Knottins provide useful scaffolds or leads for drug design

  • ‣ Knottins are exceptional in that they are very small proteins yet with particularly well-defined scaffolds and remarkably high stability.
  • ‣ Also remarkable is the fact that knottins with very similar 3D structures have virtually no sequence identity except for cysteines. This observation has led to the conclusion that the knottin scaffold is very sequence tolerant.
  • ‣ These remarkable features suggest that knottins can provide excellent lead molecules or elementary scaffold in drug design studies [Chiche et al., 2004; Craik et al., 2006; Werle et al., 2006; Moore et al., 2012], and in biotechnological applications [Cox et al., 2016; Moore et al., 2013; Glotzbach et al., 2013; Barba et al., 2012].
  • ‣ Main efforts along this way are outlined below.
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Cell internalization

Circular permutations

Computer simulations

Homology modeling



Protein engineering

Point mutations

A large number of point mutations have been performed on squash trypsin inhibitors

The trypsin selectivity of EETI-II was changed to elastase and α-chymotrypsin by modification of the P1 residue [Favel et al., 1989; Le-Nguyen et al., 1990]. Similar results were obtained for another squash inhibitor, CMTI-III [McWherter et al., 1989].
Numerous mutants of CMTI-I and CMTI-III were reported, mainly by polish groups.
CMTI-III mutants of the Arg5 P1 residue exhibit antitrypsin, antichymotrypsin or antielastase activity [Rolka et al., 1989; Rolka et al., 1991, Rozycki 1994a].
Double mutants with Tyr27-Val mutation and Glycine in position 9, 11, 14, 17, 19 or 29 reduce association equilibrium constants by 6-7 orders of magnitude [Rozycki et al., 1993].
Several mutations outside the contact region did not change the antitrypsin activity [Rozycki et al., 1994b].
while other mutations outside the binding loop did affect binding to trypsin [Jaskiewicz et al., 1998].
Mutants of CMTI-I were recently screened for inhibition of serine protease from human blood clotting system [Grzesiak et al., 2000].
Mutation one to four of four conserved hydrophobic residues in CMTI-I were replaced by alanine. Single replacements led to 5-40 times less effective trypsin inhibitors whereas the quadruple mutant was approximately 450 times less effective [Kojima et al., 1996].
The conservative Met8-Leu mutation in CMTI-I was shown to affect both stability and folding of the protein [Zhukov et al., 2000].
The impact of mutation Trp7Leu and addition of Asn25 in MCTI-II, on inhibition of factor Xa and on factor X activation, has been studied [Kamei et al., 2000].

The Potato Carboxypeptidase Inhibitor

Residues of the C-terminal tail were screened by directed mutagenesis for their role in the interaction with carboxypeptidase [Marino-Buslje et al., 2000; Molina et al., 1994].

The cyclotide kalata B1 is tolerant to point mutations

Structural studies of [W19K/P20N/V21K] kalata B1 and [P20D/V21K] kalata B1 showed that the cyclotide framework is tolerant to residue substitutions [Clark et al., 2006].