Mapping the active site of factor Xa by selective inhibitors: An NMR and MD study

Abstract

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH2 and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH2, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site.

Stabilizing of a Globular Protein by a Highly Complex Water Network: A Molecular Dynamics Simulation Study on Factor Xa

“…18 It was also shown that missing or badly placed water molecules in fXa may lead to computational artifacts. [19][20][21][22][23] fXa is one of the key enzymes in the blood coagulation cascade. [24][25][26][27][28][29][30][31][32][33][34][35] It is the joining point for the extrinsic and the intrinsic pathway of fibrin production.…”

“…In all experimental X-ray structures with resolved water molecules, it is filled with a chain of water molecules that are considered to be important for the catalytic function. − If some of these waters are not seen in a serine protease X-ray structure, this is probably due to low resolution of the experiment . It was also shown that missing or badly placed water molecules in fXa may lead to computational artifacts. − …”

Topological analysis of the complex formed between neurokinin A and the NK2 tachykinin receptor

“…TxR was positioned considering channels found with Molcad Multi‐channel, directing the fluorophore part towards the extra cellular surface, as it is charged. A local minimisation was then undertaken as described above in the Tripos force field (Fraternali et al. 1998).…”

“…All the minimisation procedure was undertaken without charge and in Amber 4.1 force field (Fraternali et al. 1998) with a maximum of 25 simplex steps coupled to a Powell (Fraternali et al. 1998) method, with an energy termination gradient of 0.05 × kcal × mol −1 × Å −1 .…”

“…A local minimisation was undertaken as described above with the entire NK2R and NKA complex. Following we optimised the placement by using a local molecular dynamic in vacuo around NKA at 300 K, NTV constant during 50 ps, with an acquisition time of 50 fs, and Shake algorithm applied on bonds involving hydrogens (Fraternali et al. 1998).…”