Mg2+ Dependence of 70 S Ribosomal Protein Flexibility Revealed by Hydrogen/Deuterium Exchange and Mass Spectrometry

The ribosome from Escherichia coli requires a specific concentration of Mg²⁺ to maintain the 70 S complex formation and allow protein synthesis, and then the structure must be stable and flexible. How does the ribosome acquire these conflicting factors at the same time? Here, we investigated the hydrogen/deuterium exchange of 52 proteins in the 70 S ribosome, which controlled stability and flexibility under various Mg²⁺ concentrations, using mass spectrometry. Many proteins exhibited a sigmoidal curve for Mg²⁺ concentration dependence, incorporating more deuterium at lower Mg²⁺ concentration. By comparing deuterium incorporation with assembly, we have discovered a typical mechanism of complexes for acquiring both stability and flexibility at the same time. In addition, we got information of the localization of flexibility in ribosomal function by the analysis of related proteins with stalk protein, tRNA, mRNA, and nascent peptide, and demonstrate the relationship between structure, assembly, flexibility, and function of the ribosome.     

Rapid Analysis of Protein Structure and Dynamics by Hydrogen/Deuterium Exchange Mass Spectrometry

An automated approach for the rapid analysis of protein structure has been developed and used to study acid-induced conformational changes in human growth hormone. The labeling approach involves hydrogen/deuterium exchange (H/D-Ex) of protein backbone amide hydrogens with rapid and sensitive detection by mass spectrometry (MS). Briefly, the protein is incubated for defined intervals in a deuterated environment. After rapid quenching of the exchange reaction, the partially deuterated protein is enzymatically digested and the resulting peptide fragments are analyzed by liquid chromatography mass spectrometry (LC-MS). The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. Additional analyses allow mapping of the free energy of folding on localized segments along the protein sequence affording unique dynamic and structural information. While amide H/D-Ex coupled with MS is recognized as a powerful technique for studying protein structure and protein–ligand interactions, it has remained a labor-intensive task. The improvements in the amide H/D-Ex methodology described here include solid phase proteolysis, automated liquid handling and sample preparation, and integrated data reduction software that together improve sequence coverage and resolution, while achieving a sample throughput nearly 10-fold higher than the commonly used manual methods.     

Conformational Dynamics of the Bovine Mitochondrial ADP/ATP Carrier Isoform 1 Revealed by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

The mitochondrial adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in cellular energy metabolism. During the transport mechanism the carrier switches between two different conformations that can be blocked by two toxins: carboxyatractyloside (CATR) and bongkrekic acid. Therefore, our understanding of the nucleotide transport mechanism can be improved by analyzing structural differences of the individual inhibited states. We have solved the three-dimensional structure of bovine carrier isoform 1 (bAnc1p) in a complex with CATR, but the structure of the carrier-bongkrekic acid complex, and thus, the detailed mechanism of transport remains unknown. Improvements in sample processing in the hydrogen/deuterium exchange technique coupled to mass spectrometry (HDX-MS) have allowed us to gain novel insights into the conformational changes undergone by bAnc1p. This paper describes the first study of bAnc1p using HDX-MS. Results obtained with the CATR-bAnc1p complex were fully in agreement with published results, thus, validating our approach. On the other hand, the HDX kinetics of the two complexes displays marked differences. The bongkrekic acid-bAnc1p complex exhibits greater accessibility to the solvent on the matrix side, whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp.     

