upon conditions described for homologous T3SS ATPases, 45, 46 temperature impact on YscN ATPase activity was first measured at a pH of 8.0 and assay temperatures were examined from 20 C to 36 C. The highest rates of ATP hydrolysis are observed between 28 C and 32 C (Figure 1A). Activity falls off steeply as the temperatures are reduced from 28 C or increased from 32 C, losing ~50% of activity when the assay temperatures reach a low of 20 C or high of 36 C. pH influence on activity was then tested by maintaining the temperature at 28 C and varying the assay pH from 6.75 to 9.25 (Figure 1B). The greatest activity levels were observed between pH 8.0-9.0 with a strong negative influence in conditions more acidic than pH 8.0. More specifically, YscN is completely inactive at pH 6.75, but displays a strong positive correlation in activity as pH increases, with maximal activity observed at pH 8.5. A modest decline in activity results from further increasing the pH to 9.25. Based on these data, all subsequent kinetic assays in this study were performed at 28 C and pH 8.0 as this supports robust YscN activity near the maximal buffering capacity of Tris (pKa 8.07). pH influence on YscN activity correlates to secondary structure content and thermal stability - Far-UV circular dichroism (CD) spectroscopy was performed on YscN in each of the pH conditions examined in the kinetic assays described above (Figure 2A). Regardless of the tested pH, each of the CD spectra exhibit minima at 208 nm and 222 nm, consistent with the dominantly α-helical secondary structure expected from this family of T3SS ATPases. 22, 47 Interestingly, the pH 8.0 and 8.5 conditions that proved optimal for ATPase activity also result in the strongest CD signals while the more “extreme” pHs of 6.75, 9.0, and 9.25 yield the weakest CD signals, perhaps a direct impact of pH on YscN secondary structure content and/or stability. To examine this impact quantitatively, the spectra were each submitted to the BeStSel CD spectral analysis server for secondary structure content calculations. 38 The secondary structure prediction results from three biological replicates are displayed in Table 1. Though the secondary structure content calculations suggest a trend in which the highest activity conditions (pH 8.0 and 8.5) contain the highest degree of α-helix content, no statistically significant differences in calculated content are observed in the tested pH conditions. YscN thermal unfolding profiles were additionally collected across the tested pH conditions (Figure 2B) and as expected, each result in a temperature dependent loss of YscN secondary structure. The melting temperatures (T M ) in each condition cluster from 38 C to 41 C (Table 1), though the unfolding curves (Figure 2B) clearly show that pH impacts the temperature at which unfolding begins (T onset ). The T onset is quantified by determining the temperature that corresponds to a transition slope of 40% of that at the inflection point (T M ). The lowest T onset value of 32 ± 3 C was seen in the pH 6.75 condition, with the highest T onset values of 37 ± 2 C, 38 ± 1 C, and 37 ± 2 C in pHs 8.0, 8.5, and 9.0, respectively. Figure 2. pH impacts YscN secondary structure content and stability. (A) Far-UV CD spectra were collected in pH conditions tested previously for impact on ATPase activity (pH 6.75 to 9.25). All collected spectra exhibit minima at 208 nm and 222 nm, consistent with significant α-helical content. Spectra collected at pH 8.0 and 8.5 demonstrate the strongest mean residue molar ellipticities, suggesting a greater proportion of defined secondary structure content compared to the other tested pH conditions. (B) Thermal unfolding profiles of YscN result from monitoring CD signal at 208 nm as the solution temperature is increased from 10 C to 90 C. Thermal unfolding profiles collected at pH 8.0 and 8.5 demonstrate sharp unfolding transitions that are initiated at higher temperatures than the other tested pHs. The spectra and unfolding profiles are representative of three independent biological replicates and the calculated secondary structure content and quantified T M and T onset values from the spectra and unfolding profiles are summarized in Table 1. Table 