Structural Analysis of the Bacterial Effector AvrA Identifies a Critical

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Structural analysis of the bacterial effector, AvrA, identifies a critical helix involved in MKK4-substrate recognition Jonathan Labriola, Yifan Zhou, and Bhushan Nagar Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00512 • Publication Date (Web): 19 Jul 2018 Downloaded from http://pubs.acs.org on July 20, 2018

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Biochemistry

Labriola et al., 7/19/18 3:13 PM

Structural analysis of the bacterial effector, AvrA, identifies a critical helix involved in MKK4substrate recognition

Jonathan M. Labriola, Yifan Zhou, and Bhushan Nagar Department of Biochemistry and Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, QC H3G 0B1, Canada

Running title: Surface helix of AvrA enables MKK4 recognition Correspondence regarding this manuscript should be sent to: Bhushan Nagar Department of Biochemistry McGill University 3649 Promenade Sir William Osler Montreal, Quebec H3G 0B1 Canada Phone: 514-398-7272, Fax: 514-398-2983 email: [email protected]

The manuscript contains 51758 characters including spaces (title page, abstract, main text, author contributions, acknowledgements, figure legends, materials and methods).

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Labriola et al., 7/19/18 3:13 PM Abstract Bacterial effector proteins are essential for the infection and proliferation of pathogenic bacteria through manipulation of host immune response pathways. AvrA is a Salmonella effector belonging to the YopJ family of acetyltransferases, which suppresses c-JUN N-terminal kinase (JNK) signaling in mammals through acetylation of mitogen activated receptor kinase kinase 4/7 (MKK4/7). Interestingly, two paralogues of AvrA exist that differ by only a single internal leucine residue, which when absent (AvrA∆L140), abrogates the ability to suppress JNK signaling. Here, we present the first crystal structure of a bacterial effector from an animal pathogen, AvrA∆L140, accompanied by a thorough biophysical characterization of both AvrA variants. The structure in complex with inositol hexaphosphate and coenzyme A reveals two closely associated domains consisting of a catalytic core that resembles the CE clan peptidases, and a wedgeshaped regulatory region that mediates co-factor and substrate binding. The loss of the putative function of AvrA∆L140 is due to its inability to interact with MKK4/7, which ultimately arises from an altered conformation of a critical helix adjacent to the active site that harbors L140. These results provide general insights into substrate recognition across the YopJ family of acetyltransferases.

Keywords YopJ acetyltransferases; crystallography; bacterial effectors; pathogen-host interactions; innate immune signaling.

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Biochemistry

Labriola et al., 7/19/18 3:13 PM Abbreviations AvrA; Anti-virulence factor A, IP6; inositol hexakisphosphate (IP6), MKK; Mitogen activated receptor kinase kinase, YopJ; Yersinia outer protein J,

Introduction The ongoing competition between microbes and their multicellular hosts has necessitated the evolution of progressively sophisticated mechanisms of infection and defense, respectively, in an effort to gain the upper hand 1. Hosts have developed the innate immune system as a first line of defense comprised of an array of pattern recognition receptors (PRRs) that specifically sense pathogen associated molecular patterns (PAMPs) to trigger signaling pathways that debilitate invading pathogens. Pathogens have countered with numerous strategies to evade these host defenses, including the delivery of ‘effector proteins’ by Type 3 secretion (T3SE) systems 2, in the case of Gram negative bacteria, into the host cell to strategically modulate immune response pathways. Among the many classes of bacterial effector proteins, exists a unique group of acetyltransferases from the Yersinia outer protein J (YopJ) superfamily. These enzymes are present in in a wide variety of plant and animal pathogens and operate by disrupting the function of immune response targets in the host cell, including transcription factors3, signaling kinases4,5, and growth/stress response proteins6,7, through acetylation of key protein residues8. YopJ acetyltransferases are not related to any known mammalian acetyltransferase and instead, show homology to the CE peptidase clan of cysteine proteases9 (~ 20% identity), which includes the Ubiquitin-like protease (Ulp), making use of a canonical catalytic triad mechanism consisting of a nucleophilic cysteine residue to break peptide bonds. However, unlike CE peptidases, YopJ

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Labriola et al., 7/19/18 3:13 PM acetyltransferase activity requires the presence of two co-factors: acetyl-CoA as the source of the acetyl group, and inositol hexakisphosphate (IP6)10, which is abundantly present in eukaryotic cells, but absent in prokaryotes, thus activating enzymatic function only after introduction into the host cell. Recently, the crystal structures of two plant pathogen YopJ acetyltransferases were reported11,12. The structure of HopZ1, a tubulin modifying YopJ from the plant pathogen Pseudomonas syringae, showed that IP6 binds to a positively charged pocket on the surface of the protein, causing conformational changes that facilitate binding of the AcCoA substrate11. The structure of PopP2 from the plant pathogen R. solaneacearum in complex with its target substrate RRS1-RWRKY, revealed additionally how PopP2 binds regions of WRKY distant from the target acetylation site, and posited a ping-pong style mechanism involving initial autoacetylation of the catalytic cysteine residue followed by transfer from the cysteine to the substrate12. Despite these advances, structures of YopJ acetyltransferases from animal pathogens, which target highly divergent substrates, have thus far remained elusive. AvrA is a YopJ acetyltransferase from the gram-negative animal pathogen Salmonella enterica, found predominantly in the serovar Typhimurium13, which can infect and live within intestinal epithelial cells and immune cells of mammals. Salmonella poses a challenge in many contexts, from food processing14,15 to healthcare16, and there is mounting evidence linking chronic infection to the development of certain types of cancer17,18. Salmonella infection induces proinflammatory immune response pathways and cytokine release, which in turn activate the proapoptotic JNK signaling pathway19-21. JNK signal transduction moves exclusively through mitogen activated receptor kinase kinases (MKKs) 4 and 7, ultimately leading to JNK phosphorylation, translocation to the nucleus, and activation of relevant transcription factors22.