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G- and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts.The ability of actin to engage in a wide range of physiological functions requires that it be subject to complex spatial and temporal control by a large array of actin-binding proteins (1–3). Such regulation demands that both the monomeric and filamentous forms of actin be able to exist in a number of different conformations. Some of these may be differentially recognized by different actin-binding proteins, and some might actually be induced by the interaction of actin with these proteins.Actin is highly conserved from yeast to humans. There is 87% sequence identity between yeast and muscle actin and 91% sequence identity between yeast and nonmuscle actins. Even with this high sequence similarity and minor differences in crystal structures, there are some major differences in yeast and muscle actin behavior. Yeast, compared with muscle actin, polymerizes faster and exchanges its bound nucleotide faster. In contrast to muscle actin, the yeast actin filament releases its P_i almost immediately after ATP hydrolysis and the filament fragments more readily (4). Yeast cells cannot survive with muscle actin as the sole actin, and with β-nonmuscle actin as the only actin, yeast cells are very sick (5). A set of biochemical data indicates that the muscle filament is less flexible than yeast (6, 7). However, conformational and structural differences that could explain these behavioral differences are unknown.The actin molecule is divided into two domains, the small domain consisting of subdomains 1 and 2 and the large domain consisting of subdomains 3 and 4. Modeling studies (8, 9) have suggested that each of the subdomains can move, and biochemical studies have suggested allosteric interactions between the subdomains. However, the nature of these interactions has not been elucidated on a molecular level.To try to gain insight into what parts of the actin molecule are important for the behavioral differences observed between yeast and muscle actin, yeast-muscle hybrid actins were constructed (10). Hybrid sub1 actin has muscle-specific residues introduced into the small domain of actin, making it muscle-like in subdomain 1 and yeastlike in subdomains 2, 3, and 4. Sub12 hybrid actin has muscle-specific residues in both subdomains 1 and 2, making it muscle-like in subdomains 1 and 2 and yeastlike in subdomains 3 and 4. These actins showed muscle-like behavior in several biochemical properties: nucleotide exchange rates, thermostability, and the nucleation phase during polymerization. Although most of the residues proposed to be involved in the interaction of actin with myosin are located in subdomains 1 and 2, both hybrids exhibited yeast actin behavior in the activation of myosin ATPase activity. The hybrid actins also exhibited higher critical concentrations than either yeast or muscle actin and formed ADP-F-actin that was less stable than either yeast or muscle actin (10).The experimental results with the hybrid actins indicated that the behavior of actin as a whole is dictated by the interaction and cross-talk of the two halves. One experimental approach to further understand the structural bases of 1) biochemical differences observed with yeast and muscle actins, 2) how introduction of muscle-specific residues affects conformational behavior of the hybrids, and 3) whether there is propagation of change and cross-talk from muscle domains into yeast domains of the hybrid actins in solution is to use NMR. However, NMR as a technique is not suitable here due to the high actin concentrations that would be needed and the propensity of actin to aggregate at these concentrations. Therefore, we utilized hydrogen-deuterium (HD)² exchange detected with mass spectrometry. This is a powerful technique that can be used to obtain information about conformational changes in proteins in solution on a global as well as a local level.A protein sample placed in a deuterated solution will exchange its amide protons of the polypeptide backbone with exchange times varying from seconds to months (11). The amount and rate of deuterium exchange will depend on factors like temperature, pD, accessible surface area, local environment, and conformational flexibility (12–15). Labeled protein samples are digested by pepsin, the protease of choice in HD experiments, because of the requirement for acidic pH in order to minimize back exchange. The resulting peptide fragments are separated by high pressure liquid chromatography and analyzed for deuterium uptake.For a peptide that is undergoing amide proton exchange, the average mass of the peptide will increase with labeling time with an increase in mass greater for the peptides that are more exposed to the solvent. The analysis of deuterium uptake for specific peptides and areas of proteins could potentially provide information about structural and conformational differences between different actin forms. Since changes in conformation among different actin states will affect deuterium uptake, HD exchange coupled with mass spectrometry should provide us with a tool to study the states of actin present in solution. HD exchange detected with mass spectrometry was previously used successfully by Chik and co-workers (16) to assess changes in muscle actin structure due to the G- to F-actin transition and the binding of phallodin to F-actin and of DNase I to G-actin. More recently, the technique has been used to assess conformational changes in the Arp2/3 complex, which contains actin like subunits (17). In this study, we used HD exchange detected with mass spectrometry to analyze differences in solution structures of yeast, muscle, sub1, and sub12 G- and F-actins.     

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-¹H/²H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex.        

