Enzyme Mechanistic Studies of NMA1982, a Protein Tyrosine Phosphatase and Potential Virulence Factor in Neisseria meningitidis

Protein phosphorylation is an integral part of many cellular processes, not only in eukaryotes but also in bacteria. The discovery of both prokaryotic protein kinases and phosphatases has created interest in generating antibacterial therapeutics that target these enzymes. NMA1982 is a putative phosphatase from Neisseria meningitidis, the causative agent of meningitis and meningococcal septicemia. The overall fold of NMA1982 closely resembles that of protein tyrosine phosphatases (PTPs). However, the hallmark C(X)5R PTP signature motif, containing the catalytic cysteine and invariant arginine, is shorter by one amino acid in NMA1982. This has cast doubt about the catalytic mechanism of NMA1982 and its assignment to the PTP superfamily. Here, we demonstrate that NMA1982 indeed employs a catalytic mechanism that is specific to PTPs. Mutagenesis experiments, transition state inhibition, pH-dependence activity, and oxidative inactivation experiments all support that NMA1982 is a genuine PTP. Importantly, we show that NMA1982 is secreted by N. meningitidis, suggesting that this protein is a potential virulence factor. Future studies will need to address whether NMA1982 is indeed essential for N. meningitidis survival and virulence. Based on its unique active site conformation, NMA1982 may become a suitable target for developing selective antibacterial drugs.


Introduction
Protein phosphorylation is considered a universal mechanism to regulate protein function during most cellular processes 1,2 .About one third of all eukaryotic proteins are phosphorylated, predominantly on serine, threonine, and tyrosine 3 .In bacteria, phosphorylation on aspartate and histidine is well characterized as part of the two-component system 4 .However, bacterial proteins are also phosphorylated on serine, threonine, and tyrosine 5,6 .Interestingly, phosphorylation on tyrosine is much more abundant in bacterial proteins (9%), relative to serine and threonine, than it is in humans (1.8%) 7 .Both protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) have been isolated from bacteria 8-10 and have been proposed as anti-bacterial drug targets [10][11][12] .
The PTP superfamily contains various subfamilies with the two largest being the classical PTPs, which only dephosphorylate phosphotyrosine (pTyr), and the dual-speci city phosphatases (DUSPs), which in addition to pTyr can also dephosphorylate serine, threonine, or non-protein substrates such as phospholipids or RNA 13,14 .All PTPs have a cysteine-based catalytic mechanism (Fig. 1), which involves the nucleophilic attack on the phosphate group by the catalytic cysteine thiolate, resulting in the cleavage of the phosphoester bond and the formation of a phospho-enzyme intermediate 15 .This rst catalytic step is followed by another nucleophilic attack of a coordinated water molecule, resulting in the hydrolysis of the sulfur-phosphorus bond, the release of inorganic phosphate, and the recovery of the enzyme.The catalytic cysteine as well as the invariant arginine, which stabilizes the transition state, are part of the PTP signature motif C(X) 5 R that forms the phosphate-binding loop, also known as the P-loop.
Catalysis is further assisted by an aspartic acid residue that functions both as the catalytic acid, donating a proton to the leaving group in the rst step, and the catalytic base, activating the water molecule in the second step of the PTP reaction.This aspartic acid is part of the WPD-loop, named after the conserved tryptophan-proline-aspartate (WPD) motif in all classical PTPs.In DUSPs, only the aspartic acid is conserved; hence, this loop is also referred to as the D-loop.The WPD-loop uctuates between an "open" conformation, allowing substrate binding, and a "closed" conformation, necessary for catalysis.
Decreased mobility of the WPD-loop can result in substantially lower catalytic activity, as shown by mutational studies 16,17 as well as allosteric inhibitors that decrease WPD-loop exibility 18 .
Neisseria meningitidis is a human pathogen responsible for causing meningococcal diseases such as meningitis and meningococcal septicemia 19 .Despite established vaccination strategies, N. meningitidis continues to be an important cause of mortality and morbidity in newborns and children in both developed and underdeveloped countries 20 .Unlike viral meningitis, meningococcal meningitis can potentially be fatal within 24 h after the rst symptoms appear 21 .While antibiotics typically provide an effective treatment, cases of N. meningitidis antibiotic resistance have been reported 22 .Thus, the identi cation of novel drug targets that can be exploited for the treatment of meningitis is an unmet medical need.Previously, we reported the crystal structure of the N. meningitidis protein NMA1982, analysis of which suggested that its overall fold shows strong similarity to the DUSP subfamily of PTPs 23 .However, the P-loop containing the hallmark C(X) 5 R PTP signature motif is shorter by one amino acid in NMA1982, forming a C(X) 4 R motif.This disparity has cast doubts about the catalytic mechanism of NMA1982 and the assignment of NMA1982 to the PTP superfamily.Here, we show that NMA1982 indeed uses a PTP-like catalytic mechanism, demonstrating that the shorter C(X) 4 R motif is suitable to perform phosphatase catalytic function.Moreover, using pH-dependence, small molecule transition state inhibition, and oxidative inactivation experiments, we con rm that NMA1982 behaves like a typical PTP.Finally, we show that NMA1982, like other virulence factors, is secreted by N. meningitidis, suggesting that this phosphatase may be important for bacterial infection.

