Biochemical characterisation of class III biotin protein ligases from Botrytis cinerea and Zymoseptoria tritici.

Biotin protein ligase (BPL) is an essential enzyme in all kingdoms of life, making it a potential target for novel anti-infective agents. Whilst bacteria and archaea have simple BPL structures (class I and II), the homologues from certain eukaryotes such as mammals, insects and yeast (class III) have evolved a more complex structure with a large extension on the N-terminus of the protein in addition to the conserved catalytic domain. The absence of atomic resolution structures of any class III BPL hinders structural and functional analysis of these enzymes. Here, two new class III BPLs from agriculturally important moulds Botrytis cinerea and Zymoseptoria tritici were characterised alongside the homologue from the prototypical yeast Saccharomyces cerevisiae. Circular dichroism and ion mobility-mass spectrometry analysis revealed conservation of the overall tertiary and secondary structures of all three BPLs, corresponding with the high sequence similarity. Subtle structural differences were implied by the different thermal stabilities of the enzymes and their varied Michaelis constants for their interactions with ligands biotin, MgATP, and biotin-accepting substrates from different species. The three BPLs displayed different preferences for fungal versus bacterial protein substrates, providing further evidence that class III BPLs have a 'substrate validation' activity for selecting only appropriate proteins for biotinylation. Selective, potent inhibition of these three BPLs was demonstrated despite sequence and structural homology. This highlights the potential for targeting BPL for novel, selective antifungal therapies against B. cinerea, Z. tritici and other fungal species.

Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches.

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88-99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.

Structure-functional changes in eNAMPT at high concentrations mediate mouse and human beta cell dysfunction in type 2 diabetes.

AIMS/HYPOTHESIS: Progressive decline in functional beta cell mass is central to the development of type 2 diabetes. Elevated serum levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) are associated with beta cell failure in type 2 diabetes and eNAMPT immuno-neutralisation improves glucose tolerance in mouse models of diabetes. Despite this, the effects of eNAMPT on functional beta cell mass are poorly elucidated, with some studies having separately reported beta cell-protective effects of eNAMPT. eNAMPT exists in structurally and functionally distinct monomeric and dimeric forms. Dimerisation is essential for the NAD-biosynthetic capacity of NAMPT. Monomeric eNAMPT does not possess NAD-biosynthetic capacity and may exert distinct NAD-independent effects. This study aimed to fully characterise the structure-functional effects of eNAMPT on pancreatic beta cell functional mass and to relate these to beta cell failure in type 2 diabetes. METHODS: CD-1 mice and serum from obese humans who were without diabetes, with impaired fasting glucose (IFG) or with type 2 diabetes (from the Body Fat, Surgery and Hormone [BodyFatS&H] study) or with or at risk of developing type 2 diabetes (from the VaSera trial) were used in this study. We generated recombinant wild-type and monomeric eNAMPT to explore the effects of eNAMPT on functional beta cell mass in isolated mouse and human islets. Beta cell function was determined by static and dynamic insulin secretion and intracellular calcium microfluorimetry. NAD-biosynthetic capacity of eNAMPT was assessed by colorimetric and fluorescent assays and by native mass spectrometry. Islet cell number was determined by immunohistochemical staining for insulin, glucagon and somatostatin, with islet apoptosis determined by caspase 3/7 activity. Markers of inflammation and beta cell identity were determined by quantitative reverse transcription PCR. Total, monomeric and dimeric eNAMPT and nicotinamide mononucleotide (NMN) were evaluated by ELISA, western blot and fluorometric assay using serum from non-diabetic, glucose intolerant and type 2 diabetic individuals. RESULTS: eNAMPT exerts bimodal and concentration- and structure-functional-dependent effects on beta cell functional mass. At low physiological concentrations (~1 ng/ml), as seen in serum from humans without diabetes, eNAMPT enhances beta cell function through NAD-dependent mechanisms, consistent with eNAMPT being present as a dimer. However, as eNAMPT concentrations rise to ~5 ng/ml, as in type 2 diabetes, eNAMPT begins to adopt a monomeric form and mediates beta cell dysfunction, reduced beta cell identity and number, increased alpha cell number and increased apoptosis, through NAD-independent proinflammatory mechanisms. CONCLUSIONS/INTERPRETATION: We have characterised a novel mechanism of beta cell dysfunction in type 2 diabetes. At low physiological levels, eNAMPT exists in dimer form and maintains beta cell function and identity through NAD-dependent mechanisms. However, as eNAMPT levels rise, as in type 2 diabetes, structure-functional changes occur resulting in marked elevation of monomeric eNAMPT, which induces a diabetic phenotype in pancreatic islets. Strategies to selectively target monomeric eNAMPT could represent promising therapeutic strategies for the treatment of type 2 diabetes.

