Mapping ERK2-MKP3 binding interfaces by hydrogen/deuterium exchange mass spectrometry

ERK2, a prototypic member of the MAPK family, plays a central role in regulating cell growth and differentiation. MKP3, an ERK2-specific phosphatase, terminates ERK2 signaling. To understand the molecular basis of ERK2 recognition by MKP3, we carried out hydrogen/deuterium exchange mass spectrometry experiments to map the interaction surfaces between the two proteins. The results show that the exquisite specificity of MKP3 for ERK2 is governed by two distinctive protein-protein interactions. To increase the "effective concentration" of the interacting molecules, the kinase interaction motif in MKP3 ((64)RRLQKGNLPVR(74)) and an MKP3-specific segment ((101)NSSDWNE(107)) bind the common docking site in ERK2 defined by residues in L(16), L(5), beta(7)-beta(8), and alpha(d)-L(8)-alpha(e), located opposite the kinase active site. In addition to this "tethering" effect, additional interactions between the (364)FTAP(367) sequence in MKP3 and the ERK2 substrate-binding site, formed by residues in the activation lip and the P+1 site (beta(9)-alpha(f) loop), L(13) (alpha(f)-alpha(g) loop), and the MAPK insert (L(14)-alpha(1L14)-alpha(2L14)), are essential for allosteric activation of MKP3 and formation of a productive complex whereby the MKP3 catalytic site is correctly juxtaposed to carry out the dephosphorylation of phospho-Thr(183)/phospho-Tyr(185) in ERK2. This bipartite protein-protein interaction model may be applicable to the recognition of other MAPKs by their cognate regulators and substrates.     

Structural basis of specific interactions of Lp-PLA2 with HDL revealed by hydrogen deuterium exchange mass spectrometry

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂), specifically Group VIIA PLA₂, is a member of the phospholipase A₂ superfamily and is found mainly associated with LDL and HDL in human plasma. Lp-PLA₂ is considered as a risk factor, a potential biomarker, a target for therapy in the treatment of cardiovascular disease, and evidence suggests that the level of Lp-PLA₂ in plasma is associated with the risk of future cardiovascular and stroke events. The differential location of the enzyme in LDL/HDL lipoproteins has been suggested to affect Lp-PLA₂ function and/or its physiological role and an abnormal distribution of the enzyme may correlate with diseases. Although a mutagenesis study suggested that a surface helix (residues 362–369) mediates the association between Lp-PLA₂ and HDL, the molecular details and mechanism of association has remained unknown. We have now employed hydrogen deuterium exchange mass spectrometry to characterize the interaction between recombinant human Lp-PLA₂ and human HDL. We have found that specific residues 113–120, 192–204, and 360–368 likely mediate HDL binding. In a previous study, we showed that residues 113–120 are important for Lp-PLA₂-liposome interactions. We now find that residues 192–204 show a decreased deuteration level when Lp-PLA₂ is exposed to apoA-I, but not apoA-II, the most abundant apoproteins in HDL, and additionally, residues 360–368 are only affected by HDL.The results suggest that apoA-I and phospholipid membranes play crucial roles in Lp-PLA₂ localization to HDL.     

Differential hydrogen/deuterium exchange mass spectrometry analysis of protein-ligand interactions

Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule-receptor interactions, this technique has also been applied to study protein-protein complexes, such as mapping antibody-antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein-ligand interactions has had an impact on biology and drug discovery.     

Vibrio cholerae toxin-coregulated pilus structure analyzed by hydrogen/deuterium exchange mass spectrometry

The bacterial pathogen Vibrio cholerae uses toxin-coregulated pili (TCP) to colonize the human intestine, causing the severe diarrheal disease cholera. TCP are long, thin, flexible homopolymers of the TcpA subunit that self-associate to hold cells together in microcolonies and serve as the receptor for the cholera toxin phage. To better understand TCP's roles in pathogenesis, we characterized its structure using hydrogen/deuterium exchange mass spectrometry and computational modeling. We show that the pilin subunits are held together by tight packing of the N-terminal alpha helices, but loose packing of the C-terminal globular domains leaves substantial gaps on the filament surface. These gaps expose a glycine-rich, amphipathic segment of the N-terminal alpha-helix, contradicting the consensus view that this region is buried in the filament core. Our results explain extreme filament flexibility, suggest a molecular basis for pilus-pilus interactions, and reveal a previously unrecognized therapeutic target for V. cholerae and other enteric pathogens.     