3D Structural comparison of NMA1982 with human PTPs
Using pairwise Dali 24 structural alignment of the crystal structures of NMA1982 and human DUSPs, we found that cyclin-dependent kinase inhibitor 3 (CDKN3), also known as kinase-associated phosphatase (KAP), is the most similar human phosphatase to NMA1982, with an amino acid identity of 15% and a Dali Z-score of 14.9 (Fig. 2a).Importantly, several conserved loops and amino acid residues critical for phosphatase activity 13 could be identi ed in NMA1982 (Fig. 2b): 1) The phosphate-binding loop (P-loop), which forms the center of the active site, is present in NMA1982, albeit shorter by one amino acid.In NMA1982 the putative catalytic cysteine (C95) and invariant arginine (R100), which both align very well with the corresponding residues in CDKN3, form a C(X) 4 R motif instead of the typical C(X) 5 R PTP signature motif.2) The general catalytic acid/base aspartate-containing WPD-loop is also present in NMA1982.D71 putatively serves the role of the catalytic acid/base.Notably, the WPD-loop in the NMA1982 crystal structure adopts an atypically open conformation (with D71 far removed from the active site), which also has been observed for several human PTPs 25 .3) NMA1982 features a typical PTP E-loop, which contains a conserved glutamic acid that coordinates the side chain of the invariant arginine in the P-loop and positions it for phosphate binding 13 .Indeed, in the NMA1982 crystal structure, the side chain carboxylic acid of E53 forms a salt bridge with the guanidinium group of R100.Collectively, our comparison with human DUSPs con rms that amino acids and loops critical for phosphatase activity are present in NMA1982.

Assessing NMA1982 phosphatase activity using Michaelis-Menten kinetics
We previously showed that NMA1982 has phosphatase activity using p-nitrophenyl phosphate as a pTyr surrogate substrate in a colorimetric assay 23 .To assess the catalytic parameters of NMA1982 more precisely in a kinetic experiment, we adapted a standard, continuous, uorescence-based phosphatase assay using 6,8-di uoro-4-methylumbelliferyl phosphate (DiFMUP) as the substrate 26,27 .Using this assay, we performed a Michaelis-Menten experiment to determine the Michaelis-Menten constant and turnover number for NMA1982 (Fig. 3a).The calculated K m value of 35 µM was well in the range of the K m values for human PTPs that we assayed under similar conditions, including the DUSPs VHX (K m = 11 µM), VHR (K m = 39 µM), and MKP-1 (K m = 40 µM); the classical PTPs PTP1B (K m = 4 µM), CD45 (K m = 43 µM), and SHP1 (K m = 52 µM); as well as the low-molecular weight PTPs LMPTP-A (K m = 63 µM) and LMPTP-B (K m = 218 µM).Thus, the Michaelis-Menten kinetic data suggest that the standard PTP substrate DiFMUP is similarly well accepted by NMA1982 and human PTPs.The turnover number for the NMA1982-catalyzed DiFMUP reaction was calculated to k cat = 4.4 x 10 − 05 s − 1 .This value was at least two orders of magnitude lower than the k cat values for the human PTPs we tested, suggesting that the catalytic conversion of the substrate is slower for NMA1982 compared to many human PTPs.Interestingly, a comparably low activity has been reported for CDKN3, the human PTP that is most similar to NMA1982 28 .

