A missense variant remote from the active site impairs stability of human phosphoglucomutase 1

Abstract

Missense variants of human phosphoglucomutase 1 (PGM1) cause the inherited metabolic disease known as PGM1 deficiency. This condition is categorised as both a glycogen storage disease and a congenital disorder of glycosylation. Approximately 20 missense variants of PGM1 are linked to PGM1 deficiency, and biochemical studies have suggested that they fall into two general categories: those affecting the active site and catalytic efficiency, and those that appear to impair protein folding and/or stability. In this study, we characterise a novel variant of Arg422, a residue distal from the active site of PGM1 and the site of a previously identified disease‐related variant (Arg422Trp). In prior studies, the R422W variant was found to produce insoluble protein in a recombinant expression system, precluding further in vitro characterisation. Here we investigate an alternative variant of this residue, Arg422Gln, which is amenable to experimental characterisation presumably due to its more conservative physicochemical substitution. Biochemical, crystallographic, and computational studies of R422Q establish that this variant causes only minor changes in catalytic efficiency and 3D structure, but is nonetheless dramatically reduced in stability. Unexpectedly, binding of a substrate analog is found to further destabilise the protein, in contrast to its stabilising effect on wild‐type PGM1 and several other missense variants. This work establishes Arg422 as a lynchpin residue for the stability of PGM1 and supports the impairment of protein stability as a pathomechanism for variants that cause PGM1 deficiency. Synopsis Biochemical and structural studies of a missense variant far from the active site of human PGM1 identify a residue with a key role in enzyme stability.

Effects of the T337M and G391V disease-related variants on human phosphoglucomutase 1: structural disruptions large and small

“…Both variants show a significant reduction in stability, with a T m approximately 6-7 C below that of the WT enzyme (Table 5). This decrease in stability exceeds that of 1-3 C seen for most other PGM1 missense variants (Lee et al, 2014), except for one remote from the active site that showed a >10 C reduction from the WT (Stiers et al, 2020).…”

“…The T337M and G391V variant proteins characterized here represent the tenth and eleventh crystal structures of PGM1-CDG missense variants to be determined (Stiers et al, 2016(Stiers et al, , 2020Stiers, Graham et al, 2017;Stiers & Beamer, 2018a). Through its ability to accommodate significant perturbation, the robust, multi-domain structure of the enzyme has enabled detailed structural characterization of its variants, making this system a paradigm for understanding the molecular impacts of mutations related to inherited metabolic disease (Beamer, 2021).…”

“…In other cases, the variant proteins proved to be quite well behaved in solution and were amenable to detailed structural characterization. More than ten crystal structures have been determined of WT PGM1 and mutant proteins, demonstrating both local and global structural perturbations (Stiers et al, 2016;Stiers, Graham et al, 2017), effects on protein dynamics (Stiers & Beamer, 2018a) and changes in stability (Stiers et al, 2020). While some common themes have emerged from these studies, it remains difficult to predict the biochemical and structural impacts on PGM1 based solely on the genotype of the variant.…”