Amide Hydrogen-deuterium Exchange Research Articles

Asymmetric viral genomic RNA egress from a spring-loaded virus through rubonucleoprotein rearrangements.

RNA viruses are metastable macromolecular assemblies that sense their environment through dynamic breathing motions. While surfaces of viral particles have been well characterized by cryo-EM, dynamic genome-capsid at the virus’ interior are poorly understood. To address this gap in understanding, we have combined cryo-EM with amide hydrogen deuterium exchange mass spectrometry (HDXMS) on turnip crinkle virus (TCV) as a model system to probe its RNA genome-capsid interface and overall energetics of the virus particle across all stages of programmed disassembly.

Perturbation-HDX reveals glycosylation sites as weakest links in dengue capsid quaternary structure.

Dengue virus (DENV) is a mosquito-borne, icosahedral flavivirus that represents a significant threat to human health worldwide. Vaccine development for dengue has long eluded scientists due to serotype and strain diversity associated with altered viral capsid dynamics. Despite being largely conserved, dengue strains show distinctive reversible fluctuations referred to as ‘breathing’. DENV capsids have T=3 icosahedral symmetry, consisting of 180 copies of out envelope (E) protein. E-protein dimer contacts confer stability of the native virus particle. To localize and rank the strengths of E-protein interfaces on the surface, we carried out urea and temperature perturbation of DENV particles analyzed using amide hydrogen-deuterium exchange mass spectrometry. Our results highlight the weakest links on the virion surface. These sites are spanning the glycosylation sites of dengue. We have isolated and characterized the viable deglycosylated mutant which shows a different temperature stability profile. These sensitive sites are prime targets for drug development and predict sites that may change due to the selective pressure that impact viral evolution.

Protein Kinase A Catalytic subunit:Smoothened interface ‐ Role of a parallel holoenzyme in negative feedback in Hedgehog Signaling

During signal transduction in the Hedgehog pathway, the G protein‐coupled receptor (GPCR) Smoothened (SMO) signals intracellularly via a novel mechanism involving sequestration of the Protein Kinase A (PKA) catalytic (C)‐subunit at the membrane. Activation of Hedgehog signaling leads to negative feedback regulation mediated through inhibition of PKA‐C by SMO at the ciliary membrane. While it is known that PKA‐C interacts with SMO at its C‐terminal proximal cytoplasmic tail domain (pCT), the dynamics of PKA‐C:SMO interactions is poorly understood. Here, we report a map of the PKA‐C:SMO interface by Amide Hydrogen Deuterium Exchange Mass spectrometry (HDXMS) over 1‐10 min timescales. SMO binding on PKA‐C resulted in global decreases in deuterium exchange (Dex). Peptides spanning the kinase activation loop (188‐198), P+1 loop (199‐211) and pseudo‐substrate recognition site between β5 and αD (129‐145) showed significant protection (>0.5 Da) when bound to SMO, identifying these regions as the SMO binding interface on PKA‐C. Interestingly, the footprint of SMO binding on PKA‐C was identical to that of the RIa regulatory subunit in PKA‐I holoenzymes. Furthermore, increased Dex (0.4 Da) was observed at the region spanning αC and β4 (83‐100), indicating that SMO binding destabilizes the active kinase conformation to allosterically inhibit PKA‐C. Correspondingly on SMO, PKA‐C binding elicited only low magnitudes of Dex protection (0.4 Da) in regions flanking the pseudo substrate site (573‐590, 622‐631). Increased Dex (0.4 Da) was observed at the sterol binding pocket (96‐112) in the extracellular Cysteine rich domain, indicating that PKA‐C binding may allosterically impact SMO activation by modulating its ability to bind sterols. A significant increase in Dex (0.9 Da) was observed at SMO helix 8 (537‐546) which is positioned parallel to the membrane, indicating that C binding promoted a disordered helix 8 conformation. We speculate that the pCT exists in a membrane‐bound or folded conformation in free SMO, but PKA‐C interaction switches the binding mode of pCT leading to a significantly Dex‐protected state. Thus, PKA‐C:SMO interactions reveal an unexpected intersection between cyclic nucleotide signaling and the Hedgehog pathway, defining a novel mechanism of PKA‐C regulation beyond the canonical PKA signalosome.

