Serpins (serine protease inhibitors) belong to the group of protease inhibitors superfamily. They irreversible inhibit proteases by undergoing to a huge conformational change to break the proteases active site. The changes in serpin structure caused by proteolysis of the reactive-center loop (RCL), which in the native state (also called the S state) has an extended conformation that protrudes from the serpin domain. During the cleavage, the amino-terminal part of the RCL inserts into the central β-sheet to form an additional β-strand. This structural rearrangement is crucial for protease inhibition and results in the so-called R state of serpin, which is more stable compared with the S state. In the final serpin – protease complex the protease remains covalently linked to the serpin.
Several crystals have been grown in commercial conditions Index (Hampton Research, USA), JBScreen, (Jena Bioscience GmbH, Germany). The vapor diffusion sitting drop method was performed with ratio protein to precipitant 1:1 and 2:1 and stored at temperature 293 K. Optimization of successful crystallization conditions was performed by variation of salt, PEG and protein concentration. Number of diffraction data sets was collected at resolution ranging from 2.0 to 3.0Å. The image processing was performed by XDS [1] software package and preliminary results shown that serpin crystals belong to the trigonal space group P3121 with unit cell parameters a=78.38 b=78.38 c=99.57 Å, α= β=90.0°γ=120.0°. The structure with identity above 50% was found. This particular structure was used as a model for molecular replacement for processed data sets. MolPrep and Phaser as well as automated BALBES pipeline were used to obtain initial models, followed by structure refinement with REFMAC5 from the CCP4 software package. Model building using Coot and refinement in Refmac5 is under way.
This research was supported by the GACR 19-14704Y, GACR 17-24341S and GAJU (04-017/2019/P).