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. 2013;8(1):e54104.
doi: 10.1371/journal.pone.0054104. Epub 2013 Jan 15.

The structure of human microplasmin in complex with textilinin-1, an aprotinin-like inhibitor from the Australian brown snake

Emma-Karin I Millers  1 , Lambro A Johnson, Geoff W Birrell, Paul P Masci, Martin F Lavin, John de Jersey, Luke W Guddat
Affiliations

The structure of human microplasmin in complex with textilinin-1, an aprotinin-like inhibitor from the Australian brown snake

Emma-Karin I Millers et al. PLoS One. 2013.

Abstract

Textilinin-1 is a Kunitz-type serine protease inhibitor from Australian brown snake venom. Its ability to potently and specifically inhibit human plasmin (K(i) = 0.44 nM) makes it a potential therapeutic drug as a systemic anti-bleeding agent. The crystal structures of the human microplasmin-textilinin-1 and the trypsin-textilinin-1 complexes have been determined to 2.78 Å and 1.64 Å resolution respectively, and show that textilinin-1 binds to trypsin in a canonical mode but to microplasmin in an atypical mode with the catalytic histidine of microplasmin rotated out of the active site. The space vacated by the histidine side-chain in this complex is partially occupied by a water molecule. In the structure of microplasminogen the χ(1) dihedral angle of the side-chain of the catalytic histidine is rotated by 67° from its "active" position in the catalytic triad, as exemplified by its location when microplasmin is bound to streptokinase. However, when textilinin-1 binds to microplasmin the χ(1) dihedral angle of this amino acid residue changes by -157° (i.e. in the opposite rotation direction compared to microplasminogen). The unusual mode of interaction between textilinin-1 and plasmin explains textilinin-1's selectivity for human plasmin over plasma kallikrein. This difference can be exploited in future drug design efforts.

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Conflict of interest statement

