Skip to main content
NCBI home page
Acta Crystallographica Section F: Structural Biology Communications logoLink to Acta Crystallographica Section F: Structural Biology Communications
. 2023 Mar 30;79(Pt 4):87–94. doi: 10.1107/S2053230X23001735

Structural and enzymatic characterization of the sialidase SiaPG from Porphyromonas gingivalis

Wen-Bo Dong a, Yong-Liang Jiang b, Zhong-Liang Zhu b, Jie Zhu b, Yang Li b, Rong Xia a,*, Kang Zhou b,*
Editor: Z S Derewendac
PMCID: PMC10071834  PMID: 36995120

The crystal structure of the sialidase SiaPG from Porphyromonas gingivalis, a pathogenic bacterium which causes human periodontal disease, is reported.

Keywords: sialidases, Porphyromonas gingivalis, crystallography, kinetic analysis, human periodontal disease

Abstract

The sialidases, which catalyze the hydrolysis of sialic acid from extracellular glycoconjugates, are a group of major virulence factors in various pathogenic bacteria. In Porphyromonas gingivalis, which causes human periodontal disease, sialidase contributes to bacterial pathogenesis via promoting the formation of biofilms and capsules, reducing the ability for macrophage clearance, and providing nutrients for bacterial colonization. Here, the crystal structure of the P. gingivalis sialidase SiaPG is reported at 2.1 Å resolution, revealing an N-terminal carbohydrate-binding domain followed by a canonical C-terminal catalytic domain. Simulation of the product sialic acid in the active-site pocket together with functional analysis enables clear identification of the key residues that are required for substrate binding and catalysis. Moreover, structural comparison with other sialidases reveals distinct features of the active-site pocket which might confer substrate specificity. These findings provide the structural basis for the further design and optimization of effective inhibitors to target SiaPG to fight against P. gingivalis-derived oral diseases.

1. Introduction

Periodontal disease is the most common oral disease in the world. As part of the human oral microbiome, the Gram-negative anaerobic bacterium Porphyromonas gingivalis plays a crucial role in the occurrence and development of perio­dontal disease (Xu et al., 2020 ▸). In addition to locating in the oral cavity, P. gingivalis can easily invade the respiratory tract, the digestive tract and the blood, causing systemic disease (Haraszthy et al., 2000 ▸; Patrakka et al., 2019 ▸; Harding et al., 2017 ▸; Dominy et al., 2019 ▸). Previous studies have also suggested that P. gingivalis is related to autoimmune disease, intrauterine growth retardation and preterm birth (Reyes et al., 2017 ▸; Loyola-Rodriguez et al., 2010 ▸).

In the treatment of periodontitis, systemic antibiotics are utilized as a common therapeutic strategy (Muniz et al., 2013 ▸). However, many periodontitis patients respond poorly to antibiotics (Colombo et al., 2009 ▸); thus, an ‘antivirulence’ therapy approach has been promoted that targets the virulence factors of specific periodontal pathogens, including P. gingivalis. Studies revealed that P. gingivalis has a number of virulence factors (Xu et al., 2020 ▸), including the well studied enzymes known as sialidases and also termed neuraminidases (Acuña-Amador et al., 2018 ▸). Not limited to P. gingivalis, sialidases are well established virulence factors in many pathogenic microorganisms, including Streptococcus pneumoniae, Propionibacterium acnes, Vibrio cholerae and Tannerella forsythia (Gualdi et al., 2012 ▸; Höfler et al., 1981 ▸; Kaisar et al., 2021 ▸). Glycoside hydrolase family 33 (GH33) represents one of the carbohydrate-active enzyme (CAZY) families; these are families of structurally related catalytic and carbohydrate-binding modules of enzymes that degrade, modify or create glycosidic bonds. GH33 contains many kinds of hydrolases, such as sialidases or neuraminidases (EC 3.2.1.18), trans-sialidases (EC 2.4.1.–), anhydrosialidases (EC 4.2.2.15), Kdo hydrolases (EC 3.2.1.124) and 2-keto-3-deoxynononic acid hydrolases/KDNases (EC 3.2.1.–). The bacterial sialidases belong to the GH33 family (EC 3.2.1.18). In particular, sialidases cleave the terminal sialic acid, a nine-carbon sugar, from various surface-exposed glycoproteins, glycolipids and polysaccharides (Taylor, 1996 ▸). The sialidases have been found to support the growth and colonization of microorganisms in mucosal ecosystems in different ways, including catabolizing sialic acid as a nutrient, exposing host ligands for adhesion, participating in biofilm formation and involving immuno­modulatory functions (Lewis & Lewis, 2012 ▸).

A previous study showed that P. gingivalis sialidase (SiaPG) can remove the terminal sialic acid from glycoconjugates on the host-cell surface, and thus P. gingivalis can bind to exposed glycoside residues (Aruni et al., 2011 ▸). The proteins desialyl­ated by SiaPG could be further hydrolyzed by proteases to generate peptides required for the asaccharolytic property of P. gingivalis. In addition, compared with the SiaPG-deficient strain, which is more susceptible to macrophage clearance (Yang et al., 2018 ▸), wild-type P. gingivalis can promote the formation of biofilms, thereby enhancing the pathogenicity of P. gingivalis (Li et al., 2012 ▸).

Although sharing less than 30% sequence homology, most sialidases have a similar domain organization, which consists of a conserved catalytic domain in addition to one or two carbohydrate-binding domains (CBDs). The catalytic domain harboring the highly conserved active-site residues shares a similar topology with a six-bladed β-propeller fold (Kim et al., 2011 ▸), while the CBDs enhance the catalytic efficiency via assisting the recognition of polysaccharide substrates (Frey et al., 2018 ▸), thereby conferring the specificity to recognize diverse substrates (Møller et al., 2021 ▸). To date, sialidase structures from different organisms have been solved (New­stead et al., 2008 ▸; Hsiao et al., 2009 ▸; Park et al., 2013 ▸; Lee et al., 2017 ▸; Kryshtafovych et al., 2021 ▸), which revealed a conserved catalytic mechanism but a varying substrate specificity in different sialidases.

Given the importance of SiaPG for pathogen–host interactions and virulence, SiaPG has potential as a therapeutic target for periodontitis. However, a lack of structural information prevents further investigation of and inhibitor design for SiaPG. In this study, we solved the crystal structure of SiaPG at a resolution of 2.1 Å. Structural analysis together with catalytic assays enabled us to clearly identify the substrate-binding pocket and the key residues required for catalytic activity. The results substantially extend our understanding of the catalysis and substrate recognition of SiaPG, which provides a structural basis for the design of inhibitors against P. gingivalis infection.

2. Materials and methods

2.1. Cloning, expression and purification of SiaPG and its mutants

The gene sequence for SiaPG was obtained from the NCBI database (Sayers et al., 2022 ▸). After removing the signal peptide region, the siaPG gene (GenBank Accession No. AUR47485.1) was codon-optimized and synthesized by Sangon Biotech. A DNA fragment encoding SiaPG residues 31–526 was PCR-amplified from the synthetic gene and cloned into a pET-28-derived vector (Novagen) using NdeI and SacI restriction enzymes to guide the production of SiaPG31–526 containing an N-terminal His6 tag. Based on the wild-type recombinant plasmid, mutants (R194A, D219A, D381A, E382A, R398A, R460A or Y488A) and a truncation (residues 31–175) were produced using a standard PCR-based strategy (the primers are listed in Supplementary Table S1). All constructs were verified by DNA sequencing (General Biol).

The recombinant plasmids were overexpressed in Escherichia coli strain PKY-206 (DE3) cells (Novagen). The cells were cultured in Luria–Bertani (LB) medium (10 g NaCl, 10 g Bacto tryptone and 5 g yeast extract in 1 l double-distilled H2O) containing 30 µg ml−1 kanamycin at 37°C. When the optical density at 600 nm (OD600) reached approximately 0.6–0.8, the cells were induced with 0.2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16°C. After cultivation for 21 h, the cells were harvested by centrifugation.

For the purification of SiaPG, the cells were resuspended in lysis buffer (20 mM Tris–HCl pH 7.5, 100 mM NaCl) and lysed via sonication on ice for 15 min. After centrifugation at 12 000g for 30 min at 4°C, the supernatant of the lysate was filtered and passed though an Ni–NTA column (GE Healthcare, Chicago, Illinois, USA) equilibrated with binding buffer (20 mM Tris–HCl pH 7.5, 100 mM NaCl). The column was washed with 40 ml binding buffer containing 30 mM imidazole. The target protein was eluted with 6 ml binding buffer containing 500 mM imidazole. The eluent was further purified by size-exclusion chromatography (SEC) on a Superdex 200 16/60 column (GE Healthcare) equilibrated with 20 mM Tris-HCl pH 7.5, 100 mM NaCl (Supplementary Fig. S1). Fractions containing the target protein were pooled and concentrated to 15 mg ml−1 by ultrafiltration (Millipore Amicon) for crystallization. The protein purity was assessed by SDS–PAGE and the protein sample was stored at −80°C.

2.2. Crystallization, X-ray data collection and structure determination

Preliminary crystallization was performed with a Mosquito robot (TTP Labtech) using commercial screening kits from Hampton Research. After initial screening, optimization of the crystals was performed by mixing 1 µl concentrated SiaPG protein (15 mg ml−1) with an equal volume of reservoir solution at 14°C using the hanging-drop vapor-diffusion method. After optimization, crystals with good diffraction quality were obtained using a reservoir solution consisting of 0.1 M Tris pH 7.0, 1.4 M ammonium tartrate dibasic. Crystals were transferred to reservoir solution supplemented with 30%(v/v) glycerol as a cryoprotectant and were flash-cooled in liquid nitrogen. Diffraction data were collected on beamline BL18U1 of the Shanghai Synchrotron Radiation Facility (SSRF) using a PILATUS3 detector at 100 K. The diffraction data were processed using HKL-2000 (Otwinowski & Minor, 1997 ▸).

The structure was solved by molecular replacement with Phaser (McCoy et al., 2007 ▸) using the structure of sialidase 26 from gut bacteria (PDB entries 6mrx for the apo form, 6mrv for the complex with DANA and 6myv for the complex with DANA-Gc; Zaramela et al., 2019 ▸) as the search model, which has 29% sequence identity with SiaPG. The model was automatically refined by REFMAC5 implemented in CCP4i (Winn et al., 2011 ▸) and manually rebuilt using Coot (Emsley et al., 2010 ▸). After several rounds of iteration of automatic and manual refinement, the R factor and R free values converged and the final model was assessed with MolProbity (Chen et al., 2010 ▸). Data-collection parameters and refinement statistics are listed in Table 1 ▸. The surface electrostatic potential was calculated using APBS (Jurrus et al., 2018 ▸). Structural figures were prepared with PyMOL (DeLano, 2002 ▸). AlphaFold (Jumper et al., 2021 ▸) was also used to predict a model of SiaPG (Supplementary Fig. S2).

