Structural insight into the mechanism of Staphylococcus aureus Stp1

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Structural insight into the mechanism of Staphylococcus aureus Stp1 phosphatase Teng Yang, Tingting Liu, Jianhua Gan, Kunqian Yu, Kaixian Chen, Wei Xue, Lefu Lan, Song Yang, and Cai-Guang Yang ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00316 • Publication Date (Web): 14 Mar 2019 Downloaded from http://pubs.acs.org on March 17, 2019

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ACS Infectious Diseases

Structural insight into the mechanism of Staphylococcus aureus Stp1 phosphatase

Teng Yang1,2,5, Tingting Liu2,3,5, Jianhua Gan4, Kunqian Yu2,3, Kaixian Chen2,3, Wei Xue1, Lefu Lan2,3,, Song Yang1,*, Cai-Guang Yang2,3,* State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Center for R&D of Fine Chemicals, Guizhou University, 2708 South Huaxi Road, Guiyang, Guizhou 550025, P. R. China 2 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, P. R. China 3 University of the Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, P. R. China 4 School of Life Sciences, Fudan University, 2005 Songhu Road, Shanghai 200433, P. R. China 5 These authors contributed equally to this work 1

*Correspondence: [email protected] (S.Y.) or [email protected] (C.-G.Y.)

Staphylococcus aureus Stp1, which belongs to the bacterial PPM (Mg2+ or Mn2+ dependent) phosphatase family, is a promising candidate for anti-virulence targeting. How Stp1 recognizes the phosphorylated peptide remains unclear, however. In order to investigate the recognition mechanism of Stp1 in depth, we have determined a series of crystal structures of S. aureus Stp1 in different states and the structural complex of Stp1 bound with a phosphorylated peptide His12. Different phosphorylated peptides, including MgrA- and GraR-derived phosphopeptides, are substrates of Stp1, which supports the function of Stp1 as a selective Ser/Thr phosphatase. In addition, interestingly, the crystal structures of R161-Stp1 variants combined with the biochemical activity validations have uncovered that R161 residue 1 / 31

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plays a key role to control the conformation switches of the flap domain in order to facilitate substrate binding and the dephosphorylation process. Our findings provide crucial structural insight into the molecular mechanism of S. aureus Stp1 phosphatase and reveal the phosphorylated peptides for biochemistry study and inhibitor screening of Stp1. Key words: Stp1 phosphatase, dephosphorylation, crystal structure, substrate recognition Staphylococcus aureus has become a global threat to public health due to its ability to cause multiple infections. These infections range from skin infections to complicated bacteremia and even acquired antibiotic resistance.1 Anti-virulence drugs, regarded as promising therapies superior to conventional antimicrobial agents, aim to attenuate the virulence of pathogens and decrease the occurrence of drug resistance rather than kill bacteria or inhibit their growth.2,3 The past decade has seen a considerable increase in attempts to clarify the biological roles and the corresponding chemical manipulation of bacterial virulence as alternative approaches to combat antimicrobial resistance,4 including the inhibition of ATP-dependent Clp protease,5-8 identification of transcriptional regulator CcpE,9 inhibition of diapophytoene desaturase,10 and development of SrtA inhibitor.11,12 Our previous study discovered that the serine/threonine kinase/phosphatase (Stk1-Stp1) pair exerted significant roles in the virulence regulation of S. aureus.13 Particularly, either deletion or mutation of stp1 gene leads to increased level of protein phosphorylation and weakened 2 / 31


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virulence, thus suggesting that Stp1 is a promising antivirulence target.13

Stp1 belongs to the bacterial PPM (Mg2+ or Mn2+ dependent) or human PP2C (protein phosphatase 2C) phosphatase family. The catalytic domains in PPM/PP2C structures are highly conserved; in fact, even the sequence conservations are relatively low.14-20 The first crystal structure of S. aureus Stp1 was determined to a 2.32 Å resolution (5F1M).21 The presence of four metal ions were observed in Stp1, which is different from other PPM/PP2C phosphatases that typically contain two or three metal ions. Several small-molecule inhibitors of Stp1 dephosphorylation were developed in order to suppress S. aureus virulence, including aurintricarboxylic acid (ATA) and its derivative Aurin, and 5,5’-Methylenedisalicylic Acid (MDSA).21,22 In mechanisms, these inhibitors were demonstrated to presumably occupy the active site for Stp1 inhibition. However, their inhibitory activities were usually evaluated by using p-nitrophenyl phosphate (pNPP) as a substrate, which the literature reveals is an artificial but widely used non-peptide substrate. Obviously, due to the absence of complex structure of Stp1 bound with native substrate of phosphopeptide, the recognition mechanism of dephosphorylation process and the biochemical validation of inhibitors have yet to be elucidated.

In addition to the catalytic active site in Stp1, the flexible flap domain shows structural mobility by adopting different conformations,14, 17, 20, 23-25 which likely participates in substrate binding and is involved in catalytic dephosphorylation as well.14 For example, 3 / 31



the contacts between Ser155 in the flap domain and Arg13 observed in Streptococcus agalactiae STP protein (SaSTP) could be a clue to understand the binding of Ser/Thr phosphatase with a dephosphorylated product.26 These interactions were observed in crystal packing but without validation by biochemical assays, which barely represented the characteristic of multiple substrates recognition since STPs are not restricted by the secondary structure of peptide. Although the dephosphorylation of tPphA was explored in the presence of different phosphopeptides,20 the recognition mechanism through the flap domain remains unclear.