Oligomerization Interface of RAGE Receptor Revealed by MS-Monitored Hydrogen Deuterium Exchange

Activation of the receptor for advanced glycation end products (RAGE) leads to a chronic proinflammatory signal, affecting patients with a variety of diseases. Potentially beneficial modification of RAGE activity requires understanding the signal transduction mechanism at the molecular level. The ligand binding domain is structurally uncoupled from the cytoplasmic domain, suggesting receptor oligomerization is a requirement for receptor activation. In this study, we used hydrogen-deuterium exchange and mass spectrometry to map structural differences between the monomeric and oligomeric forms of RAGE. Our results indicated the presence of a region shielded from exchange in the oligomeric form of RAGE and led to the identification of a new oligomerization interface localized at the linker region between domains C1 and C2. Based on this finding, a model of a RAGE dimer and higher oligomeric state was constructed.     

Dynamics and Conformational Changes in Human NEIL2 DNA Glycosylase Analyzed by Hydrogen/Deuterium Exchange Mass Spectrometry

Base excision DNA repair (BER) is necessary for removal of damaged nucleobases from the genome and their replacement with normal nucleobases. BER is initiated by DNA glycosylases, the enzymes that cleave the N-glycosidic bonds of damaged deoxynucleotides. Human endonuclease VIII-like protein 2 (hNEIL2), belonging to the helix–two-turn–helix structural superfamily of DNA glycosylases, is an enzyme uniquely specific for oxidized pyrimidines in non-canonical DNA substrates such as bubbles and loops. The structure of hNEIL2 has not been solved; its closest homologs with known structures are NEIL2 from opossum and from giant mimivirus. Here we analyze the conformational dynamics of free hNEIL2 using a combination of hydrogen/deuterium exchange mass spectrometry, homology modeling and molecular dynamics simulations. We show that a prominent feature of vertebrate NEIL2 – a large insert in its N-terminal domain absent from other DNA glycosylases – is unstructured in solution. It was suggested that helix–two-turn–helix DNA glycosylases undergo open–close transition upon DNA binding, with the large movement of their N- and C-terminal domains, but the open conformation has been elusive to capture. Our data point to the open conformation as favorable for free hNEIL2 in solution. Overall, our results are consistent with the view of hNEIL2 as a conformationally flexible protein, which may be due to its participation in the repair of non-canonical DNA structures and/or to the involvement in functional and regulatory protein–protein interactions.     

Distinct Structures of Scrapie Prion Protein (PrPSc)-seeded Versus Spontaneous Recombinant Prion Protein Fibrils Revealed by Hydrogen/Deuterium Exchange

The detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrP^Sc) are largely unknown. PrP^Sc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrP^C). One manifestation of PrP^Sc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrP^Sc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrP^Sc. Here, we compare the structures of PrP^Sc-seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ∼163–223 represents a systematically H-bonded cross-β amyloid core structure. PrP^Sc-seeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ∼145, and there is a significant degree of order within residues ∼117–133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrP^Sc-seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrP^Sc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP.Transmissible spongiform encephalopathies (TSEs),² or prion diseases, are a group of infectious neurodegenerative disorders that affect many mammalian species and include Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in cervids, and bovine spongiform encephalopathy (“mad cow” disease) (1–7). All of these diseases appear to be intimately associated with conformational conversion of the normal host-encoded prion protein, termed PrP^C, to a pathological isoform, PrP^Sc (1–5). According to the “protein-only” model, PrP^Sc itself represents the infectious prion agent (1, 8); it is believed to self-propagate by an autocatalytic mechanism involving binding to PrP^C and templating the conversion of the latter protein to the PrP^Sc state (9, 10). Although molecular details of such a mechanism of disease propagation remain largely unknown, the general principle of protein-based infectivity is supported by a wealth of experimental data (1–7).PrP^C is a monomeric glycophosphatidylinositol-linked glycoprotein that is highly protease-sensitive and soluble in nonionic detergents. High resolution NMR data show that the recombinant PrP (rPrP), a nonglycosylated model of PrP^C, consists of a flexible N-terminal region and a folded C-terminal domain encompassing three α-helices and two short β-strands (11–13). Conversely, the PrP^Sc isoform is aggregate in nature, rich in β-sheet structure, insoluble in nonionic detergents, and partially resistant to proteinase K (PK) digestion, with a PK-resistant core encompassing the C-terminal ∼140 residues (1–5, 14, 15). Little specific structural information is available, however, for this isoform beyond low resolution biochemical and spectroscopic characterization. Thus, the structure of PrP^Sc conformer(s) associated with prion infectivity remains one of the best guarded mysteries, hindering efforts to understand the molecular basis of TSE diseases.Many efforts have been made over the years to recapitulate PrP^Sc formation and prion propagation in vitro. Early studies have shown that PrP^C can be converted with remarkable species and strain specificities to a PrP^Sc-like conformation (as judged by PK resistance) simply by incubation with PrP^Sc from prion-infected animals (16, 17). The yields of these original cell-free conversion experiments were low, and no new infectivity could be attributed to the newly converted material (18). An important more recent study showed that both PrP^Sc and TSE infectivity can be amplified indefinitely in crude brain homogenates using successive rounds of sonication and incubation (19), a procedure called protein misfolding cyclic amplification (PMCA) (20). Similar amplification of the TSE infectivity was also accomplished by PMCA employing purified PrP^C as a substrate, although only in the presence of polyanions such as RNA and copurified lipids (21). Unfortunately, the quantities of infectious PrP^Sc generated by PMCA using purified brain-derived PrP^C are very small, precluding most structural studies.In contrast to brain-derived PrP^C, large scale purification can be readily accomplished for bacterially expressed rPrP, a form of PrP lacking glycosylation and the glycophosphatidylinositol anchor. The latter protein can spontaneously polymerize into amyloid fibrils, and much insight has been gained into mechanistic and structural aspects of this reaction (22–28). However, although rPrP fibrils were shown to cause or accelerate a transmissible neurodegenerative disorder in transgenic mice overexpressing a PrP^C variant encompassing residues 89–231, the infectivity titer of these “synthetic prions” was extremely low (29) or absent altogether (4). This low infectivity coincides with much shorter PK-resistant core of rPrP amyloid fibrils compared with brain-derived PrP^Sc (26, 30), raising questions regarding the relationship between these fibrils and the authentic TSE agent. In this context, an important recent development was the finding that the PrP^Sc-seeded PMCA method can be extended to rPrP, yielding protease-resistant recombinant PrP aggregates (rPrP^PMCA or rPrP-res^(Sc)) (31). These aggregates display a PK digestion pattern that is much more closely related to PrP^Sc than that of previously studied spontaneously formed rPrP fibrils, offering a potentially more relevant model for biochemical and biophysical studies. Here, we provide, for the first time, a direct insight into the structure of rPrP^PMCA. H/D exchange data coupled with MS analysis (HXMS) allowed us to identify systematically H-bonded core region(s) of these aggregates, shedding a new light on the mechanisms underlying formation of PK-resistant structures.     

Berberine Promotes Glucose Consumption Independently of AMP-Activated Protein Kinase Activation

Berberine is a plant alkaloid with anti-diabetic action. Activation of AMP-activated protein kinase (AMPK) pathway has been proposed as mechanism for berberine’s action. This study aimed to examine whether AMPK activation was necessary for berberine’s glucose-lowering effect. We found that in HepG2 hepatocytes and C2C12 myotubes, berberine significantly increased glucose consumption and lactate release in a dose-dependent manner. AMPK and acetyl coenzyme A synthetase (ACC) phosphorylation were stimulated by 20 µmol/L berberine. Nevertheless, berberine was still effective on stimulating glucose utilization and lactate production, when the AMPK activation was blocked by (1) inhibition of AMPK activity by Compound C, (2) suppression of AMPKα expression by siRNA, and (3) blockade of AMPK pathway by adenoviruses containing dominant-negative forms of AMPKα1/α2. To test the effect of berberine on oxygen consumption, extracellular flux analysis was performed in Seahorse XF24 analyzer. The activity of respiratory chain complex I was almost fully blocked in C2C12 myotubes by berberine. Metformin, as a positive control, showed similar effects as berberine. These results suggest that berberine and metformin promote glucose metabolism by stimulating glycolysis, which probably results from inhibition of mitochondrial respiratory chain complex I, independent of AMPK activation.     