Table 1. Data-collection and refinement statistics for SiaPG.

Values in parentheses are for the highest resolution shell.

Data collection
 Space group P3121
  a, b, c (Å) 131.16, 131.16, 181.17
 α, β, γ (°) 90, 90, 120
 Resolution range (Å) 56.80–2.10 (2.14–2.10)
 No. of reflections 1550917 (57812)
 No. of unique reflections 101168 (5048)
 〈I/σ(I)〉 18.5 (2.4)
  R r.i.m. † 0.033 (0.338)
 Completeness (%) 96.0 (97.3)
 CC1/2 0.99 (0.68)
 Average multiplicity 15.3 (11.5)
 Wilson B factor (Å2) 29.8
Structure refinement
 Resolution range (Å) 56.80–2.10
  R factor‡ (%)/R free § (%) 17.3/21.6
 No. of protein atoms 11623
 No. of water atoms 852
 R.m.s.d.
  Bond lengths (Å) 0.007
  Bond angles (°) 1.403
 Average B factor (Å2) 34.5
 Ramachandran plot††
  Favored regions (%) 96.0
  Allowed regions (%) 4.0
  Outliers (%) 0
 PDB code 8gn6
Open in a new tab
†

Estimated R r.i.m. = R merge[N/(N−1)]1/2, where N is the data multiplicity.

‡

R factor = Inline graphic Inline graphic , where F obs and F calc are the observed and calculated structure-factor amplitudes, respectively.

§

R free was calculated using 5% of the data that were excluded from refinement.

R.m.s.d. from ideal values.

††

Categories were defined by MolProbity.

2.3. Multi-angle light scattering

The molecular mass of SiaPG in solution was determined by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). 150 µl SiaPG solution (0.8 mg ml−1) was analyzed by size-exclusion chromatography on a small Superdex 200 Increase 10/300 GL column in 20 mM Tris–HCl pH 7.5, 100 mM NaCl. A DAWN HELEOS II light-scattering detector (Wyatt Technology) and an Optilab T-rEX refractive-index detector were used to record the data. The ASTRA 7.0.1 software (Wyatt Technology) was used to process the data. The Origin software was used to prepare the fitting curve.

2.4. Enzymatic activity assays

The activity assays were performed in reaction buffer (20 mM MES pH 6.0, 100 mM NaCl) using the purified wild-type SiaPG or its mutants with the fluorogenic derivative 4-trifluoromethylumbelliferyl-α-d-N-acetylneuraminic acid (4MU-Neu5Ac) as the substrate, based on a previous report (Frey et al., 2019 ▸). In detail, 10 µl SiaPG at 0.125 nM was added to 90 µl reaction buffer pre-incubated with the substrate 4MU-Neu5Ac at various concentrations (0, 5, 10, 20, 50, 65, 90, 120, 160, 220 and 300 µM). The reaction lasted for 1 min at 25°C and was subsequently terminated by adding 100 µl NaHCO3/Na2CO3 pH 10.5 to the reaction system. A fluorescence microplate reader with excitation at 365 nm and emission at 460 nm was used to record the fluorescence in real time. The standard curve of the product was prepared by measuring the fluorescence of 4-MU at various concentrations. By fitting the data to the Michaelis–Menten equation with nonlinear regression, the kinetic parameters of SiaPG were determined using GraphPad Prism 9 (GraphPad Software). At least three independent measurements were performed to calculate the standard deviations.

3. Results

3.1. Overall structure of SiaPG

The SiaPG crystal belonged to space group P3121 and its structure was determined by molecular replacement at 2.1 Å resolution. Each asymmetric unit contains three SiaPG molecules, and the crystal packing results in a largest buried interface of ∼1650 Å2 between two molecules. SEC-MALS analysis showed that SiaPG exists as a monomer in solution (Fig. 1 ▸ a). The atomic model of SiaPG contains residues 31–526, and the N-terminal 30-residue signal peptide was replaced by a His tag that is not visible in the density map (Fig. 1 ▸ b). Each SiaPG molecule consists of two canonical domains: a small carbohydrate-binding domain (CBD; residues 31–175) with a β-barrel fold and a catalytic sialidase domain with a typical β-propeller fold (residues 176–526) (Figs. 1 ▸ b and 1 ▸ c). The catalytic domain is formed by six sequential repeats, each of which consists of four antiparallel β-strands (Fig. 1 ▸ c). The six repeats, which are well conserved in sialidases (Taylor, 1996 ▸), pack against each other and are arranged into a six-bladed propeller forming a central cleft. Notably, as also observed in other sialidases (Yu et al., 2022 ▸; Newstead et al., 2008 ▸; Chavas et al., 2005 ▸), the catalytic domain of SiaPG has four Asp boxes in the first four blades of the β-propeller (Fig. 1 ▸ c), which may assist in protein folding (Quistgaard & Thirup, 2009 ▸). Each Asp box harbors the sequence Ser-X-Asp-X-Gly-X-Thr-Trp, which folds into a β-hairpin structure located at the periphery of the β-propeller. Similarly to the secreted bacterial sialidase (Yu et al., 2022 ▸), the electrostatic surface potential of SiaPG shows an asymmetric pattern in which the active-site pocket is mainly positive, whereas the surface remote from the active-site pocket carries a large amount of negative charge (Fig. 1 ▸ d).

