Transient Structural Dynamics of Glycogen Phosphorylase from Nonequilibrium Hydrogen/Deuterium-Exchange Mass Spectrometry.

It remains a major challenge to ascertain the specific structurally dynamic changes that underpin protein functional switching. There is a growing need in molecular biology and drug discovery to complement structural models with the ability to determine the dynamic structural changes that occur as these proteins are regulated and function. The archetypal allosteric enzyme glycogen phosphorylase is a clinical target of great interest to treat type II diabetes and metastatic cancers. Here, we developed a time-resolved nonequilibrium millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS) approach capable of precisely locating dynamic structural changes during allosteric activation and inhibition of glycogen phosphorylase. We resolved obligate transient changes in the localized structure that are absent when directly comparing active/inactive states of the enzyme and show that they are common to allosteric activation by AMP and inhibition by caffeine, operating at different sites. This indicates that opposing allosteric regulation by inhibitor and activator ligands is mediated by pathways that intersect with a common structurally dynamic motif. This mass spectrometry approach uniquely stands to discover local transient structural dynamics and could be used broadly to identify features that influence the structural transitions of proteins.

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

Allostery is a fundamental mechanism of protein activation, yet the precise dynamic changes that underlie functional regulation of allosteric enzymes, such as glycogen phosphorylase (GlyP), remain poorly understood. Despite being the first allosteric enzyme described, its structural regulation is still a challenging problem: the key regulatory loops of the GlyP active site (250' and 280s) are weakly stable and often missing density or have large b-factors in structural models. This led to the longstanding hypothesis that GlyP regulation is achieved through gating of the active site by (dis)order transitions, as first proposed by Barford and Johnson. However, testing this requires a quantitative measurement of weakly stable local structure which, to date, has been technically challenging in such a large protein. Hydrogen-deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for studying protein dynamics, and millisecond HDX-MS has the ability to measure site-localized stability differences in weakly stable structures, making it particularly valuable for investigating allosteric regulation in GlyP. Here, we used millisecond HDX-MS to measure the local structural perturbations of glycogen phosphorylase b (GlyPb), the phosphorylated active form (GlyPa), and the inhibited glucose-6 phosphate complex (GlyPb:G6P) at near-amino acid resolution. Our results support the Barford and Johnson hypothesis for GlyP regulation by providing insight into the dynamic changes of the key regulatory loops.

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

Amide hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for analyzing the conformational dynamics of proteins in a solution. Current conventional methods have a measurement limit starting from several seconds and are solely reliant on the speed of manual pipetting or a liquid handling robot. Weakly protected regions of polypeptides, such as in short peptides, exposed loops and intrinsically disordered the protein exchange on the millisecond timescale. Typical HDX methods often cannot resolve the structural dynamics and stability in these cases. Numerous academic laboratories have demonstrated the considerable utility of acquiring HDX-MS data in the sub-second regimes. Here, we describe the development of a fully automated HDX-MS apparatus to resolve amide exchange on the millisecond timescale. Like conventional systems, this instrument boasts automated sample injection with software selection of labeling times, online flow mixing and quenching, while being fully integrated with a liquid chromatography-MS system for existing standard "bottom-up" workflows. HDX-MS's rapid exchange kinetics of several peptides demonstrate the repeatability, reproducibility, back-exchange, and mixing kinetics achieved with the system. Comparably, peptide coverage of 96.4% with 273 peptides was achieved, supporting the equivalence of the system to standard robotics. Additionally, time windows of 50 ms-300 s allowed full kinetic transitions to be observed for many amide groups; especially important are short time points (50-150 ms) for regions that are likely highly dynamic and solvent- exposed. We demonstrate that information on structural dynamics and stability can be measured for stretches of weakly stable polypeptides in small peptides and in local regions of a large enzyme, glycogen phosphorylase.

HDfleX: Software for Flexible High Structural Resolution of Hydrogen/Deuterium-Exchange Mass Spectrometry Data.

Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) experiments on protein structures can be performed at three levels: (1) by enzymatically digesting labeled proteins and analyzing the peptides (bottom-up), (2) by further fragmenting peptides following digestion (middle-down), and (3) by fragmenting the intact labeled protein (top-down) using soft gas-phase fragmentation methods, such as electron transfer dissociation (ETD). However, to the best of our knowledge, the software packages currently available for the analysis of HDX-MS data do not enable the peptide- and ETD-levels to be combined; they can only be analyzed separately. Thus, we developed HDfleX, a standalone application for the analysis of flexible high structural resolution of HDX-MS data, which allows data at any level of structural resolution (intact protein, peptide, fragment) to be merged. HDfleX features rapid experimental data fitting, robust statistical significance analyses, and optional methods for theoretical intrinsic calculations and a novel empirical correction for comparison between solution conditions.

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry Approach to Correlate Local Structure and Aggregation in α-Synuclein.

In Parkinson's disease and other synucleinopathies, α-synuclein misfolds and aggregates. Its intrinsically disordered nature, however, causes it to adopt several meta-stable conformations stabilized by internal hydrogen bonding. Because they interconvert on short timescales, monomeric conformations of disordered proteins are difficult to characterize using common structural techniques. Few techniques can measure the conformations of monomeric α-synuclein, including millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS). Here, we demonstrate a new approach correlating millisecond HDX-MS data with aggregation kinetics to determine the localized structural dynamics that underpin the self-assembly process in full-length wild-type monomeric α-synuclein. Our custom instrumentation and software enabled measurement of the amide hydrogen-exchange rates on the millisecond timescale for wild-type α-synuclein monomer up to residue resolution and under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal cellular compartments. We applied an empirical correction to normalize measured hydrogen-exchange rates and thus allow comparison between drastically different solution conditions. We characterized the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Applying a correlative approach to connect molecular conformation to aggregation in α-synuclein for the first time, we found that the central C-terminal residues of α-synuclein are driving its nucleation and thus its aggregation. We provide a new approach to link the local structural dynamics of intrinsically disordered proteins to functional attributes, which we evidence with new details on our current understanding of the relationship between the local chemical environment and conformational ensemble bias of monomeric α-synuclein.

Interconversion of Unexpected Thiol States Affects the Stability, Structure, and Dynamics of Antibody Engineered for Site-Specific Conjugation.

Antibody-drug conjugates have become one of the most actively developed classes of drugs in recent years. Their great potential comes from combining the strengths of large and small molecule therapeutics: the exquisite specificity of antibodies and the highly potent nature of cytotoxic compounds. More recently, the approach of engineering antibody-drug conjugate scaffolds to achieve highly controlled drug to antibody ratios has focused on substituting or inserting cysteines to facilitate site-specific conjugation. Herein, we characterize an antibody scaffold engineered with an inserted cysteine that formed an unexpected disulfide bridge during manufacture. A combination of mass spectrometry and biophysical techniques have been used to understand how the additional disulfide bridge forms, interconverts, and changes the stability and structural dynamics of the antibody intermediate. This quantitative and structurally resolved model of the local and global changes in structure and dynamics associated with the engineering and subsequent disulfide-bonded variant can assist future engineering strategies.

Different Structural Conformers of Monomeric α-Synuclein Identified after Lyophilizing and Freezing.