Herein, we have solved four crystal structures of wild-type S. aureus Stp1 in different states, including Stp1 with and without the His tag (His-Stp1 and Stp1), Stp1 with Histag mutant (His(SE)-Stp1), and Stp1 bound with phosphopeptide (His12-Stp1). These structures enable us to design biochemical experiments to verify the selective hydrolysis of the phosphorylated Ser/Thr by Stp1 phosphatase, and clearly provide the molecular insights into the recognition mechanism. In addition, we have solved three more crystal structures of Stp1 bearing Arg161 variants, thus revealing the role of Arg161 residue in bacterial PPMs for the first time. Arg161 plays a critical role in facilitating the phosphopeptides binding and maintaining the dephosphorylation activity of Stp1.

Results The crystal structures of wild-type S. aureus Stp1 4 / 31


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Firstly, we determined the crystal structure of wild-type Stp1 in high resolutions of 1.57 Å (Figure 1A and Table S1). Four metal ions are embedded in the catalytic site (Figure 1B), which exist in accordance with the previously reported Stp1 structure.21 Of note, we performed the entire procedure of purification and crystallization of Stp1 proteins in the presence of MgCl2. Although difficult to determine, the metal ions showing in the structural complexes are most likely Mg2+. Three divalent cations (M1, M2, and M3) present the classical octahedron geometry with six coordination bonds, whereas M4 displays five coordination bonds. M4 could also have six coordinations, if one more of water presents in the network. The two catalytic metal ions M1 and M2 coordinate a common water molecule that is catalytically active and nucleophilic.27 M3 is located near the flap domain that contains α-helical segments and a flexible loop, which might play a role in both substrate binding and catalytic dephosphorylation, as suggested previously.28,29 Four aspartates, Asp37, Asp120, Asp194, and Asp233, are involved in coordination with these three metal ions (Figure S1),29 which are highly conserved in other bacterial PPM homologs, such as tPphA.20 M2 is also coordinated by way of the Gly38 backbone, which is conserved among other PPMs as well (Figure S1). M4 in Stp1 structure is distinct compared with other PPM/PP2C homologs, which also contributes to the catalytic activity of Stp1.21 Differential scanning fluorimetry (DSF) analyses show that the divalent manganese ion elevates Tm values of Stp1 in a concentration-dependent manner (Figure 1C), thus suggesting that the binding of multiple metal ions stabilizes the Stp1 conformation.

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Figure 1. Crystal structures of Stp1 without and with His-tag. (A) The overall structure of His-tag free Stp1. The protein is colored in cyan and the metal ions are colored in orange. The flap and catalytic domains are indicated. (B) Four divalent metal ions in the catalytic site present the classical octahedron geometry with multiple coordination bonds. Water molecules are presented as sphere in red, and the nucleophilic water is indicated by an arrow. (C) DSF experiment shows the effect of Mn2+ ion on Stp1 stability. (D) The structure of His-tagged Stp1 (His-Stp1). Stp1 is colored in blue and the His-tag is colored in red. Sulfate is presented in sticks. (E) Binding mode of three divalent metal ions in His-Stp1 structure. Hydrogen bonds are indicated as black dashed lines. (F) Superimposition of Stp1 and His-Stp1 structures. This was performed in PyMol. We also solved the crystal structure of His-tagged Stp1 in a resolution of 1.35 Å (Figure 1D). Interestingly, a sulfate ion (SO42-) occupies the place where the nucleophilic water molecule was bound in the wild-type Stp1 structure (Figure 1B and 1E). SO42- and phosphate ion (PO43-) possess similar spatial conformation; therefore, the His-Stp1 structure may represent the state involving phosphopeptide binding or dephosphorylation. Next, we conducted the superimposition of the Stp1 and His-Stp1 structures in order to investigate any structural changes occurring with and without sulfate binding (Figure 1F). The catalytic dephosphorylation domains aligned well 6 / 31


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while the flap domains displayed obvious differences in conformation, revealing that the flap domain is flexible and therefore may participate in substrate binding and/or dephosphorylation.

The crystal packing of His-Stp1 reveals its binding mode for phosphopeptide In order to demonstrate the mechanism underlying the conformational change observed in His-Stp1 structure, we sought to examine crystal packing. To our surprise, the well-ordered His-tag motif in one monomer partially inserted into the catalytic pocket of the other monomer of the dimeric model of His-Stp1 and made excessive contact (Figure 2A). Several hydrogen bonds were assigned between the His-tag motif and the adjacent His-Stp1 mainly through the contacts of the amino acid backbones (Figure 2B), therefore implying a sequence-independent mode for phosphopeptide recognition by Stp1. His42 is in an exceptional state, since its side-chain forms a hydrogen bond with Leu-7 backbone in the His-tag segment (Figure 2B). Furthermore, the hydrogen bonding between Ser-9 and SO42- ion could be viewed as the molecular mode for Stp1 binding with either substrate prior to dephosphorylation or the phosphate product after hydrolysis (Figure 2B).