Figure 1.

Figure 1

Open in a new tab

Overall structure of SiaPG. (a) SEC-MALS analysis of SiaPG. SiaPG solution was analyzed by size-exclusion chromatography on a Superdex 200 Increase 10/300 GL column. (b) Secondary structure of SiaPG; the carbohydrate-binding domain and catalytic domain are colored pink and cyan, respectively. (c) Topology diagram of SiaPG. (d) The electrostatic surface potential of SiaPG shown in two opposite views calculated using the APBS plugin and colored from blue (+5 kT e−1) to red (−5 kT e−1).

3.2. The active-site pocket of SiaPG

In the SiaPG structure we found extra density in the active-site pocket which could be fitted well with a molecule of tartrate, which was derived from the crystallization buffer (Fig. 2 ▸ a). The carboxyl group of tartrate is bridged by Arg194, Glu382, Arg398, Arg460 and Tyr488, whereas the carbonyl group of tartrate interacts with Arg194. Sequence alignment reveals that all of the tartrate-binding residues are highly conserved among homologs (Fig. 2 ▸ b). Therefore, the binding of tartrate in the active-site pocket is most likely to mimic the binding of sialic acid (also termed N-acetylneuraminic acid, Neu5Ac), the product of SiaPG. To further elucidate the binding pattern of Neu5Ac, docking of Neu5Ac into the active-site pocket of SiaPG was performed using HADDOCK (Honorato et al., 2021 ▸; van Zundert et al., 2016 ▸). The tartrate-binding residues and the nearby hydrophobic residues were defined as the active residues for HADDOCK simulation. The results gave 193 structures classified into five clusters, which represent 96% of the water refined models. We chose the most reliable cluster, with an overall lowest HADDOCK score of −45.7, for further analysis.

Figure 2.

Figure 2

Open in a new tab

(a) The active-site pocket of SiaPG. The tartrate and binding residues are shown as sticks. The density map of the tartrate molecule is shown as a blue mesh (F o − F c, contoured at the 2σ level). (b) Sequence alignment of sialidases from P. gingvalis, gut bacteria, P. acnes, A. fumigatus, B. caccae, B. thetaiotaomicron, P. distasonis and M. viridifaciens. The conservation pattern is colored according to Clustal Omega. Six well conserved residues are marked by black triangles. (c) Details of Neu5Ac docking to the active-site pocket of SiaPG. The Neu5Ac and binding residues are shown as sticks. (d) Electrostatic potential of the active-site pocket of SiaPG calculated using the APBS plugin and colored from blue (+5 kT e−1) to red (−5 kT e−1). (e) The relative enzymatic activities of wild-type SiaPG (WT) and its mutants against 4MU-Neu5Ac. CD-only is the catalytic domain.

In the simulated model, a molecule of Neu5Ac lies in the hydrophobic pocket in a semi-chair-like conformation and is fixed by extensive interactions (Figs. 2 ▸ c and 2 ▸ d). Similar to that of tartrate, the carboxyl group of Neu5Ac forms salt bridges with the arginine triplet Arg194, Arg398 and Arg460. The conserved nucleophilic dyad Tyr488 and Glu382, which hydrogen-bond to each other, function as nucleophilic attack residues to stabilize the putative carbon positive ion in the transition state (Newstead et al., 2008 ▸). The hydroxy group of Tyr488 points to the C8—C9 bond of Neu5Ac at a distance of 3.3 Å (Fig. 2 ▸ c), whereas Glu382, which is stabilized by Arg398, directly interacts with the O4 atom of Neu5Ac (Fig. 2 ▸ c). In addition, the C6 hydroxyl of Neu5Ac forms a hydrogen bond to Asp219 to further stabilize the binding of Neu5Ac. Asp381, which is generally not conserved in sialidases, also interacts with Neu5Ac by making two hydrogen bonds to the hydroxyl group of Neu5Ac (Fig. 2 ▸ c). Notably, beyond these hydrophilic interactions, the sugar ring of Neu5Ac is mainly stabilized by the hydrophobic residues Leu220, Val272, Leu277, Trp278 and Phe327 of SiaPG (Fig. 2 ▸ c), which form a generally hydrophobic pocket for Neu5Ac binding (Fig. 2 ▸ d).

3.3. Kinetic analysis of wild-type SiaPG and its mutants

To further investigate the roles of the putative active-site residues, we performed enzymatic activity assays against the generic fluorogenic substrate 4-trifluoromethylumbelliferyl-α-d-N-acetylneuraminic acid (4MU-Neu5Ac) using wild-type SiaPG and its mutants. The wild-type SiaPG is active against 4MU-Neu5Ac, with V max and K m values of ∼28 µM mg−1 min−1 and 74 µM, respectively (Supplementary Fig. S3), leading to a catalytic activity (k cat/K m) of ∼1000 µM min−1. Mutating any of the residues Arg194, Asp219, Asp381, Glu382, Arg398, Arg460 or Tyr488 to alanine retained only 10–30% of the catalytic activity (Fig. 2 ▸ e). Notably, mutation of the catalytic residues Glu382, Tyr488 or Asp219 of SiaPG almost completely abolished catalysis, further confirming the indispensable roles of these catalytic residues (Fig. 2 ▸ e).

4. Discussion

In this study, we solved the crystal structure of the sialidase SiaPG from P. gingivalis. It has a highly conserved catalytic domain adopting the six-bladed β-propeller fold (Figs. 3 ▸ a–3 ▸ c), which is shared among sialidases from different organisms (Zaramela et al., 2019 ▸; Telford et al., 2011 ▸; Yuan et al., 2005 ▸; Amaya et al., 2004 ▸; Guo et al., 2018 ▸). A structural similarity search using the DALI server (Holm, 2022 ▸) reveals that SiaPG is most structurally similar to sialidase 26 from gut bacteria (PDB entry 6myv), which has 29% sequence identity, yielding a root-mean-square deviation (r.m.s.d.) of 1.458 Å over 265 Cα atoms (Fig. 3 ▸ a). In addition, the catalytic domain of SiaPG also structurally resembles those of the sialidases from Streptococcus pneumoniae (PDB entry 2vw0; Xu et al., 2008 ▸), Clostridium perfringens (PDB entry 2bf6; Newstead et al., 2008 ▸) and leech (PDB entry 1sll; Luo et al., 1998 ▸). However, compared with the sialidases from S. pneumoniae and leech, SiaPG lacks an extra insertion segment (Figs. 3 ▸ b and 3 ▸ c), the function of which is still unknown.

Figure 3.

Figure 3

Open in a new tab

Comparison of the overall structure and the active-site pocket of SiaPG with homologs. SiaPG (colored pink and green) is superimposed on (a) sialidase 26 (PDB entry 6myv, gray) from gut bacteria, (b) sialidase from S. pneumoniae (PDB entry 2vw0, gray) and (c) leech sialidase (PDB entry 1sll, gray).

In addition to the conserved catalytic domain, many sialidases have extra domains described as lectins or CBDs, which are diverse in structure and organization among species. In sialidases, most CBDs precede the catalytic domains, as in P. gingivalis SiaPG, but in some sialidases they come after the catalytic domains, as in the sialidase from Micromonospora viridifaciens (PDB entry 1eut; Gaskell et al., 1995 ▸). Despite a similar overall structure of the CBDs between SiaPG and sialidase 26 (r.m.s.d. of 2.989 Å over 58 Cα atoms), they show different orientations relative to the catalytic domains. When the two catalytic domains are superimposed, the two CBDs are separated from each other by a 45° rotation (Fig. 3 ▸ a). Previous studies suggested that the CBDs have the property of binding polysaccharides, which might promote substrate binding and specificity (Frey et al., 2018 ▸). In this study, we found that without the CBD domain of SiaPG, the catalytic domain was rarely expressible in soluble fractions, and the catalytic activity of only the catalytic domain is almost completely abolished (Fig. 2 ▸ e). Indeed, structural analysis showed that the CBD makes direct interactions with the catalytic domain mainly via polar interactions (Supplementary Fig. S4). The results clearly demonstrated the essential role of CBD in catalysis and stabilization of the catalytic domain in SiaPG.

Molecular docking of Neu5Ac into the active-site pocket and sequence alignment of the catalytic domain in homologs enable us to identify the conserved active-site residues, which include Arg194, Asp219, Glu382, Arg398, Arg460 and Tyr488 (Figs. 2 ▸ a and 2 ▸ c). The three well conserved arginine residues (Arg194, Arg398 and Arg460) bind to the carboxyl group of both tartrate and Neu5Ac in the tartrate-complexed structure and in the simulated model with Neu5Ac, respectively. This arginine triplet may be directly involved in the SN2 catalytic reaction, as reported previously (Newstead et al., 2008 ▸). More importantly, the conserved catalytic residues Asp219, Tyr488 and Glu382 are indispensable for catalysis (Fig. 2 ▸ e), as Tyr488 and Glu382 form a Tyr–Glu nucleophilic dyad, whereas Asp219 acts as an acid/base catalyst, similar to as described previously (Buschiazzo & Alzari, 2008 ▸).