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Biochemistry

Labriola et al., 7/19/18 3:13 PM AvrA specifically inhibits JNK signaling by acetylating a threonine residue (T261/T275) on MKK4/7, whose phosphorylation is essential for their activation23,24. Thus, AvrA serves to reverse the host inflammatory effects of other effectors, such as apoptosis and destabilization of intestinal tight junctions20,25. Interestingly, investigation of AvrA sequences from different strains of S. Typhimurium revealed an allelic variation in the gene24. AvrA from strain S. Typhimurium strain SL1344 lacks an internal leucine residue (L140), which we refer to here as AvrA∆L140. This single residue deletion has a profound effect on AvrA function, as it can no longer suppress JNK signaling in animal models or in vitro. Transformation of the AvrA gene containing L140 into these strains restores their JNK suppression functionality. It was suggested that the presence of AvrA∆L140 may be a consequence of strong selective pressure to modify its function or that it may be active against an unidentified host target24. Here, we present a crystal structure of a YopJ acetyltransferase from an animal pathogen, AvrA∆L140, along with a biochemical and biophysical analysis of both AvrA variants. Both forms of the protein possess comparable structural and biophysical properties, including the ability to bind IP6 and auto-acetylate. Despite these similarities, we found that only AvrA can acetylate the target protein MKK4, while AvrA∆L140 cannot. The inability of AvrA∆L140 to act on MKK4 appears to originate from altered conformational and/or biophysical characteristics at the active site, which likely prevent their physical association. The crystal structure of AvrA∆L140 bound to IP6 and CoA revealed that the lack of L140 would alter the conformation of a putative substrate interacting α-helix. Taken together, these results suggest that the internal leucine plays an essential role in facilitating acetylation of the MKK4/7 substrate, and that AvrA∆L140 may have evolved to target a different substrate.

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Labriola et al., 7/19/18 3:13 PM Materials and Methods Purification of AvrA constructs and MKK4.

Expression constructs (AvrA 1 – 302; AvrA∆L140 1 - 301; MKK4 80 – 399) were synthesized by Integrated DNA technologies (Coralville, Iowa) with restriction sites for cloning into a modified pET28b vector containing a Ulp cleavable, N-terminal His-SUMO tag29. Mutants were generated by PCR using primers containing the desired mutation with iProof DNA polymerase (Bio-Rad). For expression, vectors were transformed into E. coli BL21(DE3) and plated on an LB agar plate containing 50 µg/mL kanamycin. Single colonies were picked to inoculate an overnight starter culture of LB with 50 µg/mL kanamycin. The starter culture was diluted 100x into fresh LB containing 30 µg/mL kanamycin and grown at 37 °C to OD600 0.7 – 0.9 at which time the temperature was lowered to 20°C, and expression induced by addition of 100 µM IPTG for 16 – 20h. Cells were harvested by centrifugation at 5000g for 15 min. Pellets were suspended in NiA buffer (300 mM NaCl, 10% Glycerol, 50 mM Tris pH 8.0 at 4°C) supplemented with DNAse and 100 µM PMSF. Cells were lysed in a Emulsifex C3 homogenizer running at ~15000 psi. The crude lysate was cleared by ultracentrifugation at 50,000g for 1h and the supernatant pooled and applied to a column containing Ni+ affinity resin (Thermo Scientific). The sample was washed with 5 column volumes of NiA containing 3% of NiB buffer (NiA + 1 M imidazole), and eluted with 30% NiB. Relevant fractions were pooled and Ulp added at a ratio of 1:1000 (w/w). The sample was dialyzed overnight into NiA buffer, followed by reapplication to Ni+ resin to remove free His-SUMO tag and uncleaved protein. The sample was then applied to a 5mL HiTrap Q column (GE Healthcare) in 50 mM Tris pH 8 and eluted with a 0 – 50% gradient with 1 M NaCl. Finally, the sample was applied to a Superdex 200 preparative column or a Superdex 200 increase analytical column (GE Healthcare) equilibrated with 200 mM NaCl, 15 mM HEPES pH

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Biochemistry

Labriola et al., 7/19/18 3:13 PM 7.0 buffer. Samples were concentrated to 6 mg/mL (AvrA) or 25 mg/mL (MKK4), aliquoted and stored at -80 °C until needed.

∆L140

Crystallization and structure determination of AvrA

Samples of AvrA∆L140 were concentrated to 15 – 20 mg/mL in storage buffer and ligand (IP6 and AcCoA) added at a 2-fold molar excess followed by incubation on ice for 30 min prior to crystallization. The crystallization condition was 0.1 M MES pH 5.5 – 5.8, 8% PEG 8000, 18% ethylene glycol. Crystals were typically harvested 1 – 2 days after setup and flash frozen in liquid nitrogen. Data was collected at the Canadian Light Source, beamline 08ID-1 equipped with a PILATUS3 S Series (6M) detector. Diffraction images were processed using HKL200037. The structure was solved with molecular replacement using HopZ1 (5KLP) as a search model using Phaser as implemented in Phenix38. Iterative refinement and model building were performed using Phenix39 and COOT40, respectively. Fo-Fc map was calculated by deleting IP6 or CoA from the model and running a refinement with simulated annealing. Electrostatics were calculated using the APBS plugin in Pymol.

Sequence alignment and structure superposition

Structures of Ulp, PopP2, HopZ1, and AvrA∆L140 were aligned using the cealign function in Pymol.

Structure

based

sequence

alignments

PROMALS3D (PROfile Multiple Alignment and 3D constraints) online server

41

with

were

run

using

the

predicted Local Structures

. Minor manual adjustments were made in the sequence

alignments.

HPLC - Mass spectrometry

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Labriola et al., 7/19/18 3:13 PM Mass measurements were performed on a 1260 Infinity HPLC (Aglient) attached in-line to an amaZon speed ETD ion trap MS (Bruker). For measurements of whole protein mass, purified samples in storage buffer were, if applicable, incubated with 300 µM IP6, 50 µM AcCoA, and 50 uM MKK at room temperature for 30 min. Samples were then applied to a PLRP-S (5 µM bead size, 1000 Å pore size) reverse-phase column at 80 °C. The sample was eluted with a linear gradient starting at 5% buffer A (0.1% formic acid) to 100% buffer B (acetonitrile, 0.1% formic acid).

Isothermal titration calorimetry

95 µM (AvrA) or 80 µM (AvrA∆L140) in 200 mM NaCl, 5% Glycerol, 20 mM HEPES pH 7.0 was placed in the cell. IP6 in the same buffer was placed in the syringe at concentrations of 1.2 mM (AvrA) or 1 mM (AvrA∆L140). The titration was performed in a MicroCal iTC 200 at 20 °C with a reference power of 11 µcal/sec.

Circular diochroism

Far UV circular dichroism data was collected on a Chirascan spectrophotometer (Applied Photophysics). AvrA and AvrA∆L140 were dialyzed against 50 mM sodium phosphate pH 7.0, 50 mM NaCl, and 5% glycerol. Samples were run at concentrations ranging from 0.1 – 0.5 mg/mL to ensure no appreciable concentration dependent distortions of spectra were occurring. Presented spectra are at a concentration of 0.4 mg/mL. IP6 was added to a final concentration of 500 µM. For data collection, 80 µL of sample was placed in a 0.2 mm quartz cuvette. A constant stream of nitrogen gas at 25 psi was applied to the lamp. Bandwidth was set at 1 nm with 0.5 sec collection time per data point. Scanning was set from 260 nm – 190 nm. 20 scans were collected

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Biochemistry

Labriola et al., 7/19/18 3:13 PM and averaged followed by a Savitsky-Golay smoothing function with a window size of three. Secondary structure content was calculated using the betsel server at http://bestsel.elte.hu/42.

