Streptococcus co-opts a conformational lock in human plasminogen to facilitate streptokinase cleavage and bacterial virulence (2024)

Sequence alignments of hPg and mPg/kringle domains of hPg

In order to examine the primary structural similarities and differences between hPg and mPg, their sequences were aligned (Fig.S1A). The alignments showed that the overall level of hom*ology between hPg and mPg is ∼80%. Sequences within the latent HCs (shown in black) are ∼77% hom*ologous between hPg and mPg, and sequences of the latent LCs (shown in blue) show ∼84% hom*ology. Figure. S1B provides an alignment of the five kringle domains of hPg and shows that the lysine-binding residues (highlighted in red) of hPg are highly conserved across all the lysine-binding kringles (K1_hPg, K2_hPg, K4_hPg, and K5_hPg). K3_hPg does not bind to lysine because of a key mutation in its anionic center (DGD to DGK).

hom*ogeneity, molecular weight, and Pg conformation

Parental Pgs (hPg and mPg), along with a set of chimeric Pgs, viz., mouse heavy chain (mHC)-human light chain (hLC) and human heavy chain (hHC)-mouse light chain (mLC), and a point mutant, hPg[D²¹⁹N], which eliminates by a single mutation the LBS in the K2 domain of hPg, were used in this work. Following expression of the Pgs in Drosophila Schneider S2 cells, each variant was purified by lysine-Sepharose affinity chromatography (37). Typical protein yields ranged from 7 to 21 mg/l. The purities, molecular weights, and conformations of the Pg variants were examined by SDS-PAGE, size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), and analytical ultracentrifugation (AUC).

The gel electrophoretograms (Fig.1A) show that all proteins were highly purified, with molecular weights close to their expected values. Some slight differences in mobility are observed, perhaps due to glycosylation differences. However, for precise determinations of the molecular weights of the chimeric Pgs, the purified proteins were first subjected to analysis by SEC-MALS, an example of which is shown in Figure1B, as well as sedimentation equilibrium by AUC. The results, listed in Table1, show agreement between the molecular weights using both analytical methods and the calculated molecular weights of each protein (minus the carbohydrate). These results demonstrate that highly purified full-length Pgs were obtained.

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Figure1

hom*ogeneity test and molecular weight determinations. (A) Electrophoretogram of Pg variants using 10% tris-glycine gels. Lanes marked 1 to 6 refer to the following: 1, protein marker; 2, hPg; 3, mPg, 4, mHC-hLC, 5, hHC-mLC, 6, hPg[D²¹⁹N]. (B) Examples of SEC-MALS chromatograms of representative Pgs: red, hHC-mLC; green, mHC-hLC. One hundred microliters of each Pg was injected onto a Wyatt -030S5 SEC column (7.6× 300 nm, 5 μm, 300 Å), equilibrated and eluted with PBS, pH 7.4, at a flow rate of 0.5 ml/min. The horizontal thick lines across each peak indicate the molecular weight of the Pg. SEC-MALS, size exclusion chromatography coupled to multiangle light scattering. hHC, human heavy chain; hLC, human light chain; mHC, mouse heavy chain; mLC, mouse light chain.

In the case of Pg, the sedimentation coefficient (S⁰_20,w) value is highly diagnostic of the overall conformation and activatability of Pg. We have shown in numerous studies that fully intact Pgs from all species examined in Cl^--containing buffers provide a S_20,w value of ∼5.7 S, which represents the tight (T) conformation of Pg (19, 38, 39). In this state, the LBSs of the kringles are bound to side chains of other regions of the HC, including the activation peptide (40), thereby compacting the structure. The T form of hPg is not readily activatable. Relaxation of this conformation by addition of lysine and its analogues, e.g., EACA, or removal of the 77-residue activation peptide, disrupts the side chain binding with the LBS of the kringle domains, thereby producing the highly activatable relaxed (R)-conformation with a S_20,w value that is reduced by ∼1 S (38, 39, 40, 41, 42, 43). The S_20,w values of each Pg (Table2) show small differences in the T-conformations of the different chimeric proteins, perhaps reflective of altered interactions between their HCs and LCs, but the T-conformations of the HC were retained to a large degree in all proteins since addition of EACA reduced the S_20,w by ∼1.0 S. On the other hand, in the mutant hPg[D²¹⁹N], the lowered S_20,w of 5.31 S partially disrupts the T-conformation of the hHC since a smaller additional change was found after addition of EACA. This observation has significant implications in its activation properties (vide infra).