MhuD from Mycobacterium tuberculosis: Probing a Dual Role in Heme Storage and Degradation.

The Mycobacterium tuberculosis (Mtb) heme oxygenase MhuD liberates free iron by degrading heme to the linear tetrapyrrole mycobilin. The MhuD dimer binds up to two hemes within the active site of each monomer. Binding the first solvent-exposed heme allows heme degradation and releases free iron. Binding a second heme renders MhuD inactive, allowing heme storage. Native-mass spectrometry revealed little difference in binding affinity between solvent-exposed and solvent-protected hemes. Hence, diheme-MhuD is formed even when a large proportion of the MhuD population is in the apo form. Apomyoglobin heme transfer assays showed MhuD-diheme dissociation is far slower than monoheme dissociation at ∼0.12 min-1 and ∼0.25 s-1, respectively, indicating that MhuD has a strong affinity for diheme. MhuD has not evolved to preferentially occupy the monoheme form and, through formation of a diheme complex, it functions as part of a larger network to tightly regulate both heme and iron levels in Mtb.

Hybrid mass spectrometry methods reveal lot-to-lot differences and delineate the effects of glycosylation on the tertiary structure of Herceptin®.

To quantify the measurable variations in the structure of a biopharmaceutical product we systematically evaluate three lots of Herceptin®, two mAb standards and an intact Fc-hinge fragment. Each mAb is examined in three states; glycan intact, truncated (following endoS2 treatment) and fully deglycosylated. Despite equivalence at the intact protein level, each lot of Herceptin® gives a distinctive signature in three different mass spectrometry approaches. Ion mobility mass spectrometry (IM-MS) shows that in the API, the attached N-glycans reduce the conformational spread of each mAb by 10.5-25%. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) data support this, with lower global deuterium uptake in solution when comparing intact to the fully deglycosylated protein. HDX-MS and activated IM-MS map the influence of glycans on the mAb and reveal allosteric effects which extend far beyond the Fc domains into the Fab region. Taken together, these findings and the supplied interactive data sets establish acceptance criteria with application for MS based characterisation of biosimilars and novel therapeutic mAbs.

Uncoupling conformational states from activity in an allosteric enzyme.

ATP-phosphoribosyltransferase (ATP-PRT) is a hexameric enzyme in conformational equilibrium between an open and seemingly active state and a closed and presumably inhibited form. The structure-function relationship of allosteric regulation in this system is still not fully understood. Here, we develop a screening strategy for modulators of ATP-PRT and identify 3-(2-thienyl)-L-alanine (TIH) as an allosteric activator of this enzyme. Kinetic analysis reveals co-occupancy of the allosteric sites by TIH and L-histidine. Crystallographic and native ion-mobility mass spectrometry data show that the TIH-bound activated form of the enzyme closely resembles the inhibited L-histidine-bound closed conformation, revealing the uncoupling between ATP-PRT open and closed conformations and its functional state. These findings suggest that dynamic processes are responsible for ATP-PRT allosteric regulation and that similar mechanisms might also be found in other enzymes bearing a ferredoxin-like allosteric domain.Active and inactive state ATP-phosphoribosyltransferases (ATP-PRTs) are believed to have different conformations. Here the authors show that in both states, ATP-PRT has a similar structural arrangement, suggesting that dynamic alterations are involved in ATP-PRT regulation by allosteric modulators.

