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. 2020 Apr 24;295(17):5614-5625.
doi: 10.1074/jbc.RA120.012588. Epub 2020 Mar 10.

Specificity and affinity of the N-terminal residues in staphylocoagulase in binding to prothrombin

Ashoka A Maddur  1 , Heather K Kroh  2 , Mary E Aschenbrenner  2 , Breanne H Y Gibson  2 , Peter Panizzi  3 , Jonathan H Sheehan  4 , Jens Meiler  5 , Paul E Bock  2 , Ingrid M Verhamme  6
Affiliations

Specificity and affinity of the N-terminal residues in staphylocoagulase in binding to prothrombin

Ashoka A Maddur et al. J Biol Chem. .

Abstract

In Staphylococcus aureus-caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1-325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile1-Val2 into the ProT Ile16 pocket, forming a salt bridge with ProT's Asp194, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1-246) as a probe for competitively defining the affinities of N-terminal SC(1-246) variants preselected by modeling. Using ProT(R155Q,R271Q,R284Q) (ProTQQQ), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between ∼1 and 650 nm and activation potencies ranging from 1.8-fold that of WT SC(1-246) to complete loss of function. Substrate binding to ProTQQQ caused allosteric tightening of the affinity of most SC(1-246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well-tolerated, with Val1-Val2, Ile1-Ala2, and Leu1-Val2 variants exhibiting ProTQQQ affinity and activation potency comparable with WT SC(1-246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProTQQQ levels, several variants exhibited limiting rates similar to or higher than that of WT SC(1-246). The Ile16 pocket in ProTQQQ appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile1-Val2-Thr3 might emerge with similar ProT-activating efficiency.