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Figure 2. Interactions of the His-tag segment in the catalytic domain of Stp1. (A) The structure of His-Stp1 dimer was modeled according to crystal packing in COOT. One protein is colored in blue, the other in green. His tag is colored in red. (B) Close view of the binding pattern between His tag and the catalytic domain in the adjacent HisStp1. Hydrogen bonds are indicated by black dashed lines. (C) The structure of His(SE)Stp1 dimer modeled in COOT, in which the Glu mutant is introduced in order to mimic the phosphorylated Ser. One Stp1 is colored in yellow, the other in green. (D) Close view of the interactions at the binding interface. (E) Structural superimposition of HisStp1 with His(SE)-Stp1 (left panel) and for the zoom-in view on the recognition of Glu and sulfate (right panel). This structural observation encouraged us to further elucidate the recognition mechanism of phosphopeptide by Stp1. After considering the conformation of the phosphorylated serine residue and the electrostatics character of phosphorylation site, we introduced glutamic acid to mimic the phosphorylated Ser-9,30,31 while another amino acid, such as aspartic acid, was also used to mimic the phosphorylated state.32,33 Then, the variant of Stp1-bearing S-9E mutation in His-tag segment was purified and 8 / 31


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cultured for crystallization. The structure (His(SE)-Stp1) was solved in a high resolution of 1.40 Å (Table S1). The dimeric His(SE)-Stp1 was then built in order to show the molecular recognition (Figure 2C). Similarly, the majority of hydrogen bonding observed in His(SE)-Stp1 structure is comparable with that in the wild-type His-Stp1 dimeric model, and in the four metal ions binding as well (Figures 2B and 2D). Besides, we noted an extra hydrogen bond between residue Ser-10 and Asn162, which might further enhance the affinity for binding. Obviously, the residue Glu-9 that mimics the phosphorylated Ser, forms hydrogen bonds with Gly40 and Gly41, and a salt bridge with Arg14. The SO42- ion disappeared in the His(SE)-Stp1 structure since the binding space was fully occupied by Glu-9. The structural superimposition of His-Stp1 with His(SE)-Stp1 illustrates highly identified folding for the two Stp1 proteins (Figure 2E, left panel), further indicating the usefulness of structural models for uncovering the recognition mechanism of Stp1. As revealed in the catalytic pockets of two packed dimers (Figure 2E, right cartoon), the side chain of Glu-9 occupies the space where SO42- is taken up in the His-Stp1 structure. Particularly, both Glu-9 and SO42- ion form a salt-bridge with Arg14, thus validating that Glu-9 in His(SE)-Stp1 well mimics the real phosphorylated Ser/Thr residue. Taken together, the dimeric packing observed in both His-Stp1 and His(SE)-Stp1 represents a putative recognition mode between the phosphopeptide and Stp1 phosphatase, thus uncovering the molecular mechanism at the structural level.

Catalytic activity of Stp1 towards phosphorylated Ser/Thr 9 / 31



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The interaction observed in dimeric Stp1-packing shed light on the design of phosphopeptide for Stp1. We synthesized the peptide His12 containing phosphorylated Ser by taking the sequence of His-tag segment (Table 1). The highperformance liquid chromatography (HPLC) analyses was applied for investigating dephosphorylation of His12 by Stp1. The product of His12 dephosphorylation is peptide His11 (Figure 3A and Table 1). The dephosphorylation of His12 was dependent on Stp1 concentration (Figure 3B). Steadily, the dephosphorylation of 500 µM His12 completed in 10 min in the presence of 5 µM Stp1. In addition, MDSA, a Stp1 inhibitor,22 was assayed in order to inhibit His12 dephosphorylation in a dosedependent manner (Figure 3C). Thus, the biochemical assays confirmed that the Stp1 phosphatase can recognize and dephosphorylate the peptide that contains phosphorylated Ser residue.

Table 1. The sequence of peptides used in this study. Peptide

Sequence

Peptide

Sequence

His11

GSSGLGW

MgrA1

PELSNASDKVW

His12

GSS(p)GLGW

MgrA1P2

PELSNAS(p)DKVW

His12Y(p)

GSY(p)GLGW

MgrA1P1

PELS(p)NASDKVW

His12T(p)

GST(p)GLGW

MgrA5

ALDTGTVSPLW

His12S(p)L

GSS(p)LLGW

MgrA5P

ALDT(p)GTVSPLW

His12S(p)D

GSS(p)DLGW

GraR1

TLTWQDAVVDLSK

His12S(p)K

GSS(p)KLGW

GraR1P

T(p)LT(p)WQDAVVDLSK

10 / 31


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His12S(p)Y

GSS(p)YLGW

GraR2

GDDTIFLSK

GraR2P

GDDT(p)IFLSK

Figure 3. Biochemical activity of Stp1 towards phosphopeptides. (A) HPLC analysis of dephosphorylation of His12 to yield His11 in a dose-dependent manner. (B) The timedependent nature of Stp1 dephosphorylation is illustrated by the presence of 5 μM Stp1 and 500 μM His12. (C) MDSA inhibited His12 dephosphorylation by Stp1 in a dose-dependent manner. (D) Stp1 is inactive for phosphorylated Tyr substrate. The peak with retention time of 4.30 min is His12Y(p) with molecular weight of 819.3173. (E) Shown is the LC-MS detection of Stp1 activity on phosphorylated Thr substrate. The peak with retention time of 4.12 min is the product of His12T(p) 11 / 31



dephosphorylation with a molecular weight of 677.3342. (F) Stp1 dephosphorylates MgrA-derived peptide. The peak with retention time of 4.21 min represents MgrA1P2 that contains the phosphorylated Ser, and the retention time of 4.07 min corresponds to the MgrA1 product. (G) Stp1 dephosphorylates GraR-derived substrates. The retention time for the phosphorylated Thr-containing GraR1P and GraR2P is 12.18 min and 16.26 min, respectively. The retention time of 16.52 min and 17.63 min correspond to the GraR1 and GraR2 peptide product, respectively.