Charge Mediated Compaction and Rearrangement of Gas-Phase Proteins: A Case Study Considering Two Proteins at Opposing Ends of the Structure-Disorder Continuum.

Charge reduction in the gas phase provides a direct means of manipulating protein charge state, and when coupled to ion mobility mass spectrometry (IM-MS), it is possible to monitor the effect of charge on protein conformation in the absence of solution. Use of the electron transfer reagent 1,3-dicyanobenzene, coupled with IM-MS, allows us to monitor the effect of charge reduction on the conformation of two proteins deliberately chosen from opposite sides of the order to disorder continuum: bovine pancreatic trypsin inhibitor (BPTI) and beta casein. The ordered BPTI presents compact conformers for each of three charge states accompanied by narrow collision cross-section distributions (TWCCSDN2→He). Upon reduction of BPTI, irrespective of precursor charge state, the TWCCSN2→He decreases to a similar distribution as found for the nESI generated ion of identical charge. The behavior of beta casein upon charge reduction is more complex. It presents over a wide charge state range (9-28), and intermediate charge states (13-18) have broad TWCCSDN2→He with multiple conformations, where both compaction and rearrangement are seen. Further, we see that the TWCCSDN2→He of the latter charge states are even affected by the presence of radical anions. Overall, we conclude that the flexible nature of some proteins result in broad conformational distributions comprised of many families, even for single charge states, and the barrier between different states can be easily overcome by an alteration of the net charge. Graphical Abstract ᅟ.

Hybrid Mass Spectrometry Approaches to Determine How L-Histidine Feedback Regulates the Enzyzme MtATP-Phosphoribosyltransferase.

MtATP-phosphoribosyltransferase (MtATP-PRT) is an enzyme catalyzing the first step of the biosynthesis of L-histidine in Mycobacterium tuberculosis, and proposed to be regulated via an allosteric mechanism. Native mass spectrometry (MS) reveals MtATP-PRT to exist as a hexamer. Conformational changes induced by L-histidine binding and the influence of buffer pH are determined with ion mobility MS, hydrogen deuterium exchange (HDX) MS, and analytical ultracentrifugation. The experimental collision cross-section (DTCCSHe) decreases from 76.6 to 73.5 nm2 upon ligand binding at pH 6.8, which correlates to the decrease in CCS calculated from crystal structures. No such changes in conformation were found at pH 9.0. Further detail on the regions that exhibit conformational change on L-histidine binding is obtained with HDX-MS experiments. On incubation with L-histidine, rapid changes are observed within domain III, and around the active site at longer times, indicating an allosteric effect.

Use of a charge reducing agent to enable intact mass analysis of cysteine-linked antibody-drug-conjugates by native mass spectrometry.

Antibody-drug-conjugates (ADC) are a growing class of anticancer biopharmaceuticals. Conjugation of cysteine linked ADCs, requires initial reduction of mAb inter-chain disulfide bonds, as the drugs are attached via thiol chemistry. This results in the active mAb moiety being transformed from a covalently linked tetramer to non-covalently linked complexes, which hinders precise determination of drug load with LC-MS. Here, we show how the addition of the charge reducing agent triethylammonium acetate (TEAA) preserves the intact mAb structure, is well suited to the study of cysteine linked conjugates and facilitates easy drug load determination by direct infusion native MS.

Molecular Insights into the Thermal Stability of mAbs with Variable-Temperature Ion-Mobility Mass Spectrometry.