Proteins' Evolution upon Point Mutations.

As the reader must be already aware, state-of-the-art protein folding prediction methods have reached a smashing success in their goal of accurately determining the three-dimensional structures of proteins. Yet, a solution to simple problems such as the effects of protein point mutations on their (i) native conformation; (ii) marginal stability; (iii) ensemble of high-energy nativelike conformations; and (iv) metamorphism propensity and, hence, their evolvability, remains as an unsolved problem. As a plausible solution to the latter, some properties of the amide hydrogen-deuterium exchange, a highly sensitive probe of the structure, stability, and folding of proteins, are assessed from a new perspective. The preliminary results indicate that the protein marginal stability change upon point mutations provides the necessary and sufficient information to estimate, through a Boltzmann factor, the evolution of the amide hydrogen exchange protection factors and, consequently, that of the ensemble of folded conformations coexisting with the native state. This work contributes to our general understanding of the effects of point mutations on proteins and may spur significant progress in our efforts to develop methods to determine the appearance of new folds and functions accurately.

Design for Solubility May Reveal Induction of Amide Hydrogen/Deuterium Exchange by Protein Self-Association

Structural heterogeneity often constrains the characterization of aggregating proteins to indirect or low-resolution methods, obscuring mechanistic details of association. Here, we report progress in understanding the aggregation of Adnectins, engineered binding proteins with an immunoglobulin-like fold. We rationally design Adnectin solubility and measure amide hydrogen/deuterium exchange (HDX) under conditions that permit transient protein self-association. Protein–protein binding commonly slows rates of HDX; in contrast, we find that Adnectin association may induce faster HDX for certain amides, particularly in the C-terminal β-strand. In aggregation-prone proteins, we identify a pattern of very different rates of amide HDX for residues linked by reciprocal hydrogen bonds in the native structure. These results may be explained by local loss of native structure and formation of an inter-protein interface. Amide HDX induced by self-association, detected here by deliberate modulation of propensity for such interactions, may be a general phenomenon with the potential to expose mechanisms of aggregation by diverse proteins.

Achieving a realistic native protein ensemble by HDX-MS and computational modeling

Amide hydrogen-deuterium exchange (HDX) measurements pioneered in the 1950s and developed extensively by Englander in the 1980s (1), still give us more information than we understand about protein motions. Some backbone amides exchange within milliseconds, whereas others may take months. How amides exchange in a folded protein and why some exchange so quickly and others so slowly has puzzled biophysicists for several generations. What exactly are the underlying physicochemical phenomena that result in amide exchange and how do the rates of exchange relate to structural dynamics? Early investigators, including Ken Dill and Hue Sun Chan, worked in statistical mechanical lattice models of proteins to try to answer this question (2,3).

High Resolution Structural Analysis Of ALS‐Associated Mutant SOD1 Inclusion Bodies

Inclusion bodies (IBs) – insoluble structures that can form upon overexpression of proteins- have wide relevance in research, industrial settings, and disease. In addition to their use in protein purification, IBs provide a tractable model to elucidate the molecular mechanisms of protein aggregation in cells, as also occurs in neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Parkinson's, and Alzheimer's diseases. The aggregation of Cu, Zn superoxide dismutase (SOD1) variants is associated with ALS, yet the relationships between mutant characteristics and disease properties remain obscure. Here, we report a systematic investigation of SOD1 IBs using two powerful complementary methods to analyze the structures of these cellular aggregates and their changes upon mutation. Quenched amide hydrogen-deuterium exchange (HDX) measurements by NMR for individual residues throughout SOD1 reveal IB structural features and similarities, whereas quantitative conformation specific antibody binding assays highlight notable differences in surface features between mutant IBs. Taken together, these data provide a valuable new high-resolution view of cellular aggregation. These powerful HDX and quantitative antibody binding methods are widely applicable to other proteins to define the molecular determinants of their aggregation and solubility.