Amide hydrogen/deuterium exchange & MALDI-TOF mass spectrometry analysis of Pak2 activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry(1,2,3). The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein(4). H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection(2), instead of a HPLC/ESI (electrospray ionization)-MS system(5,6). The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called γ-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A(2;) (sPLA(2;)). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation(7,8,9).     

The structural basis of serpin polymerization studied by hydrogen/deuterium exchange and mass spectrometry

The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type alpha(1)-antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the alpha(1)-antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of beta-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into beta-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of beta-sheet C. On the basis of our results, we propose a mechanism for polymerization in which beta-strand 1C is displaced from the rest of beta-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of beta-sheet A, and allows for subsequent polymerization.     

High-sensitivity mass spectrometry for imaging subunit interactions: hydrogen/deuterium exchange

In recent years, advances in mass spectrometry have provided unprecedented knowledge of protein expression within cells. It has become apparent that many proteins function as macromolecular complexes. Structural genomics programs are determining the fold of these proteins at an increasing rate and electron microscopic tomography potentially provides a means to determine the location of these complexes within the cell. A complete understanding of the molecular mechanism of these proteins requires detailed information on the interactions and dynamics within the complex. Recent advances in mass spectrometry now make it possible to use hydrogen/deuterium exchange to detect intersubunit interfaces and dynamics within supramolecular complexes.     

Protein conformation in amorphous solids by FTIR and by hydrogen/deuterium exchange with mass spectrometry

Solid-state hydrogen/deuterium exchange (ssHDX) with electrospray ionization mass spectrometry (ESI-MS) and Fourier transform infrared (FTIR) spectroscopy were used to assess protein conformation in amorphous solids. Myoglobin, lysozyme, β-lactoglobulin, ribonuclease A, E-cadherin 5, and concanavalin A were co-lyophilized with carbohydrates (trehalose, raffinose, and dextran 5000), linear polymers (polyvinyl alcohol and polyvinyl pyrrolidone) or guanidine hydrochloride (negative control). For ssHDX, samples were exposed to D₂O vapor at 33% relative humidity and room temperature, and then reconstituted at low temperature (4°C) and pH 2.5 and analyzed by ESI-MS. Peptic digestion of selected proteins was used to provide region-specific information on exchange. FTIR spectra were acquired using attenuated total reflectance. FTIR and ssHDX of intact proteins showed preservation of structure by raffinose and trehalose, as indicated by FTIR band intensity and protection from exchange. ssHDX of peptic digests further indicated that these protective effects were not exerted uniformly along the protein sequence but were observed primarily in α-helical regions, a level of structural resolution not afforded by FTIR. The results thus demonstrate the utility of HDX with ESI-MS for analyzing protein conformation in amorphous solid samples.     

Conformational analysis of Epac activation using amide hydrogen/deuterium exchange mass spectrometry

Exchange proteins directly activated by cAMP (Epac) play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Rap. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen/deuterium exchange and structural modeling. Our studies show that cAMP induces significant conformational changes that lead to a spatial rearrangement of the regulatory components of Epac and allows the exposure of the catalytic core for effector binding without imposing significant conformational change on the catalytic core. Homology modeling and comparative structural analyses of the cAMP binding domains of Epac and cAMP-dependent protein kinase (PKA) lead to a model of Epac activation, in which Epac and PKA activation by cAMP employs the same underlying principle, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different.     

Mapping of discontinuous conformational epitopes by amide hydrogen/deuterium exchange mass spectrometry and computational docking

Understanding antigen-antibody interactions at the sub-molecular level is of particular interest for scientific, regulatory, and intellectual property reasons, especially with increasing demand for monoclonal antibody therapeutic agents. Although various techniques are available for the determination of an epitope, there is no widely applicable, high-resolution, and reliable method available. Here, a combination approach using amide hydrogen/deuterium exchange coupled with proteolysis and mass spectrometry (HDX-MS) and computational docking was applied to investigate antigen-antibody interactions. HDX-MS is a widely applicable, medium-resolution, medium-throughput technology that can be applied to epitope identification. First, the epitopes of cytochrome c-E8, IL-13-CNTO607, and IL-17A-CAT-2200 interactions identified using the HDX-MS method were compared with those identified by X-ray co-crystal structures. The identified epitopes are in good agreement with those identified using high-resolution X-ray crystallography. Second, the HDX-MS data were used as constraints for computational docking. More specifically, the non-epitope residues of an antigen identified using HDX-MS were designated as binding ineligible during computational docking. This approach, termed HDX-DOCK, gave more tightly clustered docking poses than stand-alone docking for all antigen-antibody interactions examined and improved docking results significantly for the cytochrome c-E8 interaction.     