The LC of hPg is responsible for the sensitivity to activation by SK2b

In studying the activation properties of the Pgs, the generation of Pm was coupled in a single reaction to hydrolysis of the chromogenic substrate, S2251, and the continual release of p-nitroaniline was monitored by A_405nm to provide an initial rate of activation (44). Figure2A shows the activation rate profiles by SK2b for the parental Pgs (hPg and mPg), chimeric Pgs, and the point mutant hPg[D²¹⁹N]. Consistent with our previous report (15), the rate of activation of hPg by SK2b is extremely slow in Cl^- buffers in the absence of PAM. A notable exception is found for hPg[D²¹⁹N], in which a key residue of the LBS of K2_hPg was inactivated by mutagenesis, and led to a greatly enhanced activation rate by SK2b in the absence of PAM.

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Figure2

Activation of Pg variants. Assays were conducted in 10 mM Na⁺-Hepes/150 mM NaCl, pH 7.4, at 25 °C. The reaction mixtures contained 0.2 μM Pg, 0.25 mM S2251(H-D-Val-Leu-Lys-pNA), 0 or 250 nM PAM. The reaction was accelerated with (A) 5 nM SK2b; (B) 5 nM SK1. The release of p-nitroaniline was continuously monitored by A_405nm for 90 min. The initial velocities of each Pg activation were calculated from linear regions of plots of A_405nmvs t². (C) Generation of an active site in the 1:1 SK2b–Pg complexes. Pg (5 μM) was incubated with 0 or 5 μM PAM and 200 μM MUGB in 0.1 M phosphate/0.1 M NaCl pH 6.0. Complex formation was initiated by adding SK2b (10 μM). The relative fluorescence units (RFU) of methylumbelliferone generated upon SK2b–Pg complex formation, accompanied by cleavage of MUGB, was measured by excitation at 323 nm and emission at 445 nm. The extrapolation of the steady-state curve to zero at 0 (dashed line) and 5 μM PAM (thick line) indicates the pre–steady-state concentration of methylumbelliferone. MUGB, 4-methylumbelliferyl 4-guanidinobenzoate-HCl; PAM, plasminogen-binding group A streptococcal M-protein.

The rate of hPg activation by SK2b was highly stimulated by PAM (Fig.2A). No form of Pg containing the mLC showed any propensity for activation by SK2b, with or without PAM. The stimulatory effect of PAM was observed on the activation of chimeric mHC-hLC, but was somewhat slower than that of hPg, possibly due to the two critical differences in residues 220 and 222 between the hHC and mHC (25). A single mutation in hPg, viz., [D²¹⁹N], abolished the stimulatory effect of PAM, as reflected by the greatly enhanced activation rate of this variant in the absence of PAM, which reached the same level as that of hPg/PAM and hPg[D²¹⁹N]/PAM (Fig.2A).

Because there is a coinheritance between SK2b and PAM that enables effective activation of hPg in strains of GAS that secrete SK2b (15), it is relevant to further understand the reason for the slow rate of activation of mHC-hLC as it relates to SK and PAM. Therefore, we investigated Pg activation by SK1, a class of SK not present in Pattern D GAS, that effectively activates hPg regardless of the presence of PAM (15, 45). Figure2B clearly shows that the rates of activation of hPg, mHC-hLC, and hPg[D²¹⁹N] by SK1 in the absence of PAM were faster than those of PAM-stimulated activations with SK2b. Rate enhancements by PAM were also observed for hPg, and mHC-hLC activations, but were understandably smaller than those of SK2b. Nevertheless, mHC-hLC, similar to the data obtained for the SK2b activation, could not achieve rates comparable to that of hPg and hPg[D²¹⁹N] under the same conditions.

To more fully probe the reasons for the lack of activation of mPg by SK, we investigated the ability of mPg to form the initial active site in the SK–Pg complex that is essential to further activation of Pg by SK. Here, we determined that the single turnover fluorometric substrate, 4-methylumbelliferyl 4-guanidinobenzoate-HCl (MUGB), is cleaved by a stoichiometric complex SK2b/hPg, SK2b/mHC-hLC, and SK2b/hPg[D²¹⁹N] to 19%, 18%, and 66%, respectively, in the absence of PAM, and to 78%, 33%, and 86%, respectively, in the presence of saturating [PAM]. SK2b/mPg and SK2b/hHC-mLC did not form an active site with or without PAM (Fig.2C). This lack of initial active site formation within the mLC prevented further activation of Pg.