The aggregation of protein-based therapeutics such as monoclonal antibodies (mAbs) can affect the efficacy of the treatment and can even induce effects that are adverse to the patient. Protein engineering is used to shift the mAb away from an aggregation-prone state by increasing the thermodynamic stability of the native fold, which might in turn alter conformational flexibility. We have probed the thermal stability of three types of intact IgG molecules and two Fc-hinge fragments by using variable-temperature ion-mobility mass spectrometry (VT-IM-MS). We observed changes in the conformations of isolated proteins as a function of temperature (300-550 K). The observed differences in thermal stability between IgG subclasses can be rationalized in terms of changes to higher-order structural organization mitigated by the hinge region. VT-IM-MS provides insights into mAbs structural thermodynamics and is presented as a promising tool for thermal-stability studies for proteins of therapeutic interest.

Distinguishing Loss of Structure from Subunit Dissociation for Protein Complexes with Variable Temperature Ion Mobility Mass Spectrometry.

The thermal stability and strength of interactions in proteins are commonly measured using isothermal calorimetry and differential scanning calorimetry providing a measurement that averages over structural transitions that occur as the proteins melt and dissociate. Here, we apply variable temperature ion mobility mass spectrometry (VT-IM-MS) to study the effect of temperature on the stability and structure of four multimeric protein complexes. VT-IM-MS is used here to investigate the change in the conformation of model proteins, namely, transthyretin (TTR), avidin, concanavalin A (conA), and human serum amyloid P component (SAP) at elevated temperatures prior, during, and after dissociation up to 550 K. As the temperature of the buffer gas is increased from 300 to 350 K, a small decrease in the collision cross sections ((DT)CCS(He)) of protein complexes from the values at room temperature is observed, and is associated with complex compaction occurring close to the reported solution T(m). At significantly higher temperatures, each protein complex undergoes an increase in (DT)CCS(He) and in the width of arrival time distributions (ATD), which is attributed to extensive protein unfolding, prior to ejection of a highly charged monomer species. This approach allows us to decouple the distinct gas phase melting temperature (T(m)) from the temperature at which we see subunit dissociation. The thermally induced dissociation (TID) mechanism is observed to initially proceed via the so-called "typical" (CID) dissociation route. Interestingly, data collected at higher analysis temperature suggests that the TID process might be adapting more "atypical" dissociation route.

Insights into the conformations of three structurally diverse proteins: cytochrome c, p53, and MDM2, provided by variable-temperature ion mobility mass spectrometry.

Thermally induced conformational transitions of three proteins of increasing intrinsic disorder-cytochrome c, the tumor suppressor protein p53 DNA binding domain (p53 DBD), and the N-terminus of the oncoprotein murine double minute 2 (NT-MDM2)-have been studied by native mass spectrometry and variable-temperature drift time ion mobility mass spectrometry (VT-DT-IM-MS). Ion mobility measurements were carried out at temperatures ranging from 200 to 571 K. Multiple conformations are observable over several charge states for all three monomeric proteins, and for cytochrome c, dimers of significant intensity are also observed. Cytochrome c [M + 5H](5+) ions present in one conformer of CCS ∼1200 Å(2), undergoing compaction in line with the reported Tmelt = 360.15 K before slight unfolding at 571 K. The more extended [M + 7H](7+) cytochrome c monomer presents as two conformers undergoing similar compaction and structural rearrangements, prior to thermally induced unfolding. The [D + 11H](11+) dimer presents as two conformers, which undergo slight structural compaction or annealing before dissociation. p53 DBD follows a trend of structural collapse before an increase in the observed collision cross section (CCS), akin to that observed for cytochrome c but proceeding more smoothly. At 300 K, the monomeric charge states present in two conformational families, which compact to one conformer of CCS ∼1750 Å(2) at 365 K, in line with the low solution Tmelt = 315-317 K. The protein then extends to produce either a broad unresolved CCS distribution or, for z > 9, two conformers. NT-MDM2 exhibits a greater number of structural rearrangements, displaying charge-state-dependent unfolding pathways. DT-IM-MS experiments at 200 K resolve multiple conformers. Low charge state species of NT-MDM2 present as a single compact conformational family centered on CCS ∼1250 Å(2) at 300 K. This undergoes conformational tightening in line with the solution Tmelt = 348 K before unfolding at the highest temperatures. The more extended charge states present in two or more conformers at room temperature, undergoing thermally induced unfolding before significant structural collapse or annealing at high temperatures. Variable-temperature IM-MS is here shown to be an exciting approach to discern protein unfolding pathways for conformationally diverse proteins.