Stp1 is a selective Ser/Thr phosphatase The S. aureus Stp1 phosphate was identified to exert a dephosphorylation function on phosphopeptide that contains phosphorylated Ser residue. We wondered whether Stp1 could dephosphorylate the polypeptides that have other phosphorylated residues. Different polypeptides that contain phosphorylated Ser, Thr, and Tyr residues were assayed, respectively (Table 1). HPLC coupled with mass spectrometry (LC-MS) assay was performed for validation of dephosphorylation activities of Stp1 on these peptide substrates. As shown in Figure 3D, the molecular weight of the major peak with a retention time of 4.30 min is 819.3173, which corresponds to peptide His12Y(p), suggests that the Stp1 phosphate has minimal dephosphorylation effect on the phosphorylated Tyr residue. As expected, a peptide-bearing phosphorylated Thr could be completely dephosphorylated by Stp1 (Figure 3E). We next wondered whether the context of the phosphorylated peptide sequence might affect Stp1 on its activity. Similarly, LC-MS analysis showed that the dephosphorylation of various His12S(p) phosphopeptides by Stp1 steadily completed to yield the corresponding dephosphorylated products (Table 1 and Figure S2A-D), indicating that the residue next to the phosphorylated Ser has a minimal effect on the enzyme activity of Stp1. In 12 / 31


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conclusion, Stp1 can selectively dephosphorylate the phosphorylated Ser/Thr rather than the phosphorylated Tyr in a sequence-independent manner.

Stp1 dephosphorylates the MgrA- and GraR-derived phosphopeptides. Kinase-phosphatase pair Stk1/Stp1 regulates the phosphorylation of the conserved residue Cys in SarA/MgrA family proteins, mediating bacterial virulence and antibiotic resistance.13 In addition to the phosphorylated Cys residue, the corresponding phosphorylated Ser/Thr segments in MgrA and the phosphorylated Thr segments in GraR also could be the substrates of Stp1.34,35 For the purpose of validating the biochemical activity of Stp1 comprehensively, we assayed Stp1 dephosphorylation on the peptide MgrA1P2, which has the speculated phosphorylated segments of MgrA protein (Table 1). As expected, we observed that the dephosphorylation of the MgrAderived phosphopeptide substrate proceeded in both time- and dose-dependent manners in the LC-MS detection (Figure 3F). Either prolonging time or increasing the dose of Stp1 significantly improved MgrA1P2 dephosphorylation. In addition, two more MgrA-derived phosphorylated peptides, MgrA1P1 and MgrA5P, were also efficiently dephosphorylated by Stp1 (Table 1 and Figure S2E-S2F). Only a single peak was monitored in the reaction system, and which corresponds to MgrA1 (retention time 4.07 and m/z 1245.6139) and MgrA5 (retention time 4.78 and m/z 1159.6106), respectively. Moreover, we have verified that Stp1 could efficiently dephosphorylate two phosphopeptides derived from the GraR protein using the HPLC-based assay (Table 1). The phosphopeptide GraR1P (retention time of 12.18 min) is steadily 13 / 31



dephosphorylated by 2 M Stp1 to yield GraR1 (retention time of 16.52 min), and similarly, GraR2P (retention time of 16.26 min) is completely dephosphorylated to GraR2 (retention time of 17.63 min) in 10 min (Figure 3G). Therefore, the phosphopeptide bearing the amino acid sequence of MgrA or GraR protein was validated as an efficient substrate of Stp1, which could be used as a real substrate rather than the non-polypeptide substrate such as pNPP. How Stp1 hydrolyzes phosphorylated MgrA or GraR protein under biological conditions remains to be explored, however.

Crystal structure of Stp1 bound with His12 Isothermal titration calorimetry (ITC) measurement was performed in order to investigate the binding between the phosphopeptide His12 and Stp1. The analysis clearly showed a reasonable binding affinity between His12 and Stp1 (Kd = 6.20 ± 0.08 μM) and an enthalpy change (Δ H = 2675 ± 41.58 cal/mol) that accompanied the binding process (Figure 4A). To further investigate the interaction pattern, a structural complex of Stp1 bound with His12 was determined in a high resolution of 1.57 Å (Table S1 and Figure 4B, left panel). The phosphopeptide His12 remains phosphorylated under the crystallized condition, and GSS(p) residues of His12 were traceable in the electron density map. As similarly observed in the dimeric packing of the His-Stp1 structure, the GSS(p) peptide forms hydrogen bonds with Stp1 mainly through backbone interactions of several residues (Figure 4C, top panel), thus suggesting a sequence-independent mode for recognizing phosphorylated Ser/Thr by Stp1 14 / 31


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phosphatase. The recognition of the phosphorylated Ser residue is quite certain in the structural complex, since the phosphate group directly contacts with side chains of Arg14, Asp37, and Asp233 (Figure 4C, bottom panel), which clearly defines the binding site for phosphorylated Ser/Thr in Stp1 phosphatase. In addition, the phosphate motif in His12 extensively coordinates with the two metal ions (M1 and M2) in the structural complex (Figure 4C, bottom panel). This binding site might also be shared by other bacterial PPMs when considering the highly conserved amino acid sequences in these catalytic cores (Figure S1). The interaction mode presented in the structural complex of Stp1/His12 provides the molecular insight necessary for understanding the recognition mechanism of phosphorylated Ser by Stp1 in depth.