LBS residue D²¹⁹ of hPg is critical for PAM binding

A key residue of the LBS of the K2 domain, D²¹⁹, was mutated to determine whether PAM binding was also ablated. If so, this would provide evidence that only the LBS of K2 was a determining factor in PAM binding to intact hPg. Figure3 illustrates the surface plasmon resonance (SPR) sensorgrams generated for the interactions between the various Pgs and PAM. The kinetic parameters are summarized in Table3. All Pg variants had K_D values for PAM in the low nM range, indicating high-affinity interactions with PAM. The K_D value of mPg is 14 nM, a value ∼10-fold higher than that of hPg. These differences in K_D values are reflections of the ∼5.5-fold faster off-rate (k_off), of mPg upon binding to PAM, as compared to that of hPg. A swap in the LC between mPg and hPg did not decrease the k_off of mHC-hLC, as this variant had similar kinetic parameters to mPg. On the other hand, the hHC-mLC chimera binds PAM as tightly as hPg (K_D ∼3 nM). The binding affinities of both mHC-hLC and hHC-mLC chimeras, as compared to their parental Pgs, indicate that the LC plays a minimal role in the interaction of Pg with PAM. The important mutation present in the variant hPg[D²¹⁹N] completely ablated the PAM binding site, demonstrating that PAM binding to hPg is primarily dependent on an intact LBS of K2_hPg.

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Figure3

SPR-based binding of Pg variants to PAM at 25 °C. PAM was immobilized on a CM-5 chip. The concentrations of Pg variants used in the titrations are indicated in each plot. Sensorgrams representing the experimental data are shown in black lines, and best-fit curves are shown in red lines. The data were fit using a 1:1 Langmuir model. PAM, plasminogen-binding group A streptococcal M-protein; SPR, surface plasmon resonance.

Binding of Pg to SK

SPR experiments were also carried out to determine the influence of the HC on the binding of SK to the LC of hPg. We have shown in a previous study that the K_D of the hPg[S⁷⁴¹A] (hPg in which the active site serine was replaced with alanine to prevent the conversion of hPg to hPm)–SK2b interaction is ∼two orders of magnitude weaker than that of SK1, as reflected in their K_D values (15). In the current study, the binding data of hPg determined in the presence of the rapid single turnover serine colorimetric protease inhibitor, p-nitrophenyl-p’guanidinobenzoate which functions similarly to the fluorimetric substrate, MUGB, and also prevents the conversion of hPg to hPm within the SK complex, thus halting further activation, agree with our initial findings of an ∼100-fold higher K_D due to a slower binding k_on of SK2b (Fig.4, Table4) than SK1 (Fig.5, Table5). This difference is also exhibited by the chimeric Pgs, all of which interacted with the both forms of SK. For SK1, the dissociation constants of all the Pg variants are in the very low nanomolar range (1–6 nM). A distinction, however, exists between the binding rate constants of Pgs with the hLC versus the mLC. SK1 dissociates ∼10x faster upon binding to mPg than its binding to hPg. This faster k_off appeared to be structurally compensated for in hHC-mLC, suggesting that stable binding between SK1 and Pg is determined by the LC and may be somewhat modulated by the Pg conformation induced by the HC. This k_off difference was not observed for the SK2b–Pg complexes. The difference between SK2b and SK1 is likely due to amino acid sequence variations in their β-domains, which have been shown earlier to be responsible for the higher k_on with corresponding high-affinity binding between SK1 and hPg (17). Overall, the binding data for the chimeric Pg variants with SK2b and SK1 show that, beyond slow association or rapid dissociation rates, the lack of activation of mPg and hHC-mLC, along with the delayed activation of mHC-hLC, are affected by properties other than simple direct binding. Most likely, the conformational rearrangement needed to form the initial active site does not occur with the mLC.

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Figure4

SPR-based binding of SK2b to Pg variants at 25 °C. Each Pg variant was immobilized on a CM-5 chip. The concentrations of SK are indicated in each plot. The sensorgrams represent the binding of SK2b to the immobilized Pg. The experimental data are shown in black lines, and best-fit curves are shown in red lines. The data were fit using 1:1 Langmuir model. SPR, surface plasmon resonance.

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Figure5

SPR-based binding of SK1 to Pg variants at 25 °C. Each Pg variant was immobilized on a CM-5 chip. The concentrations of SK are indicated in each plot. Sensorgrams representing the binding of SK1. The experimental data are shown in black lines, and best-fit curves are shown in red lines. The data were fit using 1:1 Langmuir model. SPR, surface plasmon resonance.