Protein Structure Prediction from NMR Hydrogen-Deuterium Exchange Data.

Amide hydrogen-deuterium exchange (HDX) has long been used to determine regional flexibility and binding sites in proteins; however, the data are too sparse for full structural characterization. Experiments that measure HDX rates, such as HDX-NMR, have far higher throughput compared to structure determination via X-ray crystallography, cryo-EM, or a full suite of NMR experiments. Data from HDX-NMR experiments encode information on the protein structure, making HDX a prime candidate to be supplemented by computational algorithms for protein structure prediction. We have developed a methodology to incorporate HDX-NMR data into ab initio protein structure prediction using the Rosetta software framework to predict structures based on experimental agreement. To demonstrate the efficacy of our algorithm, we examined 38 proteins with HDX-NMR data available, comparing the predicted model with and without the incorporation of HDX data into scoring. The root-mean-square deviation (rmsd, a measure of the average atomic distance between superimposed models) of the predicted model improved by 1.42 Å on average after incorporating the HDX-NMR data into scoring. The average rmsd improvement for the proteins where the selected model rmsd changed after incorporating HDX data was 3.63 Å, including one improvement of more than 11 Å and seven proteins improving by greater than 4 Å, with 12/15 proteins improving overall. Additionally, for independent verification, two proteins that were not part of the original benchmark were scored including HDX data, with a dramatic improvement of the selected model rmsd of nearly 9 Å for one of the proteins. Moreover, we have developed a confidence metric allowing us to successfully identify near-native models in the absence of a native structure. Improvement in model selection with a strong confidence measure demonstrates that protein structure prediction with HDX-NMR is a powerful tool which can be performed with minimal additional computational strain and expense.

HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1.

Annexin-A1 (ANXA1) belongs to a class of highly homologous Ca2+-dependent phospholipid-binding proteins. Its structure consists of a core region composed of four homologous repeats arranged in a compact, hydrolysis-resistant structure and an N-terminal region with a Ca2+-dependent conformation. ANXA1 is involved in several processes, including cell proliferation, apoptosis, metastasis, and the inflammatory response. Therefore, the development of antibodies blocking selected regions on ANXA1 holds great potential for the development of novel therapeutics treating inflammatory and cancer diseases. Here, we report the interaction site between an ANXA1-specific antibody known to inhibit T cell activation without adverse cytotoxic effects and ANXA1 using amide hydrogen–deuterium exchange mass spectrometry (HDX-MS). For the epitope determination, we applied two bottom-up HDX-MS approaches with pepsin digestion in solution and immobilized on beads. Both strategies revealed the interaction region within domain III of ANXA1 in Ca2+-bound conformation. The antibody-binding region correlates with the hydrophobic binding pocket of the N-terminal domain formed in the absence of calcium. This study demonstrates that even cryptic and flexible binding regions can be studied by HDX-MS, allowing a fast and efficient determination of the binding sites of antibodies which will help to define a mode of action profile for their use in therapy.

SARS-CoV-2 S protein:ACE2 interaction reveals novel allosteric targets.

The spike (S) protein is the main handle for SARS-CoV-2 to enter host cells via surface angiotensin-converting enzyme 2 (ACE2) receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, using amide hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations, we have mapped the S:ACE2 interaction interface and uncovered long-range allosteric propagation of ACE2 binding to sites necessary for host-mediated proteolysis of S protein, critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 Å away while dampening dynamics of the stalk hinge (central helix and heptad repeat [HR]) regions ~130 Å away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the prefusion state. Our findings provide a dynamics map of the S:ACE2 interface in solution and also offer mechanistic insights into how ACE2 binding is allosterically coupled to distal proteolytic processing sites and viral-host membrane fusion. Thus, protease docking sites flanking the S1/S2 cleavage site represent alternate allosteric hotspot targets for potential therapeutic development.