Figure 4. Characterization of the interaction between Stp1 and phosphopeptide His12. (A) ITC assay to measure the binding between Stp1 protein and His12 peptide. (B) Structural complex of Stp1 bound with His12. The GSS(p) segment in His12 could be traceable in the electron density map and colored in green. (C) Showing of the close view for the interaction mode of His12 in Stp1 binding site. Hydrogen bonds and coordination bonds are indicated as black dashed lines.

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Role of R161 in bacterial PPMs The bacterial PPMs typically contain two functional domains, the flap domain and the catalytic domain. The underlying mechanism for connection between these two domains is not fully understood. We have noticed conformational differences of the flap domains between the structures of Stp1 and His-tagged Stp1 (Figure 1F and 5A), which might be attributed to the presence of N-terminal His-tag motif mimicking peptide-binding by Stp1 phosphatase. Of note, the side-chain conformation of Arg161 in the apo-form of Stp1 is flexible, since the electron density was poorly mapped (Figure 5B, left panel). We carefully performed more structural superimpositions and compared the differences. Of particular note are the conformations of the side-chain in Arg161, which vary in different Stp1 states (Figure 5B). In the His-Stp1 structure, Arg161 formed hydrogen bonds by contacting the backbone of Ala155 as well as the side chain of Asp198 (Figure 5B, left panel). These two residues are located at the helix of the flap domain and the helix of the catalytic domain, respectively. While the connecting disappeared in the structure of Stp1, wherein the side-chain conformation of Arg161 is disordered. Similarly, R161 also tightly hold the flap domain and the catalytic domain closer in space by interacting with both backbones of Phe156 and Asp198 in the His(SE)-stp1 structure (Figure 5B, right panel). Of course, Arg161, which is located on a loop motif, functions as a bridge in immobilizing the flap domain and in order to create contact with the catalytic domain. Moreover, site-directed mutagenesis and the evaluation of phosphatase activity were performed in order to further validate the proposed role of Arg161. The variants of R161A and R161E lost 16 / 31


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phosphatase activities almost completely (Figure 5C). When the basic Arg161 residue was mutated to neutral Leu, we found 65% loss of phosphatase activity. As expected, R161K variant largely retained the catalytic dephosphorylation as wild-type Stp1 phosphatase. In short, these results validated the fundamental role of Arg161 in both structural and biochemical aspects.

Figure 5. Structural and biochemical characterization to reveal the molecular role of Agr161. (A) Structural superimposition of Stp1 (cyan), His-Stp1 (blue), and His(SE)-Stp1 (yellow) to show the conformational differences of the flap domain in Stp1 structures. (B) Close view to show the connection between the flap domain and catalytic domain through interactions mediated by Arg161. The hydrogen bonds are indicated by black dashed lines. (C) Enzyme activity of Arg161 variants. Phosphopeptide His12 was used as a substrate for the assay. The assays were performed in triplicate, and the error bar is indicated. (D) The alignment of crystal structures of Arg161 variants of Stp1. The following color mode was applied, gray for R161A, magenta for R161E, and green for R161K, respectively, and orange for metal ions. (E) The structural superimposition of Lys161 mutant and wild-type Stp1. In order to further characterize the role of Arg161 in Stp1, we sought to perform structural studies of the Arg161 variants. Three crystal structures were determined in 17 / 31



high resolutions, including R161A, R161E, and R161K variants (Table S1 and Figure 5D). Interestingly, the flap domain in the structures of R161A and R161E mutants could not be traceable in the electron density maps, indicating that the conformations of the flap domain are highly flexible in these two mutants. On the other hand, the conformation of the flap domain in Lys161 variant of Stp1 is relatively fixed, although the side chain of Lys161 is also observably disordered. The entire protein folding of Lys161 mutant is almost identical with that of wild-type Stp1, in which the side chain of Arg161 is also disordered (Figure 5E). This structural evidence strongly supports that the basic residue of Arg participates in stabling the conformation of the flap domain in Stp1.

We identified that Arg161 in S. aureus Stp1 functions as a bridge residue in order to impact the connection between the flap domain and the catalytic domain on the basis of both structural and biochemical results. In order to check whether this is a general principle utilized by different bacterial PPMs, we performed sequence alignment of bacterial PPM homologies including Stp1, SaSTP (2PK0),26 tPphA (5ITI),17 PstP (1TXO),25 and MspP (2JFR).36 As illustrated in Figure S1, Arg161 is highly conserved, except for the Lys161 in SaSTP. In addition, we performed structural superimposition in order to explore the conformation of Arg161 in bacterial PPMs (Figure S3A). Almost all the corresponding Arg or Lys residues stay away from the catalytic domains (Figure S3B), probably because of the absence of binding with the phosphopeptides. Such an open conformation may facilitate the recruitment of the substrate or release of the 18 / 31


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dephosphorylated product. Interestingly, the structural complex of MspP (2JFR),36 in which phosphate ion binding partially represents binding with substrate, is similar to our His-Stp1 structure, where a sulfate ion binding occurs in the active site (Figure S3C). In addition, the conformation of Arg152 in MspP is highly identical to that of Arg161 in the His-Stp1 structure (Figure S3C). These structural elements commonly shared by MspP and His-Stp1 suggest a coordinated effect between the flap domain and the catalytic

domain

for

PPMs

dephosphorylation.

Furthermore,

structural

superimpositions of S. aureus Stp1 and human PP2Cs containing PPM1A (1A6Q),27 PPM1B (2P8E),37 and PPM1K (2IQ1)37 revealed that the human flap conformation is distinct from bacterial ones (Figure S4), suggesting that human PP2Cs and bacterial PPMs use different mechanisms.

Discussion There are urgent but unmet needs to find new antibacterial agents as alternatives to conventional antibiotics in order to deal with the emerging antimicrobial resistance of S. aureus. The Stp1 phosphatase represents a novel antivirulence target. Because the substrate of Stp1 was not identified yet, the non-natural substrate pNPP was mainly used in the screening and validation of the inhibitory activity of small-molecule inhibitors for Stp1. To this end, the characterization of phosphorylated peptide substrate for Stp1 is of particular significance.

Our work has uncovered the molecular mechanism of the phosphopeptide recognition 19 / 31



by Stp1 and provides the correspondent substrate for the biochemical validation of Stp1. It has been achieved by solving several crystal structures of Stp1 representing the different states. It is unsurprising to observe multiple divalent metal ions embedded in the catalytic dephosphorylation site; it is, after all, common for most bacterial PPMs to bind these metal ions in a classical octahedron geometry in particular with six coordination bonds. To our surprise, the crystal packing into dimeric His-Stp1, for the first time, clearly revealed a putative binding mode for the recognition of phosphorylated peptide by Stp1 phosphatase. The major interactions occurred between the backbone of the peptide and Stp1, thus indicating a nonspecific sequence selectivity. That is well-correlated with the functional characterization of Stp1 as a global Ser/Thr phosphatase. Indeed, the biochemical assays of Stp1 dephosphorylation have proved that Stp1 is an efficient phosphatase selective to phosphorylated Ser/Thr substrates in both artificial peptides and MgrAand GraR-derived peptides. Due to lack of an ideal structural complex of Stp1 with phosphorylated MgrA or GraR, the mechanism of how Stp1 recognizes and dephosphorylates the phosphorylated domain within the entire protein needs further investigation. Furthermore, Stp1 selectively dephosphorylates phosphorylated Ser/Thr rather than phosphorylated Tyr, probably because the steric hindrance of phosphorylated Tyr is much larger than phosphorylated Ser/Thr while the catalytic pocket of Stp1 is not enough to accommodate phosphorylated Tyr. The structural complex of Stp1 bound with phosphopeptide was also solved in high resolution, which further demonstrates the recognition mode of phosphorylated Ser motif by Stp1 in 20 / 31


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the presence of divalent metal ions. These discoveries reveal the molecular mechanism of substrate recognition of Stp1 and provide the foundation for substituting artificial non-polypeptide substrates with real ones, which may facilitate the screening and validation of Stp1 inhibitors in order to validate the antivirulence target.

In addition, our study has demonstrated a connective role of Arg161 in the regulation of the conformation of the flap domain, which could be involved in the substratebinding and dephosphorylation process. This mechanism might be commonly shared by other bacterial PPMs. The residue Arg is located in a flexible loop of the flap domain in bacterial PPM homologies with two conformations. One is pointing away from the catalytic domain, which facilitates either substrate recruitment or dephosphorylated product release; the other acts as a bridge that immobilizes the flap domain in order to connect with the catalytic domain. These two conformations could be distinguished in the representative structures of wild-type Stp1 with and without the His-tag motif, respectively. As expected, enzyme activity assays show that variants of R161A and R161E mutation of Stp1 lose enzyme activity almost completely, while the R161K variant retains its phosphatase activity as wild-type Stp1, thus further suggesting the key role of Arg161 for Stp1 function. The enzyme activity differences among Stp1 variants may be due to the interactions between the basic residue and flap domain, whereas acidic or natural residue cannot contribute such interactions on the flap domain. However, the molecular process of conformational transition of Arg161 or 21 / 31



flap domain has not been shown and needs further exploration.

The known small-molecule inhibitors of Stp1 are typically carboxylic acids bearing multiple negative charges, such as ATA derivatives and MDSA. These moderately active inhibitors are thought to occupy active site and therefore compete for binding with the highly-charged phosphorylated peptide, which usually are usually PAINS compounds. Alternatively, the development of allosteric inhibitor that modulates the conformational changes of the flap domain could be excited in order to inhibit Stp1, since our study has illustrated a novel mechanism that the conformational control of the flap domain could significantly influence Stp1 on substrate-binding and eventually dephosphorylation process.