Competing Interests: QRxPharma did provide some funding for this project. This partnership does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Progress curves for p-nitroaniline production by plasmin, plasma kallikrein and trypsin in the presence of textilinin-1.
(a) 0.5 nM plasmin in the presence of 0, 2, 5, 10, 15, 20, 40 and 50 nM textilinin-1; (b) 1.0 nM plasma kallikrein in the presence of 0, 3, 6, 12 and 24 nM textilinin-1; and (c) 0.5 nM trypsin in the presence of 0, 2, 3, 4, 6, 11, 19 and 21 nM textilinin-1. For clarity of presentation only a selection of the data points are shown. The fitted lines were obtained by simultaneous regression analysis of each set of curves, using the numerical integration software DynaFit and the one-step model in Scheme 1. The correlation coefficients in each set were >0.99. The resulting parameter estimates (kon and koff) and their associated error estimates are shown in Table 1.
Figure 2
Figure 2. Electron density maps for the microplasmin-textilinin-1 complex.
(a) Stereo representation of the 2Fo-Fc electron density (contoured at 1.3 σ) for the interface between textilinin-1 and microplasmin. (b) Stereo representation of the 2Fo-Fc electron density (contoured at 1.3 σ) for the region around R17(textilinin-1) and S741 (microplasmin). (c) omit Fo-Fc electron density map after simulated annealing contoured at 3σ for R17(textilinin-1) and S741 (microplasmin). Textilinin-1 and microplasmin are identified by purple and green carbon atoms, respectively. Bond angles centered around the carbonyl carbon for the tetrahedral intermediates refined to values in the range 103.0° to 118.3° with an average value of 109.3° for both complexes in the asymmetric unit.
Figure 3
Figure 3. Superimposition of the two complexes in the asymmetric unit of the microplasmin-textilinin-1 crystal structure.
The textilinin-1 molecules differ by 8° in their docking angles with microplasmin.
Figure 4
Figure 4. Interactions between microplasmin and textilinin-1.
(a) ChemDraw representation of the interactions between microplasmin (black) and textilinin-1 (blue). Hydrogen bonds are shown as red dashed lines. Dashed green semi-circles represent van der Waals attractions. A closer than van der Waals approach of the -OH nucleophile of S741 towards the carbonyl carbon of R17 is depicted as a black dashed line. (b and c) Connolly and electrostatic surface of microplasmin and interactions with textilinin-1 (as observed in complex A in the asymmetric unit of the crystal structure). The canonical loop, including residues P15 to F20 of textilinin-1, and the secondary loop, including residues I36 to C40 are drawn as a stick models. In (c) the side-chain of F37 is removed for clarity.
Figure 5
Figure 5. Sequence alignment for the canonical and secondary loops of textilinin-1 and aprotinin.
The residue colored red is the P1 site and cyan the P1' site.
Figure 6
Figure 6. Sequence alignment for the subsites for plasmin and plasma kallikrein and the 99-loop.
The 99-loop is a section of polypeptide located behind the catalytic histidine in plasma kallikrein. Similar loops are present in tissue kallikrein, also referred to as KLK1 , and trypsin. In plasmin, this loop is missing and none of the near by amino acid residues occupy this space. A “/” represents a break in the polypeptide sequence.
Figure 7
Figure 7. The catalytic triad of microplasmin and interactions with textilinin-1.
Microplasmin, as it appears in the textilinin-1 complex, is identified by the blue carbon atoms and textilinin-1 is coloured green. The locations of the side-chain of H603 of microplasmin when bound to streptokinase (carbon atoms in white) (pdb code 1BML) and in the microplasminogen structure (carbon atoms in yellow) are overlayed. If the side-chain of H603 is located in the former orientation it would be within 1 Å of the side-chain of V18 and therefore a steric clash would exist.
Figure 8
Figure 8. Electron density maps for the trypsin-textilinin-1 complex.
(a) Stereo representation of the 2Fo-Fc electron density (contoured at 1.5 σ) for the interface between textilinin-1 and trypsin. (b) omit Fo-Fc electron density map after simulated annealing contoured at 3σ for R17 (textilinin-1) and S195 (trypsin) Textilinin-1 and trypsin are identified by purple and green carbon atoms, respectively. Bond angles centered around the carbonyl carbon for the tetrahedral intermediates refined to values in the range 93.0° to 119.1° with an average value of 108.6° for this complex.
Figure 9
Figure 9. Structural images for the trypsin-textilinin-1 complex.
(a) Interactions between textilinin-1 (blue) and trypsin (black). Hydrogen bonds are shown as red dashed lines. Dashed green semi-circles represent van der Waals attractions. A closer than van der Waals approach of the hydroxyl nucleophile of S195 towards the carbonyl carbon of R17 is depicted by a black dashed line. (b) Connolly and electrostatic surface of trypsin and interactions with textilinin-1 (stick model with green carbon atoms). (c) Connolly and electrostatic surface of trypsin and interactions with aprotinin (stick model with magenta carbon atoms). (d) Superimposition of the trypsin-textilinin-1 and trypsin-aprotinin complexes. Trypsin is shown as a Connolly and electrostatic surface. Textilinin-1 is in green and aprotinin in magenta.
Figure 10
Figure 10. Superimposition of the crystal structure of a molecule of free textilinin-1 and the microplasmin-textilinin-1 complex.
Microplasmin is shown as a Connolly and electrostatic surface. Free textilinin-1 is in blue and textilinin-1 as it exists in the microplasmin complex is in green.
Figure 11
Figure 11. Superimposition of the catalytic subunits of plasma kallikrein and microplasmin from the microplasmin-textilinin-1 complex.
The 99-loop makes a close approach to H603 and to the secondary binding loop of textilinin-1. The side-chains of Y94 and S97 and the backbone atoms of E98 and G99 of plasma kallikrein make steric clashes with H603 in this conformation. Plasma kallikrein is in brown and microplasmin (blue) from the microplasmin-textilinin-1 is in blue.
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This work was supported by an ARC-Linkage grant funded by the Australian Research Council and by QRxPharma Pty Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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