Insights into Phosphorylation-Induced Protein Allostery and Conformational Dynamics of Glycogen Phosphorylase via Integrative Structural Mass Spectrometry and In Silico Modeling

Abstract

Allosteric regulation plays a fundamental role in innumerable biological processes. Understanding its dynamic mechanism and impact at the molecular level is of great importance in disease diagnosis and drug discovery. Glycogen phosphorylase (GP) is a phosphoprotein responding to allosteric regulation and has significant biological importance to glycogen metabolism. Although the atomic structures of GP have been previously solved, the conformational dynamics of GP related to allostery regulation remain largely elusive due to its macromolecular size (∼196 kDa). Here, we integrated native top-down mass spectrometry (nTD-MS), hydrogen–deuterium exchange MS (HDX-MS), protection factor (PF) analysis, molecular dynamics (MD) simulations, and allostery signaling analysis to examine the structural basis and dynamics for the allosteric regulation of GP by phosphorylation. nTD-MS reveals differences in structural stability as well as oligomeric state between the unphosphorylated (GPb) and phosphorylated (GPa) forms. HDX-MS, PF analysis, and MD simulations further pinpoint the structural differences between GPb and GPa involving the binding interfaces (the N-terminal and tower–tower helices), catalytic site, and PLP-binding region. More importantly, it also allowed us to complete the missing link of the long-range communication process from the N-terminal tail to the catalytic site caused by phosphorylation. This integrative MS and in silico-based platform is highly complementary to biophysical approaches and yields valuable insights into protein structures and dynamic regulation.

Allosteric Regulation of Glycogen Phosphorylase by Order/Disorder Transition of the 250′ and 280s Loops

“…A notable HDX uptake difference of 1.7 Da was identified in the peptide containing residues Arg309 and Arg310, which play a crucial role in the G6P allosteric site interactions, as shown in the crystallographic model of the binding epitope, Figure3. Despite this, the results showed that the stability of the α8 helix (residues 307−326) remained unchanged within the conventional measurement time frame, in agreement with prior studies 21. This suggests that part of the binding epitope for G6P, as determined by crystallography, is not captured in HDX-MS data when measured within the standard time window (i.e., > 10 s).These findings suggest that the N-terminus of the α8 helix and the preceding 280s loop are highly sensitive to allosteric modulation and that the Arg309 and Arg310 residues play a crucial role in these structural changes.This may represent direct mediation of allostery from the nucleotide site via the rigid α8 helix to the active site, although it is unclear how the partial release of the 280s entropic gate can manifest as inhibitory.…”

Online Fully Automated System for Hydrogen/Deuterium-Exchange Mass Spectrometry with Millisecond Time Resolution

“…To further estimate the advances achieved with the ms2min system, we compared the information content available from the conventional (≥seconds) and the millisecond time range (Figure D) (the gray shaded region denotes the conventionally acquired time window data). Data from the ms2min in the 1–300 s time domain were similar to published values from CTC-PAL experiments (given limitations in the interpretation from variation in the labeling conditions and LC–MS systems) . We found certain regions in this large enzyme that have little to no information content by HDX-MS when resolved at the seconds-minutes time window, but almost complete kinetics were obtained when the HDX window was extended by 3 orders of magnitude, down to 50 ms.…”

“…Data from the ms2min in the 1−300 s time domain were similar to published values from CTC-PAL experiments (given limitations in the interpretation from variation in the labeling conditions and LC−MS systems). 54 We found certain regions in this large enzyme that have little to no information content by HDX-MS when resolved at the seconds-minutes time window, but almost complete kinetics were obtained when the HDX window was extended by 3 orders of magnitude, down to 50 ms. Insights into the structural changes upon Ser14 phosphorylation were attained, though it is evident that expansion of the time window stands to detect structural perturbations more sensitively.…”

Fundamentals of HDX-MS

“…The sample may then undergo mild fragmentation by either an ECD or ETD to cleave labile interdomain bonds, providing additional ‘domain’ level resolution [ 54–56 ]. While top-down experiments will not provide local resolution of mass shifts, the technique is utilized to compare global deuterium incorporation rates and observe interspecies differences that may occur, such as the influence of post-translational modifications, as well as it will aid in characterizing conformationally heterogenous samples [ 57–59 ].…”