Fibrillation of Human Calcitonin and Its Analogs: Effects of Phosphorylation and Disulfide Reduction

Some therapeutic peptides self-assemble in solution to form ordered, insoluble, β-sheet-rich amyloid fibrils. This physical instability can result in reduced potency, cause immunogenic side effects, and limit options for formulation. Understanding the mechanisms of fibrillation is key to developing rational mitigation strategies. Here, amide hydrogen-deuterium exchange with mass spectrometric analysis (HDX-MS) coupled with proteolytic digestion was used to identify the early stage interactions leading to fibrillation of human calcitonin (hCT), a peptide hormone important in calcium metabolism. hCT fibrillation kinetics was sigmoidal, with lag, growth, and plateau phases as shown by thioflavin T and turbidity measurements. HDX-MS of fibrillating hCT (pH 7.4; 25°C) suggested early involvement of the N-terminal (1–11) and central (12–19) fragments in interactions during the lag phase, whereas C-terminal fragments (20–32 and 26–32) showed limited involvement during this period. The residue-level information was used to develop phosphorylated hCT analogs that showed modified fibrillation that depended on phosphorylation site. Phosphorylation in the central region resulted in complete inhibition of fibrillation for the phospho-Thr-13 hCT analog, whereas phosphorylation in the N-terminal and C-terminal regions inhibited but did not prevent fibrillation. Reduction of the Cys1-Cys7 disulfide bond resulted in faster fibrillation with involvement of different hCT residues as indicated by pulsed HDX-MS. Together, the results demonstrate that small structural changes have significant effects on hCT fibrillation and that understanding these effects can inform the rational development of fibrillation-resistant hCT analogs.

Synergistic Allostery in Multiligand-Protein Interactions

Amide hydrogen-deuterium exchange mass spectrometry is powerful for describing combinatorial coupling effects of a cooperative ligand pair binding at noncontiguous sites: adenosine at the ATP-pocket and a docking peptide (PIFtide) at the PIF-pocket, on a model protein kinase PDK1. Binding of two ligands to PDK1 reveal multiple hotspots of synergistic allostery with cumulative effects greater than the sum of individual effects mediated by each ligand. We quantified this synergism and ranked these hotspots using a difference in deuteration-based approach, which showed that the strongest synergistic effects were observed at three of the critical catalytic loci of kinases: the αB-αC helices, and HRD-motif loop, and DFG-motif. Additionally, we observed weaker synergistic effects at a distal GHI-subdomain locus. Synergistic changes in deuterium exchange observed at a distal site but not at the intermediate sites of the large lobe of the kinase reveals allosteric propagation in proteins to operate through two modes. Direct electrostatic interactions between polar and charged amino acids that mediate targeted relay of allosteric signals, and diffused relay of allosteric signals through soft matter-like hydrophobic core amino acids. Furthermore, we provide evidence that the conserved β-3 strand lysine of protein kinases (Lys111 of PDK1) functions as an integrator node to coordinate allosteric coupling of the two ligand-binding sites. It maintains indirect interactions with the ATP-pocket and mediates a critical salt bridge with a glutamate (Glu130) of αC helix, which is conserved across all kinases. In summary, allosteric propagation in cooperative, dual-liganded enzyme targets is bidirectional and synergistic and offers a strategy for combinatorial drug development.