Influence of Domain Interactions on Conformational Mobility of the Progesterone Receptor Detected by Hydrogen/Deuterium Exchange Mass Spectrometry

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Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG₁ and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG–FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG–FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG–FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution.Antibodies and variants thereof constitute the fastest growing category of therapeutic agents, and currently more than 30 immunoglobulins (Igs)¹ have been approved for the treatment of cancer, immunological diseases, and infectious diseases (1). The success of therapeutic monoclonal antibodies (mAbs) is based on the ability to specifically target diverse antigens and activate immunological effector responses. An Ig is a “dimer of a dimer” consisting of light chains and heavy chains in which each light chain is linked to a heavy chain and the light–heavy dimers are connected by disulfide bridges to form the intact antibody. IgG is the most prevalent Ig isotype in plasma and is the most commonly used isotype for therapeutic antibodies because of its strong ability to induce antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (2). The IgG₁ subtype is a 150 kDa Y-shaped glycoprotein. Its stem and arms are referred to as the fragment crystallizable (Fc) and fragment antigen binding (Fab) regions, respectively. The Fab region is composed of a variable (V) and constant (C) domain from both the light chain and the heavy chain (V_L, C_L, V_H, C_H1). Antigen binding is achieved through three highly variable complementary determining regions in each variable domain (V_L and V_H) of the Fab region. The Fc region is composed of additional constant domains of the heavy chain (C_H2 and C_H3); it mediates antibody-dependent cellular cytotoxicity through interaction with Fcγ receptors (3, 4) and activates complement-dependent cytotoxicity through interaction with C1q (5). The Fc region also interacts with the neonatal Fc receptor (FcRn), which regulates the maintenance of antibody levels in plasma and thus the half-life of endogenous and recombinant monoclonal antibodies (6). The interaction between IgG and FcRn displays a characteristic pH dependence that is the basis for the function of FcRn in IgG recycling (7). FcRn rescues and recycles IgG from lysosomal degradation by binding with low micromolar affinity to internalized IgG in the slightly acidic late endosome of, for example, vascular endothelial cells (pH < 6.5). The IgG is rescued from intracellular degradation as the IgG–FcRn complex returns to the cell surface, where the IgG is released into circulation as FcRn binding is abolished in the neutral pH of plasma (6). FcRn-mediated IgG recycling contributes to the long catabolic half-life of endogenous and therapeutic antibodies of ∼22 days (8).The FcRn is a heterodimer of an MHC-class-I-like heavy chain and a β₂-microglobulin (β₂m) light chain. The FcRn heavy chain (α-chain) is composed of three structural domains, α₁, α₂, and α₃, followed by a transmembrane region and a cytoplasmic domain. The three-dimensional structure of FcRn is similar to that of MHC class I molecules in which domains α₁ and α₂ are stacked against domain α₃ and β₂m (9, 10). The pH dependence of the IgG–FcRn interaction is attributed to highly conserved residues in both FcRn and IgG (10). The first crystal structures of rat FcRn and rat Fc revealed that FcRn binds to the C_H2 and C_H3 domains of the IgG Fc region—specifically, C_H2 residues 252–254 and 309–311, as well as C_H3 residues 434–436 (11, 12). Several positively charged histidines in the IgG C_H2 and C_H3 domains (H310, H433, H435, and H436; the latter is not found in humans) interact with acidic residues E117, E132, W133, E135, and D137 in the FcRn α₂ domain, accounting for the pH-sensitive nature of the IgG–FcRn interaction. The interface is also composed of a hydrophobic core around Fc I253 that interacts with FcRn W133 and the N-terminal I1 residue of the β₂m, which has been proposed to contact Fc residues 309–311. The interaction of FcRn and IgG occurs in a 2:1 stoichiometry, where two FcRn molecules bind to one IgG through binding sites on each heavy chain (12). Two distinct binding modes have been suggested in which the FcRn molecules bind in a