NMA1982 putative catalytic P-loop and WPD-loop mutagenesis studies
The putative P-loop in NMA1982, consisting of the C95-R-T-G-T-R100 sequence, is one amino acid shorter than the typical PTP P-loop containing the C(X) 5 R signature motif.Superposition of the NMA1982 crystal structure with crystal structures of human DUSPs suggested that the putative catalytic cysteine C95 and the putative invariant arginine R100 in NMA1982 align very well with the corresponding residues in human DUSPs (shown for CDKN3 in Fig. 1b).To test whether C95 and R100 are necessary for NMA1982 phosphatase activity, we generated and assessed the NMA1982 C95S and R100A mutant proteins.Since the corresponding residues are absolutely essential for enzymatic activity in all known PTPs 29 , we expected the C95S and R100A mutants to be inactive.Indeed, both mutants showed no detectable phosphatase activity in the DiFMUP assay, even at protein concentrations as high as 60 µM (~ 6-times higher than we used for assaying wt NMA1982).These data demonstrate that C95 and R100 of the C(X) 4 R motif are essential for NMA1982 phosphatase activity.
Next, we tested whether the putative catalytic acid/base D71 in the WPD-loop is critical for NMA1982 activity.Previous studies have shown that the mutation of the catalytic acid/base Asp to Ala typically results in a signi cant loss of phosphatase activity 30 .However, the degree of this loss in activity can vary widely among the PTPs and depends on whether other, nearby amino acids can compensate for the absence of Asp by providing a similar functionality 31 .To test the role of D71 for NMA1984 activity, we generated recombinant NMA1982 D71A mutant protein.We then assessed phosphatase activity of the mutant using a Michaelis-Menten kinetic assay with DiFMUP as the substrate.We found a substantial drop of 50% in the catalytic e ciency (k cat /K m ) for the D71A mutant protein compared to wt NMA1982, demonstrating that D71 is important for catalysis (Table 1).Collectively, our data demonstrate that the putative catalytic amino acid residues in NMA1982 are indeed crucial for catalysis.Given the relatively low activity we observed for wt NMA1982, we analyzed the NMA1982 crystal structure for additional insights Interestingly, we found that the side chain of R136 of the α6 helix, a residue that is unique to NMA1982 compared to human PTPs, forms multiple hydrogen bond interactions with the backbone oxygen atoms of A69, R70, and I72 of the WPD-loop (Fig. 3b).Because restriction of the WPDloop dynamics can result in decreased PTP activity [16][17][18] , we hypothesized that the interactions between R136 and the WPD-loop residues could potentially interfere with WPD-loop dynamics, and thus limit enzymatic activity.To test this hypothesis, we generated the NMA1982 R136A mutant protein and measured its phosphatase activity using the DiFMUP assay.Notably, we found that the R136A mutant had a 7.4-times higher catalytic e ciency than wt NMA1982, and that this increase was mainly due to an increase in k cat (Table 1).This suggested that the interactions of R136 with the WPD-loop residues limit turnover in NMA1982, most likely by restricting WPD-loop dynamics.To further support this hypothesis, we tested the effect of the D71A mutation on the activity of the R136A mutant compared to wt NMA1982.
If the greater activity of R136A was due to an increase in WPD-loop exibility, we expected that the D71A mutation would cause a greater drop in catalytic e ciency in the R136A mutant compared to wt.Indeed, the D71A mutation had a 6-times greater effect on catalytic turnover for the R136A mutant compared to wt NMA1982 (Table 1).Our results suggest that intramolecular interactions restrict WPD-loop mobility and therefore limit enzymatic activity of NMA1982.These data further demonstrate the importance of the WPD-loop for NMA1982 activity, and hence provide additional proof for NMA1982 utilizing a PTP catalytic mechanism.