Conformational dynamics of free and catalytically active thermolysin are indistinguishable by hydrogen/deuterium exchange mass spectrometry

Conformational dynamics are thought to be a prerequisite for the catalytic activity of enzymes. However, the exact relationship between structural fluctuations and function is not well understood. In this work hydrogen/deuterium exchange (HDX) and electrospray ionization mass spectrometry (ESI-MS) are used for exploring the conformational dynamics of thermolysin. Amide HDX reflects the internal mobility of proteins; regions that undergo frequent unfolding-refolding show faster exchange than segments that are highly stable. Thermolysin is a zinc protease with an active site that is located between two lobes. Substrate turnover is associated with hinge bending that leads to a closed conformation. Product release regenerates the open form, such that steady-state catalysis involves a continuous closing/opening cycle. HDX/ESI-MS with proteolytic peptide mapping in the absence of substrate shows that elements in the periphery of the two lobes are most mobile. A comparison with previous X-ray data suggests that these peripheral regions undergo quite pronounced structural changes during the catalytic cycle. In contrast, active site residues exhibit only a moderate degree of backbone flexibility, and the central zinc appears to be in a fairly rigid environment. The presence of both rigid and moderately flexible elements in the active site may reflect a carefully tuned balance that is required for function. Interestingly, the HDX behavior of catalytically active thermolysin is indistinguishable from that of the free enzyme. This result is consistent with the view that catalytically relevant motions preexist in the resting state and that enzyme function can only be performed within the limitations given by the intrinsic dynamics of the protein. The data presented in this work indicate the prevalence of stochastic elements in the function of thermolysin, rather than supporting a deterministic mechanism.     

Analysis of oligomeric stability of insulin analogs using hydrogen/deuterium exchange mass spectrometry

Insulin analog products for subcutaneous injection are prepared as solutions in which insulin analog molecules exist in several oligomeric states. Oligomeric stability can affect their onset and duration of action and has been exploited in designing them. To investigate the oligomeric stability of insulin analog products having different pharmacokinetics, we performed hydrogen/deuterium exchange mass spectrometry (HDX/MS), which is a rapid method to analyze dynamic aspects of protein structures. Two rapid-acting analogs (lispro and glulisine) incorporated deuteriums more and faster than recombinant human insulin, whereas a long-acting analog (glargine) and two intermediate-acting preparations (protamine-containing formulations) incorporated them less and more slowly. Kinetic analysis revealed that the number of slowly exchanged hydrogens (D(s)) (k<0.01 min(-1)) accounted for the difference in HDX reactivity among analogs. Furthermore, we found correlations between HDX kinetics and pharmacokinetics reported previously. Their maximum serum concentration (C(max)) was linearly correlated with D(s) (r=0.88) and the number of maximum exchangeable hydrogens (D(∞)) (r=0.89). The maximum drug concentration time (t(max)) was also correlated with reciprocals of D(s) and D(∞) (r=0.86 and r=0.96, respectively). Here we demonstrate the ability of HDX/MS to evaluate oligomeric stability of insulin analog products.     

Local dynamics measured by hydrogen/deuterium exchange and mass spectrometry of creatine kinase digested by two proteases

Hydrogen/deuterium exchange coupled to mass spectrometry has been used to investigate the structure and dynamics of native dimeric cytosolic muscle creatine kinase. The protein was incubated in D₂O for various time. After H/D exchange and rapid quenching of the reaction, the partially deuterated protein was cleaved in parallel by two different proteases (pepsin or type XIII protease from Aspergillus saitoi) to increase the sequence coverage and spatial resolution of deuterium incorporation. The resulting peptides were analyzed by liquid chromatography coupled to mass spectrometry. In comparison with the 3D structure of MM-CK, the analysis of the two independent proteolysis deuteration patterns allowed us to get new insights into CK local dynamics as compared to a previous study using pepsin [Mazon et al. Protein Science 13 (2004) 476-486]. In particular, we obtained more information on the kinetics and extent of deuterium exchange in the N- and C-terminal extremities represented by the 1-22 and 362-380 pepsin peptides. Indeed, we observed a very different behaviour of the 1-12 and 13-22 type XIII protease peptides, and similarly for the 362-373 and 374-380 peptides. Moreover, comparison of the deuteration patterns of type XIII protease segments of the large 90-126 pepsin peptide led us to identify a small relatively dynamic region (108-114).     