Thermal denaturation

Thermal melt experiments were performed according to the protocol from the protein thermal shift kit (Invitrogen, cat# 4461146). Briefly, ~ 5 µM of AvrA or AvrA∆L140 were mixed with dye with or without 500 µM IP6 in 20 mM HEPES, pH 7.0, 200 mM NaCl, and 5% glycerol. The melting experiment was performed in a StepOnePlus qPCR thermocycler (Applied Bioscience) starting with a 2 min incubation at 25 °C followed by a temperature ramp to 99 °C. Fluorescence signal was plotted versus temperature and Tm was calculated by taking the peak of the 1st derivative of this plot.

Internal tryptophan fluorescence

100 µL of 1 µM of AvrA, AvrA∆L140, and L140A in buffer containing 20 mM HEPES pH 7.0, 200 mM NaCl, 5% glycerol were added to separate wells of a black half area 96-well plate (corning). Samples were excited at 280 nm and emission was measured at 355 nm with a 325 nm cutoff in a Spectramax M5 (Molecular Devices). Initial measurements were taken in the absence of IP6 (Fo). Thereafter increasing concentrations of IP6 were added to the wells followed by a 10 min incubation and fluorescence measurement (I). The resulting curve was fit to an exponential to determine minimum fluorescence upon IP6 saturation (Fmin). The change in fluorescence at a given IP6 concentration (∆F) and maximum change in fluorescence upon IP6 saturation (∆Fmax) was determined by: (1)

∆I = Fo – F

(2)

∆I = Fo - Fmin

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∆I/∆Imax was

plotted

versus

IP6

concentration.

Results AvrA∆L140 is unable to catalyze acetylation of the MKK kinase domain To understand the origin of the stark physiological difference between AvrA (Uniprot: A0A0C5PR83) and AvrA∆L140 (Uniprot: E8XKZ3), we began by bacterially expressing and purifying both forms of the enzyme and testing their ability to acetylate MKK4 in vitro. Investigation of gene databanks show two annotations for the AvrA gene due to an ambiguous start site. Du et al. confirmed that the entry associated with the 287 amino acid product is the correct one, which is the amino acid numbering used herein24. The MKK4 kinase domain (residues 80 – 399), which contains the AvrA acetylation site T26126, was also purified. Using LC-MS with separation on a C18 reverse phase column connected to an ESI ion-trap MS to determine acetylation status, we found that incubating AvrA with MKK4, IP6, and AcCoA for 30 minutes led to robust acetylation of MKK4 at a single site (Fig. 1A). The site corresponds to the known target residue in MKK4, T26123, as mutation of this residue abolishes acetylation (Fig. 1B). In contrast, AvrA∆L140 was unable to appreciably acetylate MKK4 under the same reaction conditions.

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Labriola et al., 7/19/18 3:13 PM YopJ acetyltransferases are known to become auto-acetylated on an internal residue during the course of target acetylation10,27,28, although the reason for this and the site of

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Figure 1. Whole protein mass spectra to determine acetylation status. Contents of each reaction are shown to the left of each curve. (A) Purified MKK4 (80-399) in the presence of top: IP6 and unlabeled AcCoA; middle, IP6, AcCoA, and AvrA; bottom: IP6, AcCoA, and AvrA∆L140. (B) T261A mutant of MKK4 is unable to be acetylated. (C) Auto-acetylation status of top: AvrA; middle: AvrA∆L140; bottom: AvrA C172A catalytically dead mutant. Black lines are for the protein alone, grey lines are for the protein incubated with IP6 and AcCoA. Theoretical masses are listed.

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Labriola et al., 7/19/18 3:13 PM acetylation is not yet unclear. Studies of PopP2 have suggested that the acetylation site is a conserved lysine27 (K224 in AvrA), whereas studies of HopZ1 identified two conserved serines as the auto-acetylation site28. Recent structural analyses of PopP2, however, suggested that the auto-acetylation site is the catalytic cysteine12. Thus, akin to the thioester intermediate that occurs during proteolysis by the cysteine protease Ulp29, the catalytic cysteine of YopJ acetyltranferases may form a transient and labile thioacetyl intermediate. To understand whether the inability of AvrA∆L140 to acetylate MKK4 was due to a defect in the auto-acetylation step, we measured its auto-acetylation activity in the presence of IP6 and AcCoA compared to AvrA. Under the same reaction conditions for the MKK4 activity measurement, we found that both forms are able to auto-acetylate (Fig. 1C). As expected, mutation of the catalytic cysteine (C172A) abolished auto-acetylation. Interestingly, we observed multiple auto-acetylation sites on both AvrA variants: AvrA∆L140 showed at least two sites, while AvrA showed at least three. Additionally, a higher proportion of AvrA∆L140 was singly auto-acetylated, whereas AvrA autoacetylation was more evenly distributed over the sites. The relevance and identity of these sites is not entirely clear and will require further investigation (see discussion). Thus, the inability of AvrA∆L140 to acetylate MKK4 does not appear to arise from a defect in auto-acetylation per se, although we do note that the auto-acetylation ability of AvrA∆L140 is modulated somewhat.

Both AvrA and AvrA∆L140 bind IP6 IP6 is an essential co-factor for YopJ acetyltransferase function as there is no enzymatic activity in its absence. In HopZ1 and YopJ, binding of IP6 was shown to induce secondary structure and conformational changes that rigidify the proteins10,11. Thus, we sought to determine if both AvrA and AvrA∆L140 were capable of binding IP6 using isothermal titration calorimetry. Both

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Labriola et al., 7/19/18 3:13 PM homologues could bind IP6 with low µM affinity, although surprisingly, AvrA∆L140 displayed a 2.5-fold higher affinity (Fig. 2A and B). However, given that the concentration of IP6 in mammalian cells is estimated to be in the 10 - 50 µM range30, the difference in affinity is probably not physiologically relevant, although if IP6 concentration were a limiting factor, it should favor AvrA∆L140 function. Thus, a defect in IP6 binding does not underlie the lack of function of AvrA∆L140 towards MKK4.

