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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 Ã… 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 H<sub>3</sub>O<sup>+</sup>
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 Mn<sup>2+</sup> or
Mg<sup>2+</sup> ions for activity. Whereas the crystal structure of
human PP2Cα displayed two tightly bound Mn<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup>, 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
Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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