Beyond the conserved active-site residues mentioned above, structural analysis of SiaPG reveals distinct features of the active-site pocket compared with its homologs. Firstly, Asp381 in SiaPG, which is proposed to directly interact with Neu5Ac (Fig. 3 ▸ a), is substituted by Thr397 in sialidase 26, which shows no interaction with the substrate (Zaramela et al., 2019 ▸). Secondly, gut bacteria sialidase 26, S. pneumoniae NanB and leech sialidase all have a tryptophan (Trp507 in sialidase 26, Trp674 in S. pneumoniae NanB and Trp734 in leech sialidase) located close to the carboxyl group of the substrate (Figs. 3 ▸ a–3 ▸ c). In S. pneumoniae NanB a loop harboring Trp674 is positioned close to the active-site pocket, which creates a shielding effect of bulky hydrophobic side chains, and thereby confers a preference for α-2,3-linked substrates (Xu et al., 2008 ▸; Luo et al., 1998 ▸). However, in SiaPG the corresponding loop is much shorter and flips away from the active-site pocket, in which this tryptophan is substituted by a cluster of acidic residues, including Glu505, Asp506, Asp507 and Glu508 (Fig. 3 ▸ b). These acidic residues in SiaPG greatly increase the hydrophilic property of the active-site pocket, which might be related to the distinct substrate-binding pattern of SiaPG. In addition, compared with the completely capped substrate-binding pocket in other sialid­ases (Telford et al., 2011 ▸; New­stead et al., 2008 ▸; Xu et al., 2008 ▸), SiaPG has an incomplete capped pocket formed by Leu220, Val272, Leu277, Trp278 and Phe327. This atypical pocket may confer the distinct substrate-binding pattern given the fact that SiaPG shows no apparent substrate preference for α2,3- or α2,6-linked Neu5Ac (Frey et al., 2019 ▸).

In summary, we present the crystal structure of SiaPG complexed with the product mimic tartrate. Together with a credible product-binding simulation and kinetic analysis of mutants, we establish the atomic details and the key residues required for the enzymatic activity of SiaPG. Our findings provide a structural basis for studies of the substrate specificity of SiaPG and the future design of inhibitors to eradicate P. gingivalis-derived oral diseases.

Supplementary Material

PDB reference: SiaPG, 8gn6

Supplementary Figures and Table. DOI: 10.1107/S2053230X23001735/dw5234sup1.pdf

f-79-00087-sup1.pdf (458.3KB, pdf)

Acknowledgments

We appreciate the staff of beamline BL18U1 at the Shanghai Synchrotron Radiation Facility. Author contributions were as follows. KZ, RX and YC designed and supervised the project. W-BD, JZ and YL designed and performed the experiments. Z-LZ and Y-LJ collected the X-ray data and solved the structure. W-BD, Z-LZ and Y-LJ analyzed the data. W-BD, KZ and Y-LJ prepared the manuscript. All authors discussed the data and read the manuscript. The authors declare no competing interests.

Funding Statement

This work was funded by a grant from Anhui Health Research Project (AHWJ2022b017). Y-LJ thanks the Youth Innovation Promotion Association of Chinese Academy of Sciences for their support (Membership No. 2020452).

