Resolving DJ-1 Glyoxalase Catalysis Using Mix-and-Inject Serial Crystallography at a Synchrotron.

DJ-1 (PARK7) is an intensively studied protein whose cytoprotective activities are dysregulated in multiple diseases. DJ-1 has been reported as having two distinct enzymatic activities in defense against reactive carbonyl species that are difficult to distinguish in conventional biochemical experiments. Here, we establish the mechanism of DJ-1 using a synchrotron-compatible version of mix-and-inject-serial crystallography (MISC), which was previously performed only at XFELs, to directly observe DJ-1 catalysis. We designed and used new diffusive mixers to collect time-resolved Laue diffraction data of DJ-1 catalysis at a pink beam synchrotron beamline. Analysis of structurally similar methylglyoxal-derived intermediates formed through the DJ-1 catalytic cycle shows that the enzyme catalyzes nearly two turnovers in the crystal and defines key aspects of its glyoxalase mechanism. In addition, DJ-1 shows allosteric communication between a distal site at the dimer interface and the active site that changes during catalysis. Our results rule out the widely cited deglycase mechanism for DJ-1 action and provide an explanation for how DJ-1 produces L-lactate with high chiral purity.

Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography.

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography.

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

Human IgE monoclonal antibody recognition of mite allergen Der p 2 defines structural basis of an epitope for IgE cross-linking and anaphylaxis in vivo.

Immunoglobulin E (IgE) antibody is a critical effector molecule for adaptive allergen-induced immune responses, which affect up to 40% of the population worldwide. Allergens are usually innocuous molecules but induce IgE antibody production in allergic subjects. Allergen cross-linking of IgE bound to its high affinity receptor (FcεRI) on mast cells and basophils triggers release of histamine and other mediators that cause allergic symptoms. Little is known about the direct allergen-IgE antibody interaction due to the polyclonal nature of serum IgE and the low frequency of IgE-producing B cells in blood. Here, we report the X-ray crystal structure of a house dust mite allergen, Der p 2, in complex with Fab of a human IgE monoclonal antibody (mAb) isolated by hybridoma technology using human B cells from an allergic subject. This IgE mAb, 2F10, has the correct pairing of heavy and light chains as it occurs in vivo. Key amino acids forming the IgE epitope on Der p 2 were identified. Mutation of these residues ablated their functional ability to cross-link IgE in a mouse model of passive systemic anaphylaxis. These analyses revealed an important conformational epitope associated with the IgE antibody repertoire to a major mite allergen.