Keywords: Staphylococcus aureus (S. aureus); affinity; clot formation; coagulation; coagulation factor; competitive equilibrium binding; endocarditis; equilibrium binding; fibrin; kinetics; ligand-binding protein; prothrombin; specificity; staphylocoagulase (SC); virulence factor.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Full-length staphylocoagulase from S aureus Newman D2 Tager 104 showing different regions of the protein. The full-length protein contains a 26-residue signal sequence, the D1 and D2 domains, a central region, and a C-terminal repeat region consisting of a pseudo-repeat (PR) and seven repeats.
Figure 2.
Figure 2.
The SC(1–325)·Pre2 complex. A, the molecular surface of Pre2 is shown colored by electrostatic potential, with the ATA-PPACK (N-(sulfanylacetyl)-d-phenylalanyl-N-[(2S,3S)-6-{[amino(iminio)methyl]amino}-1-chloro-2-hydroxyhexan-3-yl]-l-prolinamide) inhibitor displayed as gray sticks in the active site. SC(1–325) is displayed in ribbon mode, with the N-terminal D1 domain colored yellow and the D2 domain colored gold. B, the complex above is rotated ∼90º from the standard orientation to show the insertion of the N-terminal SC peptide Ile1-Val2-Thr3 into the Ile16-binding pocket of Pre2, triggering activation. This figure was constructed with UCSF Chimera using the X-ray crystal structure 1NU9.pdb (16).
Figure 3.
Figure 3.
Characterization of SC(1–246)-BODIPY and its complex with ProTQQQ. A, SDS-PAGE showing the purity of the labeled probe, SC(1–246)-BODIPY. 5 μg of the labeled probe was separated under reduced (lane 2) and nonreduced (lane 4) conditions. Lane 1, protein standards; lane 3, blank. B, native gel electrophoresis was used to show the SC(1–246)-BODIPY·ProTQQQ complex formation. ProTQQQ (2.5 μm, lane 1) was incubated with SC(1–246)-BODIPY (0-fold (lane 2), 0.5-fold (lane 3), 1.0-fold (lane 4), and 1.5-fold (lane 5) excess of ProTQQQ) for 15–30 min at 25 °C, and the complex was separated on native PAGE at 4 °C.
Figure 4.
Figure 4.
Equilibrium binding of SC(1–246)-BODIPY and WT SC(1–246) to ProTQQQ. A, C, E, and G, SC(1–246)-BODIPY (29 nm (○) and 502 nm (●)) titrated with ProTQQQ, four separate batches. B, titration of 29 nm SC(1–246)-BODIPY (○); a mixture of 50 nm SC(1–246)-BODIPY and 50 nm WT SC(1–246) (▵); 502 nm SC(1–246)-BODIPY (●); and a mixture of 502 nm SC(1–246)-BODIPY and 500 nm WT SC(1–246) (▴) with ProTQQQ. D, F, and H, titrations of SC(1–246)-BODIPY (29 nm (○) and 502 nm (●)) and a mixture of 502 nm SC(1–246)-BODIPY and 500 nm WT SC(1–246) (▴) with ProTQQQ. Titrations shown in B, D, F, and H were performed with the corresponding, separate ProTQQQ preparations as in A, C, E, and G. The SC(1–246)-BODIPY titration data were analyzed by the quadratic binding equation to obtain the affinity, stoichiometry, and maximum fluorescence intensity (ΔFmax/Fo). Titration data of the probe and competitor were analyzed simultaneously by the cubic binding equation to obtain the affinity and stoichiometry of the competitor, WT SC(1–246) (Table 1).
Figure 5.
Figure 5.
Competitive binding of SC(1–246) N-terminal double mutants that bind tightly to ProTQQQ. Titrations of the probe, SC(1–246)-BODIPY (29 nm (○) and 502 nm (●)), with ProTQQQ from Fig. 4 served as reference curves for competitive titrations of SC(1–246) mutants. The probe concentration for all of the competitive titrations (▵, ▴) in A–H was 50 nm. Concentrations of competing SC(1–246) mutants were as follows: V1V2 0.90 μm (A), I1A2 0.74 μm (B), L1V2 0.75 μm (C), I1T2 2.24 μm (D), I1W2 0.52 μm (▵) and 9.97 μm (▴) (E), T1V2 6.16 μm (F), L1T2 9.55 μm (G), and L1Q2 3.95 μm (H). Titrations of probe and competitor were simultaneously analyzed by the cubic binding equation to obtain KC, stoichiometry, and maximum fluorescence intensity (ΔFmax/Fo) of the SC(1–246) mutants (Tables 2 and 3).
Figure 6.
Figure 6.
Competitive binding titrations of SC(1–246) N-terminal double mutants that bind weakly to ProTQQQ. Titrations of the probe, SC(1–246)-BODIPY (29 nm (○) and 502 nm (●)) with ProTQQQ from Fig. 4 served as reference curves for competitive titrations of SC(1–246) mutants. The probe concentration for all of the competitive titrations (▴) in A–F was 50 nm. Concentrations of competing SC(1–246) mutants were as follows: G1G2 8.12 μm (A), R1R2 9.06 μm (B), K1A2 9.50 μm (C), E1T2 9.04 μm (D), R1Q2 10.30 μm (E), and E1S2 11.38 μm (F). Titrations of probe and competitor were simultaneously analyzed by the cubic binding equation to obtain KC, stoichiometry, and maximum fluorescence intensity (ΔFmax/Fo) of the SC(1–246) mutants (Tables 2 and 3).
Figure 7.
Figure 7.
Competitive binding titrations of SC(1–246) N-terminal triple mutants with ProTQQQ. Titrations of the probe, SC(1–246)-BODIPY (29 nm (○) and 502 nm (●)) with ProTQQQ from Fig. 4 served as reference curves for competitive titrations of SC(1–246) mutants. The probe concentration for all of the competitive titrations (▴) in A–G was 50 nm. Concentrations of competing SC(1–246) mutants were as follows: I1I2V3 2.84 μm (A), R1H2W3 7.59 μm (B), F1L2Q3 5.49 μm (C), E1S2W3 5.74 μm (D), D1D2Y3 7.65 μm (E), E1L2K3 5.77 μm (F), and G1G2G3 6.44 μm (G). Titrations of probe and competitor were simultaneously analyzed by the cubic binding equation to obtain KC, stoichiometry, and maximum fluorescence intensity (ΔFmax/Fo) of the SC(1–246) mutants (Table 4).
Figure 8.
Figure 8.
Gibbs free energy of the SC(1–246) N-terminal double and triple mutants for binding to ProTQQQ. ΔG values were calculated using KD values obtained from equilibrium binding, as described under “Experimental Procedures.”
Figure 9.
Figure 9.
Correlation between REU and ΔG values calculated from equilibrium binding.
Figure 10.
Figure 10.
Activation of ProTQQQ by SC(1–246) WT or double mutants. ProTQQQ (1, 10, or 20 nm) and SC(1–246) WT (black circles) and a representative selection of mutants (navy blue, I1A2; red, L1T2; dark green, L1Q2; brown, V1V2; purple, A1K2; green, A1W2; cyan, K1A2; yellow, Q1K2; eggplant, E1S2) were incubated for 10 min at 25 °C, and the reaction was initiated by adding S-2238. Activation of ProTQQQ was measured by the relative rates of increase in absorbance at 405 nm, and weak-binding mutants with low activation potency were titrated up to 1300 nm. The data were analyzed as described under “Experimental Procedures.”
Figure 11.
Figure 11.
Binding site complementarity between SC(1–325) and prethrombin 2. Native residues I1V2T3 of SC are shown in the Ile16 pocket of prothrombin 2. The steric complementarity excludes the possibility of adding additional bulk to the N terminus of SC without generating energetically unfavorable clashes with neighboring residues.
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