Conclusions

The four molecular dynamics simulations of the wild type and three mutant Antennapedia homeodomain-DNA complexes provide information for the comparison of the four systems.

1.

The root mean square deviation test suggests that in all of the runs the protein backbone was stable. The mutation in the DNA though resulted in less stable complexes, possibly as a result of unresolved steric conflicts. This instability is especially noteworthy in the double mutant complex, where after 160 ps, the DNA appears to begin to dissociate at its termini (see Figures 3.1, 3.2, 3.3). Another reason for such a behaviour could be the intrinsic property of these simulations which did not take into account counterions present in vivo or in solution. This problem is to be tackled in a future project.

2.

Analysis of direct specific contacts between the macromolecules indicates that the wild type and doubly mutated complexes show greater binding affinity than the two other simulated complexes, since there are more direct contacts in the former systems than in the latter. Residues 47 and 50 follow the same pattern and form significantly more hydrogen bonds and hydrophobic contacts in wild type and double mutant complexes (only residue 50 in the latter).

3.

However, the other two residues do not follow such a pattern, especially Asn51 seems to replace the function of Gln50 in the complex with mutated protein and forms twice as many direct contacts comparing to the other three systems. Since Asn51 is strictly conserved among all homeodomains, this indicates a loss in binding specificity. In the complex with the mutant protein, the recognition helix was distorted at residue 51 (see Figure 3.7).

4.

Base pair 7 forms significantly more contacts in the wild type and double mutant complexes to residue 50, than base pair 6, whereas they are contacted more or less evenly in the other two complexes. This could imply that base pair 7 is more important for recognition than base pair 6. Base pair 6 also forms more contacts to Gln50 thatn to Lys50.

5.

The number of water mediated contacts does not reveal any significant overall difference between the complexes, yet, residue 50 forms most bridges in the wild type complex and Asn51--in the complex with the mutated protein.

6.

There are twice as many water molecules in the interfaces of the ``wrong'' complexes in comparison to the wild type and double mutant systems. The waters in the latter complexes are also less mobile and spend more time in the interface being involved in hydrogen bonding to one or both macromolecules.

7.

While water molecules are in the interface they are more likely to hydrogen-bond to the DNA than to the protein. This is in conformity with studies of free DNA in solution, which indicate that water may be regarded as being a part of the DNA. The bonding to the protein is more stable in wild type and double mutant complexes, whereas in the other two it is more occasional.

As a final conclusion of this study one can state that it agrees with the earlier biochemical investigation of a closely related system [7]. I.e. the Gln50$\to$Lys mutated Antennapedia homeodomain binds with high affinity to the DNA sequence GGATTA as compared to the wild type protein, which binds preferably to the DNA sequence CCATTA.