AvrA and AvrA∆L140 possess similar secondary structure To determine if the lack of the leucine residue somehow affects the fold of AvrA∆L140, we attempted to analyze it using gel-filtration chromatography and circular dichroism (CD) spectroscopy. Gel-filtration profiles for both purified homologues showed single symmetrical peaks at roughly the same retention volume (~33 kDa), suggesting that they are folded and monomeric in solution (Fig. 2C). CD spectra of AvrA and AvrA∆L140 in the absence of IP6 revealed that the secondary structure content of both were very similar (Fig. 2D and Table S1). However, spectra recorded in the presence of IP6 showed an increase in helical content in AvrA while AvrA∆L140 showed an increase in turn content (Table S1). Thus, AvrA∆L140 appears to fold properly into a structure similar to AvrA, and its lack of function cannot be attributed to any gross structural perturbations or misfolding, though IP6 induced structural changes show slight deviations.

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Figure 2. Biophysical characterization of AvrA and AvrA∆L140. Isothermal titration calorimetry experiments of IP6 binding to (A) AvrA∆L140 and (B) AvrA. Curves were fit in the Origin software to determine binding constant (Kd) and number of binding sites (n). Errors are derived from fitting statistics. (C) Gel-filtration (S200 increase, GE) profiles of AvrA, AvrA∆L140, and AvrA L140A. (D) CD-specta of AvrA (dashed lines) and AvrA∆L140 (solid lines) in the absence (grey lines) and presence (black lines) of 500 µM IP6. Protein concentrations are 0.3 mg/mL in a buffer with 50 mM sodium phosphate, pH 7.0 and 5% glycerol.

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Biochemistry

Labriola et al., 7/19/18 3:13 PM Table 1. Summary of results from fluorescence-based thermal denaturation experiments AvrA -IP6 36.5 ± 0.13 °C* *

AvrA∆L140

AvrA L140A +IP6 -IP6 +IP6 -IP6 +IP6 39.5 ± 0.1 °C 38.3 ± 0.2 °C 43.5 ± 0.2 °C 46.9 ± 0.1 °C 48.2 ± 0.1 °C

Errors are standard deviations from three independent measurements.

AvrA is less stable than AvrA∆L140 Since AvrA and AvrA∆L140 appear to be structurally and functionally similar, we next questioned whether they possess similar stabilities through thermal denaturation experiments using a probe that increases in fluorescence upon binding to exposed protein hydrophobic regions. This allowed us to quantitatively monitor protein unfolding as hydrophobic regions become exposed upon denaturation. From these measurements, a melting curve as a function of temperature could be constructed, from which the temperature at which 50% of the sample is denatured (Tm), could be determined. Initial experiments carried out in the absence of IP6 revealed high fluorescence intensities before initiating the temperature ramp, an effect that was more pronounced in AvrA compared to AvrA∆L140 (Figs. S1A and B). Although this significantly reduced the dynamic range of the experiment, Tm values could still be estimated. The results, summarized in Table 1, showed that in the absence of IP6, AvrA had lower thermal stability than AvrA∆L140. Addition of IP6 dropped the starting fluorescence signals significantly, suggesting that accessible hydrophobic regions of both proteins become inaccessible to the probe upon IP6 binding, consistent with the results of our CD experiments of both AvrA homologues (Fig. 2D, Table S1), and with previous observations of HopZ1 and YopJ acetyltransferases10,11. This allowed for more accurate determination of Tm of both homologues and, as in the absence of IP6, AvrA had lower thermal stability compared to AvrA∆L140 (Table 1).

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Labriola et al., 7/19/18 3:13 PM Structure of AvrA∆L140 from S. Typhimurium in complex with IP6 and CoA To gain further insight into the structural characteristics that underlie the enzymatic differences between AvrA and AvrA∆L140, we attempted to determine their crystal structures. Unfortunately, AvrA did not crystallize alone or in the presence of the IP6 and CoA ligands. However, crystals of full-length, wild-type AvrA∆L140, including the N-terminal 15 amino acids previously believed to be part of the gene product, were obtained (PDB: 6BE0), allowing its structure to be determined at 2.4 Å resolution with molecular replacement using the structure of HopZ1 as a search model (Table 2). The first 36 residues of the 301 amino acid construct were not visible in the electron density map, presumably due to disorder. Thus, the first 21 amino acids of the bona fide AvrA∆L140 gene product are disordered in our structure. Although the structure at first glance appears as a single large domain, careful inspection reveals the presence of two closely associated domains (Fig. 3). The regulatory region comprised of flanking N- and C-terminal segments (residues 1 – 45 and 220 – 287) forms a wedge-shaped domain comprised of 8 α-helices and a β-hairpin at the apex of the wedge (Fig. 3A). The intervening residues (46 – 219) form the catalytic domain of AvrA∆L140, composed of a central 5stranded mixed β-sheet abutted by two α-helices on one face and another three α-helices on the other (Fig. 3A). The regulatory domain straddles the catalytic domain burying a total of 1560 Å2 between them. The catalytic domain is structurally homologous to the CE clan catalytic core (Fig. 3A). As observed in CE clan peptidases like Ulp, the catalytic triad residues of AvrA∆L140, H109, E128, and C171 (H109, E128, and C172 in AvrA) coalesce at one end of the β-sheet in the catalytic domain (Fig. 3A).

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Biochemistry

Labriola et al., 7/19/18 3:13 PM Table 2. Crystallographic data collection and model refinement statistics. Data Collection Space group Unit cell a, b, c (Å) Unit cell α,β,γ (°) X-ray wavelength (Å) Resolution range (Å) Total reflections Unique reflections Multiplicity Completeness (%) Rmeas Rpim mean I/σ(I) CC 1/2 Wilson B factor (Å2) Refinement Molecules per ASU Resolution range (Å) Reflections used Reflections for R(free) R(work) (%) R(free) (%) Non-hydrogen atoms protein ligands water Average B-factor (Å2) protein ligands water RMSD bond lengths (Å) RMSD bond angles (°) Ramachandran favored (%) Ramachandran allowed (%) Ramachandran outliers (%) Rotamer outliers (%) Clashscore

P3121 62.9, 62.9, 187.6 90, 90, 120 0.98 50 - 2.44 (2.48 – 2.44)* 178086 16757(534) 10.6 (10.3) 99.8 (100) 6.6 2.2 32.0(1.2) (0.61) 32.3 1 50 - 2.44(last shell) 14840 1275 18.7 23.1 2237 2109 63 65 46.6 46.5 64.4 33.4 0.003 0.65 97 3 0 0.4 0.46

*Data in parentheses are for highest resolution shell.