1. YscN secondary structure content and thermal stability pH α-Helix (%) β-Sheet (%) Turn (%) Other (%) T M (C) T onset (C) 6.75 18 ± 1 19 ± 2 17 ± 1 47 ± 1 39 ± 1 32 ± 3 7 20 ± 1 18 ± 1 17 ± 1 46 ± 2 39 ± 1 34 ± 1 7.25 20 ± 3 19 ± 2 17 ± 1 45 ± 3 38 ± 1 33 ± 1 7.5 20 ± 3 18 ± 1 17 ± 1 44 ± 4 38 ± 1 34 ± 1 8 22 ± 3 18 ± 3 18 ± 1 42 ± 3 40 ± 1 37 ± 2 * 8.5 23 ± 6 18 ± 2 17 ± 1 42 ± 6 41 ± 2 38 ± 1 * 9 20 ± 9 22 ± 5 17 ± 1 42 ± 5 41 ± 1 37 ± 2 * 9.25 20 ± 5 23 ± 5 17 ± 1 40 ± 3 41 ± 2 36 ± 2 a YscN secondary structure content was calculated from the far-UV CD spectra using the BeStSel CD spectral analysis server. YscN secondary structure thermal stability was monitored by recording the CD signal at 208 nm as the temperature increased from 10 C to 90 C. The melting temperature (T M ) was determined by fitting the thermal unfolding data to a 4-parameter logistic sigmoidal function and identifying the infection point of each unfolding curve. The melting onset temperature (T onset ) was determined as the temperature at which the slope of the thermal denaturation profile is 40% of that at the inflection point. All values are reported as the mean ± standard deviation resulting from three independent biological replicates at each indicated pH value. *indicates statistical significance vs the corresponding value from pH 6.75 (one-way ANOVA and Dunnett’s test, p ≤ 0.05). YscN ATPase activity requires homo- oligomerization – High-resolution structures often prove invaluable in describing enzyme function; however, no structure of YscN was previously available. Here, YscN constructs and crystallization conditions were explored to solve a 2.0 Å resolution X-ray structure of YscN Δ1-92 (Figure 3A, Table 2). As truncation of the N-terminal oligomerization domain of other T3SS ATPases was required for their crystallization, 22, 47, 48 we generated a similar construct for this study by truncating the first 92 residues from YscN. Notable features in the catalytic core of the YscN structure (Figure 3A) include a Rossmann fold and a Walker A motif, or P-loop, predicted to be essential for phosphate binding, as well as a catalytic Walker B motif, each consistent with its ATPase function. 49, 50 A more complete picture of the active site is seen in the model presented in Figure 3B where two monomeric YscN crystal structures are aligned to adjacent protomers of a high-resolution cryo-EM structure of the T3SS ATPase EscN from E. coli (PDB: 6NJO, RMSD 0.985 Å). This alignment shows that, like in the EscN complex, the YscN active site is located at the interface between YscN protomers, evidenced by the presence of catalytic active site residues spanning the protomer interface. These residues include Lysine 175 and Glutamate 198, which are highly conserved in T3SS ATPases and essential for activity, 47 and Arginine 359, which extends into the ATP binding site from an adjacent YscN oligomer to stabilize the negatively charged ATP substrate and position it for nucleophilic attack by the catalytic water observed in the model. This “arginine finger” has been observed in several additional ATPases, including F1 ATP synthase, Spa47, and FliI. 22, 48, 51 Because the arginine is contributed by an adjacent protomer in this YscN model, yet recombinant YscN demonstrated robust ATPase activity in vitro (Figure 1), the active YscN must form at least a homo-dimer to provide this completed active site. To test this model and the importance of R359 in YscN ATPase function, R359 was mutated to alanine to generate YscN R359A . YscN R359A was recombinantly expressed and purified and malachite green ATPase assays were performed, confirming a complete loss of ATPase activity by this mutant (Figure S1). Figure 3. 2.0 Å X-ray crystal structure of the Yersinia T3SS ATPase YscN. (A) The crystal structure of YscN Δ1–92 is shown with coloration corresponding to the predicted domains designated in the included schematic of the YscN sequence. The core catalytic ATPase domain is colored gray, the Walker A (P-loop) and B motifs are shown in orange and purple, respectively, the C-terminal domain is in cyan, and the truncated N-terminal oligomerization domain is represented by a dashed line. (B) YscN modeled as an activated homo-oligomer, based on the structure of the enteropathogenic Escherichia coli homolog EscN, where ADP is bound in the active site formed at the interface between protomers. 