Defining the interacting regions between apomyoglobin and lipid membrane by hydrogen/deuterium exchange coupled to mass spectrometry

Sperm whale myoglobin can be considered as the model protein of the globin family. The pH-dependence of the interactions of apomyoglobin with lipid bilayers shares some similarities with the behavior of pore-forming domains of bacterial toxins belonging also to the globin family. Two different states of apomyoglobin bound to a lipid bilayer have been characterized by using hydrogen/deuterium exchange experiments and mass spectrometry. When bound to the membrane at pH 5.5, apomyoglobin remains mostly native-like and interacts through alpha-helix A. At pH 4, the binding is related to the stabilization of a partially folded state. In that case, alpha-helices A and G are involved in the interaction. At this pH, alpha-helix G, which is the most hydrophobic region of apomyoglobin, is available for interaction with the lipid bilayer because of the loss of the tertiary structure. Our results show the feasibility of such experiments and their potential for the characterization of various membrane-bound states of amphitropic proteins such as pore-forming domains of bacterial toxins. This is not possible with other high-resolution methods, because these proteins are usually in partially folded states when interacting with membranes.     

Enzyme conformational dynamics during catalysis and in the 'resting state' monitored by hydrogen/deuterium exchange mass spectrometry

This work reports the use of electrospray mass spectrometry for studying the conformational dynamics of enzymes by amide hydrogen/deuterium exchange (HDX) measurements. A rapid-mixing quench-flow approach allows comparisons to be made between the HDX kinetics of free enzymes with those under steady-state conditions. Experiments carried out on carboxypeptidase B in the absence of substrate and in the presence of saturating concentrations of hippuryl-Arg result in HDX kinetics that are indistinguishable. This finding implies that the conformational dynamics that mediate HDX are not significantly different in the resting state of the enzyme and during substrate turnover.     

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

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

Characterizing protein structure in amorphous solids using hydrogen/deuterium exchange with mass spectrometry

Elucidating protein structure in amorphous solids is central to the rational design of stable lyophilized protein drugs. Hydrogen/deuterium (H/D) exchange with electrospray ionization mass spectrometry was applied to lyophilized powders containing calmodulin (17 kDa) and exposed to D(2)O vapor at controlled relative humidity (RH) and temperature. H/D exchange was influenced by RH and by the inclusion of calcium chloride and/or trehalose in the solid. The effects were not exhibited uniformly along the protein backbone but occurred in a site-specific manner, with calcium primarily influencing the calcium-binding loops and trehalose primarily influencing the alpha-helices. The results demonstrate that the method can provide quantitative and site-specific structural information on proteins in amorphous solids and on changes in structure induced by protein cofactors and formulation excipients. Such information is not readily available with other techniques used to characterize proteins in the solid state, such as Fourier transform infrared, Raman, and near-infrared spectroscopy.     

Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.     

Interaction of group IA phospholipase A2 with metal ions and phospholipid vesicles probed with deuterium exchange mass spectrometry

Deuterium exchange mass spectrometric evaluation of the cobra venom (Naja naja naja) group IA phospholipase A 2 (GIA PLA 2) was carried out in the presence of metal ions Ca (2+) and Ba (2+) and phospholipid vesicles. Novel conditions for digesting highly disulfide bonded proteins and a methodology for studying protein-lipid interactions using deuterium exchange have been developed. The enzyme exhibits unexpectedly slow rates of exchange in the two large alpha-helices of residues 43-53 and 89-101, which suggests that these alpha-helices are highly rigidified by the four disulfide bonds in this region. The binding of Ca (2+) or Ba (2+) ions decreased the deuterium exchange rates for five regions of the protein (residues 24-27, 29-40, 43-53, 103-110, and 111-114). The magnitude of the changes was the same for both ions with the exception of regions of residues 24-27 and 103-110 which showed greater changes for Ca (2+). The crystal structure of the N. naja naja GIA PLA 2 contains a single Ca (2+) bound in the catalytic site, but the crystal structures of related PLA 2s contain a second Ca (2+) binding site. The deuterium exchange studies reported here clearly show that in solution the GIA PLA 2 does in fact bind two Ca (2+) ions. With dimyristoylphosphatidylcholine (DMPC) phospholipid vesicles with 100 microM Ca (2+) present at 0 degrees C, significant areas on the i-face of the enzyme showed decreases in the rate of exchange. These areas included regions of residues 3-8, 18-21, and 56-64 which include Tyr-3, Trp-61, Tyr-63, and Phe-64 proposed to penetrate the membrane surface. These regions also contained Phe-5 and Trp-19, proposed to bind the fatty acyl tails of substrate.