The KNOTTIN database

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].
  • Main efforts along this way are outlined below.
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Cell internalization

Circular permutations

Computer simulations

Homology modeling

Libraries

Mutations

Protein engineering

Libraries

Knottins provide interesting structural scaffolds for constrained libraries

The use of nonimmunoglobulin scaffolds, including knottin-based scaffolds, to engineer novel binding molecules has been reviewed by Binz et al. in 2005 and by Hosse et al. in 2006.

Sequence randomization, phage-display and amplification-selection technics allow selection of optimal binders of protein targets. The cellulose-binding domain of the fungal enzyme cellobiohydrolase I has been used as a structural scaffold. Although this two-disulfide domain contains the CSB elementary motif also contained by knottins, it is not a knottin and does not display the typical "disulfide through disulfide knot". Seven residues, located on the surface of the domain, were varied by random mutation of the gene. The repertoire was cloned for display on filamentous bacteriophage, and selected for binding to cellulose or alkaline phosphatase. Binders of cellulose and alkaline phosphatase were isolated [Smith et al., 1998]. More recently, the two-disulfide Cystine Stablized Beta-sheet motif from a squash inhibitor (The 'Min-23' peptide) has been used succesfully to select 21 new specific binders on 7 different targets. Min-23 is one of the smallest known structural scaffold for efficient phage display and selection [Souriau et al., 2005].

The mRNA display is another approach to identify peptide sequences involved in macromolecular recognition. A constrained peptide library has been built using this technic based on the Knottin EETI-II, where the six residues in the first loop were randomized. The constrained library was screened against the natural target of wild-type EETI-II. Selected sequences revealed minimal consensus sequences of PR(I,L,V)L for the inhibitory loop of EETI-II and the wild-type sequence, PRILMR, was selected with the highest frequency [Baggio et al., 2002].