35 In Zoom is a closer view of the YscN active site model where Lys175 and Glu198 side chains from one protomer (gray), Arg395 from the adjacent protomer (slate), and a catalytic water are in proximity to the bound ADP modeled from its location in the published EscN Cryo-EM structure. 35 To verify that elimination of activity by the YscN R359A mutant resulted from the exchange of the intended sidechain and not via unintended impact on YscN structure, a 2.50 Å X-ray structure of YscN Δ1-92, R359A was also solved (Figure S1, Table 2). When aligned to YscN Δ1-92, WT , it is clear that the overall protein structure is unaltered by the mutation (Figure S1, Cα RMSD 0.195Å). Furthermore, YscN Δ1-92, R359A appropriately maintains the presence and positioning of the catalytic E198 and K175 residues despite elimination of the R359 sidechain, confirming the importance of the arginine and YscN oligomerization in ATPase activity (Figure S1, Figure 3B) . Recombinant YscN activity relies on transient homo-oligomer formation - The absolute reliance of YscN on the presence of interfacial active site residues contributed from adjacent protomers supports an oligomerization-based activation of YscN. Interestingly, however, the retention volume of pure YscN fractions isolated by size exclusion chromatography suggest that it is primarily monomeric, despite demonstrating robust activity in vitro (Figure 1). Notably, a modest population of large, ATPase inactive, aggregate YscN species elutes in the column void volume and fractions of YscN containing contaminating E. coli proteins elute just prior to fractions of pure YscN. Thus, this study exclusively focuses on the highly pure YscN isolated from SEC. Sedimentation velocity analytical ultracentrifugation confirmed that this YscN population is nearly exclusively monomeric with a sedimentation coefficient of 3.37 ± 0.02 Svedbergs and a friction ratio of 1.43 ± 0.05 (Figure 4). Importantly, the YscN AUC analysis was performed at concentrations exceeding those of the activity assays to ensure that a concentration-dependent formation of higher order species was not overlooked in this assessment (16 µM – 32 µM for AUC and 1.75 µM for kinetics). The N-terminal truncation construct of YscN engineered for crystallization (YscN Δ1-92 ) was additionally assessed by AUC with the c(s) vs (s) distribution displaying a sharp, symmetric peak corresponding to a sedimentation coefficient of 2.60 ± 0.01 Svedbergs and a friction ratio of 1.49± 0.03, as expected for monomers of this truncated construct (Figure S2). Table 2. YscN data collection, phasing and refinement statistics. Data collection X-ray source Home source SSRL 9-2 Wavelength (Å) 1.54 0.9795 Space group P 1 P 1 Cell dimensions a, b, c (Å) 46.6, 59.9, 65.1 46.6, 60.5, 65.1 α, β, γ (°) 94.0, 89.0, 99.0 94.5, 89.9, 98.8 Resolution (Å) a 50.00-2.00 (2.07-2.00) 50.00-2.50 (2.59-2.50) No. of reflections 44287 (4011) 22012 (2119) CC½ (outer shell) 0.914 0.909 I/σ 17.9 (3.7) 8.9 (4.0) Completeness (%) 94.6 (86.4) 90.7 (87.4) Redundancy 3.8 3.7 Refinement Resolution (Å) 26.8 – 2.0 46.1 – 2.5 R work /R free 0.164/0.219 0.196/0.255 No. of molecules 2 2 No. of atoms 5675 5298 Protein 5218 5194 Ligand/ion 37 32 Water 420 72 B-factors 34.03 48.21 Protein 33.67 48.19 Ligand/ion 48.81 61.13 RMSD Bond lengths (Å) 0.006 0.002 Bond angles (°) 0.85 0.49 Ramachandran Preferred (%) 98.26 97.82 Outliers (%) 0.00 0.00 PDB code 9E58 9DMD a Values in parenthesis are for the highest resolution shell Figure 4. Recombinant YscN is predominantly monomeric. (A) A 280 absorbance scans of YscN monitored during SV-AUC. (B) Representative residuals from fitting YscN sedimentation to a continuous c(s) distribution model. (C) YscN c(s) versus (S) sedimentation coefficient distribution. YscN sediments predominantly as a single species with a sedimentation coefficient of 3.37 ± 0.02 Svedbergs, a friction ratio of 1.43 ± 0.05, and a calculated molecular mass of 55 ± 3 kDa, all consistent with monomeric YscN. The sedimentation coefficient, friction ratio, and calculated molecular