title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: CNRS, UMR5048, Université Montpellier 1 et 2, Centre de Biochimie Structurale, 34090 Montpellier, France. Annie.Heitz@cbs.cnrs.fr
authors.forename: Annie
authors.initials: A
authors.lastname: Heitz
authors.forename: Olga
authors.initials: O
authors.lastname: Avrutina
authors.forename: Dung
authors.initials: D
authors.lastname: Le-Nguyen
authors.forename: Ulf
authors.initials: U
authors.lastname: Diederichsen
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Hernandez
authors.forename: Jérôme
authors.initials: J
authors.lastname: Gracy
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
authors.forename: Laurent
authors.initials: L
authors.lastname: Chiche
mesh_terms: D003198:Computer Simulation; D003500:Cyclization; D047169:Cyclotides / Q000737:chemistry*; D047168:Cystine Knot Motifs / Q000235:genetics*; D015195:Drug Design; D047028:Macrocyclic Compounds / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation
chemical_list: D047169:Cyclotides; D047028:Macrocyclic Compounds; C409923:trypsin inhibitor MCoTI-II