Orthogonal coiled-coils.html

De novo design of orthogonal coiled-coil forming segments

One of the main challenges we were faced with was to ensure exclusivity of the pairing. Our selected heterodimeric coiled-coil-forming segments had to satisfy the following requirements:

• any coiled-coil-forming segment must form coiled-coil with exactly one segment
• any other combination is forbidden/significantly disfavored
  

In this way selected system of coiled-coil forming segments is called orthogonal.
Additionally we have to ensure that selected proteins are not too long (in order to avoid bending as well as decreased energetic difference between desired and undesired pairs) but still have approximately the same length to form symmetrical structures if not otherwise required by some special design.
As mentioned in the introduction, coiled-coils can be parallel or antiparallel as well as homodimers and heterodimers. These are also features we have to consider when planning (i.e. we identify a parallel pair but have to also check that neither of proteins forms more stable parallel/antiparallel homodimer/heterodimer).

Based on the published rules for the prediction of stability of coiled-coils (Hageman et al., 2008) we prepared a program to predict the stability of each tested pair. More about software (download and abstract) follow link ________

In order to restrict the number of possible combinations to manageable we limited the variability at defined positions to a limited set of residues that have been previously shown to stabilize the interactions. We started by choosing the most appropriate amino acids for the every position in sequence, similar as reported by (Bromley et al., 2009). Generally we followed the rule that hydrophobic residues at positions “a” or “d” and opposite charge at positions “e” and “g” on the equivalent positions between the two helices stabilize, whereas burial of polar residues (Asn) at positions “a” or “d” and the same charge at positions “e” and “g” destabilize the structure (see Introduction). In this simplified model we considered a limited number of variable residues only at positions that significantly affect the stability (“a”, “d”, “e”, “g”) and neglecting the effect on other positions (positions “b”, “c”, “f”). It has to be noted that the parameters to predict stability have been extracted from parallel dimers of basic leucine zipper (bZIP) peptides, therefore the predictions for this type of interaction are most reliable. Although we can probably estimate also contributions to the stability of antiparallel orientation, we tried to use experimentally parallel orientations whenever it was possible.

At the N-terminal segment of each coiled-coil segment we introduced a Ser-Pro-Glu-Asp tetrapeptide, which is composed of contributions of the preferred residues at the N-termini of coiled-coil helices (Sun et al., 2000; Cochran et al., 2001; Penel et al.,l 1999). Side chain of those residues can form hydrogen bond with polypeptide backbone of the residues in the fist turn, interact with a dipole moment of helix and have conformational properties compatible with the beginning of a helix and at the same time serve to prevent formation of an extended helix, that might form between the consecutive coiled-coil forming segments.

To predict coiled-coil interactions we examined two algorithms (see Hagemann et al., 2009; Fong et al., 2006) and in the end chose to follow Hageman et al.: There are many factors that affect stability: hydrophobic burial, propensity, solubility, electrostatic interaction of flanking residues and others. This algorithm considers three factors: core, electrostatic and propensity which seem to have the key role in CC formation. The calculation is shown in following scheme (shown on a fictional protein pair)(Figure 1):

Figure 1

Identification of a set of orthogonal coiled-coil pairs

Figure 2

Figure 3