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. Owingto their small size and their high stability, knottins are considered as excellent leads or scaffolds indrug design. Two knottin families contain macrocyclic compounds, namely the cyclotides and thesquash 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-tailcyclization is intuitively expected to improve bioactivities by increasing stability and loweringflexibility as well as sensitivity to proteolytic attack. Results : In this paper, we report data on solution structure, thermal stability, and flexibility asinferred from NMR experiments and molecular dynamics simulations of a linear squash inhibitorEETI-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 orflexibility in standard conditions, raising the question as to why few squash inhibitors have evolvedinto cyclic compounds. The simulations revealed however that the cyclization increases resistanceto 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 impactmight come from incorporation of an additional loop sequence, that can contribute to theminiprotein specificity and affinity, rather than from an increase in conformational rigidity orprotein stability. Unfolding simulations showed however that cyclization is a stabilizing factor instrongly denaturing conditions. This information should be useful if one wants to use the squashinhibitor scaffold in drug design.