Page 19 of 39


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 40


Page 20 of 39


Page 21 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry


Figure 3. Crystal structure of AvrA∆L140. (A) Top: schematic showing the boundaries of the regulatory (dirty violet) and catalytic (teal) domains. Bottom: ribbon representations of the structure with transparent surface overlaid. The IP6 and CoA ligands are shown as sticks. Strands and helices are labeled. Position 140 is indicated by a red arrow on the bottom panel. Inset shows close-up of the Cys-His-Glu catalytic triad residues superimposed onto the HopZ1 (purple, PDB:5KLP), Ulp (grey, PDB:1EUV) and PopP2 (yellow, PDB:5W3X) structures. Top-right cartoon shows topology diagram of AvrA∆L140’s secondary structure organization. β-strands are annotated with numbers and α-helices by letters. Position 140 on helix αE is indicated by a red arrow. (B) Simulated annealing Fo-Fc omit map of CoA (top) and IP6 (bottom) binding sites contoured at 3.5σ. (C) Electrostatic surface representation of AvrA∆L140 depicting the positively charged (blue) binding pockets of IP6 (top) and CoA (bottom). Contour levels are +/- 5 kT. (D) Surface representation showing binding of both ligands (sticks) at the interface of the catalytic and regulatory domains and on opposing sides of the protein. Yellow patch indicates the location of the catalytic cysteine. Inset shows close-up of the CoA (top) and IP6 binding sites (bottom). A dotted line indicates the direction the CoA pantothenate arm would take towards the active site. Binding site residues are shown as sticks. Ordered water molecules are shown as red spheres.

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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 40

Labriola et al., 7/19/18 3:13 PM While the disposition of the catalytic triad is conserved in HopZ1 (Fig. 3A), comparison of the surface surrounding the catalytic cysteine reveals marked differences. The cysteine of HopZ1 lies at the base of a shallow, narrow groove lined by A172, V173, and S213 (Fig. S2B and C), whereas the cysteine of AvrA∆L140 lies at the base of a wider, more accessible concave surface lined by A130, A131, and S167-S169 (Fig. S2D). This difference is a consequence of the S168 side chain being oriented away from the cysteine and the less bulky A131 side chain, contributing to a more accessible active site (Fig. S2A). Moreover, despite the overall similarity between the AvrA∆L140, HopZ1, and PopP2 structures we identified sites of significant variation (Fig. 4A-C). Site 1 is at the N-terminus between β2 and β3 while Site 2 is at the C-terminus between helices αF and αG. For both, large insertions in PopP2 and HopZ1 replace shorter loops in AvrA∆L140 (Fig. 4D). Site 3 involves a helix (helix αE) adjacent to the active site and Site 4 is the region this helix packs against (αG-αF loop) on the catalytic domain, which have altered conformations and variable secondary structural elements, respectively, amongst the proteins.

AvrA∆L140 interactions with IP6 and CoA AvrA∆L140 was crystalized in the presence of IP6 and CoA. Unambiguous difference electron density was observed for both IP6 and CoA allowing us to model them accurately (Fig. 3B). The positions of both agree with what was observed in the HopZ1 and PopP2 structures. A large cluster of mainly positively charged residues bind IP6 (K67, Y68, K180, K181, K223, H224, K247, K248, R256, K276) in a groove at the interface between the catalytic and regulatory domains (Fig. 3C and D). The highly conserved K223 (K224 in AvrA), implicated in PopP2

Page 22 of 39


Page 23 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Labriola et al., 7/19/18 3:13 PM auto-acetylation27, is buried and makes intimate interactions with IP6, suggesting that it may not

Figure 4 A

D

AvrA

αA

αC

β1

4 AvrA YopJ VopJ PopP2 HopZ1a AvrBst Ulp

1

23 21 29 161 62 69 21

LN----------TEALTRIVERLESEII-DGSW--IHISYEETDLEMMPFLVAQANKKYPELNLKFVMSV SLIS--------NEELKNIITQLETDIS-DGSW--FHKNYSRMDVEVMPALVIQANNKYPEMNLNLVTSP LM--------------------DDAIRQGKQL---PKDTAAANDIALMDDFIAIANQKKEGLNAHFFR-QV----------TGFLSGALGKLQALSAQNMDPELAQFRVLDVDRAIMPLLIVAENARNPGLNLVPLHMD RHA---------RGPIKAYGQAALDRAS-------GAVSFAELDATHLDAMVYIENQRNPGLNLKHFRDH NSSNNIPY------EMRMYAEAALSAAK-HGS----SEAITKADVENKYYLAHAYNERFPELHLSCHDSA SRENTQLMNRDNIEITVRDFKTLAP-------------RRW-LNDTIIEFFMKYIEKSTPNTVAFNSFFY

αD 3 AvrA YopJ VopJ PopP2 HopZ1a AvrBst Ulp

2

HopZ1

B

80 80 74 221 119 128 77

αE

β5

124 124 120 269 166 169 127

αF

Site 3

1

3

AvrA YopJ VopJ PopP2 HopZ1a AvrBst Ulp

125 125 121 270 167 170 128

ILFEPAACSAFG------PALLALRTKAALER----EQLPDCYFAMVELDIQRSSSECGIFSLALAKKLQ ILFEPANFNSMG------PAMLAIRTKTAIER----YQLPDCHFSMVEMDIQRSSSECGIFSFALAKKLY IGIEPVTMNSLG——————ASMLAIRLQSVCKRE-----LPETSLVIMETDMQRSQGECLMFSLFLVKKMH IVVDPLRKEKDESAY---VDY-ADNVNMEF--------GEHAKCAFIPVDIQKSFFDCRILSLSLALKMH IITEPAVIVGARYQQ---LQR-HNLTLEDLSE----SGVPLSQVAIIETQAQKTSDDCVMYSLNYAIKAH LVIEPALAYGMKDGEIKVMAG-YETLGKNVQN----CLGENGDMAVIQLGAQKSLFDCVIFSLNMALCAY GYVDSLSNGP-NAMS---FAI-LTDLQKYVMEESKHTIGEDFDLIHLDCPQQPNGYDCGIYVCMNTLYGS

αF

αK

183 184 179 333 228 234 192

αG

Site 4

2

C

β3 Site 2

------------------HELVSSIKET-RME-GVESARFLVNM-----GSSGIHISVVDFRVMDGKTSV ------------------LDLSIEIKNV-IEN-GVRSSRFIINM-----GEGGIHFSVIDYKHINGKTSL ------------SPLDMVNYVKSLIPSE------DTTARFVVNM-----GSGGIHCIAVDCAIKNGKCSL --MAEDEEVRTQPPMAGSRHIAEFVASA-----RPGRYRAVID--------DGSHTRAADIRKDASGTSV ------------------YYLIQALQSD-----GPSAFRAIFPQTCPETGQTLKHHVMADVRLHQGAPTI ------------------QSFFSEFMTS-----EKQAWRSIVR-----LSPSSMHHAAIDVRFKDGKRTM TNLSE------RGY----QGVRRWMKRKKTQIDKLDKIFTPIN-------LNQSHWALGIIDLKK--KTI