DECA, A Comprehensive, Automatic Post-processing Program for HDX-MS Data*

Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) has become widely popular for mapping protein-ligand interfaces, for understanding protein-protein interactions, and for discovering dynamic allostery. Several platforms are now available which provide large data sets of amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) data. Although many of these platforms provide some down-stream processing, a comprehensive software that provides the most commonly used down-stream processing tools such as automatic back-exchange correction options, analysis of overlapping peptides, calculations of relative deuterium uptake into regions of the protein after such corrections, rigorous statistical analysis of the significance of uptake differences, and generation of high quality figures for data presentation is not yet available. Here we describe the Deuterium Exchange Correction and Analysis (DECA) software package, which provides all these downstream processing options for data from the most popular mass spectrometry platforms. The major functions of the software are demonstrated on sample data.

HDX-MS reveals orthosteric and allosteric changes in apolipoprotein-D structural dynamics upon binding of progesterone.

Apolipoprotein-D is a glycosylated tetrameric lipocalin that binds and transports small hydrophobic molecules such as progesterone and arachidonic acid. Like other lipocalins, apolipoprotein-D adopts an eight-stranded β-barrel fold stabilized by two intramolecular disulphide bonds, with an adjacent α-helix. Crystallography studies of recombinant apolipoprotein-D demonstrated no major conformational changes upon progesterone binding. Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) reports structural changes of proteins in solution by monitoring exchange of amide hydrogens in the protein backbone with deuterium. HDX-MS detects changes in conformation and structural dynamics in response to protein function such as ligand binding that may go undetected in X-ray crystallography, making HDX-MS an invaluable orthogonal technique. Here, we report an HDX-MS protocol for apolipoprotein-D that solved challenges of high protein rigidity and low pepsin cleavage using rigorous quenching conditions and longer deuteration times, yielding 85% sequence coverage and 50% deuterium exchange. The relative fractional deuterium exchange of ligand-free apolipoprotein-D revealed apolipoprotein-D to be a highly structured protein. Progesterone binding was detected by significant reduction in deuterium exchange in eight peptides. Stabilization of apolipoprotein-D dynamics can be interpreted as a combined orthosteric effect in the ligand binding pocket and allosteric effect at the N-terminus and C-terminus. Together, our experiments provide insight into apolipoprotein-D structural dynamics and map the effects of progesterone binding that are relayed to distal parts of the protein. The observed stabilization of apolipoprotein-D dynamics upon progesterone binding demonstrates a common behaviour in the lipocalin family and may have implications for interactions of apolipoprotein-D with receptors or lipoprotein particles. Statement: We reveal for the first time how apolipoprotein-D, which is protective in Alzheimer's disease, becomes more ordered when bound to a molecule of steroid hormone. These results significantly extend the understanding of apolipoprotein-D structure from X-ray crystallography studies by incorporating information on how protein motion changes over time. To achieve these results an improved protocol was developed, suitable for proteins similar to apolipoprotein-D, to elucidate how proteins change flexibility when binding to small molecules.

Uncovering metastability and disassembly hotspots in whole viral particles

Viruses are metastable macromolecular assemblies that toggle between multiple conformational states through molecular rearrangements that are critical for mediating viral host entry. Viruses respond to different host specific environmental cues to form disassembly intermediates for the eventual release of genomic material required for replication. Although static snapshots of these intermediates have been captured through structural techniques such as X-ray crystallography and cryo-EM, the mechanistic details of these conformational rearrangements underpinning viral metastability have been poorly understood. Amide hydrogen deuterium exchange mass spectrometry (HDXMS) is a powerful tool that measures hydrogen bonding propensities to probe changes in the dynamics of different macromolecular interactions. Chaotropic agents such as urea can be used to disrupt hydrogen bonds between different subunits, thereby ranking regions of the virus that are critical in maintaining viral stability. By controlled urea denaturation with HDXMS, we have identified specific loci in a Turnip Crinkle Virus (TCV) model showing increased deuterium exchange with even minimally disruptive concentrations of urea. These loci represent dynamic disassembly hotspots. These hotspots are predominantly present at the quaternary contacts at the 3-fold and 5-fold axes. This approach can be applied to detect vulnerabilities in virus icosahedral structures to uncover the molecular mechanism of viral disassembly.

