Shaolong Zhu

Hyperphosphorylation of intrinsically disordered tau protein induces an amyloidogenic shift in its conformational ensemble.

Tau is an intrinsically disordered protein (IDP) whose primary physiological role is to stabilize microtubules in neuronal axons at all stages of development. In Alzheimers and other tauopathies, tau forms intracellular insoluble amyloid aggregates known as neurofibrillary tangles, a process that appears in many cases to be preceded by hyperphosphorylation of tau monomers. Understanding the shift in conformational bias induced by hyperphosphorylation is key to elucidating the structural factors that drive tau pathology, however, as an IDP, tau is not amenable to conventional structural characterization. In this work, we employ a straightforward technique based on Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) and Hydrogen/Deuterium Exchange (HDX) to provide a detailed picture of residual structure in tau, and the shifts in conformational bias induced by hyperphosphorylation. By comparing the native and hyperphosphorylated ensembles, we are able to define specific conformational biases that can easily be rationalized as enhancing amyloidogenic propensity. Representative structures for the native and hyperphosphorylated tau ensembles were generated by refinement of a broad sample of conformations generated by low-computational complexity modeling, based on agreement with the TRESI-HDX profiles.

Conformational Dynamics and Allostery in Pyruvate Kinase.

Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the “domain rotation model” suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the “rigid body reorientation model” proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the β-strands within the (β/α)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site.

Differential Mobility Spectrometry-Hydrogen Deuterium Exchange (DMS-HDX) as a Probe of Protein Conformation in Solution.

AbstractDifferential mobility spectrometry (DMS) is an ion mobility technique that has been adopted chiefly as a pre-filter for small- to medium-sized analytes (<1 000 Da). With the exception of a handful of studies that employ an analogue of DMS—field asymmetric waveform ion mobility spectroscopy (FAIMS)—the application of DMS to intact biomacromolecules remains largely unexplored. In this work, we employ DMS combined with gas-phase hydrogen deuterium exchange (DMS-HDX) to probe the gas-phase conformations generated from proteins that were initially folded, partially-folded, and unfolded in solution. Our findings indicate that proteins with distinct structural features in solution exhibit unique deuterium uptake profiles as function of their optimal transmission through the DMS. Ultimately we propose that DMS-HDX can, if properly implemented, provide rapid measurements of liquid-phase protein structural stability that could be of use in biopharmaceuticals development. Graphical Abstractᅟ

Suppressing allostery in epitope mapping experiments using millisecond hydrogen / deuterium exchange mass spectrometry

ABSTRACT Localization of the interface between the candidate antibody and its antigen target, commonly known as epitope mapping, is a critical component of the development of therapeutic monoclonal antibodies. With the recent availability of commercial automated systems, hydrogen / deuterium eXchange (HDX) is rapidly becoming the tool for mapping epitopes preferred by researchers in both industry and academia. However, this approach has a significant drawback in that it can be confounded by ‘allosteric’ structural and dynamic changes that result from the interaction, but occur far from the point(s) of contact. Here, we introduce a ‘kinetic’ millisecond HDX workflow that suppresses allosteric effects in epitope mapping experiments. The approach employs a previously introduced microfluidic apparatus that enables millisecond HDX labeling times with on-chip pepsin digestion and electrospray ionization. The ‘kinetic’ workflow also differs from conventional HDX-based epitope mapping in that the antibody is introduced to the antigen at the onset of HDX labeling. Using myoglobin / anti-myoglobin as a model system, we demonstrate that at short ‘kinetic’ workflow labeling times (i.e., 200 ms), the HDX signal is already fully developed at the ‘true’ epitope, but is still largely below the significance threshold at allosteric sites. Identification of the ‘true’ epitope is supported by computational docking predictions and allostery modeling using the rigidity transmission allostery algorithm.

Enhanced Binding Affinity via Destabilization of the Unbound State: A Millisecond Hydrogen–Deuterium Exchange Study of the Interaction between p53 and a Pleckstrin Homology Domain

The incorporation of intrinsically disordered domains enables proteins to engage a wide variety of targets, with phosphorylation often modulating target specificity and affinity. Although phosphorylation can clearly act as a chemical driver of complexation in structured proteins, e.g., by abrogating or permitting new charge-charge interactions, the basis for enhancement of the hydrophobically driven interactions that are typical of disordered protein-target complexation is less clear. To determine how phosphorylation can positively impact target recruitment in disordered domains, we have examined the interaction between the disordered N-terminal transactivation domain (TAD) of p53 and the pleckstrin homology (PH) domain of p62. Using time-resolved electrospray ionization with hydrogen-deuterium exchange, we demonstrate that phosphorylation has little effect on the conformation of the p53 TAD when it is bound to the PH domain but instead increases the degree of conformational disorder in the unbound state. We propose that this increase in the degree of disorder creates a wider free energy gap between the free and bound states, providing a target-independent mechanism for enhanced binding when the phosphorylated and unphosphorylated p53-target complexes have similar free energies.

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

TARGETING OLIGOMERIC PROTEIN SPECIES WITH SMALL MOLECULES

Marcia Taylor, Scott Banfield, Christopher Barden, Bin Deng, Kunal Keskar, Erhu Lu, Luhze Pan, Mark Reed, Kurt Stover, Braden Sweeting, Yanfei Wang, Derek J. Wilson, Fan Wu, Arun Yadav, Seung-Pil Yang, Shaolong Zhu, Donald F. Weaver, Treventis Corporation, Toronto, ON, Canada; Treventis Corporation, Halifax, NS, Canada; Krembil Research Institute, Toronto, ON, Canada; York University, Toronto, ON, Canada; Treventis, Toronto, ON, Canada; Center for Research inMass Spectrometry, Toronto, ON, Canada. Contact e-mail: dweaver@uhnres.utoronto.ca