Understanding the mechanisms behind amyloid protein aggregation in diseases, such as Parkinson's and Alzheimer's disease, is often hampered by the reproducibility of in vitro assays. Yet, understanding the basic mechanisms of protein misfolding is essential for the development of novel therapeutic strategies. We show here, that for the amyloid protein α-synuclein (aSyn), a protein involved in Parkinson's disease (PD), chromatographic buffers and storage conditions can significantly interfere with the overall structure of the protein and thus affect protein aggregation kinetics. We apply several biophysical and biochemical methods, including size exclusion chromatography (SEC), dynamic light scattering (DLS), and atomic force microscopy (AFM), to characterize the high molecular weight conformers formed during protein purification and storage. We further apply hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to characterize the monomeric form of aSyn and reveal a thus far unknown structural component of aSyn at the C-terminus of the protein. Furthermore, lyophilizing the protein greatly affected the overall structure of this monomeric conformer. We conclude from this study that structural polymorphism may occur under different storage conditions, but knowing the structure of the majority of the protein at the start of each experiment, as well as the factors that may influence it, may pave the way to an improved understanding of the mechanism leading to aSyn pathology in PD.

Rate of Asparagine Deamidation in a Monoclonal Antibody Correlating with Hydrogen Exchange Rate at Adjacent Downstream Residues.

Antibodies are an important class of drugs, comprising more than half of all new FDA approvals. Therapeutic antibodies must be chemically stable both in storage and in vivo, following administration to patients. Deamidation is a major degradation pathway for all natural and therapeutic proteins circulating in blood. Here, the linkage between deamidation propensity and structural dynamics is investigated by examining two antibodies with differing specificities. While both antibodies share a canonical asparagine-glycine (NG) motif in a structural loop, this is prone to deamidation in one of the antibodies but not the other. We found that the hydrogen-exchange rate at the adjacent two amides, often the autocatalytic nucleophiles in deamidation, correlated with the rate of degradation. This previously unreported observation was confirmed upon mutation to stabilize the deamidation lability via a generally applicable orthogonal engineering strategy presented here. We anticipate that the structural insight into chemical degradation in full-length monoclonal antibodies and the high-resolution hydrogen-exchange methodology used will have broad application across biochemical study and drug discovery and development.

Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.

Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.

Repeat protein engineering: creating functional nanostructures/biomaterials from modular building blocks.

There is enormous interest in molecular self-assembly and the development of biological systems to form smart nanostructures for biotechnology (so-called 'bottom-up fabrications'). Repeat proteins are ideal choices for development of such systems as they: (i) possess a relatively simple relationship between sequence, structure and function; (ii) are modular and non-globular in structure; (iii) act as diverse scaffolds for the mediation of a diverse range of protein-protein interactions; and (iv) have been extensively studied and successfully engineered and designed. In the present review, we summarize recent advances in the use of engineered repeat proteins in the self-assembly of novel materials, nanostructures and biosensors. In particular, we show that repeat proteins are excellent monomeric programmable building blocks that can be triggered to associate into a range of morphologies and can readily be engineered as stimuli-responsive biofunctional materials.

Fibrous nanostructures from the self-assembly of designed repeat protein modules.

Single-protein-chain superhelical filaments are obtained from monomeric repeat proteins by controlling the chemistry and solvent exposure at their terminal interfaces. The assembly was achieved in aqueous solution, at neutral pH value, and at room temperature. The building block was a recombinantly engineered designed tetratricopeptide repeat protein. Directed head-to-tail self-assembly was driven by genetically encoded orthogonal native chemical ligation.

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions.

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

Mechanistic insights into the rational design of masked antibodies.

Although monoclonal antibodies have greatly improved cancer therapy, they can trigger side effects due to on-target, off-tumor toxicity. Over the past decade, strategies have emerged to successfully mask the antigen-binding site of antibodies, such that they are only activated at the relevant site, for example, after proteolytic cleavage. However, the methods for designing an ideal affinity-based mask and what parameters are important are not yet well understood. Here, we undertook mechanistic studies using three masks with different properties and identified four critical factors: binding site and affinity, as well as association and dissociation rate constants, which also played an important role. HDX-MS was used to identify the location of binding sites on the antibody, which were subsequently validated by obtaining a high-resolution crystal structure for one of the mask-antibody complexes. These findings will inform future designs of optimal affinity-based masks for antibodies and other therapeutic proteins.

