Comparison of the flap regions of wild-type FIV protease, 12X FIV protease, and wild-type HIV protease. (a) In the wild-type FIV protease, residues positioned at the top and tips of the flaps are not able to form stabilizing interactions. (b) In the 12X mutant Asparagine 55 has been mutated to Methionine and Glycine 62 has been mutated to Phenylalanine, allowing the formation of an intra-flap packing contact between these two residues and an electrostatic interaction between Sδ of Methionine 55 and Nη of Arginine 64. Two additional substitutions in the flap regions of 12X FIV protease, Valine 59 to Isoleucine, and Lysine 63 to Isoleucine, result in the formation of an inter-flap packing contact between the isoleucines (Isoleucine 59 ... Isoleucine 63'). The introduction of stabilizing contacts due to these mutations increases the overall stability of the closed conformation of the flaps. (c) The stabilizing contacts formed as a result of the 12X flap mutations closely resemble those seen in the structure of wild-type HIV in complex with TL-3. The side chain of Methionine 46 is packed between the side chains of Phenylalanine 53 and Lysine 55 in the wild-type HIV protease, just as Methionine 55 is packed between the side chains of Phenylalanine 62 and Arginine 64 in 12X FIV protease (b). Also as in 12X FIV protease, an inter-flap packing contact is formed in HIV protease between Isoleucine 50 and Isoleucine 54'.