A mass-spectrometry-based framework to define the extent of disorder in proteins.

In the past decade, mass spectrometry (MS) coupled with electrospray ionization (ESI) has been extensively applied to the study of intact proteins and their complexes, often without the requirement of labels. Solvent conditions (for example, pH, ionic strength, and concentration) affect the observed desolvated species; the ease of altering such extrinsic factors renders ESI-MS an appropriate method by which to consider the range of conformational states that proteins may occupy, including natively folded, disordered and amyloid. Rotationally averaged collision cross sections of the ionized forms of proteins, provided by the combination of mass spectrometry and ion mobility (IM-MS), are also instructive in exploring conformational landscapes in the absence of solvent. Here, we ask the following question: "If the only technique you had was ESI-IM-MS, what information would it provide on the structural preferences of an unknown protein?" We have selected 20 different proteins, both monomeric and multimeric, ranging in mass from 2846 Da (melittin) to 150 kDa (Immunoglobulin G), and we consider how they are presented to a mass spectrometer under different solvent conditions. Mass spectrometery allows us to distinguish which of these proteins are structured (melittin, human beta defensin 1, truncated human lymphotactin, Cytochrome C, holo hemoglobin-α, ovalbumin, human transthyretin, avidin, bovine serum albumin, concanavalin, human serum amyloid protein, and Immunoglobulin G) from those that contain at least some regions of disorder (human lymphotactin, N-terminal p53, α-Synuclein, N-terminal MDM2, and p53 DNA binding domain) or denatured due to solvent conditions (ubiquitin, apo hemoglobin-α, apo hemoglobin-ß) by considering two experimental parameters: the range of charge states occupied by the protein (Δz) and the range of collision cross sections in which the protein is observed (ΔCCS). We also provide a simple model to predict the difference between the collision cross sections of the most compact and the most extended form of a given protein, based on the volume of the amino acids it contains. We compare these calculated parameters with experimental values. In addition, we consider the occupancy of conformations based on the intensities of ions in the mass spectra. This allows us to qualitatively predict the potential energy landscape of each protein. Our empirical approach to assess order or disorder is shown to be more accurate than the use of charge hydropathy plots, which are frequently used to predict disorder, and could provide an initial route to characterization. Finally, we present an ESI-IM-MS methodology to determine if a given protein is structured or disordered.

Dynamics of intact immunoglobulin†G explored by drift-tube ion-mobility mass spectrometry and molecular modeling.

Collision cross-sections (CCS) of immunoglobulins G1 and G4 have been determined using linear drift-tube ion-mobility mass spectrometry. Intact antibodies and Fc-hinge fragments present with a larger range of CCS than proteins of comparable size. This is rationalized with MD simulations, which indicate significant in vacuo dynamics between linked folded domains. The IgG4 subclass presents over a wider CCS range than the IgG1 subclass.

Mass spectrometry based tools to investigate protein-ligand interactions for drug discovery.

The initial stages of drug discovery are increasingly reliant on development and improvement of analytical methods to investigate protein-protein and protein-ligand interactions. For over 20 years, mass spectrometry (MS) has been recognized as providing a fast, sensitive and high-throughput methodology for analysis of weak non-covalent complexes. Careful control of electrospray ionization conditions has enabled investigation of the structure, stability and interactions of proteins and peptides in a solvent free environment. This critical review covers the use of mass spectrometry for kinetic, dynamic and structural studies of proteins and protein complexes. We discuss how conjunction of mass spectrometry with related techniques and methodologies such as ion mobility, hydrogen-deuterium exchange (HDX), protein footprinting or chemical cross-linking can provide us with structural information useful for drug development. Along with other biophysical techniques, such as NMR or X-ray crystallography, mass spectrometry provides a powerful toolbox for investigation of biological problems of medical relevance (204 references).