References

  1. Acuña-Amador, L., Primot, A., Cadieu, E., Roulet, A. & Barloy-Hubler, F. (2018). BMC Genomics, 19, 54. [DOI] [PMC free article] [PubMed]
  2. Amaya, M. F., Watts, A. G., Damager, I., Wehenkel, A., Nguyen, T., Buschiazzo, A., Paris, G., Frasch, A. C., Withers, S. G. & Alzari, P. M. (2004). Structure, 12, 775–784. [DOI] [PubMed]
  3. Aruni, W., Vanterpool, E., Osbourne, D., Roy, F., Muthiah, A., Dou, Y. & Fletcher, H. M. (2011). Infect. Immun. 79, 2779–2791. [DOI] [PMC free article] [PubMed]
  4. Buschiazzo, A. & Alzari, P. M. (2008). Curr. Opin. Chem. Biol. 12, 565–572. [DOI] [PubMed]
  5. Chavas, L. M. G., Tringali, C., Fusi, P., Venerando, B., Tettamanti, G., Kato, R., Monti, E. & Wakatsuki, S. (2005). J. Biol. Chem. 280, 469–475. [DOI] [PubMed]
  6. Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. & Richardson, D. C. (2010). Acta Cryst. D66, 12–21. [DOI] [PMC free article] [PubMed]
  7. Colombo, A. P., Boches, S. K., Cotton, S. L., Goodson, J. M., Kent, R., Haffajee, A. D., Socransky, S. S., Hasturk, H., Van Dyke, T. E., Dewhirst, F. & Paster, B. J. (2009). J. Periodontol. 80, 1421–1432. [DOI] [PMC free article] [PubMed]
  8. DeLano, W. L. (2002). PyMOL. http://www.pymol.org.
  9. Dominy, S. S., Lynch, C., Ermini, F., Benedyk, M., Marczyk, A., Konradi, A., Nguyen, M., Haditsch, U., Raha, D., Griffin, C., Holsinger, L. J., Arastu-Kapur, S., Kaba, S., Lee, A., Ryder, M. I., Potempa, B., Mydel, P., Hellvard, A., Adamowicz, K., Hasturk, H., Walker, G. D., Reynolds, E. C., Faull, R. L. M., Curtis, M. A., Dragunow, M. & Potempa, J. (2019). Sci. Adv. 5, eaau3333. [DOI] [PMC free article] [PubMed]
  10. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486–501. [DOI] [PMC free article] [PubMed]
  11. Frey, A. M., Satur, M. J., Phansopa, C., Honma, K., Urbanowicz, P. A., Spencer, D. I. R., Pratten, J., Bradshaw, D., Sharma, A. & Stafford, G. (2019). Microbiology, 165, 1181–1197. [DOI] [PMC free article] [PubMed]
  12. Frey, A. M., Satur, M. J., Phansopa, C., Parker, J. L., Bradshaw, D., Pratten, J. & Stafford, G. P. (2018). Biochem. J. 475, 1159–1176. [DOI] [PubMed]
  13. Gaskell, A., Crennell, S. & Taylor, G. (1995). Structure, 3, 1197–1205. [DOI] [PubMed]
  14. Gualdi, L., Hayre, J. K., Gerlini, A., Bidossi, A., Colomba, L., Trappetti, C., Pozzi, G., Docquier, J.-D., Andrew, P., Ricci, S. & Oggioni, M. R. (2012). BMC Microbiol. 12, 200. [DOI] [PMC free article] [PubMed]
  15. Guo, T., Héon-Roberts, R., Zou, C., Zheng, R., Pshezhetsky, A. V. & Cairo, C. W. (2018). J. Med. Chem. 61, 11261–11279. [DOI] [PubMed]
  16. Haraszthy, V. I., Zambon, J. J., Trevisan, M., Zeid, M. & Genco, R. J. (2000). J. Periodontol. 71, 1554–1560. [DOI] [PubMed]
  17. Harding, A., Robinson, S., Crean, S. & Singhrao, S. K. (2017). J. Alzheimers Dis. 58, 337–348. [DOI] [PubMed]
  18. Höfler, U., vom Gloor, M. & Nicolai, H. (1981). Zentralbl. Bakteriol. Mikrobiol. Hyg. A Med. Mikrobiol. Infekt. Parasitol. 250, 122–126. [PubMed]
  19. Holm, L. (2022). Nucleic Acids Res. 50, W210–W215. [DOI] [PMC free article] [PubMed]
  20. Honorato, R. V., Koukos, P. I., Jiménez-García, B., Tsaregorodtsev, A., Verlato, M., Giachetti, A., Rosato, A. & Bonvin, A. (2021). Front. Mol. Biosci. 8, 729513. [DOI] [PMC free article] [PubMed]
  21. Hsiao, Y. S., Parker, D., Ratner, A. J., Prince, A. & Tong, L. (2009). Biochem. Biophys. Res. Commun. 380, 467–471. [DOI] [PMC free article] [PubMed]
  22. Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., Bridgland, A., Meyer, C., Kohl, S. A. A., Ballard, A. J., Cowie, A., Romera-Paredes, B., Nikolov, S., Jain, R., Adler, J., Back, T., Petersen, S., Reiman, D., Clancy, E., Zielinski, M., Steinegger, M., Pacholska, M., Berghammer, T., Bodenstein, S., Silver, D., Vinyals, O., Senior, A. W., Kavukcuoglu, K., Kohli, P. & Hassabis, D. (2021). Nature, 596, 583–589. [DOI] [PMC free article] [PubMed]
  23. Jurrus, E., Engel, D., Star, K., Monson, K., Brandi, J., Felberg, L. E., Brookes, D. H., Wilson, L., Chen, J., Liles, K., Chun, M., Li, P., Gohara, D. W., Dolinsky, T., Konecny, R., Koes, D. R., Nielsen, J. E., Head–Gordon, T., Geng, W., Krasny, R., Wei, G. W., Holst, M. J., McCammon, J. A. & Baker, N. A. (2018). Protein Sci. 27, 112–128. [DOI] [PMC free article] [PubMed]
  24. Kaisar, M. H., Bhuiyan, M. S., Akter, A., Saleem, D., Iyer, A. S., Dash, P., Hakim, A., Chowdhury, F., Khan, A. I., Calderwood, S. B., Harris, J. B., Ryan, E. T., Qadri, F., Charles, R. C. & Bhuiyan, T. R. (2021). mSphere, 6, e01232-20. [DOI] [PMC free article] [PubMed]
  25. Kim, S., Oh, D. B., Kang, H. A. & Kwon, O. (2011). Appl. Microbiol. Biotechnol. 91, 1–15. [DOI] [PubMed]
