Computational Structural Analysis of POLG Variants R627Q and W748S with Model-Variability Controls

Abstract

Background: Comparisons between independently predicted wild-type and missense-variant protein structures can generate mechanistic hypotheses, but small apparent differences may reflect model-selection variability rather than mutation-specific effects. Methods: Human mitochondrial DNA polymerase gamma (POLG; UniProt P54098) variants p.Arg627Gln (R627Q) and p.Trp748Ser (W748S) were evaluated using five AlphaFold2-PTM network-model outputs per condition generated with one random seed under matched ColabFold settings. Ten pairwise wild type comparisons at each site described between-network model-selection variability. Variant effects were summarized across five within-network wild-type-versus-variant comparisons using rotation-invariant local C-alpha pair distances and local displacement after global and local alignment. Because these comparison designs differ, the wild-type distribution was used as context rather than a mutation-effect null. Wild-type cryo-EM structure 9GGF was used for contact and interface mapping. Experimental A467T and G848S structures 9GGE and 9GGC provided contextual benchmarks. Results: R627Q measurements fell within the range of between-network wild-type differences: its median mean local pair-distance change was 0.170 angstrom, compared with a wild-type median of 0.170 angstrom, and its locally aligned displacement was 0.265 versus 0.248 angstrom. W748S showed higher median values (0.168 versus 0.132 angstrom for pair-distance change; 0.236 versus 0.182 angstrom for locally aligned displacement), but the ranges overlapped and the comparison-design asymmetry precluded a calibrated mutation-effect percentile. Experimental A467T and G848S comparisons produced local changes of similar magnitude. In 9GGF, R627 and W748 directly shared a local microenvironment, with a minimum heavy-atom distance of 3.53 angstrom. R627 also formed short polar-contact candidates with D629 and D743, whereas W748 occupied a hydrophobic packing environment containing Y622 and F750. Both sites were more than 18 angstrom from nucleic acid, more than 30 angstrom from POLG2, and more than 33 angstrom from PZL-A in a ligand-bound structure. Conclusions: Available AlphaFold2 comparisons do not establish a mutation-specific structural deformation for either variant. Experimental-structure mapping supports testable physicochemical hypotheses involving a shared R627-W748 microenvironment - loss of an arginine-centered polar network for R627Q and disruption of a buried aromatic environment for W748S - but not direct DNA, POLG2, or PZL-A contact mechanisms. Matched control substitutions and independent seeds are required to calibrate small mutation-associated structural deltas.