NMA1982 inhibition, pH and temperature dependence, and oxidative inactivation studies
To provide additional evidence that NMA1982 behaves like a typical PTP, we tested the enzyme's response to sodium orthovanadate (Na 3 VO 4 ), a general PTP inhibitor that resembles inorganic phosphate and binds into the catalytic pocket as a transition state analog 32 .Using our established DiFMUP assay, we found that orthovanadate inhibited NMA1982 activity in a dose-dependent manner with an IC 50 value of 161 µM (Fig. 4a).Thus, the potency of orthovanadate against NMA1982 was comparable to its potency against other human PTPs (Reference 33 and our unpublished data).Next, we tested the pH dependence of the NMA1982 phosphatase reaction.We subjected wt NMA1982 to catalytic rate measurements under various pH conditions and found that NMA1982 activity peaked at pH 6 (Fig. 4b).
These data agreed with a PTP peak activity that is typically observed between pH 5 and 6 34 .Next, we tested how various temperatures affected NMA1982 activity.A close structural relative of NMA1982 is SsoPTP from Sulfolobus solfataricus, which exhibits peak phosphatase activity at 90°C 35 .As shown in Fig. 4c, NMA1982 exhibited peak activity around 37°C, and activity was substantially reduced at 50°C, and completely absent at 80°C.Thus, NMA1982 was most active at temperatures at which eukaryotic PTPs show peak activity.Finally, we assessed NMA1982 activity in oxidative inactivation and rescue studies.Because PTP activity depends on the catalytic cysteine in the reduced state, and hydrogen peroxide treatment results in thiol oxidation and PTP inactivation 36 , we tested whether NMA1982 activity was similarly sensitive to oxidation.Thus, we preincubated NMA1982 with various concentrations of hydrogen peroxide for 1 or 2 h and subsequently determined catalytic activity compared to a non-treated control (Fig. 4d).Our data demonstrated that hydrogen peroxide treatment decreased NMA1982 activity, and that this decrease was time-and dose-dependent.Since the cysteine thiol can be oxidized to several oxidation states that vary in terms of reversibility with reducing agents such as dithiothreitol (DTT) 37 , we also tested whether DTT treatment after peroxide treatment can rescue NMA1982 activity (Fig. 4e).Our data showed that DTT treatment could partially rescue NMA1982 activity, indicating the presence of both thiol-reversible and -irreversible oxidation states.Higher peroxide concentrations (≥ 400 µM) and longer treatment tended to cause more irreversible oxidation of NMA1982.Collectively, our studies demonstrate that NMA1982 behaves like a typical PTP when subjected to a transition state inhibitor, various pH and temperature conditions, or oxidizing agents.

N. meningitidis NMA1982 secretion studies
PTPs encoded by bacteria have been shown to be secreted by many pathogens during infection, particularly by intracellular pathogens that can thereby directly target eukaryotic effectors 38 .Extracellular pathogens such as N. meningitidis are capable of secreting various proteins and virulence factors to promote their growth, for example, to allow bio lm formation or to target the host response 39,40 .Furthermore, N. meningitidis can target host cells by secreting toxins that are endocytosed, such as the C2 fragment of the neisserial heparin-binding antigen (NHBA), which is released upon proteolysis 41 , or PorB porin, which is released via outer membrane vesicles (OMVs) 42,43 .The similarity of NMA1982 to eukaryotic PTPs strongly suggests that it may act as a virulence factor.We therefore assessed the secretion of NMA1982 in the model organism N. meningitidis 8013, which expresses the NMA1982 ortholog NMV0640 (Fig. S2).A derivative of the wt NEM8013 strain lacking NMV0640 was engineered as a control (ΔNMV0640).Both wt and ΔNMV0640 strains were grown with agitation in rich medium before the cells and supernatant were separated, collected, and processed by SDS-Page and immunoblot analysis (Fig. 5).The antibodies raised against NMA1982 clearly detected NMV0640 in both cell lysate and supernatant of the wt NEM8013 strain, whereas NMV0640 was not detected in the ΔNMV0640 control strain.As expected, PilE, the main component of type IV pili, was also recovered in the supernatant of the bacteria.The cytosolic marker NADP glutamate dehydrogenase and the outer membrane marker RMP4 were both absent from the supernatant fraction, con rming that there was no contamination of the supernatant fraction.Thus, our data demonstrate that N. meningitidis secretes NMA1982 during growth.Three secretion pathways are known to be active in N. meningitidis: the autotransporter, the two-partner secretion (TPS), and the type I secretion systems (T1S) 44 .The T1S is devoted to the secretion of the iron-regulated proteins FrpA/C only.Secretory proteins of the autotransporter and TPS rst cross the inner membrane via the general secretion (Sec) or the twin arginine translocation (Tat) pathways.Periplasmic and outer membrane components also rely on the Sec and Tat systems to translocate through the cytosolic membrane.We therefore looked for a signal peptide in the sequence of NMA1982 that would indicate secretion by these pathways.Using both PrediSi 45 and SignalP6.0 46, no known peptide signals were found, suggesting that NMA1982 is secreted via a Tat and Sec independent pathway.