mass represent the mean values ± SDs from three independent replicates performed at varying concentrations. Insight into how monomeric YscN species support catalysis was gained by first collecting a substrate concentration-dependent activity profile for a single concentration of YscN (1.75 μM) and fitting the data to the Michaelis Menten equation to calculate a K M, app of 1.3 ± 0.4 mM and a V max, app of 0.35 ± 0.05 µmol P i /min/mg YscN (Figure 5A). The impact of YscN concentration on its specific activity was then examined to assess oligomer-driven activation (Figure 5B). Holding substrate concentration constant while varying YscN concentration resulted in a sigmoidal specific activity response curve to a concentration of approximately 3 μM YscN, beyond which increasing the YscN concentration did not further increase specific activity (Figure 5B). This response is consistent with oligomerization activation of an enzyme as the slope of such an Figure 5 Kinetic analysis of YscN ATPase activity. (A) Substrate concentration-dependent ATPase activity was assessed by plotting initial reaction velocities as a function of ATP concentration. Data were fit to the Michaelis-Menten equation to determine the kinetic parameters K M, app and V max, app . (B). The effect of YscN concentration on its specific activity was determined at three substrate concentrations. Triplicate kinetic measurements were taken and each data set fit to a 4-parameter logistic sigmoidal function. The mean ± SD of the calculated Hill coefficients are 5.4 ± 1.2, 6.4 ± 1.0, and 4.8 ± 1.4 for 300 µM, 600 µM, and 1200 µM ATP, respectively. assay would be zero if no enzyme oligomerization-dependent activation were present. In addition, because some T3SS ATPases demonstrate a substrate dependence on enzyme oligomer formation and others are substrate independent, 35, 46, 52 YscN concentration dependence on specific activity was quantified at 300 µM, 600 µM, and 1200 µM ATP concentrations. Each of these conditions demonstrated statistically similar enzyme concentration response profiles (Hill slopes, one-way ANOVA) with the specific activity maxima scaling with substrate concentration, as expected (Figure 5B). Together, these data suggest that complexes containing at least two YscN proteins and one ATP must form for ATP hydrolysis to occur, with the formation of the complex relying on “chance interactions” supported by YscN concentration in an ATP-independent manner. T3SS ATPase inhibitors display varied efficacy against YscN - Small molecule T3SS ATPase inhibitors are highly-anticipated for their potential as anti-infective therapeutics against bacteria expressing one or more T3SSs as essential virulence factors. 17, 20, 53 We previously used in silico methods to identify a series of small molecule inhibitors against the Shigella T3SS ATPase Spa47 and show that many were not only effective against Spa47, but also against homologous T3SS ATPases, including EscN and FliI. 17 Having developed robust activity assays and solving the YscN structure, we were equipped to extend these inhibition analyses to YscN. Importantly, these inhibitors were originally designed to bind within the modeled protomer interface of homo-oligomeric Spa47, avoiding the broadly conserved ATPase active site and associated concerns with toxicity against eukaryotic cells. 17 These inhibitors were subsequently characterized against stable Spa47 oligomers, with many efficiently inhibiting activity. To determine whether these inhibitors were also effective against monomeric T3SS ATPase species, here we examined the ATPase activity of monomeric YscN and Spa47 in the presence of these compounds as Spa47 can be isolated as both ATPase active monomers and oligomers (Figure S3). 