Construction, expression, and purification of recombinant proteins

Pg and Pg chimeras. Parental Pg expression plasmids, hPg-pMT-Puro and mPg-pMT-Puro, were generated by insertion of the cDNAs encoding these proteins into the multiple cloning site of pMT-Puro as described (25, 54). All mutations were constructed from these two parental plasmids. The chimeric Pgs, viz., mHC-hLC (mouse heavy chain–human light chain) and hHC-mLC (human heavy chain–mouse light chain), were generated by overlapping primer-mediated restriction-free cloning (55), with details provided earlier (25). hPg[D²¹⁹N] (human Pg with a [D²¹⁹N] mutation) was constructed with the QuikChange XL site-directed mutagenesis kit (Stratagene). Nucleotide sequencing of each Pg verified the integrity of the constructs. Drosophila Schneider S2 cells were then transfected with the various Pg/pMT-Puro plasmids. Positive colonies were screened by puromycin resistance, and the Pgs were expressed as previously described (56).

After induction with CuSO₄, all Pg variants were purified from their culture supernatants using lysine-Sepharose affinity chromatography (37). Protein concentrations of the Pgs were determined by A_280nm, using the following extinction coefficients (M^-1cm^-1): hPg, 152,200; mPg, 162,630; mHC-hLC, 159,650; hHC-mLC, 155,180; and hPg[D²¹⁹N], 152,200. The values were calculated from their amino acid sequence by ExPASy. The purity and hom*ogeneity of the Pgs were assessed by SDS-PAGE using 10% Tris-glycine gels.

SK. Detailed descriptions of plasmid construction, expression from Escherichia coli, and purification of recombinant SK proteins have been provided in a previous study (15). Briefly, gDNAs encoding sk1 and sk2b were cloned from GAS isolate NS53 (from M. Walker, Queensland, Au) and AP53 (from G. Lindahl, Lund, SE), respectively, using standard methodology. The SKs were cloned into pCR2.1-TOPO (Invitrogen) and sequenced. The sk1 and sk2a genes (minus the signal sequences) were amplified by Phusion Hot Start High-Fidelity DNA Polymerase (New England Biolabs), digested with BamH1 (inserted through the reverse PCR primers), and ligated into PshA1/BamH1-digested pET42a (EMD4Biosciences). The final plasmids contained [glutathione S-transferase-(His)₆-Factor Xa (FXa) cleavage site-sk1/sk2b]. Each plasmid was transformed into BL21/DE3 (New England Biolabs) cells for expression. After induction with isopropyl β-d-1-thiogalactopyranoside, SKs were purified on a Ni+-charged His-bind column, eluted with imidazole, and finally cleaved with FXa (ERL) to yield purified SK with an intact amino-terminus (16).

PAM. PAM, without the N-terminal signal peptide and C-terminal LPXTG cell wall anchor region, was cloned by PCR from GAS-AP53 gDNA. A (His)₆-tag was engineered into the reverse primer for PAM for further facile purification by Ni+-based affinity chromatography. The detailed steps have been described earlier (57).

SEC-MALS analysis

Molecular weights of Pg variants were determined using an Agilent 1260 Infinity II HPLC system connected to a Wyatt WTC-030S5 (7.6 nm× 300 nm, 5 μm, 300A°) SEC column. The SEC column was coupled to a mini DAWN Treos II light scattering detector and an Optilab T-rex differential refractive index detector (Wyatt Technology Corporation). Pg (100 μl) at a concentration of 1 mg/ml was injected into the SEC column at a flow rate of 0.5 ml/min using PBS, pH 7.4, as the running buffer. Light scattering and refractive index signals of eluted Pg peaks were recorded by respective detectors. The refractive index increment (dη/dc) was set to 0.185 ml/g. The data were analyzed for molar mass determinations using Astra 7.0 software (Wyatt Technology Corporation).

Analytical ultracentrifugation

Sedimentation velocity experiments were carried out using absorption optics at 20 °C with an initial Pg A_280nm of 0.75 (∼0.4 mg/ml) in a Beckman Optima XL-1 analytical ultracentrifuge. The buffer employed was 50 mM sodium phosphate/100 mM NaCl, pH 7.4. Samples were run in three 2-channel centerpieces with sample buffer in the reference channels and Pg in the same buffer in the sample channels. The cells were centrifuged at 30,000 rpm and scans were recorded every 3 min for 12 h. Curves generated from the scans were analyzed for the S_app using Optima XL-A/XL-I software (Beckman coulter). The buffer viscosity and density and the partial specific volume of each Pg were calculated from their compositions using ExPASY. These values were used for correction of the S_app to the S_20,w. Since the protein concentration is so low, the S_20,w values are essentially equal to the S⁰_20,w values. The effect of EACA on the S_20,w of Pg was determined in sample buffers containing 100 mM EACA. The S_20,w is reported as mean of three values± the SD.