β4

4

β2

Site 2

79 79 73 220 118 127 76


PopP2

4

184 185 180 334 229 235 193

LEFMNLVKIHEDNICERLCG------------------------EEPFLPSDKADRYLPVSFYKHTQGAQ IERDSLLKIHEDNIKGILSD------------------------GENPLPHDKLDPYLPVTFYKHTQGKK KECDEFQYLHDKNINRELPLTQGL----------------------IVSVK-DADSLLPPSLMKHTQSPN DKDDAFAAFHETLRNGGDPSHHVSRAQ-------------QTEELGATLVL-DGAPLVDARMMKHGQAAS KNAAQFDDIHHGLQHGTLST-ESESRARTTLGALEASSSYSVMHEGAHAA--FGADVLPVDFYKHGASLT QKDSVFDNLHDCLRRNVRCF-SSGE-------------RKSILHKNIEFI--EGDKFLPPIFYKHSHSRG AD-------------------------------------------------------AP--LDF------

αI

229 230 226 389 295 288 199

αL Site 1

3 1


230 231 227 390 296 289 200

RLNEYVEANPAAGSSIVNK----KNETLYERFDNNAVMLN---------DKKLSISAHKKRIAEYKSLLK RLNEYLNTNPQGVGTVVNK----KNETIVNRFDNNKSIVD---------GKELSVSVHKKRIAEYKTLLK RLQKYLEMRPEAMNCVVNK----KGETLKTRQQRHITTIEL-----GEKTVSYSNSIEQKRIKEAKGLLN SVSRYLGNHPEQSTVPVNK----RNETLGERTTRHLVKRKVRNRSGETKEITFSNSVEQKRIALLNRAAS QAYYLMKRPDGRMAGRVNSEGHSEAENLVQRNQAFRVKRRELLDDETPSNTQFSASIDGFRLQEIKRVLA VVGEFISNQPEYAHKNVSTGRTNPSEDLSERVENFRVR---------RGDLSYSMSIEASRLRKIRKTIE ----------------------------------------------------DYKDAIRMRRFIAHLILT

2

Page 23 of 39


285 287 287 463 365 349 217

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 40


Figure 4. Comparison of structures and sequences of YopJ acetyltransferases. Structures of (A) AvrA∆L140, (B) HopZ1, and (C) PopP2, with 4 circled regions that display significant structural deviations between them. (D) Sequence alignment of YopJ acetyltransferases. For Ulp, HopZ1 and PopP2, the alignment with AvrA∆L140 is structure-based. Secondary structure elements are depicted along the top row. Segments that significantly deviate in structure are shaded and colored correspondingly as the circled regions in A-C. Arrows show the location of catalytic triad residues (gold) and residues involved in binding IP6 (red) and CoA (blue) in AvrA∆L140.

Page 24 of 39


Page 25 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Labriola et al., 7/19/18 3:13 PM be a site of auto-acetylation. Although the configuration of IP6 binding is very similar to that observed in HopZ1, the number of interacting residues, their identities, and their precise positions vary somewhat. The CoA substrate is also located at the interface between the catalytic and regulatory domains, but approximately on the opposing side of the protein relative to IP6 (Fig. 3C and D). Only the adenosine moiety and two of its phosphates at the 3’- and 5’- position could be modeled as the remaining phosphate and pantothenate arm at the 5’-position were not visible in the electron density map (Fig. 3B), presumably due to disorder. The indole ring of adenosine is wedged between a hydrophobic pocket in the regulatory domain and a loop (Lβ5-αF) in the catalytic domain that leads to the active site cysteine. The corresponding loop in HopZ1 was shown to undergo a shift upon CoA binding, exposing the active site cysteine, which is otherwise largely buried in the absence of CoA (Fig. S2)11. In AvrA∆L140, the loop is shifted even further towards CoA, completely exposing the catalytic cysteine (Fig. S2D). The position of CoA puts it about 19 Å away from the catalytic cysteine, an appropriate distance for the pantothenate arm to reach it for acetyl transfer intramolecularly.

Location of L140 and the role of Helix E. That L140 is the sole difference between the two forms of AvrA indicates that it plays a key role in facilitating transfer of the acetyl group to the MKK4 substrate. From the context of the AvrA∆L140 structure, the L140 insertion of AvrA would land within helix αE, an amphipathic, surface exposed segment in the catalytic domain (Fig. 5A and Fig. 4A). One face of this helix makes hydrophobic interactions and packs against strand β5 and the αG-αF loop of the catalytic

Page 25 of 39


Biochemistry

Labriola et al., 7/19/18 3:13 PM domain (Fig. 5A). Insertion of L140 in AvrA would shift the register of the residues along the

Figure 5 A β5

F207

F207

αG-αF M159

M159

A140

β5

A140

F207

F207 αG-αF

M159

B

M159

A140L

A140L

C

150

Absorbance (mAU)

1.0

120

max

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 40

90 60 30

L140N

0 5.0

10.0

15.0

20.0

KD

0.5

AvrA

4.2 µM

5.9 µM

L140A

9.1 µM

0 0

20

Retention Volume (mL)

40

60

80

[IP6] µM

D MKK4 (36683.22 Da) Acetyl group (+43 Da)

AvrA L140A (33789.83 Da) Acetyl group (+43 Da) Ac

+0

+1

+2

Ac

33790.28

+0 36684.13

+1 36725.76

33832.73

L140A IP6 AcCoA

33874.66

33700 33750 33800 33850 33900 33950 34000

L140A IP6 AcCoA MKK4 36600

Mass (Da)

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36650

36700

36750

Mass (Da)

36800

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Biochemistry


Figure 5. Location of position 140 on helix αE in AvrA∆L140. (A) Helix αE is outlined in the red box. The top inset shows A140 in AvrA∆L140 and interacting residues on strand β5 and loop αGαF in wall-eyed stereo view. The bottom inset shows mutation of A140 in AvrA∆L140 structure to a Leu. (B) Gel-filtration profile of AvrA L140N mutant. (C) Binding curves of IP6 to AvrA constructs measured by internal tryptophan fluorescence. (D) Mass spectra of AvrA L140 in the presence of IP6 and CoA (left). Mass spectra of MKK4 in the presence of AcCoA, IP6, and AvrA L140A (right).

