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. 2012 Nov 1;68(Pt 11):1289-93.
doi: 10.1107/S1744309112039085. Epub 2012 Oct 26.

Structure of the Recombinant BPTI/Kunitz-type Inhibitor rShPI-1A From the Marine Invertebrate Stichodactyla Helianthus

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Structure of the Recombinant BPTI/Kunitz-type Inhibitor rShPI-1A From the Marine Invertebrate Stichodactyla Helianthus

Rossana García-Fernández et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. .
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Abstract

The BPTI/Kunitz-type inhibitor family includes several extremely potent serine protease inhibitors. To date, the inhibitory mechanisms have only been studied for mammalian inhibitors. Here, the first crystal structure of a BPTI/Kunitz-type inhibitor from a marine invertebrate (rShPI-1A) is reported to 2.5 Å resolution. Crystallization of recombinant rShPI-1A required the salt-induced dissociation of a trypsin complex that was previously formed to avoid intrinsic inhibitor aggregates in solution. The rShPI-1A structure is similar to the NMR structure of the molecule purified from the natural source, but allowed the assignment of disulfide-bridge chiralities and the detection of an internal stabilizing water network. A structural comparison with other BPTI/Kunitz-type canonical inhibitors revealed unusual ϕ angles at positions 17 and 30 to be a particular characteristic of the family. A significant clustering of ϕ and ψ angle values in the glycine-rich remote fragment near the secondary binding loop was additionally identified, but its impact on the specificity of rShPI-1A and similar molecules requires further study.

Figures

Figure 1
Figure 1
(a) DLS analysis of the rShPI-1A aggregation state in solution after concentration by different techniques. Lyophilization (sample A) induced rShPI-1A oligomerization, while large inhomogeneous aggregates are formed by ultrafiltration (sample B). For monomeric ShPI-1 a theoretical hydrodynamic radius (R h) of 1.6 nm was calculated. (b) In the presence of equimolar concentrations of trypsin, rShPI-1A disaggregates in solution A (4.0 mM) owing to complex formation, while the heterogeneous radius distribution in solution B (3.5 mM) is not affected. DLS analysis of free trypsin is shown for comparison. The colour code corresponds to the relative frequency of particles characterized by a specific radius in solution, with dark red being the highest and blue the lowest.
Figure 2
Figure 2
(a) Superposition of the three-dimensional structure of free rShPI-1A (blue) and the average NMR structure of ShPI-1 purified from the natural source (grey). The canonical (P3–P3′) and secondary (Ile32–Gly37) binding loops are highlighted in red and orange, respectively, while the conserved disulfide bridges are shown in yellow stick representation. Residues with backbone r.m.s.d.s of more than 1.7 Å are labelled. (b) Internal water-coordination sites near the binding loops of rShPI-­1A. The shifted water molecule (87) observed in rShPI-1A is shown in grey. Dashed lines represent water-mediated hydrogen bonds. The unusal right-handed conformation of the Cys12–Cys36 disulfide bridge is well defined by the electron density (grey mesh, 2σ).
Figure 3
Figure 3
(a) Structure-based multiple sequence alignment of BPTI/Kunitz-type canonical domains. The domain names and the PDB codes are shown on the left, while the sequence identity (%), the number of equivalent/total compared Cα atoms and the resulting r.m.s.d. values are displayed on the right. The canonical (P3–P3′) and secondary binding loops (residues 32–37 in rShPI-1A) are highlighted in boxes. (b) Cα deviations (Å) of the analyzed BPTI/Kunitz-type canonical domains compared with rShPI-1A (from Ala0 to Arg54 in rShPI-1A numbering). Line identifiers correspond to the associated sequences of the BPTI/Kunitz-type domains shown in (a).

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