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, 9 (1), 3870

Reactive Centre Loop Dynamics and Serpin Specificity

Affiliations

Reactive Centre Loop Dynamics and Serpin Specificity

Emilia M Marijanovic et al. Sci Rep.

Abstract

Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational lability of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as α1-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Stability and inhibitory activity of conserpin-AATRCL. (A) RCL sequence alignment indicating which residues of conserpin were replaced with the corresponding residues in α1-AT; (B) Variable temperature thermal melt of conserpin-AATRCL, heating to 95 °C (black line) and cooling to 35 °C (red line), measured by CD at 222 nm; (C) Spectral scan before (black line) and after (red line) variable temperature thermal melt; (D) Variable temperature thermal melt in the presence in 2 M GdnHCl (heating to 95 °C; black line, cooling: red line); (E) Inhibitory activity assay and (F) SI against trypsin (n = 3); (G) A cropped SDS-PAGE showing a serpin:protease complex formed between HNE and AAT, but less complex formed between HNE and conserpin-AATRCL. From left to right: 1. Molecular weight markers (kDa); 2. α1-AT alone; 3. 1:1 ratio of α1-AT: HNE; 4. 2:1 ratio of α1-AT:HNE; 5. HNE alone; 6. conserpin-AATRCL alone; 7. 1:1 ratio of conserpin-AATRCL:HNE; 8. 2:1 ratio of conserpin-AATRCL:HNE. The full length SDS-PAGE gel is presented in Fig. S1.
Figure 2
Figure 2
Structure and electrostatics of conserpin-AATRCL. (A,D) X-ray crystal structure of native state conserpin-AATRCL represented as a cartoon. The breach and shutter regions are marked with black broken circles. (BF) A comparison of electrostatic potential surfaces (blue = +ve, red = −ve) of (B,E) conserpin-AATRCL and (C,F) α1-AT. Both conserpin-AATRCL and α1-AT feature a large electropositive surface centred around the loop connecting strands 2 and 3 of β-sheet B (s2B and s3B) (B,C). A large surface patch between helix D and the RCL, highlighted with yellow broken circles, has a generally positive potential in conserpin-AATRCL (E), and negative potential in α1-AT (F).
Figure 3
Figure 3
Electrostatic potential surfaces of the RCL differs between conserpin-AATRCL and α1-AT. While we have grafted the α1-AT RCL (cartoon) from P7−P2’ onto conserpin (surface), the electrostatic surface potential between conserpin-AATRCL and α1-AT differs beneath the RCL. (A) In conserpin-AATRCL, the region below the RCL contains a large electropositive potential, while in α1-AT (B), the corresponding region is more neutral in charge. (C) ConSURF conservation scores for the serpin superfamily, mapped onto the surface of α1-AT as colours from forest green (highly conserved) to brick red (highly variable). This depicts poor conservation (red) of residues 201−202 and 223−225 of α1-AT, suggesting that these residues may be responsible for contributing to protease specificity within the serpin family.
Figure 4
Figure 4
Electrostatic compatibility between serpin and protease. (A) Electrostatic surfaces of a modeled complex between trypsin and conserpin-AATRCL, and (D) between HNE and conserpin-AATRCL. Associated complexes are separated into individual proteins by rotating each molecule by 90° around the horizontal axis in the plane of the paper (clockwise for the top molecules, anti-clockwise for the bottom molecules). (B) Electrostatic surface for the active site of trypsin and (E) HNE shows that trypsin has a more electronegative binding cleft than HNE. Comparing this to the electrostatic surface of (C, F idem.) conserpin-AATRCL suggests a greater electrostatic compatibility between trypsin, particularly the electropositive surface below the RCL. However, the electropositive surface of S3–S4 binding pocket in HNE suggests there may be a charge repulsion with the electropositive surface potential of conserpin-AATRCL at P6-P3.
Figure 5
Figure 5
The dynamics of the RCL is important for inhibition. Snapshots of conformations of the RCL from the MD runs at 50 ns intervals overlaid on static structure for the rest of the molecule, showing that (A) conserpin prefers an extended-hinge RCL conformation, (B) α1-AT prefers a bent-hinge RCL conformation, and (C) conserpin-AATRCL occupies both of these conformations. the increased flexibility of the lower RCL region (residues 342314-352323) relative to both conserpin and α1-AT. (D) Root mean square fluctuation (RMSF) calculated for the RCL region from the molecular dynamics simulations shows that the conserpin-AATRCL (red) has lower flexibility than conserpin (black) in the 353324-362333 region but a higher flexibility in the 342314-352323 region than conserpin and α1-AT (blue) (α1-AT numbering), reflecting the structural differences between the two conformational clusters occupied by the RCL.
Figure 6
Figure 6
RCL conformational cluster determination by principal component analysis. To describe the motion of the RCL across all simulations, principal component vectors were determined for all RCL backbone conformations. (A) The trajectories of each RCL (α1-AT: blue, conserpin: black, conserpin-AATRCL: red) are projected on the first 2 PC axes, and (B) these conformations were grouped into 9 clusters. For (C) conserpin, (D) conserpin-AATRCL and (E) α1-AT, representative RCL backbone conformations for the clusters explored by each serpin over the course of the MD simulations, are shown atop a serpin body (grey cartoon α1-AT (PDB: 3NE4)).

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