Computational protocol: The role of substrate specificity and metal binding in defining the activity and structure of an intracellular subtilisin

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Protocol publication

[…] Peptide substrates. Synthetic substrates succinyl-XXXX-para-nitroanilide (Suc-XXXX-pNA with XXXX representing either FAAF, AAPF, AAPNle (Nle – Norleucine), AAPL, AAPM, AAVA, AAPK, AAPE and YVAD) were purchased from Sigma–Aldrich (Poole, Dorset, UK) or Bachem (Bubendorf, Switzerland). Peptides were dissolved in either water or DMF before being diluted into buffer.ISP enzyme kinetics. The various forms of ISP were purified by Ni2+ affinity chromatography as described previously [,]. The Michaelis–Menten kinetic parameters KM, kcat and kcat/KM were determined for each of the Suc-XXXX-pNA substrates. The concentrations of ISP used for each peptide were 0.02 μM (FAAF), 0.2 μM (AAPF) and 1 μM for the remaining peptides. Enzyme rates were measured in buffer containing 50 mM Tris–HCl pH 8.0, 0.5 M ammonium sulphate and 1 mM CaCl2 at 25 °C. Enzymatic activity was determined by measuring the increase in absorbance at 405 nm, due to hydrolysis of the amino acid–pNA peptide bond, using a Varian Cary 50 Bio uv/vis spectrophotometer. The initial rates measured as A405/min were converted into velocity (μM−1 min−1) for each substrate concentration using the molar absorbance coefficient for pNA (9800 M−1 cm−1 at 405 nm). Data were analysed using the GraphPadPrism software. [...] All molecular modelling studies were performed on a MacPro dual 2.66 GHz Xeon running Ubuntu 10.04. The ISP structure used was PDB code 2XRM, with Ala250 mutated in silico back to Ser. Hydrogen atoms were added to the protein using the protonat 3D function of Molecular Operating Environment (MOE) ( The peptide structure was built using MOE and minimized using the AMBER99 force field until a rmsd gradient of 0.05 kcal mol−1 Å−1 was reached. The docking simulations were performed using FlexX ( The best scored conformation that presented the scissile bond between F1 and A1′ in proximity of Ser250 was kept for further studies. Molecular dynamics was then performed with Gromacs 4.5 [] on the protease/peptide complex. The structure was solvated in a periodic octahedron simulation, providing a minimum of 9.0 Å of water between the protein surface and any periodic box edge and appropriately neutralised. The system was minimized and a position restrained molecular dynamic run was carried out for 150 ps, at 300 K in a NTP environment, using the GROMOS96 force field. Finally, a 5 ns production simulation was conducted, using the same conditions.Proteolysis of protein substrates. Digestion of bovine serum albumin (BSA), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) (all from Sigma–Aldrich) as substrates was performed by the addition of 2 μM ISP (2 μM) to 38 μg of each substrate in 50 mM Tris–HCl pH 8.0, 1 mM calcium chloride and 0.5 M ammonium sulphate. The substrate protein digestion was analysed before and after heat treatment at 95 °C for 5 min. Aliquots were taken at time intervals of 0, 1, 5, 10, 30 and 60 min and digestion analysed by reducing 12.5% (w/v) SDS–PAGE.Effect of EDTA, CaCl2 and urea. The calcium dependency of ISP proteolytic activity was measured as above but with the buffer (50 mM Tris–HCl pH 8.0, 0.5 M ammonium sulphate) supplemented with either CaCl2 (1 mM) or ethylenediaminetetraacetic acid (EDTA) (0.01 mM, 0.1 mM or 1 mM). Rate of Suc-FAAF-pNA (50 μM) hydrolysis was measured as described above using 0.1 μM of ISP. To measure EDTA dependent self-proteolytic stability, ISP or proISPS250A was incubated in the presence or absence of 50 mM EDTA and aliquots taken at time intervals of 0, 10, 30 and 60 min and analysed using reducing 12.5% (w/v) SDS–PAGE. Circular dichroism spectroscopy and size exclusion chromatography (using a Superdex 200 10/300 GL column) were performed as described previously [,] with the solutions supplemented with concentrations of CaCl2 or EDTA as outlined in the text. For the denaturation studies, the buffers were supplemented with the appropriate amount of urea indicated in the main text and samples left to equilibrate for at least 5 h. […]

Pipeline specifications

Software tools GraphPad Prism, MOE, GROMACS
Applications Miscellaneous, Circular dichroism spectroscopy
Organisms Bacillus clausii
Diseases Hamartoma Syndrome, Multiple
Chemicals Calcium