Effects of Distal Mutations on the Structure, Dynamics and Catalysis of Human Monoacylglycerol Lipase

An understanding of how conformational dynamics modulates function and catalysis of human monoacylglycerol lipase (hMGL), an important pharmaceutical target, can facilitate the development of novel ligands with potential therapeutic value. Here, we report the discovery and characterization of an allosteric, regulatory hMGL site comprised of residues Trp-289 and Leu-232 that reside over 18 Å away from the catalytic triad. These residues were identified as critical mediators of long-range communication and as important contributors to the integrity of the hMGL structure. Nonconservative replacements of Trp-289 or Leu-232 triggered concerted motions of structurally distinct regions with a significant conformational shift toward inactive states and dramatic loss in catalytic efficiency of the enzyme. Using a multimethod approach, we show that the dynamically relevant Trp-289 and Leu-232 residues serve as communication hubs within an allosteric protein network that controls signal propagation to the active site, and thus, regulates active-inactive interconversion of hMGL. Our findings provide new insights into the mechanism of allosteric regulation of lipase activity, in general, and may provide alternative drug design possibilities

Molecular Dynamics Simulations of Native Protein Charging via Proton Transfer during Electrospray Ionization with Grotthuss Diffuse H₃O⁺

Unraveling the mechanism by which native proteins are charged through electrospray ionization (ESI) has been the focus of considerable research because observable charge states can be correlated to biophysical characteristics, such as protein folding and, thus, solution conformation. Difficulties in characterizing electrosprayed droplets have catalyzed the use of molecular dynamics (MD) to provide insights into the mechanisms by which proteins are charged and transferred to the gas phase. However, prior MD studies have utilized metal ions, primarily Na+, as charge carriers, even though proteins are primarily detected as protonated ions in the mass spectra. Here, we propose a modified MD protocol for simulating discrete Grotthuss diffuse H3O+ that is capable of dynamically altering amino-acid protonation states to model electrospray charging and gaseous ion formation of model proteins, ubiquitin, and myoglobin. Application of the protocol to the evaporation of acidic droplets enables a molecular perspective of H3O+ coordination and proton transfer to/from proteins, which is unfeasible with the metal charge carriers used in previous MD studies of ESI. Our protocol recreates experimentally observed charge-state distributions and supports the charge residue model (CRM) as the dominant mechanism of native protein ionization during ESI. Additionally, our results suggest that protonation is highly specific to individual residues and is correlated to the formation of localized hydrated regions on the protein surface as droplets desolvate. Considering the use of discrete H3O+ instead of Na+, the developed protocol is a necessary step toward developing a more comprehensive model of protein ionization during ESI

Effect of pH on In-Electrospray Hydrogen/Deuterium Exchange of Carbohydrates and Peptides

Carbohydrates are critical for cellular functions as well as an important class of metabolites. Characterizing carbohydrate structures is a difficult analytical challenge due to the presence of isomers. In-electrospray hydrogen/deuterium exchange mass spectrometry (in-ESI HDX-MS) is a method of HDX that samples the solvated structure of carbohydrates during the ESI process and requires little to no instrument modification. Traditionally, solution-phase HDX is utilized with proteins to sample conformational differences, and pH is a critical parameter to monitor and control due to the presence of both acid- and base-catalyzed mechanisms of exchange. For In-ESI HDX, the pH surrounding the analyte changes before and during labeling, which has the potential to affect the rate of labeling for analytes. Herein, we alter the pH of spray solutions containing model carbohydrates and peptides, perform in-ESI HDX-MS, and characterize the deuterium uptake trends. Varying pH results in altered D uptake, though the overall trends differ from the expected bulk-solution trends due to the electrospray process. These findings show the utility of varying pH prior to in-ESI HDX-MS for establishing different extents of HDX as well as distinguishing labile functional groups that are present in different analytes

Automated Removal of Phospholipids from Membrane Proteins for H/D Exchange Mass Spectrometry Workflows

Membrane proteins are currently the most common targets for pharmaceuticals. However, characterization of their structural dynamics by hydrogen/deuterium exchange mass spectrometry (HDX-MS) is sparse due to insufficient automated methods to handle full-length membrane proteins in lipid bilayers. Additionally, membrane lipids used to mimic the membrane environment and to solubilize membrane proteins can impair chromatography performance and cause ion suppression in the mass spectrometer. The workflow discussed herein advances HDX-MS capabilities and other MS applications for membrane proteins by providing a fully automated method for HDX-MS analysis based on a phospholipid removal scheme compatible with robotic handling. Phospholipids were depleted from protein samples by the addition of zirconium oxide beads, which were subsequently removed by inline filtration using syringeless nanofilters. To demonstrate this method, single-pass transmembrane protein FcγRIIa (CD32a) expressed into liposomes was used. Successful depletion of phospholipids ensured optimal liquid-chromatography–mass-spectrometry performance, and measurement of peptides from the transmembrane domain of FcγRIIa indicated phospholipids associated with this region were either not present or did not shield the transmembrane domain from digestion by pepsin. Furthermore, amino acid sequence coverage provided by this method was suitable to enable future measurement of structural dynamics of ectodomain, transmembrane domain, and endodomain of FcγRIIa. Moreover, this method is the first to enable fully automated HDX-MS on full-length transmembrane proteins in lipid bilayers, a notable advancement to facilitate understanding of membrane proteins, development of pharmaceuticals, and characterization for regulatory agencies

Conformational Changes in Active and Inactive States of Human PP2Cα Characterized by Hydrogen/Deuterium Exchange–Mass Spectrometry

PPM serine/threonine protein phosphatases function in signaling pathways and require millimolar concentrations of Mn²⁺ or Mg²⁺ ions for activity. Whereas the crystal structure of human PP2Cα displayed two tightly bound Mn²⁺ ions in the active site, recent investigations of PPM phosphatases have characterized the binding of a third, catalytically essential metal ion. The binding of the third Mg²⁺ to PP2Cα was reported to have millimolar affinity and to be entropically driven, suggesting it may be structurally and catalytically important. Here, we report the use of hydrogen/deuterium exchange–mass spectrometry and molecular dynamics to characterize conformational changes in PP2Cα between the active and inactive states. In the presence of millimolar concentrations of Mg²⁺, metal-coordinating residues in the PP2Cα active site are maintained in a more rigid state over the catalytically relevant time scale of 30–300 s. Submillimolar Mg²⁺ concentrations or introduction of the D146A mutation increased the conformational mobility in the Flap subdomain and in buttressing helices α1 and α2. Residues 192–200, located in the Flap subdomain, exhibited the greatest interplay between effects of Mg²⁺ concentration and the D146A mutation. Molecular dynamics simulations suggest that the presence of the third metal ion and the D146A mutation each produce distinct conformational realignments in the Flap subdomain. These observations suggest that the binding of Mg²⁺ to the D146/D239 binding site stabilizes the conformation of the active site and the Flap subdomain

Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab fragment of NISTmAb

Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein–ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A) from 15 laboratories. Laboratories reported ∼89 800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (∼78 900 centroids), giving ∼100% coverage, and ∼10 900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨sLab⟩ ≤ (0.15 ± 0.01) Da (1σx̅). All laboratories achieved ⟨sLab⟩ ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) °C and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Laboratories(tHDX) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at THDX = 25 °C exhibited reproducibility of σreproducibility25C cohort(tHDX) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements