Bifidobacterial PK Protein Structural Models

The structural models of the PK (or XFPK) protein from several bifidobacteria species (viz. B. breve, B. bifidum, B. animalis, B. reuteri) were generated using homology modelling. The secondary structure analyses on the selected homolog sequences were initially performed via PSIPRED v3.3 web server [39 (link)].The crystalized B. longum PK structure (PDB ID: 3AI7) was utilized as a template and the comparative modeling was carried out using MODELLER v9.11 [40 ]. Initially, 200 models were generated and ranked/selected using Discrete Optimized Protein Energy (DOPE) scores [41 (link)]. The secondary structure elements in the regions containing CSIs were examined and compared with results of the PSIPRED analysis to ensure their reliability. The stereo-chemical properties of the final models were assessed using three independent servers: RAMPAGE, ERRAT, and Verify3D [42 (link)], [43 (link)] [44 (link),45 (link)]. These tools use a dataset of highly refined structures to evaluate the statistical significance of models based on the conformation, location, and the environment of each amino acid in the sequence, as well as the model’s overall structural stability. The superimposition of the validated models with the template structures was carried out using PyMOL (Version 1.7.4; Schrödinger, LLC.) to examine the structure and location of identified CSIs in the PK (or XFPK) structures..
Identification of the macromolecular interface formed between the individual subunit and the residues in the CSIs that are involved in subunit-subunit interactions was determined by submitting the three-dimensional coordinate file of the B. longum PFK dimeric structure to the PDBePISA server using default parameters (Version 1.48)[46 (link)].