46 Eight of the ten tested inhibitors reduced ATPase activity of monomeric Spa47, with candidates 1870 and 5765 effective against neither monomeric nor oligomeric Spa47 (Figure 6). Interestingly, inhibitor 4967 inhibited YscN activity by approximately 80%, though none of the other 9 tested compounds proved effective against YscN. Figure 6. Effect of small molecule inhibitors on monomeric YscN and Spa47. The inhibitors originally designed to target the protomer interface of homo-oligomeric Spa47 species were tested against monomeric YscN (black) and monomeric Spa47 (gray). The inhibitors remain largely effective against monomeric Spa47, with eight of ten significantly inhibiting ATPase activity. Only inhibitor 4967, however, significantly reduces activity of YscN (~80% inhibition). Data are plotted as the mean ± SD of three independent biological replicates. *indicates a statistically significant difference in activity relative to the uninhibited control of the same isozyme (one-way ANOVA and Dunnett’s test p ≤ 0.05). Discussion T3SS ATPases are essential for T3SS function as they drive the assembly of the extracellular portion of the injectisome and support protein effector secretion. 54, 55 As seen for many other T3SS-expressing bacteria, Yersinia spp. rely on their T3SS ATPase (YscN) for proper T3SS function and virulence. 9, 19 In this study, we conducted a detailed structural and biochemical characterization of YscN, describing its kinetic characteristics with respect to environmental conditions, homo-oligomerization, and susceptibility to T3SS ATPase inhibitors recently designed against the Shigella T3SS ATPase, Spa47. Despite its strong sequence and structural homology to numerous T3SS ATPases, we uncovered intriguing biochemical properties of YscN, including narrow pH and temperature activity profiles, a homo-oligomeric activation mechanism, and a distinct inhibitor profile, suggesting evolutionary adaptations that finely tune its function in Yersinia virulence and highlight critical considerations as a target for anti-infective therapeutics. Our work here with YscN began with successful expression and purification of the recombinant enzyme and identification of robust conditions that support ATPase activity. Doing so uncovered both pH and temperature influences that suggest YscN has evolved to support its virulence pathway. More specifically, YscN was maximally active at pH 8.5, with no detectable activity at pH 6.75 (Figure 1A) and a sharp temperature dependence with greatest activity between 28 C and 32 C (Figure 1B). Of course, cytoplasmic buffering makes it challenging to know the extent that environmental pH alters that of the Yersinia cytoplasm and YscN activity within it. However, even modest reduction in pH has significant negative impact on in vitro YscN activity (Figure 1B), supporting an opportunity for in vivo T3SS regulation. Considering pathogenesis of Y. pesitis specifically, this means that YscN activity (and T3SS activity) is tempered in the environmental conditions encountered in the proventriculus of host fleas where the T3SS is not required (pH ~6.5-7.0, 20 C - 26 C) 56, 57 and may be enhanced when transferred from the flea to a mammalian host. Once introduced to the host, many of the bacteria will be engulfed by macrophages, which will attempt to destroy them via phagosome acidification (early phagosome pH ~6.0-6.5, late phagosome pH ≤ 5). 58 Given our findings, this acidification may inhibit YscN and prevent secretion of T3SS effectors that inhibit phagosome maturation, and ultimately facilitate phagosomal escape. Connor and colleagues showed, however, that T3SS-independent recruitment of host Rab1b to the Y. pestis containing vacuoles (YCV) prevents early acidification, seemingly protecting YscN/T3SS function and allowing necessary T3SS effector secretion. 59 In addition to pH, YscN activity is not surprisingly also influenced by temperature, with maximal activity of the purified enzyme observed between 28 C and 32 C (Figure 1A). Interestingly, while Yersinia T3SS protein expression is supported at 37 C and secretion is optimally active under these human host cell temperatures, 60, 61 we found that purified YscN activity decreases significantly when the temperature reaches 36 C (Figure 1A). Differences between optimal in vitro temperatures and in vivo conditions are not uncommon and in fact, T3SS ATPase homologs from P. syringae (HrcN) and S. flexneri (Spa47), each display optimal in vitro activity below the 37 C temperatures they encounter during a human infection, suggesting that unaccounted protein interactions likely provide in vivo stabilization. 