Computational protocol: Isolation, Cloning and Structural Characterisation of Boophilin, a Multifunctional Kunitz-Type Proteinase Inhibitor from the Cattle Tick

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Protocol publication

[…] Diffraction data to 2.35 Å were collected from a single cryo-cooled (100 K) crystal on a MAR CCD detector at ESRF beamline ID14-EH3. Data were processed with MOSFLM , scaled with SCALA , and reduced with tools from the CCP4 package . Data collection and refinement statistics are summarised in .The structure was solved by molecular replacement using AMoRe with data in the 10-4.0 Å resolution range. Two proteinase molecules were located using the coordinates of unliganded bovine α-thrombin from PDB entry 1MKX as search model. All attempts to locate the Kunitz domains of boophilin by molecular replacement, using truncated models of either BPTI (1BPI) or ornithodorin (1TOC) were unsuccessful.The initial electron density maps, computed after rigid body refinement of the proteinase molecules with CNS v. 1.1 , displayed additional density for most of the inhibitor molecules. Two cycles of manual rebuilding of the proteinase models on a graphic workstation using TurboFRODO (Bio-Graphics, France), followed by positional refinement with CNS resulted in significant improvement of the electron density maps. This allowed manual docking of a truncated model of BPTI close to thrombin exosite I for each of the complexes. Further crystallographic refinement alternated with manual model building until complete interpretation of the electron density maps. The final isotropically refined model obtained with CNS was further refined with REFMAC_5 using TLS displacement parameters.The final model comprises residues TF1P to TR15 and TI16 to TD243 of one thrombin molecule (chains A and B, respectively) and residues TP1N to TE14L and TI16 to TD243 of the other proteinase moiety (chains C and D, respectively). Both boophilin molecules (chains E and F) were modelled from BQ16 to BM142. The side chains of TE14L (chain A), TK236 (chain B), TK1I, TK14D (chain C), TK87, TR93, TK97, TE113 (chain D), BE75, BE110 (chain E), and of BK86 (chain F) are not well defined in the electron density maps; we have assigned an occupancy value of zero to these side chains. The model comprises also three phosphate and two sodium ions, 453 water molecules and an N-acetyl-glucosamine sugar moiety attached to TN60G in chain D. [...] Sequence similarity searches were performed with the program FastA from the Wisconsin package (version 9.0, Genetics Computer Group, Madison, Wisconsin), and alignments were prepared with ALSCRIPT . Major antigenic peptides were predicted with the Kolaskar & Tongaonkar method (http://bio.dfci.harvard.edu/Tools/antigenic.pl). Models of the thrombin·boophilin complex were built based on the co-ordinates of (E192Q)thrombin·BPTI (1BTH) and thrombin·ornithodorin (1TOC) complexes, and were subjected to energy minimization with CNS. The amblin model was built by applying homology-based methods as implemented in the automatic SWISS-MODEL server (http://swissmodel.expasy.org) , using the structure of the Kunitz inhibitor bikunin as template . Structure representations were generated with PyMOL (http://www.pymol.org) or SETOR . […]

Pipeline specifications

Software tools iMosflm, CCP4, CNS, SWISS-MODEL, PyMOL
Databases ExPASy
Applications Small-angle scattering, Protein structure analysis
Organisms Bos taurus, Rhipicephalus microplus, Escherichia coli
Chemicals Guanidine