symmetric or asymmetric fashion to the Fc. In symmetric models FcRns bind to opposite sites on the Fc, whereas in the asymmetric models two FcRn molecules form a homodimer with only one FcRn molecule binding the Fc directly (6, 11). The extracellular domains of rat and human FcRn have 68% sequence identity and are structurally similar (9, 10). The first crystal structure of human FcRn in complex with an engineered human Fc fragment (Fc-YTE) as well as human serum albumin was published recently (13) and showed a binding mode similar to that of rodent IgG–FcRn variants, with the exception of the additional interaction sites caused by substitutions in the Fc domain. To the best of our knowledge, no crystal structures of full-length human IgG and human FcRn are currently available.From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize the pharmacokinetics and thus ultimately the efficacy of therapeutic monoclonal antibodies. The goal of FcRn modulation is typically prolongation of the in vivo half-life in order to reduce dosing frequency and ultimately the cost of treatment. However, a shorter half-life can also be desirable, for example, for antibody–toxin conjugates or antibodies used in bioimaging (6). Several engineered therapeutic mAb variants with improved in vitro FcRn binding affinity and extended in vivo half-life have been generated via mutation of residues in the Fc domain (14–19). For example, the engineered variants of palivizumab (M252Y/S254T/T256E) (15, 16) and bevacizumab (M428L/N434S) (17) show 10- and 11-fold increases in relative FcRn affinity that result in increases of the in vivo half-life in cynomolgus monkeys of 4- and 3-fold, respectively. Mutation can also impact half-life negatively: mAb engineering can improve FcRn affinity at both pH 6 and 7.5 such that the pH-dependent release of IgGs is prohibited, leading to increased IgG clearance (16). Interestingly, post-translational modifications such as oxidation of conserved methionines in the C_H2 and C_H3 domains of IgG₁ and IgG₂ have been shown to affect FcRn affinity negatively. Antibody oxidation that can occur during production or storage significantly reduces FcRn binding in vitro (20, 21), which also translates to a reduced in vivo half-life in human FcRn transgenic mice models (22). The molecular origins of the effect of post-translational modifications on the IgG–FcRn interaction are, however, unclear. Further, the impact of FcRn binding on the conformational properties and dynamics of IgG in solution is currently not well understood.In this study we investigated the interaction between human FcRn and two variants of a full-length IgG₁ by means of hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS). HDX-MS has become a popular approach for studying protein dynamics and interactions (23–27), as the technique provides access to proteins at native solution conditions with modest sample requirements. Amide HDX rates in native proteins are highly influenced by higher order structure: fully solvated (non-hydrogen-bonded) amides exchange rapidly, whereas structurally protected (hydrogen-bonded) amides exchange up to 7 orders of magnitude slower (28, 29). Protein interactions can be studied and mapped via HDX-MS, as binding events can perturb HDX rates as solvation and hydrogen bonding changes directly in the binding interface or indirectly in conformationally linked regions. The structural resolution of a classic peptide-level HDX-MS experiment is dependent on the generation of overlapping peptides by acid-stable proteases, such as pepsin, typically used in HDX-MS workflows. More recently, the use of gas-phase fragmentation of deuterated peptides with ETD (30–33) has become a viable option for sublocalizing deuterium uptake to short peptide stretches or even individual amino acids, thus increasing the spatial resolution of the classical bottom-up HDX-MS method.Here, we used HDX-MS to probe the solution-phase interactions of human FcRn with a full-length recombinant human IgG₁ and its deglycosylated variant. Our results allowed us to map antibody and FcRn regions that displayed changes in HDX upon complex formation and examine the impact of antibody glycosylation on FcRn binding. Additionally, by coupling ETD to the HDX-MS workflow in a targeted manner, we obtained high-resolution information on the HDX of individual sites that became protected upon IgG₁–FcRn complex formation.     