46, 62 Regardless, these findings provided us the opportunity to directly assess the correlation between YscN structural thermal stability and these activity data using far-UV circular dichroism (Figure 2). Interestingly, these CD thermal stability experiments show that YscN secondary structure stability is strongly influenced by pH and that the stability profiles correlate to the collected pH activity profiles (Figure 1B, Table 1). For example, the lowest T onset of 33 C was observed at pH 6.75, where no ATPase activity was detected. As the pH increased to 7.0, 7.25, and 7.5, T onset rose to approximately 34 C, with ATPase activity clustering around 0.05 µmol ADP/min/mg YscN. The most thermally stable conditions (T onset values from 37 C to 38 C) were recorded at pHs 8.0, 8.5, and 9.0 coinciding with the peak ATPase activity of approximately 0.095 µmol ADP/min/mg YscN. These findings suggest that pH impacts on YscN activity are the result of destabilizing the protein structure. The unexpectedly narrow pH and temperature ranges supporting optimal activity further suggests that YscN may have evolved a means of utilizing its environment to regulate its activity during the Yersinia spp. infection lifecycle. It is well understood that T3SS ATPases form homo-oligomeric ring-like structures at the base of the injectisome and that their ATPase activity is required for efficient protein secretion. 35, 63, 64 It is less clear, however, what the specific roles of cytoplasmic T3SS ATPase proteins are when not associated with the injectisome, necessitating consideration of both forms. Importantly, different stoichiometries of recombinantly expressed T3SS ATPase homologs suggest differences among their soluble forms and in factors driving their activation. In this study, recombinant YscN was purified as predominantly monomers (Figure 4). In contrast, both the Shigella T3SS ATPase Spa47 and the Pseudomonas T3SS ATPase HrcN are simultaneously purified as monomeric and oligomeric species, with both demonstrating robust ATPase activities. 22, 62 EscN from enteropathogenic E. coli and FliI from the Salmonella flagellar T3SS purify as ATPase active homo-hexamers following exposure to nucleotide. 35, 45, 65 In each of these cases, oligomerization appears to support ATPase activation though formation of an interfacial active site, similar to what is seen at the α/β subunit interfaces in F 1 ATP synthase. 66 A non-linear increase in ATPase activity was observed as a function of enzyme concentration (Figure 5B), suggesting a dependence on YscN/YscN interactions. Solving a 2.0 Å crystal structure of monomeric YscN illustrated a putative active site interface by modeling a YscN oligomer after the homo-hexameric EscN cryoEM structure. 35 This model predicts that the YscN active site requires the contribution of two key residues from one side of the interface and contribution of another residue from a separate protomer located across the interfacial active site. Consistent with oligomeric F 1 and EscN structures, YscN K175 (in the Walker A motif) appears to stabilize the negative charge of ATP and support substrate interaction and E198 activates and coordinates a nucleophilic water molecule necessary for hydrolysis (Figure 3). 35, 66 R359 is contributed by an adjacent protomer where it stabilizes the negatively charged ATP and properly positions it for nucleophilic attack. Schneewind and coworkers have previously shown the importance of K175 on YscN activity 19 and an ATPase inactive R359A mutant generated in this study further underscores YscN’s reliance on an interfacial active site containing each of these three critical residues (Figure S1). Interestingly, despite observing a strong non-linear activity dependence on YscN concentration (Figure 5B), we were never able to convert monomeric YscN to a stable oligomeric YscN complex (presence or absence of nucleotide). Together, this suggests that the active site(s) of YscN form transiently between two or more monomers, which interact only briefly to catalyze ATP hydrolysis. While it is feasible that these brief interactions may also catalyze necessary ATP hydrolysis within the Yersinia cytoplasm, it is more likely that this barrier to hydrolysis serves as a mechanism to mitigate unnecessary and wasteful ATP consumption by cytoplasmic YscN stores. Interactions with other T3SS proteins such as YscL and YscK may then support ATPase activity through active site formation in hetero-oligomeric complexes found within the cytoplasm and ultimately as homo-oligomers incorporated into the base of the T3SS apparatus sorting platform. 