  26. Kryshtafovych, A., Moult, J., Albrecht, R., Chang, G. A., Chao, K., Fraser, A., Greenfield, J., Hartmann, M. D., Herzberg, O., Josts, I., Leiman, P. G., Linden, S. B., Lupas, A. N., Nelson, D. C., Rees, S. D., Shang, X., Sokolova, M. L., Tidow, H. & AlphaFold2 Team (2021). Proteins, 89, 1633–1646. [DOI] [PMC free article] [PubMed]
  27. Lee, Y., Youn, H.-S., Lee, J.-G., An, J. Y., Park, K. R., Kang, J. Y., Ryu, Y. B., Jin, M. S., Park, K. H. & Eom, S. H. (2017). Biochem. Biophys. Res. Commun. 486, 470–475. [DOI] [PMC free article] [PubMed]
  28. Lewis, A. L. & Lewis, W. G. (2012). Cell. Microbiol. 14, 1174–1182. [DOI] [PubMed]
  29. Li, C., Kurniyati, Hu, B., Bian, J., Sun, J., Zhang, W., Liu, J., Pan, Y. & Li, C. (2012). Infect. Immun. 80, 3–13. [DOI] [PMC free article] [PubMed]
  30. Loyola-Rodriguez, J. P., Martinez-Martinez, R. E., Abud-Mendoza, C., Patiño-Marin, N. & Seymour, G. J. (2010). J. Oral Microbiol. 2, 5784. [DOI] [PMC free article] [PubMed]
  31. Luo, Y., Li, S.-C., Chou, M.-Y., Li, Y.-T. & Luo, M. (1998). Structure, 6, 521–530. [DOI] [PubMed]
  32. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658–674. [DOI] [PMC free article] [PubMed]
  33. Møller, M. S., El Bouaballati, S., Henrissat, B. & Svensson, B. (2021). J. Biol. Chem. 296, 100638. [DOI] [PMC free article] [PubMed]
  34. Muniz, F. W., de Oliveira, C. C., de Sousa Carvalho, R., Moreira, M. M., de Moraes, M. E. & Martins, R. S. (2013). Eur. J. Pharmacol. 705, 135–139. [DOI] [PubMed]
  35. Newstead, S. L., Potter, J. A., Wilson, J. C., Xu, G., Chien, C. H., Watts, A. G., Withers, S. G. & Taylor, G. L. (2008). J. Biol. Chem. 283, 9080–9088. [DOI] [PMC free article] [PubMed]
  36. Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307–326. [DOI] [PubMed]
  37. Park, K. H., Kim, M. G., Ahn, H. J., Lee, D. H., Kim, J. H., Kim, Y. W. & Woo, E. J. (2013). Biochim. Biophys. Acta, 1834, 1510–1519. [DOI] [PubMed]
  38. Patrakka, O., Pienimäki, J. P., Tuomisto, S., Ollikainen, J., Lehtimäki, T., Karhunen, P. J. & Martiskainen, M. (2019). J. Am. Heart Assoc. 8, e012330. [DOI] [PMC free article] [PubMed]
  39. Quistgaard, E. M. & Thirup, S. S. (2009). BMC Struct. Biol. 9, 46. [DOI] [PMC free article] [PubMed]
  40. Reyes, L., Phillips, P., Wolfe, B., Golos, T. G., Walkenhorst, M., Progulske-Fox, A. & Brown, M. (2017). J. Oral Microbiol. 9, 1374153. [DOI] [PMC free article] [PubMed]
  41. Sayers, E. W., Bolton, E. E., Brister, J. R., Canese, K., Chan, J., Comeau, D. C., Connor, R., Funk, K., Kelly, C., Kim, S., Madej, T., Marchler-Bauer, A., Lanczycki, C., Lathrop, S., Lu, Z., Thibaud-Nissen, F., Murphy, T., Phan, L., Skripchenko, Y., Tse, T., Wang, J., Williams, R., Trawick, B. W., Pruitt, K. D. & Sherry, S. T. (2022). Nucleic Acids Res. 50, D20–D26. [DOI] [PMC free article] [PubMed]
  42. Taylor, G. (1996). Curr. Opin. Struct. Biol. 6, 830–837. [DOI] [PubMed]
  43. Telford, J. C., Yeung, J. H., Xu, G., Kiefel, M. J., Watts, A. G., Hader, S., Chan, J., Bennet, A. J., Moore, M. M. & Taylor, G. L. (2011). J. Biol. Chem. 286, 10783–10792. [DOI] [PMC free article] [PubMed]
  44. Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A. & Wilson, K. S. (2011). Acta Cryst. D67, 235–242. [DOI] [PMC free article] [PubMed]
  45. Xu, G., Potter, J. A., Russell, R. J., Oggioni, M. R., Andrew, P. W. & Taylor, G. L. (2008). J. Mol. Biol. 384, 436–449. [DOI] [PubMed]
  46. Xu, W., Zhou, W., Wang, H. & Liang, S. (2020). Adv. Protein Chem. Struct. Biol. 120, 45–84. [DOI] [PMC free article] [PubMed]
  47. Yang, X., Pan, Y., Xu, X., Tong, T., Yu, S., Zhao, Y., Lin, L., Liu, J., Zhang, D. & Li, C. (2018). Front. Cell. Infect. Microbiol. 8, 100. [DOI] [PMC free article] [PubMed]
  48. Yu, A. C. Y., Volkers, G., Jongkees, S. A. K., Worrall, L. J., Withers, S. G. & Strynadka, N. C. J. (2022). Glycobiology, 32, 162–170. [DOI] [PMC free article] [PubMed]
  49. Yuan, P., Thompson, T. B., Wurzburg, B. A., Paterson, R. G., Lamb, R. A. & Jardetzky, T. S. (2005). Structure, 13, 803–815. [DOI] [PubMed]
  50. Zaramela, L. S., Martino, C., Alisson-Silva, F., Rees, S. D., Diaz, S. L., Chuzel, L., Ganatra, M. B., Taron, C. H., Secrest, P., Zuñiga, C., Huang, J., Siegel, D., Chang, G., Varki, A. & Zengler, K. (2019). Nat. Microbiol. 4, 2082–2089. [DOI] [PMC free article] [PubMed]
  51. Zundert, G. C. P. van, Rodrigues, J. P. G. L. M., Trellet, M., Schmitz, C., Kastritis, P. L., Karaca, E., Melquiond, A. S. J., van Dijk, M., de Vries, S. J. & Bonvin, A. M. J. J. (2016). J. Mol. Biol. 428, 720–725. [DOI] [PubMed]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

PDB reference: SiaPG, 8gn6

Supplementary Figures and Table. DOI: 10.1107/S2053230X23001735/dw5234sup1.pdf

f-79-00087-sup1.pdf (458.3KB, pdf)

Articles from Acta Crystallographica. Section F, Structural Biology Communications are provided here courtesy of International Union of Crystallography

RESOURCES