Activation of ClpP Protease by ADEP Antibiotics: Insights from Hydrogen Exchange Mass Spectrometry

The bacterial protease ClpP consists of 14 subunits that assemble into two stacked heptameric rings. The central degradation chamber can be accessed via axial pores. In free ClpP, these pores are obstructed by the N-terminal regions of the seven subunits at either end of the barrel. Acyldepsipeptides (ADEPs) are antibacterial compounds that bind in hydrophobic clefts surrounding the pore region, causing the pores to open up. The ensuing uncontrolled degradation of intracellular proteins is responsible for the antibiotic activity of ADEPs. Recently published X-ray structures yielded conflicting models regarding the conformation adopted by the N-terminal regions in the open state. Here, we use hydrogen/deuterium exchange (HDX) mass spectrometry to obtain complementary insights into the ClpP behavior with and without ADEP1. Ligand binding causes rigidification of the equatorial belt, accompanied by destabilization in the vicinity of the binding clefts. The N-terminal regions undergo rapid deuteration with only minor changes after ADEP1 binding, revealing a lack of stable H-bonding. Our data point to a mechanism where the pore opening mechanism is mediated primarily by changes in the packing of N-terminal nonpolar side chains. We propose that a “hydrophobic plug” causes pore blockage in ligand-free ClpP. ADEP1 binding provides new hydrophobic anchor points that nonpolar N-terminal residues can interact with. In this way, ADEP1 triggers the transition to an open conformation, where nonpolar moieties are clustered around the rim of the pore. This proposed mechanism helps reconcile the conflicting models that had been put forward earlier.     

Structure and Dynamics of NBD1 from CFTR Characterized Using Crystallography and Hydrogen/Deuterium Exchange Mass Spectrometry

The ΔF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and ΔF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because ΔF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and ΔF508 constructs, and the ΔF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide ¹H/²H exchange rates in matched F508 and ΔF508 constructs reveal that ΔF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the ΔF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-ΔF508 structures but completely solvent exposed in all ΔF508 structures. These results reinforce the importance of the perturbation ΔF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.     

AMPK上游激酶——肿瘤抑制因子LKB1

AMPK是哺乳动物细胞中高度保守的蛋白质,是细胞的“代谢感受器”。AMPK的活化需要上游激酶(AMPKK)对AMPKα亚基活化环上Thr172进行磷酸化来完成。最近的研究发现,肿瘤抑制因子LKB1可以磷酸化Thr172进而激活AMPK,因此认为它是AMPKK家族的成员。     

Nucleotide- and Activator-Dependent Structural and Dynamic Changes of Arp2/3 Complex Monitored by Hydrogen/Deuterium Exchange and Mass Spectrometry

Arp2/3 complex plays a central role in the de novo nucleation of filamentous actin as branches on existing filaments. The complex must bind ATP, protein activators [e.g., Wiskott-Aldrich syndrome protein (WASp)], and the side of an actin filament to form a new actin filament. Amide hydrogen/deuterium exchange coupled with mass spectrometry was used to examine the structural and dynamic properties of the mammalian Arp2/3 complex in the presence of both ATP and the activating peptide segment from WASp. Changes in the rate of hydrogen exchange indicate that ATP binding causes conformational rearrangements of Arp2 and Arp3 that are transmitted allosterically to the Arp complex (ARPC)1, ARPC2, ARPC4, and ARPC5 subunits. These data are consistent with the closure of nucleotide-binding cleft of Arp3 upon ATP binding, resulting in structural rearrangements that propagate throughout the complex. Binding of the VCA domain of WASp to ATP-Arp2/3 further modulates the rates of hydrogen exchange in these subunits, indicating that a global conformational reorganization is occurring. These effects may include the direct binding of activators to Arp3, Arp2, and ARPC1; alterations in the relative orientations of Arp2 and Arp3; and the long-range transmission of activator-dependent signals to segments proposed to be involved in binding the F-actin mother filament.     

