In silico three-dimensional protein structure mapping and analysis of variations

A three-dimensional (3D) protein structure mapping and analysis approach (developed and validated by Lloyd and McGeehan) [38–40] was used to predict the functional impact of all non-synonymous identified in mtDNA protein coding regions.

Briefly, the human mtDNA-encoded protein sequences were used to identify the most homologous OXPHOS complexes I, III, IV and V available from the RCSB PDB (Research Collaboratory for Structural Bioinformatics Protein Data Bank; http://www.rcsb.org/). Then, the best quality, latest and most similar complex structures were choosen; complex I – [PDB:5XTD] (Homo sapiens) [41]; complex III – [PDB:5XTE] (Homo sapiens) [41]; complex IV [PDB:5Z62] (Homo sapiens) [42] and complex V [PDB:5ARA] (Bos taurus) [43].

The atomic co-ordinates of each complex were downloaded from the PDB and opened using PyMol (the PyMOL Molecular Graphics System/Version 1.8; distributed by Schrödinger, LLC, NY, USA). All identified non-synonymous variation sites were mapped to their amino acid locations on the protein structures using PyMol. COOT software (Crystallographic Object-Oriented Toolkit) was also used to create PDB files containing all non-synonymous variations identified. PDB files including the variations (created by COOT) were also opened with PyMol, and compared with the wild-type structures. Protein structures were displayed in a cartoon style, and each variation was identified and displayed in the stick and sphere format.

Detailed analysis of the potential effect of each variation on protein structure was performed by examining the type of amino acid change, as well as its location within the protein. Factors taken into consideration included: proximity to important catalytic regions, such as active sites, binding pockets and subunit interfaces.