Proapoptotic Cyclic Peptide BC71 Targets Cell-Surface GRP78 and Functions as an Anticancer Therapeutic in Mice

Glucose regulated protein 78 kDa (GRP78) is a recently emerged target for cancer therapy and a biomarker for cancer prognosis. Overexpression of GRP78 is observed in many types of cancers, with the cell-surface GRP78 being preferentially present in cancer cells and cancer blood vessel endothelial cells. Isthmin (ISM) is a secreted high-affinity proapoptotic protein ligand of cell-surface GRP78 that suppresses angiogenesis and tumor growth in mice. The C-terminal AMOP (adhesion-associated domain in MUC4 and other proteins) domain of ISM is critical in mediating its interaction with human umbilical vein endothelial cells (HUVECs). In this work, we report novel cyclic peptides harboring the RKD motif in the ISM AMOP domain that function as proapoptotic ligands of cell-surface GRP78. The most potent peptide, BC71, binds to GRP78 and converge to tumor in mice. Intravenous administration of BC71 suppressed xenograft tumor growth in mice as a single agent, with significant reduction in tumor angiogenesis and upsurge in apoptosis. Fluorescent-labeled BC71 accumulates in tumor in mice by targeting cell-surface GRP78. We show that BC71 triggers apoptosis via cell-surface GRP78 and activates caspase-8 and p53 signaling pathways in HUVECs. Using amide hydrogen-deuterium exchange mass spectrometry (HDXMS), we identified that BC71 preferentially binds to ATP-bound GRP78 via amino acid residues 244–257 of GRP78. Hence, BC71 serves as a valuable prototype for further development of peptidomimetic anticancer drugs targeting cell-surface GRP78 as well as PET imaging agents for cancer prognosis.

Partial Intrinsic Disorder Governs the Dengue Capsid Protein Conformational Ensemble.

The 11 kDa, positively charged dengue capsid protein (C protein) exists stably as a homodimer and colocalizes with the viral genome within mature viral particles. Its core is composed of four alpha helices encompassing a small hydrophobic patch that may interact with lipids, but approximately 20% of the protein at the N-terminus is intrinsically disordered, making it challenging to elucidate its conformational landscape. Here, we combine small-angle X-ray scattering (SAXS), amide hydrogen-deuterium exchange mass spectrometry (HDXMS), and atomic-resolution molecular dynamics (MD) simulations to probe the dynamics of dengue C proteins. We show that the use of MD force fields (FFs) optimized for intrinsically disordered proteins (IDPs) is necessary to capture their conformational landscape and validate the computationally generated ensembles with reference to SAXS and HDXMS data. Representative ensembles of the C protein dimer are characterized by alternating, clamp-like exposure and occlusion of the internal hydrophobic patch, as well as by residual helical structure at the disordered N-terminus previously identified as a potential source of autoinhibition. Such dynamics are likely to determine the multifunctionality of the C protein during the flavivirus life cycle and hence impact the design of novel antiviral compounds.

