Computer-aided discrimination between active and inactive mutants of the N-terminal domain of the bacteriophage lambda repressor.

Binding of the N-terminal domain of the lambda repressor to DNA is coupled to dimerization. Hydrophobic interactions between helix-5 and helix-5' drive the packing at the dimer interface. We have carried out computations of the conformational energy of packing of the fifth helices (and of the helix-4-loop-helix-5 portions) of variants of the lambda repressor operator binding domain, using an ECEPP/3-based packing algorithm. Here, we report the results for 26 mutants chosen among those that hve been characterized experimentally. We find that the relative orientation of the fifth helices for active mutants is very similar to the wild-type. The fifth helices of the inactive mutants have a significantly different relative orientation. This result illustrates that a unique specific orientation pattern of helix-5 relative to helix-5' is required for dimerization-coupled DNA binding activity. This finding is further supported by computational studies of the whole N-terminal domain of ten variants that showed that the active mutants, including the wild-type protein, have similar values of the number of contacts between the two monomers in the dimer, involving two amino acid residues of the fifth helices (positions 84 and 87 in each monomer). A decrease in the number of such contacts abolishes DNA-binding activity. Furthermore, all active mutants have their "DNA-recognition helices", numbers 3 and 3' positioned so that they can fit in the DNA operator like those of the wild-type protein, while some inactive mutants exhibit a substantial change in the relative orientation of their recognition helices.