In summary, we report here a series of Stp1 structures in high resolutions to provide structural evidence for demonstrating the mechanism of Stp1 recognition on phosphorylated peptides. The combination of ITC assays, HPLC analyses, and structural characterizations validated that Stp1 selectively recognizes phosphorylated Ser/Thr peptides and efficiently proceeds dephosphorylation in vitro. In addition, the connective role of Arg161 between the flap domain and the catalytic domain was proposed and validated for the first time to our knowledge, and which is also conserved among other bacterial PPMs. Our findings provide significant and urgent structural insight into the molecular mechanism of S. aureus Stp1 phosphatase and offer the phosphorylated peptides for biochemical and inhibitory study of bacterial 22 / 31


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PPMs.

MATERIALS AND METHODS Materials. Unless stated otherwise, all chemicals were purchased from Sigma-Aldrich. Peptides were synthesized by GL Biochem Ltd. Protein expression and purification. Wild-type Stp1 gene was cloned into vector Pet28a. The expression of the N-His tagged Stp1 proteins was induced with 0.1 mM IPTG in E. coli BL21 (DE3) Gold strains. The cells were collected by centrifugation and re-suspended in 30 mL Ni-NTA buffer A (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 50 mM

imidazole,

2

mM

MgCl2,

1

mM

DTT)

with

protease

inhibitor

phenylmethanesulfonyl fluoride (PMSF). The samples were then purified by NI-NTA (GE Healthcare) with the gradient washing using buffer B (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 400 mM imidazole, 2 mM MgCl2, 1 mM DTT). Target eluted fractions were further purified by Superdex 75 gel-filtration chromatography (GE Healthcare) with a desalting buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM MgCl2, 1 mM DTT). The fractions were collected, concentrated and estimated by 12% (wt/vol) SDSPAGE. The purity of protein is more than 90%. Crystallization, data collection, and structure determination. Crystallization of purified Stp1 protein was performed using the hanging drop vapor diffusion method. 1 μL protein (15 mg/mL) was mixed with 1 μL crystallization buffer at 18 °C. The diffracting crystal was crystallized in a buffer containing 50 mM Tris-HCl (pH 8.0) and 100 mM NaCl. The crystallization conditions are as following, 0.05 M MgCl2, 0.1 M 23 / 31



HEPES (pH 7.5), and 30% PEG 550 for Stp1, Stp1/His12, and Stp1R159K, respectively; 0.2 M Li2SO4, 0.1 M Tris (pH 8.0), and 30% PEG 3350 for His-Stp1; 0.2 M MgCl2, 0.1 M Tris (pH 8.0), and 30% PEG 4000 for His(SE)-Stp1; 0.2 M NaCl, 0.1 M HEPES (pH 7.5), and 25% PEG 3350 for Stp1R159A; 0.1 M HEPES (pH 7.5), and 25% PEG 3350 for Stp1R159E. The crystals were transferred into cryoprotectant solutions containing 80% crystallization solution and 20% (v/v) glycerol, and flash-frozen in liquid nitrogen. Diffraction data were collected at Shanghai Synchrotron Radiation Facility (SSRF) beamline 17U. All X-ray data were processed using HKL2000 program suite38 and converted to structure factors within the CCP4 program.39 The statistics of data processing and refinement are summarized in Table S1. Isothermal titration calorimetry (ITC). All ITC measurements were recorded at 25°C using a MicroCal ITC (GE Healthcare). Proteins and peptides are dialyzed or dissolved in the same buffer containing 50 mM Tri-HCl (pH 8.0), 100 mM NaCl and 5 mM MnCl2 before use. The buffer was strictly degassed. 24 injections were performed by injecting 2 μL 500 μM peptide into a sample well containing 35 μM proteins. The concentration of the proteins and peptides were estimated with absorbance spectroscopy using the extinction coefficients OD280nm and OD260nm, respectively. Origin 7.0 software supplied with the instrument was used to analyze the ITC titration data.40 Differential scanning fluorimetry (DSF). The measurements were performed on the Prism 7500 real-time PCR system (ABI) using SYPRO orange (Invitrogen) as dye. Each sample containing 2 μM Stp1, 5 × SYPRO dye, and Mn2+ ion with different concentrations, was heated from 25 °C to 95 °C in 1% ramp rate. Fluorescence 24 / 31


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intensity was monitored at the wavelength of 492 nm for excitation and 610 nm for emission, respectively.8 Melting temperature (Tm) of protein was calculated by Protein Thermal Shift Software (Life Technologies), and was further processed in Origin 8.0. Phosphatase assay using pNPP as substrate. Phosphatase activity was measured at room temperature using a continuous method based on the detection of pnitrophenol yielded from pNPP. Briefly, reactions were conducted in 200 μL mixture containing 0.3 μM Stp1 proteins in buffer (10 mM Tris-HCl (pH 7.5), 5 mM MnCl2, 1 mM EDTA, 0.02% β-ME), followed by the addition of pNPP to a final concentration of 1 mM and incubated for 15 min. DMSO was used as the control. The reaction was quenched by the addition of 20 μL 5 M NaOH to measure absorbance at 405 nm. The experimental data was processed by GraphPad Prism software. All reactions were performed in triplicate. LC-MS based phosphatase assay. A 100 µL reaction mixture containing 50 mM TrisHCl (pH 8.0), 100 mM NaCl, 2 mM MnCl2, 1 mM DTT and 0.5 mM peptides was incubated at 20 °C for 10 min in the presence of 2 μM Stp1 proteins. The reaction was terminated by adding 5 mM EDTA to quench the enzymatic activity. An LC-30AD liquid chromatographic system (Shimadzu, Kyoto, Japan) coupled to a Triple Quad 5500 mass spectrometer (AB Sciex, Concord, ON, Canada) was used for acquiring LC-MS/MS data. Analytes were separated on an Eclipse Plus C18 column (100 mm × 4.6 mm I.D., 3.5 μm; Agilent, USA). The mobile phases used for isocratic elution were 85% (A) 0.1% formic acid and 15% (B) 0.1% formic acid in acetonitrile. The flow rate was 0.6 mL/min. 25 / 31