19, 67, 68 Studies considering the impact of such interactions are underway and will extend the findings presented here to further dissect the distinct populations of YscN and the mechanism(s) of their activation. Small molecule T3SS ATPase inhibitors are highly-anticipated for their potential as anti-infective therapeutics against bacteria expressing one or more T3SSs as essential virulence factors. In previous studies, we used in silico methods to identify a series of small molecule inhibitors against the Shigella T3SS ATPase Spa47, and demonstrated that, in addition to Spa47, these inhibitors were effective against homologous T3SS ATPases, including EscN and FliI. 17 Importantly, these compounds were originally designed as noncompetitive inhibitors to bind within the modeled protomer interface of homo-oligomeric Spa47, avoiding the broadly conserved ATPase active site and associated concerns with toxicity against eukaryotic cells. Having developed robust activity assays for YscN and solving its structure, we were equipped to extend these inhibition analyses to YscN. Thus, we examined the impact of these previously identified T3SS ATPase small molecule inhibitors on the activity of YscN. Interestingly, of the ten compounds examined, only one (4967) significantly inhibited the ATPase activity of monomeric YscN (Figure 6). In contrast, eight of these inhibitors reduced the ATPase activity of monomeric Spa47 with the two inhibitors (1870 and 5765) that were ineffective against monomeric Spa47 also ineffective against oligomeric Spa47. 17 Together, these data show that inhibitors active against multiple oligomeric states can be designed and that a pan-effective T3SS inhibitor is feasible, but also caution that consideration of each ATPase target will be required to realize this aim. Considering the stark difference observed in inhibitor efficacy for YscN and other tested T3SS ATPases is an important opportunity toward the development of pan-effective small molecule inhibitors. This is challenged by the lack of available inhibitor bound structures, but our modeling of inhibitor binding sites on the apo YscN and Spa47 structures suggests one possible explanation for these differences. Docking calculations suggest that the inhibitors cluster at one of two sites formed at the interface of oligomeric YscN and Spa47 models in a manner that is mutually exclusive and determined by the position of a loop, which we term the “gating-loop.” In Spa47, the gating-loop is observed in two distinct conformations, giving rise to the two predicted inhibitor binding sites (“upper” and “lower” sites). In YscN, the gating-loop appears to be less mobile and only supports access to the lower binding site (Figure S3) with its position in both molecules of the crystal asymmetric unit superimposable (RMSD = 0.046 Å) suggesting that its positioning is not driven by crystal contacts since the two regions are in different crystal packing environments. Comparison of the YscN and Spa47 gating-loop sequences further supports the idea that the YscN gating-loop is more constrained than Spa47 as the YscN loop sequence contains pairs of adjacent proline residues near the loop termini while Spa47 only has single prolines in each of these positions (Figure S3). The double proline arrangement in YscN may impose additional constraint on loop dynamics, restricting access to the loop conformation that allows access to the upper binding site observed in some Spa47 structures (Figure S3A and S3B). Thus, one explanation for the differences in inhibitor efficacy between YscN and Spa47 may be that the upper site is the productive inhibitor binding site and is available in Spa47 as the gating loop dynamically samples both positions, but remains inaccessible in YscN. Future studies interrogating gating-loop dynamics, its impact on inhibitor binding affinity and location, and specific interactions between T3SS ATPases