Platform Dependencies in Bottom-up Hydrogen/Deuterium Exchange Mass Spectrometry

Hydrogen-deuterium exchange mass spectrometry is an important method for protein structure-function analysis. The bottom-up approach uses protein digestion to localize deuteration to higher resolution, and the essential measurement involves centroid mass determinations on a very large set of peptides. In the course of evaluating systems for various projects, we established two (HDX-MS) platforms that consisted of a FT-MS and a high-resolution QTOF mass spectrometer, each with matched front-end fluidic systems. Digests of proteins spanning a 20–110 kDa range were deuterated to equilibrium, and figures-of-merit for a typical bottom-up (HDX-MS) experiment were compared for each platform. The Orbitrap Velos identified 64% more peptides than the 5600 QTOF, with a 42% overlap between the two systems, independent of protein size. Precision in deuterium measurements using the Orbitrap marginally exceeded that of the QTOF, depending on the Orbitrap resolution setting. However, the unique nature of FT-MS data generates situations where deuteration measurements can be inaccurate, because of destructive interference arising from mismatches in elemental mass defects. This is shown through the analysis of the peptides common to both platforms, where deuteration values can be as low as 35% of the expected values, depending on FT-MS resolution, peptide length and charge state. These findings are supported by simulations of Orbitrap transients, and highlight that caution should be exercised in deriving centroid mass values from FT transients that do not support baseline separation of the full isotopic composition.Hydrogen-deuterium exchange mass spectrometry (HDX-MS)¹ provides a powerful means to study the link between protein structure and function (1). The method involves a chemical process in which labile hydrogens within a protein are exchanged with hydrogen from bulk water. When D₂O is used in place of H₂O, a mass shift results at every point of exchange, but it is the backbone amide hydrogens that offer exchange rates on a measurable timescale (2, 3). Measuring an amide hydrogen exchange rate can provide access to conformational dynamics, stability, and the interaction characteristics in that location of structure (4, 5). H/D exchange rates have be used to explore mechanisms of protein folding (6), determine the allosteric impact of post-translational modifications and ligand binding (7, 8), define truncation points for enhancing crystallization success (9), and they have also found a role in mapping interactions between proteins (10). Applications have stepped outside of primary research to include the characterization of protein drugs for stability and similarity testing (11–13). The capacity to provide such information has attracted increased attention from regulatory bodies and is generating a push for standardizing HDX methods.Mass spectrometers are very effective tools for measuring exchange rates, from whole proteins down to the individual amide levels. Classical methods of rate measurement have used NMR (3), but mass spectrometry offers all the advantages of speed, sensitivity and scale that have made the tool so useful in proteomics. Measurements at the peptide level provide an important intermediate resolution. As with bottom-up proteomics, rendering deuterated proteins into smaller peptides through digestion provides opportunities to analyze protein systems of considerable complexity, and at the same time support analysis at higher structural resolution through MS/MS methods (14, 15). A considerable amount of effort has been applied by the research community and instrument manufacturers to produce instrument configurations that are suitable for managing the many processing steps required for labeling, digesting, separating and introducing deuterated peptides into the mass spectrometer (16). This has been supported by parallel efforts to develop software tools for the detection of deuterated peptides and the extraction of deuteration data (17–19).A successful application of the bottom-up HDX-MS method requires a full peptide sequence map of the protein, so that deuteration rates at every point in protein structure can be quantified and related back to structure. Once the peptide is identified, the primary measurement is the peptide centroid mass of the deuterated state, relative to the unlabeled state. It requires intensity measurements for a minimum of two peaks in the isotopic cluster to determine when the centroid mass changes (20), although most often the full distribution is quantified in HDX-MS applications. As the range of applications continues to grow, particularly in the regulatory area, it is important to better understand how various elements of the HDX platform deliver the essential data (21, 22). In the current study, we are interested in the contribution of the mass spectrometer alone. Most users of the HDX-MS method are migrating from low resolution to high resolution systems, operated in a single-stage MS mode. This includes FT-MS and higher-resolution QTOF platforms, therefore in this study, we explore how an LTQ Orbitrap Velos (Thermo) and a 5600 TripleTOF (AB Sciex) influence the measurement of deuteration data for proteins of increasing size. Identical front-end fluidic systems and protein digests, as well as back-end analysis procedures, allow us to perform a direct comparison of performance in areas of sequence mapping, centroid measurement precision and centroid mass accuracy. We demonstrate that the Orbitrap system returns greater sequencing depth and marginally better precision than the 5600, however the measurement accuracy is strongly influenced by destructive interference arising from unequal mass defects between ¹³C and ²H. This has implications for any application that involves centroid mass determinations, beyond HDX-MS.