Discussion
N. meningitidis causes systemic meningococcal disease, the mortality rate of which, even with optimal treatment, is still about 10% 21 .Here, we investigated an N. meningitidis protein that is highly similar to eukaryotic PTPs, enzymes that are considered crucial for many essential cellular processes.We show that NMA1982 uses a catalytic mechanism that is speci c to PTPs, involving a highly conserved phosphate-binding loop that forms the catalytic center.This loop is usually seven amino acids in length and contains the hallmark C(X) 5 R signature motif.In NMA1982 the P-loop is shorter by one amino acid and contains a novel C(X) 4 R motif.Despite being shorter, the P-loop can accommodate a PTP substrate equally well as demonstrated by the similar K m values of the pTyr mimetic DiFMUP for NMA1982 and human PTPs.In NMA1982, the catalytic cysteine and invariant arginine of the P-loop occupy space in the 3D structure that is similar to human PTPs.This ensures that the catalytic mechanism is not compromised.Given the existence of several bacterial NMA1982 homologs containing a C(X) 4 R motif (Fig. S3), other phosphatases with that motif may exist in bacteria.Notably, a conserved glycine within the P-loop of eukaryotic PTPs is also 100% conserved in all bacterial homologs (G98 in NMA1982).The relatively low catalytic activity of NMA1982 is not unprecedented among the PTPs.The structurally closest human enzyme, CDKN3, exhibits a similarly low turnover number 28 .Interestingly, CDKN3 activity, which critically regulates cell cycle progression, is itself regulated by protein-protein interactions, resulting in a substantial increase of phosphatase activity under physiological conditions 47,48 .Similarly, activity of NMA1982 might be enhanced in vivo.Our data demonstrates that NMA1982 is a PTP, enzymes that are essential for most cellular processes, not only in eukaryotes but also in bacteria.Importantly, we show that NMA1982, like known essential virulence factors, is secreted by N. meningitidis.The absence of a signal peptide suggests that NMA1982 is secreted by an atypical secretion machinery or via incorporation in OMVs as has been described for PorB.Future studies will need to address the question whether NMA1982 is indeed crucial for N. meningitidis survival and virulence, and thus may validate NMA1982 as a novel therapeutic target for the treatment of meningococcal diseases.Human PTPs have become attractive drug targets for many serious conditions, most notably cancer [49][50][51] .Compared to human PTPs, the shorter P-loop in NMA1982 results in a unique active site conformation, which may allow for the development of small molecule inhibitors with selectivity for the N. meningitidis protein.

Materials and Methods
Site-directed mutagenesis, protein expression and puri cation.
Utilizing our previously described pSpeedET plasmid containing the gene encoding NMA1982 23 , a QuickChange site-directed mutagenesis kit (Stratagene) was used for generating the NMA1982 mutants D71A, C95S, R100A, R136A, and R136A-D71A.Mutations were con rmed by DNA sequencing.The plasmids were transformed into Rosetta 2 (DE3) competent cells.Protein was expressed in LB media by induction with 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG).Wild-type and mutant NMA1982 proteins were puri ed as previously described 23 .The purity of the proteins was con rmed to be > 95% using SDS-PAGE and Coomassie staining.Differential scanning calorimetry (DSC) was used to con rm that all proteins were folded.Recombinant VHX, VHR, PTP1B, LMPTP-A, and LMPTP-B were expressed and puri ed as described previously 52,53 .Recombinant MKP-1 was purchased from Upstate.Recombinant CD45 and SHP1 were purchased from Biomol.
The uorescence emission was determined at two time points, 0 and 30 min, using an FLx800 micro plate reader (Bio-Tek Instruments, Inc.) with an excitation wavelength of 360 nm and an emission wavelength of 460 nm.The nonenzymatic hydrolysis of the substrate was corrected by measuring the control without addition of enzyme.NMA1982 activity of each sample was determined from the difference in emission intensity between the 0-and 30-min time points.

Table 1
Kinetic parameters of wild-type and mutant NMA1982 proteins.