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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 40

Labriola et al., 7/19/18 3:13 PM helix, most likely perturbing its position and/or dynamics. Indeed, as described above, the position of this helix diverges between AvrA∆L140, HopZ1 and PopP2 (Fig. 4A-C). Moreover, as shown in the structure of PopP2 in complex with its substrate WRKY, the equivalent helix (αD) is involved in substrate recognition (Fig. 4C)12. Thus, we suggest that the functional differences observed between AvrA and AvrA∆L140 are due to structural differences in helix αE arising from the L140 insertion, which likely affects recognition of MKK4/7 and concomitantly their acetylation. To test this assertion, we created several L140 mutations in AvrA and characterized their structural, functional, and biophysical properties. Interestingly, mutation of L140 to a charged or polar residue (Lys, Asp or Asn) resulted in the protein aggregating as judged by gel-filtration chromatography (Fig. 5B), likely caused by misfolding. This suggests that L140 is probably on the buried face of helix αE in AvrA, possibly at a position similar to A140 in AvrA∆L140 (Fig. 5A). Modeling of a leucine at this position results in several clashes with the βG-αF segment, which would probably destabilize this region (Fig. 5A), though owing to the hydrophobic nature of leucine, presumably prevents misfolding and aggregation as observed in the polar mutations. We speculate that this destabilization is what contributes to the reduced thermal stability of AvrA relative to AvrA∆L140 we observed. We next mutated L140 in AvrA to an alanine, hypothesizing that this would yield a protein with a biophysical and functional profile similar to that of AvrA∆L140. Indeed, AvrA L140A displayed a similar gel-filtration profile (Fig. 2C), IP6 binding affinity (Fig. 5C), and CD spectra (Fig. 2D and Table S1) to AvrA and AvrA∆L140. Interestingly, however, AvrA L140A displayed a helical content intermediary between AvrA∆L140/AvrA and IP6 bound AvrA. Furthermore, there was no appreciable change in the CD spectra of AvrA L140A upon IP6

Page 28 of 39


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Biochemistry

Labriola et al., 7/19/18 3:13 PM binding (Fig. 2D, Table S1). Unexpectedly, AvrA L140A showed significantly higher thermal stability (~10°C higher) (Table 1) compared to either AvrA or AvrA∆L140, and a small gain in thermal stability upon IP6 binding, which is consistent with our observation that there is little change in secondary structure content in AvrA L140A upon IP6 binding. Additionally, we did not observe high initial fluorescence intensity in the absence of IP6 in AvrA L140A as was observed for other AvrA constructs (Fig. S1A). Taken together, these observations suggest that previously accessible regions of AvrA and AvrA∆L140, which become buried upon IP6 binding, are not accessible in AvrA L140A even in the absence of IP6. Taken together, these results suggest that the L140A mutation induces structural changes in AvrA akin to changes induced by IP6 binding in the natural homologues (see discussion). To further test the importance of helix αE in the acetylation of MKK4, we aligned the AvrA sequence with those of YopJ from Y. pestis and VopJ from V. parahaemolyticus, both of which target MKKs more broadly4,31 (Fig. 6A). From this, we identified two surface exposed hydrophobic residues (F135 and L141) and an arginine (R142) conserved among the MKK acetylating YopJs (AvrA, VopJ, and YopJ). We subsequently mutated these residues in AvrA and tested their structural and functional properties. All mutants appeared properly folded by gelfiltration chromatography (Fig. 6B) in contrast to the L140 polar mutants, suggesting these residues likely reside on the surface exposed face of helix αE. All mutants maintained autoacetylation activity, although it was diminished in the arginine mutation, and all mutants were unable to appreciably acetylate MKK4, though very small levels of acetylation were observed in the leucine and arginine mutations (Figs. 6C and 6D). These results demonstrate the importance of helix αE with regard to AvrA’s ability to acetylate MKK4, but not auto-acetylate, suggesting that this helix is important for direct binding to MKK4 (Fig. 6E).

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Biochemistry


Figure 6 A AvrA YopJ VopJ

C FGPALLALRTKAALER MGPAMLAIRTKTAIER LGASMLAIRLQSVCKR

Ac

D AvrA F135S (33771.81 Da) +0 +1 +2

Ac

33773.92

E MKK4 (36683.22 Da) Acetyl group (+43 Da) +0 +1 36684.13

AvrA

36725.76

33818.92

L141 IP6 AcCoA F135S

Ac

A140

IP6 AcCoA F135S MKK4

33858.81

C171

AvrA L142N (33832.86 Da) +0 +1 +2 33839.17

F135

R142

33882.13

33920.53

IP6 AcCoA L142N

B 12

Ac

IP6 AcCoA L142N MKK4

AvrA

AvrA R143A (33746.80 Da) +0 +1 +2 33749.36

10

Absorbance (mAU)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 40

L140 L140

8 6

C172 R143A

4

L142N 2

F135S

0 10.0

15.0

20.0

Elution Volume (mL)

IP6 AcCoA R143A

33794.92

33838.52

IP6 AcCoA L142N MKK4

33700 33750 33800 33850 33900 33950 34000

Mass (Da)

Page 30 of 39

36600

T261 36650

36700

36750

Mass (Da)


36800

MKK4

Page 31 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry


Figure 6. Role of helix αE in MKK acetylation. (A) Top shows sequence alignment of AvrA with YopJ and VopJ. Position 140 in AvrA is indicated with a red box. Mutated residues are shown in blue boxes. Bottom is a cartoon representation of the mutated residues. (B) Gelfiltration profiles of αE mutants. Constructs are indicated on the left. (C) mass spectra of constructs in the presence of IP6 and AcCoA. (D) Mass spectra of MKK4 in the presence of AcCoA, IP6, and indicated AvrA constructs. (E) Model suggesting a potential role for helix αE in AvrA function.