The mass spectrometer was operated in the negative MRM mode.

ACCESSION NUMBERS

Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB, www.pdb.org) under accession ID code 6IHL, 6IHR, 6IHS, 6IHT, 6IHU, 6IHV, and 6IHW for the structure of Stp1, His-Stp1, His(SE)-Stp1, Stp1/His12, Stp1R161A, Stp1R161E, and Stp1R161K, respectively. Supplemental Information Supplemental Information includes four figures, one table, and Supplemental Procedures can be found with this article online.

Author contributions

C.-G.Y. and S.Y. conceived the project. C.-G.Y. designed the research and wrote the paper with help from T.Y. and T.L.. T.Y. performed most of the experiments. K.Y., K.C., W.X., and L.L. provided funding support and/or technical assistance. J.G. processed the X-ray data and refined the crystal structures. All authors reviewed the results and approved the manuscript.

Notes

The authors declare no competing financial interest. Acknowledgements We thank all beamline staff at the 17U and 18U of Shanghai Synchrotron Radiation 26 / 31


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Facility, and S.F. Reichard, MA for editing the manuscript. This study was supported by the Science and Technology Commission Shanghai Municipality (17XD1404400), the National Natural Science Foundation of China (81661138004, 81861138046, 21725801, and 21877021). References (1) Di Gregorio, S., Fernandez, S., Cuirolo, A., Verlaine, O., Amoroso, A., Mengin-Lecreulx, D., Famiglietti, A., Joris, B., and Mollerach, M. (2017) Different Vancomycin-Intermediate Staphylococcus aureus Phenotypes Selected from the Same ST100-hVISA Parental Strain. Microb. Drug Resist., 23, 4450 DOI 10.1089/mdr.2016.0160. (2) Totsika, M. (2017) Disarming pathogens: benefits and challenges of antimicrobials that target bacterial virulence instead of growth and viability. Future Med. Chem., 9, 267-269 DOI 10.4155/fmc2016-0227. (3) Allen, R. C., Popat, R., Diggle, S. P., and Brown, S. P. (2014) Targeting virulence: can we make evolution-proof drugs? Nat. Rev. Micro., 12, 300-308 DOI 10.1038/nrmicro3232. (4) Zhou, L. L., and Yang, C. G. (2019) Chemical Intervention on Staphylococcus aureus Virulence. Chin. J. Chem., 37, 183-193 DOI 1002/cjoc.201800470. (5) Zhang, J., Ye, F., Lan, L., Jiang, H., Luo, C., and Yang, C. G. (2011) Structural switching of Staphylococcus aureus Clp protease: a key to understanding protease dynamics. J. Biol. Chem., 286, 37590-37601 DOI 10.1074/jbc.M111.277848. (6) Ye, F., Li, J. H., and Yang, C. G. (2017) The development of small-molecule modulators for ClpP protease activity. Mol. Biosyst., 13, 23-31 DOI 10.1039/c6mb00644b. (7) Ye, F., Zhang, J., Liu, H. C., Hilgenfeld, R., Zhang, R. H., Kong, X. Q., Li, L. C., Lu, J. Y., Zhang, X. L., Li, D. H., Jiang, H. L., Yang, C. G., and Luo, C. (2013) Helix Unfolding/Refolding Characterizes the Functional Dynamics of Staphylococcus aureus Clp Protease. J. Biol. Chem., 288, 17643-17653 DOI 10.1074/jbc.M113.452714. (8) Ni, T. F., Ye, F., Liu, X., Zhang, J., Liu, H. C., Li, J. H., Zhang, Y. Y., Sun, Y. Q., Wang, M. N., Luo, C., Jiang, H. L., Lan, L. F., Gan, J. H., Zhang, A., Zhou, H., and Yang, C. G. (2016) Characterization of Gain-of-Function Mutant Provides New Insights into ClpP Structure. ACS Chem. Biol., 11, 1964-1972 DOI 10.1021/acschembio.6b00390. (9) Li, H., Ding, Y., and Lan, L. (2015) Transcriptional profiling of CcpE-regulated genes in 27 / 31



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Figure 1. Crystal structures of Stp1 without and with His-tag.


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Figure 2. Interactions of the His-tag segment in the catalytic domain of Stp1.



Figure 3. Biochemical activity of Stp1 towards phosphopeptides.


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Figure 4. Characterization of the interaction between Stp1 and phosphopeptide His12.



Figure 5. Structural and biochemical characterization to reveal the molecular role of Agr161.


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For Table of Contents Use Only


Structural insight into the mechanism of Staphylococcus aureus Stp1

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