and potentially pan-effective inhibitors will be required to shed light on this correlation. Additionally, structural, kinetic, and molecular dynamics studies focusing on inhibitor 4967 will likely uncover important considerations for inhibitor design as it has thus far proven effective against several T3SS ATPase homologs, including YscN. Ultimately, this study provides critical insights into the structural and biochemical properties of the Yesinia T3SS ATPase YscN. We identified a narrow range of temperatures and pH conditions for robust enzymatic activity and present a possible physiological basis for these tight constraints by examining the Yersinia infection life cycle. Kinetic studies and high-resolution crystal structures of wild-type YscN and an arginine-finger mutant (R359A) provide evidence of an interfacial active site which is further corroborated by the observation of YscN’s non-linear activity response to enzyme concentration. It is possible such oligomer-dependent activity may reduce futile ATP hydrolysis by monomeric (and cytoplasmic) YscN populations, ensuring YscN activation occurs upon incorporation into the T3SS injectisome. Finally, small molecule inhibition studies of both YscN and Spa47 support the feasibility of pan-effective T3SS ATPase inhibitors with these studies demonstrating that subtle structural and dynamic differences among T3SS ATPase homologs may critically influence the effectiveness of candidate inhibitors, ultimately underscoring the need to independently investigate the breadth of targeted ATPase homologs. Taken together, these novel findings provide important kinetic insight into YscN activity and underscore similarities and differences among T3SS homologs that further our understanding of their divergent evolution to support the infection niches of the pathogens expressing them. Data availability. Main text data are contained within this article and structure coordinates are deposited in the RCSB Protein Data Bank (PDB IDs: 9E58 and 9DMD). Supplemental data are in the Supporting Information document. Correspondence and requests for materials should be addressed to the corresponding author (nick.dickenson@usu. edu). Conflict of interest . The authors declare that they have no conflicts of interest with the contents of this article. Funding and additional information. This work was supported in part by National Institutes of Health Grant R15AI124108 and R. Gaurth Hansen endowment funds to N. E. D. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Abbreviations AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride AUC, Analytical ultracentrifugation CDC, Centers for Disease Control and Prevention CBD, Chitin-binding domain CD, Circular dichroism DMSO, Dimethyl sulfoxide DTT, Dithiothreitol EPEC, Enteropathogenic Escherichia coli IPTG, Isopropyl β-D-1-thiogalactopyranoside LB, Luria Broth PCR, Polymerase chain reaction PEG, Polyethylene glycol PDB, Protein Data Bank P i , Inorganic phosphate RMSD, Root mean square deviation S, Svedberg unit TB, Terrific Broth T3SA, Type three secretion apparatus T3SS, Type three secretion system T M , Melting temperature T onset , Thermal unfolding onset temperature WHO, World Health Organization References 1. World Health Organization. Newsroom, Plague 2022. 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Information & Authors Information Version history V1 Version 1 19 November 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords bacterial pathogenesis enzyme kinetics infectious disease inhibitors type iii secretion system (t3ss) x-ray crystallography Authors Affiliations Samuel Barker A Utah State University View all articles by this author Porter Ellis K Utah State University View all articles by this author Andrew Hammer Utah State University View all articles by this author Sean Johnson 0000-0001-7992-2494 Utah State University View all articles by this author Nicholas Dickenson 0000-0003-1572-6077 [email protected] Utah State University View all articles by this author Metrics & Citations Metrics Article Usage 203 views 121 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Samuel Barker A, Porter Ellis K, Andrew Hammer, et al. 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