Role of viral RNA in virion stability and disassembly

Viruses are metastable macromolecular entities that are assembled into compact particles. Entry into a target host cell initiates quaternary conformational changes in response to distinct host‐specific environmental changes including temperature, pH, osmolality, divalent cation concentrations, which promote disassembly and release of the virion's genome into the host cell. This host stimulus‐triggered disassembly is critical for initiating the infectious phase of the particle. How viruses use their quaternary coat protein structures as thermodynamic sensors for detecting entry into a conducive host cell environment is largely unknown. The process of viral disassembly is largely impervious to static structural methods as these only capture end‐state conformations. Amide Hydrogen Deuterium Exchange Mass Spectrometry (HDXMS) can be used to monitor whole viral particle dynamics in solution. The disassembling virion represents the virus at its most vulnerable state and is consequently a transient dynamic intermediate. A map of the quaternary structural changes in this state offers critical insights into cryptic epitopes as novel vaccine targets. Our research has pioneered structural mass spectrometry characterization of temperature and divalent cation‐dependent conformational changes on Dengue virus and Turnip Crinkle Virus (TCV) and mapped the specific disassembly processes in these two model virus systems. We have extended these studies to deconstruct the whole viral particle‐antibody (DENV2‐2D22) complex. Our results reveal the high intrinsic dynamics of the RNA packing capsid C‐protein which underpins viral transition from compact to expanded states in the process of disassembly. We have also uncovered new disassembly intermediates triggered by varying monovalent and divalent cation concentrations. Viral disassembly processes in TCV and Dengue reveal conformations of disassembly intermediates which represent powerful new targets for therapeutic antibody design as well as offer fundamental insights into strategically timed viral disassembly processes which can be extended to all viruses.Support or Funding InformationSingapore Ministry of Education Tier 3 grantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Changes in Protein Dynamics in Escherichia coli SufS Reveal a Possible Conserved Regulatory Mechanism in Type II Cysteine Desulfurase Systems.

In the Suf Fe-S cluster assembly pathway, the activity of the cysteine desulfurase, SufS, is regulated by interactions with the accessory sulfotransferase protein, SufE. SufE has been shown to stimulate SufS activity, likely by inducing conformational changes in the SufS active site that promote the desulfurase step and by acting as an efficient persulfide acceptor in the transpersulfuration step. Previous results point toward an additional level of regulation through a "half-sites" mechanism that affects the stoichiometry and affinity for SufE as the dimeric SufS shifts between desulfurase and transpersulfuration activities. Investigation of the covalent persulfide intermediate of SufS by backbone amide hydrogen-deuterium exchange mass spectrometry identified two active site peptides (residues 225-236 and 356-366) and two peptides at the dimer interface of SufS (residues 88-100 and 243-255) that exhibit changes in deuterium uptake upon formation of the intermediate. Residues in these peptides are organized to form a conduit between the two active sites upon persulfide formation and include key cross-monomer interactions, suggesting they may play a role in the half-sites regulation. Three evolutionarily conserved residues at the dimer interface (R92, E96, and E250) were investigated by alanine scanning mutagenesis. Two of the substituted enzymes (E96A and E250A SufS) resulted in 6-fold increases in the value of KSufE, confirming a functional role. Re-examination of the dimer interface in reported crystal structures of SufS and the SufS homologue CsdA identified previously unnoticed residue mobility at the dimer interface. The identification of conformational changes at the dimer interface by hydrogen-deuterium exchange confirmed by mutagenesis and structural reports provides a physical mechanism for active site communication in the half-sites regulation of SufS activity. Given the conservation of the interface interactions, this mechanism may be broadly applicable to type II cysteine desulfurase systems.

Hydrogen deuterium exchange reveals changes to protein dynamics of recombinant human erythropoietin upon N- and O- desialylation

Recombinant human erythropoietin (EPO) is a therapeutic glycoprotein widely used for treating anemia. EPO glycans carry extensive sialylation and the level of the modification is known to affect receptor binding, protein stability and pharmacokinetics. Nonetheless, a detailed understanding of the effects of sialylation on EPO conformation and dynamics is still lacking. Here we investigate the changes to EPO dynamics following enzymatic trimming of terminal sialic acid by amide hydrogen deuterium exchange mass spectrometry (HDX-MS). The results revealed that desialylation enhances structural flexibility near the glycosylation sites, with greater effects observed around the O-glycosylation site relative to the N-glycosylation sites. The affected regions are surface-exposed loops connecting the helix bundle, which do not appear to reduce the thermostability of the molecule as revealed from melting measurement. Our findings demonstrate the feasibility of HDX-MS technique in deciphering the function of specific type of glycosylation that can provide novel insights into the role of sialylation on protein therapeutics.