Table of contents
  1. Ankyrin
  2. Protein-Protein Docking
Ankyrin architecture

          Ankyrin proteins are superhelical structures containing capping repeat (N- and C-cap) and a varying number of internal repeats (Fig. 1A - 1F). An individual internal repeat folded into L-shape characteristic consists of a β-turn, two antiparallel α-helices, and a loop coresponding to 33 amino acids (Fig. 1G - 1J). A stack of repeats interacts with its partner via protruding β-turn and the first α-helix seemed the concave surface (green curve in Fig. 1C) of the L-shaped repeat. An alignment-based consensus ankyrin, called DARPins (Fig. 1J), illustrates that 6 out of 33 residues are randomized residues which is made up of solvent-exposed residues representing the potential interaction to specifically partner. Other residues are conserve residues composing of hydrophobic and hydrophilic residues. The hydrophobic residues pointing into the core of the middle ankyrin allow for structural stability (Fig. 1I). Moreover, the β-turn is stabilized through the hydrogen bonding between its main-chain residues and aspartate side-chain at position 1. The capping repeats, composed of hydrophilic residues, functions to seal the hydrophobic core of a stacking repeats leading to their stability.

Figure 1 Ankyrin architecture viewed from the front (A, D and G), the top (B, E and H) and the side (C, F and I) part. Ankyrin consists of a stack of internal repeat (gray) flanking by N-cap (purple) and C-cap (cyan) as shown in A-F. An individual internal repeat (G-J) is built from 33 amino acids containing six randomized postions as shown in red. The blue in I and J are hydrophobic residues making structural stability.

Advantages of ankyrin

  • Since the molecular weight of individual repeat is above 3.5 kDa, their molecular weight ranges 14 to 21 kDa (commonly, four to six repeats found in ankyrin). Comparing to antibody size, ankyrin size is one tenth of a conventional IgG antibody or one third of a Fab fragment. It facilitates to reach targets through tissue penetration.

  • Because of lacking disulfide cross-link, the functional ankyrin can be expressed in cytoplasm of Escherichia coli (E. coli) with providing purified ankyrin protein in milligram amount in simple fask. Moreover, the stability of ankyrin allows for large-scale, rapid, and low-cost production. It is available for producing a huge yeilds of ankyrin is needed.
  • The high target-binding affinity of ankyrin can be designed by selection technology i.e. robosome or phage display in which the pM to low nM is selected in a few round.

  • Engineering residues to improve binding affinity or to enhance specificity or sensivity to target can be performed on randomized residues while the structure is not destroyed because of the high stability of conserved residues.

  • Since the stability of ankyrin does not permit the flexible peptide shown, the proteolytic digestion has not been detected in any experiment. They, including no aggregation, are the reason for low immunogenicity. Moreover, appearing the ankyrin fragments in circulation is possible for immunological tolerance. Comparing with antibody, ankyrin does not contain Fc-domain, therefore the effector function is absence in ankyrin.
   Protein-Protein Docking
         The protein-protein docking is a computational method to predict complex structures of two unbound proteins. The docking is often performed in two stages: an initial stage and a refinement stage, as shown in Fig. 2.
The initial stage provides possible orientations of two proteins together with their scores which are generated from a searching method and a scoring function. The searching method is an algorithm for rapidly finding all possible orientations by treating proteins as rigid bodies and moving smaller protein around bigger protein in a six-dimensional (6-D) space (3 rotational and 3 translational degrees of freedom). The most popular method is the Fast Fourier Transform (FFT), while others are Monte Carlo sampling, genetic algorithms, and geometric hashing. For scoring function, it is a score value calculated from possible orientation is given. The approach for scoring function is based on shape complementarity concerning surface shape of protein or interaction energy such as electrostatic, van der waals or hydrophobic. The scoring function is used for selection the complexes for further refining or analysis.
In the refinement stage, a set of selected complexes from the initial stage are performed by minimization the side chain and recalculate the scoring function.
        To obtain the correct complexes, biological information is used for eliminating the false positive i.e. defining the residues known to be binding site.
Figure 2 Overview of the protein-protein docking stage.