Knottin cyclization: impact on structure and dynamics

Abstract

Background: Present in various species, the knottins (also referred to as inhibitor cystine knots) constitute a group of extremely stable miniproteins with a plethora of biological activities. Owing to their small size and their high stability, knottins are considered as excellent leads or scaffolds in drug design. Two knottin families contain macrocyclic compounds, namely the cyclotides and the squash inhibitors. The cyclotide family nearly exclusively contains head-to-tail cyclized members. On the other hand, the squash family predominantly contains linear members. Head-to-tail cyclization is intuitively expected to improve bioactivities by increasing stability and lowering flexibility as well as sensitivity to proteolytic attack.

Results: In this paper, we report data on solution structure, thermal stability, and flexibility as inferred from NMR experiments and molecular dynamics simulations of a linear squash inhibitor EETI-II, a circular squash inhibitor MCoTI-II, and a linear analog lin-MCoTI. Strikingly, the head-to-tail linker in cyclic MCoTI-II is by far the most flexible region of all three compounds. Moreover, we show that cyclic and linear squash inhibitors do not display large differences in structure or flexibility in standard conditions, raising the question as to why few squash inhibitors have evolved into cyclic compounds. The simulations revealed however that the cyclization increases resistance to high temperatures by limiting structure unfolding.

Conclusion: In this work, we show that, in contrast to what could have been intuitively expected, cyclization of squash inhibitors does not provide clear stability or flexibility modification. Overall, our results suggest that, for squash inhibitors in standard conditions, the circularization impact might come from incorporation of an additional loop sequence, that can contribute to the miniprotein specificity and affinity, rather than from an increase in conformational rigidity or protein stability. Unfolding simulations showed however that cyclization is a stabilizing factor in strongly denaturing conditions. This information should be useful if one wants to use the squash inhibitor scaffold in drug design.

Figure 1

The knottin fold. (Top) Stereoscopic view of a schematic representation of MCoTI-II, a head-to-tail cyclized squash inhibitor. The head-to-tail linker is shown in blue. Disulfide bridges are shown as ball-and-stick representations. The cystine knot is shown in green (the disulfide macrocycle) and orange (the penetrating disulfide). β-strands are shown as flat arrows and the 3₁₀-helix turn is shown in magenta. Cysteines are numbered. (Bottom) Sequences of the squash inhibitors used in this work. Numbering follows MCoTI-II. The disulfide bridge coloring scheme follows the one used in the structure. The colors used in the three-dimensional structure are shown using a colored line below the MCoTI-II sequence. Peptide cyclization is displayed as a black line for MCoTI-II.

Figure 2

NMR data summary for lin-McoTI. Data on sequential and medium range NOE connectivities, ³J_HN-Hα coupling constants and slowly exchanging amide protons observed for lin-MCoTI are summarized. The height of the bars correspond to the strength of the NOEs. The values of the ³J_HN-Hα coupling constants are indicated by ↓ (< 4 Hz) and ↑ (> 8.5 Hz). Open and filled squares indicate backbone amide protons that were still observed after 3 and 24 h, respectively, in ²H₂O. The deviations from random coil values for the ¹³C chemical shifts of Cα of lin-MCoTI are plotted at the bottom of the figure.

Figure 3

Stereoview of the 20 lowest energy solution structures of lin-McoTI. Structures have been superimposed for their Cα atoms. The coloring scheme is as follows: whole backbone and proline, black; hydrophobic and aromatic residues, green; polar residues, magenta; acidic residues, red, basic residues, blue; disulfide bridges, orange. Cysteines and N- and C-termini are labeled.

Figure 4

Root mean square deviation from the NMR conformation along the MD simulations. Reported values are for backbone atoms (N, Cα, C, O) of residues 8 to 33 at 300 K (green), 400 K (blue) and 500 K (red). Conformations were superimposed for residue ranges 8–33 (heavy colors) and 15–33 (light colors).

Figure 5

Root mean square positional atomic fluctuations in the 300 K, 400 K and 500 K MD simulations. Reported values are for backbone atoms (N, Cα, C, O) and per residue: MCoTI-II (green line), lin-MCoTI (blue line), EETI-II (red line).

Figure 6

Stereoview of average structures from the 300 K simulations. Structures were superimposed on top of the EETI-II X-ray structure (PDB ID: 1w7z[39]), shown in grey, for backbone atoms of residues 15–33. EETI-II is shown in green, MCoTI-II in red, and lin-MCoTI in blue. Cysteines and N- and C-termini of lin-MCoTI are labeled. Disulfide bridges are shown as orange ball-and-stick representations.

Figure 7

Thermal unfolding curves. The fraction unfolded calculated from the chemical shift (see Methods) is plotted as a function of temperature. Protein identification is as follows: Min-23 (purple, ◆), EETI-II (red, ■), MCoTI-II (green, ●), lin-MCoTI (blue, ▲).

Figure 8

Q-scores of conformations explored in the unfolding simulations. The minimized starting NMR conformation is used as the reference native structure. For each compound, the Q-score evolution is shown at 300 K (green), 400 K (blue) and 500 K (red).

Figure 9

Variation of the mean Root Mean Square deviations and Q-scores with temperature. MCoTI-II is shown as green lines, lin-MCoTI as blue lines and EETI-II as red lines.