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Page 32 of 40

Labriola et al., 7/19/18 3:13 PM Discussion Our crystal structure of AvrA∆L140 from Salmonella revealed that it shares many similarities with HopZ1 and PopP2, including the overall fold, position of the catalytic triad, and locations of the IP6 and CoA binding sites. Investigations on the role of IP6 in both HopZ1 and PopP2 found that it is responsible for inducing structural changes leading to formation of the AcCoA and substrate binding sites11,32. Molecular dynamics simulations from these studies11 revealed that residues on a loop connecting two β-strands in the regulatory domain bind IP6, and in doing so, constrain the dynamics of this loop. This rigidification is relayed through a helix to a pair of β-strands on the opposite side of the protein, which make up part of the AcCoA binding site as well as a portion of the substrate binding site (Fig. 4; Site 1). Based on the structural conservation of these regions in AvrA∆L140, and our biophysical measurements showing that AvrA∆L140 is capable of binding IP6 and CoA, we predict both homologues of AvrA will behave similarly with regards to the regulatory role of IP6 on substrate binding. Our LC-MS results indicated that both AvrA∆L140 and AvrA can auto-acetylate at multiple sites within the protein. Mass spectrometry analysis of HopZ1 revealed auto-acetylation on two conserved serine residues28. Additionally, initial studies on PopP2 suggested that it autoacetylates a conserved lysine residue27. However, the structure of PopP2 in the presence of AcCoA showed no additional density corresponding to a potential acetyl group on this lysine, but instead additional density was observed on the catalytic cysteine. Moreover, this lysine is conserved across YopJs and is involved in IP6 binding, resulting in it being buried by IP6 in all known YopJ family structures. In all cases, mutation of these residues resulted in loss of function of these proteins. Thus, our mass spectrometry results are consistent with the identification of multiple acetylation sites, though our data do not indicate the identity of these sites. Upon

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Biochemistry

Labriola et al., 7/19/18 3:13 PM discovery that YopJ acetyltransferases were related to cysteine peptidases such as Ulp, which rely on formation of a peptidyl-thiol intermediate to catalyze hydrolysis of the peptide bond29, it was speculated that YopJ may use its catalytic cysteine in a similar way, that is, formation of a thio-ester with the acetyl group of AcCoA followed by transfer to the substrate33. This model is consistent with structural and biochemical analysis PopP2 acetylation of its WRKY transcription factor substrate12. Our results resolves these seemingly convergent observations, in that it is possible that the acetyl group of AcCoA is first transferred to the catalytic Cys and subsequently to other sites on the enzyme. Thus, we speculate that the main mass spectrometry autoacetylation peak observed is due to transfer of the acetyl group to the cysteine. As for the remaining sites of auto-acetylation, their relevance and identity is at present unclear. For example both the auto-acetylated lysine of PopP2 and the serines of HopZ1 lie at the IP6 binding site. Presumably acetylation of these residues would impact IP6 binding, and possibly serve as as a regulatory mechanism, given that IP6 binding is critical for the structural integrity and function of YopJ acetyltransferases. Interestingly, the observation that auto-acetylation sites are not conserved between YopJ members suggests that the mechanism by which auto-acetylation impacts function may be divergent. Understanding to what extent auto-acetylation regulates YopJ acetyltransferase function and its mode of action among YopJ members regulates enzyme will require further investigation. The structure of AvrA∆L140 revealed that the L140 insertion of AvrA resides within helix αE, and given that both variants possess similar CD spectra in the absence of IP6, we do not expect any significant structural changes upon insertion of L140. Mutational analysis of helix αE and our structural observations suggested that position 140 in both AvrA homologues is buried (Fig. 5), and thus the chemical nature of the side chain at that position influences the structural

Page 33 of 39


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Page 34 of 40

Labriola et al., 7/19/18 3:13 PM and biophysical properties of those homologues both globally and locally. Indeed, upon IP6 binding each homolog seem to display slightly divergent secondary structural changes, with AvrA showing a slight increase in helical content (Table S1). Given that L140 lies on helix αE it is tempting to speculate that IP6 induces a more extended helix αE in AvrA compared to AvrA∆L140. Unexpectedly, mutation the L140A mutation in AvrA dramatically increased its thermal stability (Table 1), alters secondary structural content in the absence of IP6 (Table S1), and renders inaccessible regions of the protein that are otherwise accessible in the wild-type (Fig. S1A). We also observed that this mutation renders AvrA less sensitive to structural changes that normally occur upon IP6 binding, even though L140A still binds IP6 at a comparable affinity to both wild-type AvrA homologues (Fig. 5C). Thus, it appears that the L140A mutation induces structural changes that mimic those induced by IP6 in AvrA, although only to an intermediate degree, as AvrA L140 without IP6 displays some structural and biophysical characteristics akin to the IP6 bound AvrA homologues, but still cannot acetylate MKK4. Taken together, it appears that position 140 in AvrA is intimately involved in IP6 allostery. Given this, previous work that shows IP6 induces substrate binding sites in HopZ1 and PopP211,32, and that the analogous helix to αE in PopP2 (Fig. 4C) is involved in substrate binding, we hypothesize that L140 and helix αE are critical in modulating the nature of the MKK4 binding site in AvrA (Fig. 6E). Since AvrA∆L140 is a folded, functional enzyme which retains its ability to act as an acetyltransferase, yet is no longer able to acetylate MKK4/7 due to disruption of the MKK4/7 binding site, one obvious question is, has AvrA∆L140 evolved to target another substrate? It is possible that a non-functional AvrA∆L140 simply serves to modulate the infectivity of strains that carry it, however, this notion is challenged by the observation that this gene persists in the

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Biochemistry

Labriola et al., 7/19/18 3:13 PM SL1344 strain of Salmonella typhimurium and is expressed under certain conditions34-36. The observations in this study that the presence of L140 plays a critical role in facilitating binding of MKK4 and does not significantly affect other structural or functional attributes of AvrA∆L140, suggests that this mutation may serve to alter the substrate specificity of this variant. Further studies will be required to determine whether such a substrate exists.

Acknowledgements We would like to thank Diego Alonzo from Dr. Martin Schmeing’s lab for help with designing the mass spectrometry method. Research described in this paper was performed using beamline 08ID-1 at the Canadian Light Source, which is supported by the Canada Foundation for Innovation, Natural Sciences and Engineering Research Council of Canada, the University of Saskatchewan, the Government of Saskatchewan, Western Economic Diversification Canada, the National Research Council Canada, and the Canadian Institutes of Health Research. In particular, we would like to thank Shaun Labiuk for his assistance in with remote data collection at the Canadian Light Source.

Author Contributions JML and BN conceived the project and designed the experiments. JML conducted the experiments. YZ helped with ITC data collection and BN helped with solution of AvrA∆L140 structure. JML and BN wrote and edited the manuscript.

Declaration of interest The authors have no conflict of interest to declare.

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Labriola et al., 7/19/18 3:13 PM Supporting Information •

Raw fluorescence data of thermal stability experiments.

•

Comparison of active sites structures of HopZ1, PopP2, and AvrA∆L140.

•

Estimation of secondary structure content based on CD spectral measurements.

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AvrA∆L140 1 2 3 4

Biochemistry

AvrA Page 40 of 40

A140 C171

L140 L140

C172 ACS Paragon Plus Environment T261

MKK4

Structural Analysis of the Bacterial Effector AvrA Identifies a Critical

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