Engineering and exploiting synthetic allostery of NanoLuc luciferase.

Allostery enables proteins to interconvert different biochemical signals and form complex metabolic and signaling networks. We hypothesize that circular permutation of proteins increases the probability of functional coupling of new N- and C- termini with the protein's active center through increased local structural disorder. To test this we construct a synthetically allosteric version of circular permutated NanoLuc luciferase that can be activated through ligand-induced intramolecular non-covalent cyclisation. This switch module is tolerant of the structure of binding domains and their ligands, and can be used to create biosensors of proteins and small molecules. The developed biosensors covers a range of emission wavelengths and displays sensitivity as low as 50pM and dynamic range as high as 16-fold and could quantify their cognate ligand in human fluids. We apply hydrogen exchange kinetic mass spectroscopy to analyze time resolved structural changes in the developed biosensors and observe ligand-mediated folding of newly created termini.

Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity.

As an intrinsically disordered protein, monomeric alpha-synuclein (aSyn) occupies a large conformational space. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. We observe that the more exposed the N-terminus and the beginning of the NAC region of aSyn are, the more aggregation prone monomeric aSyn conformations become. Solvent exposure of the N-terminus of aSyn occurs upon release of C-terminus interactions when calcium binds, but the level of exposure and aSyn's aggregation propensity is sequence and post translational modification dependent. Identifying aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein.

Conformational dynamics of the molecular chaperone Hsp90 in complexes with a co-chaperone and anticancer drugs.

The molecular chaperone Hsp90 is essential for the correct folding, maturation and activation of a diverse array of client proteins, including several key constituents of oncogenic processes. Hsp90 has become a focus of cancer research, since it represents a target for direct prophylaxis against multistep malignancy. Hydrogen-exchange mass spectrometry was used to study the structural and conformational changes undergone by full-length human Hsp90beta in solution upon binding of the kinase-specific co-chaperone Cdc37 and two Hsp90 ATPase inhibitors: Radicicol and the first-generation anticancer drug DMAG. Changes in hydrogen exchange pattern in the complexes in regions of Hsp90 remote to the ligand-binding site were observed indicating long-range effects. In particular, the interface between the N-terminal domain and middle domains exhibited significant differences between the apo and complexed forms. For the inhibitors, differences in the interface between the middle domain and the C-terminal domain were also observed. These data provide important insight into the structure of the biologically active form of the protein.

C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction.

Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.

Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.

Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.

Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase.

Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp-Phe-Gly (DFG) motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so-called 'DFG flip'. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a 'DFG-out' state. Here we use conformationally selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the αC-ß4 loop and 'molecular brake' regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1.

Understanding the relationship between biotherapeutic protein stability and solid-liquid interfacial shear in constant region mutants of IgG1 and IgG4.

Relative stability of therapeutic antibody candidates is currently evaluated primarily through their response to thermal degradation, yet this technique is not always predictive of stability in manufacture, shipping, and storage. A rotating disk shear device is proposed that produces defined shear conditions at a known solid-liquid interface to measure stability in this environment. Five variants of IgG1 and IgG4 antibodies were created using combinations of two discrete triple amino acid sequence mutations denoted TM and YTE. Antibodies were ranked for stability based on shear device output (protein decay coefficient, PDC), and compared with accelerated thermal stability data and the melting temperature of the CH2 domain (Tm 1) from differential scanning calorimetry to investigate technique complimentarity. Results suggest that the techniques are orthogonal, with thermal methods based on intramolecular interaction and shear device stability based on localized unfolding revealing less stable regions that drive aggregation. Molecular modeling shows the modifications' effects on the antibody structures and indicates a possible role for Fc conformation and Fab-Fc docking in determining suspended protein stability. The data introduce the PDC value as an orthogonal stability indicator, complementary to traditional thermal methods, allowing lead antibody selection based on a more full understanding of process stability.