Computational prediction of (alpha-1antitrypsin)-elastase Michaelis complex reveals an exosite contributing to high-affinity interaction

Neutrophil elastase (NE) is a protease released by activated neutrophils during an inflammatory response and has an anti-microbial function, but when unregulated exerts proteolytic activity on elastin and other extracellular matrix components. Alpha-1-antitrypsin (AAT), an abundant plasma serpin, acts as an important regulator by irreversibly inhibiting NE. The protective activity of AAT is highlighted by the development of early onset emphysema in patients with alpha-1-antitrypsin deficiency. Here the structure of the Michaelis complex formed by AAT and NE was studied by computational methods. Standard molecular dynamics and essential dynamics techniques were used to simulate the docking process using, as a model, the crystal structure of the Pittsburgh variant of AAT in complex with trypsin. A good candidate for the AAT-NE structure was found and was demonstrated to satisfy the geometric conditions required for enzymatic activity, and to be stable over time in the range of hundreds of nanoseconds. Analysis of the interaction interface reveals key contacts including a strong double hydrogen bond involving the P3 residue of AAT and several hydrophobic contacts in the central part of the reactive center loop of AAT. A region located upstream strand S4C of AAT, comprising three acidic residues (D202, E199, E204), was found to strongly interact with Arg 147 of NE. Recombinant AAT variants in which all three acidic residues were mutated to either alanine or serine as well as the single D202R mutant showed reduced association rate to NE and confirmed the predicted acidic region of AAT as an exosite contributing to high affinity interaction to NE.