For molecular weight determinations, sedimentation equilibrium experiments were conducted at 25 °C. The proteins were diluted to final A_280nm of ∼0.1, loaded into six-channel centerpieces, and centrifuged at 20,000 rpm. Scans were recorded hourly until sedimentation equilibrium was attained. Apparent weight average molecular weights for each protein were calculated following data analysis with OptimaXL-A/XL-I software (Beckman Coulter). At least triplicate data were collected for each Pg, and values are reported as mean±SD.

Activation of Pg

Pg activation assays were performed in 96-well microtiter plates using the chromogenic substrate, H-D-Val-L-Leu-L-Lys-p-nitroanilide (S2251; Chromogenix), to monitor the continuous generation of Pm at 25 °C through release of p-nitroaniline from S2251. A 200-μl assay mixture in each sample well contained final concentrations of 0.2 μM Pg/0.25mM S2251/0 or 0.25 μM PAM, in 10 mM Na⁺-Hepes/150 mM NaCl, pH 7.4. The reaction was accelerated byaddition of either 5 nM SK2b or 5 nM SK1. Three independent experiments were performed for a given Pg at a specific PAM concentration. The data were analyzed using GraphPad Prism, version 8.0. Initial velocities of activation were calculated as the slope of the linear region of a plot of A_405nm against t².

For experiments designed to measure the extent of active site formation in equimolar SK2b–Pg complexes, studies using a fluorometric single turnover substrate MUGB were employed. Pg (5 μM) in 0.1 M sodium phosphate/0.1 M NaCl, pH 6.0, was placed in black 96-well microtiter plates. MUGB was added to a final concentration of 200 μM, and the reaction was accelerated by 10 μM SK2b, with and without 5 μM PAM. Excitation and emission wavelengths were 323 nm and 445nm, respectively. The reaction progressed until a steady state (∼10 mins) was achieved in the fluorescence intensity of methylumbelliferone generated. Amounts of the methylumbelliferone generated were estimated by extrapolation of the fluorescent steady state curve to zero, thus providing only the presteady state value. The ratio of [methylumbelliferone]/[Pg] present in the well× 100 was taken as the % active site formation in Pg.

A standard curve was constructed by hydrolyzing known concentrations of MUGB with 0.1 M NaOH, followed by adjustment of pH to 6.0 using 1M NaH₂PO₄.

Surface plasmon resonance

Association (k_on) and dissociation (k_off) rate constants for the binding of Pg to PAM were determined at 25 °C in a BIAcore X100 (GE Healthcare) system, using HBS-EP⁺ (10 mM Na⁺-Hepes/150 mM NaCl/3 mM EDTA/0.05% polysorbate 20, pH 7.4) as the running buffer for immobilization and binding experiments. PAM (2.5 μg/ml) in 10 mM NaOAc, pH 4.5, was covalently immobilized on a CM-5 chip to a target level of up to 250 response units using the amine coupling kit (BIAcore AB). Prior to immobilization, the surface of the chip was first activated by 0.2 M ethyl-N-dimethylaminopropyl carbodiimide and 0.05 M N-hydrosuccinimide at a flow rate of 5 μl/min. PAM was then injected into flow cell-2 (FC2) to allow immobilization, after which excess nonbound sites on the chip were blocked using 1 M ethanolamine, pH 8.5. Flow cell-1 (FC1) was prepared as a blank cell by omitting the PAM injection. At least three independent binding experiments were conducted for each Pg variant at concentrations ranging from 0 to 50 nM, except for hPg[D²¹⁹N] which was used at concentrations up to 300 nM. Each cycle during binding consists of an association step in which a single concentration of Pg was injected to both FC1 and FC2 at a flow rate of 30 μl/min for 180 s, a dissociation step in which the running buffer was allowed to flow for 300 s, and finally a regeneration step in which 10 mM glycine-HCl, pH 1.5, was injected to prepare the chip surface for the next cycle. Signals from FC1 were subtracted from FC2, and the sensorgrams were best-fit with a 1:1 binding model using BIAevaluation software version 3.0. Dissociation constants (K_D) were calculated from the average values of k_off/k_on. Error values were reported as standard deviations from mean values.

In order to determine the kinetic parameters for the interaction of SK and chimeric Pgs, immobilization of each Pg was achieved by amine coupling on CM-5 chips to target levels of ∼1200 response units as described (15). Binding experiments were conducted at SK1 and SK2b concentrations ranging from 0 to 0.25 μΜ and 0 to 2 μM, respectively, using HBS-EP+/50μΜ p-nitrophenyl-p’-guanidinobenzoate, which functions similarly to MUGB to maintain the SK–hPg complex as the analyte buffer.

This article contains supporting information.

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