Computational protocol: A combination of mutational and computational scanning guides the design of an artificial ligand-binding controlled lipase

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Protocol publication

[…] Evolutionary coupling analysis was carried out for lipases using the BsLA sequence (Uniprot ID: P37957) as input sequence for the EVcouplings webserver (www.evfold.org). For the generation of the alignment the JackHHMer software (5 interations), implemented as part of the EVcouplings webserver, was utilized, to search the Uniprot database for sequences similar to BsLA. We ran an unrestrained search not limiting the number of sequences in the alignment, which retrieved 149.524 sequences with an E-value cutoff of 10E-3, covering 168 out of 181 residues of the query BsLA sequence. In a subsequent restrained run we limited the number of sequences in the alignment to 20.000 while using the same E-value cutoff. This search produced an alignment containing 20.000 sequences covering 176 out of 181 residues of the query sequence. Covariation information was inferred employing the plmDCA (pseudolikelihood maximization for Potts models with direct coupling analysis algorithm), implemented in the EVcouplings webserver. Evolutionary constraints (EC) values were mapped onto the B-factor field of the BsLA X-ray structure (PDB ID: 1I6W) and visualized by using Pymol v1.7.0.0 (Schrödinger Inc., NY, USA). [...] SAXS was measured of CitAP-BsLA (0.5 to 5.0 mg/mL, 10 mM glycine buffer pH 10, 10 mM NaCl (±citrate), 10 °C sample temperature) at the beamline BM29 at the ESRF. Measured data were scaled by the concentration. The excluded Porod volume was calculated with the program DATPOROD and the molecular mass was estimated by using the reported protein density of 0.588 g/mL. The distance distribution function P(r) was determined using the program DATGNOM. In total 20 ab initio models were generated using the program DAMMIF, averaged and the filtered model was used. The envelope function was determined using the SITUS package. [...] The detailed strategy for modelling of the dimeric CitAP-BsLA complex is summarized in the . CitAP-BsLA monomer models were built with the program BUNCH of the ATSAS package. In all cases, template coordinates were taken from the PDB structures with IDs 2J80 (CitAP) and 1I6W (BsLA). The linker connecting the CitAP and BsLA domains in the monomer, the His6 tag and all other remaining missing residues of the fusion protein were modelled as Cα traces during the fitting procedure. Afterwards, the Cα traces were extended to all-atom models with the web server MaxSprout. As the all-atom extension of prolines failed, these were modelled by a superposition of a template proline residue onto the proline backbone obtained from MaxSprout. Dimer models were either built manually, by superimposing the corresponding monomer models onto the dimeric crystal structure of CitAP-PAS (PDB-ID 2J80), or were assembled ab initio by oligomerizing the monomer models using the program SASREF optimizing the dimer orientation against SAXS data at high protein concentration (100% dimer). Further details are given in the and . The quality of all models was evaluated with the program CRYSOL. CRYSOL computes theoretical scattering curves and compares these to the experimental data. As quality indicator for each model the χ values computed by CRYSOL were used, which present a measure for the discrepancy between theoretical and experimental curves. In order to improve the initial models, a 100 ns molecular dynamics (MD) simulation was performed for each dimeric assembly and a theoretical scattering curve was calculated for every 200 ps snapshot of each trajectory and fitted against the experimental data using CRYSOL. Details can be found in the . […]

Pipeline specifications

Software tools EVcouplings, plmDCA, DCA, PyMOL, DAMMIF, Situs, ATSAS, SASREF, CRYSOL
Applications Small-angle scattering, Protein structure analysis
Organisms Bacillus subtilis, Klebsiella pneumoniae