Evaluating the Conformation and Binding Interface of Cap-Binding

Nov 7, 2013 - John P. O'Brien , Wenzong Li , Yan Zhang , and Jennifer S. Brodbelt. Journal of the American Chemical Society 2014 136 (37), 12920-12928...
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Evaluating the Conformation and Binding Interface of Cap-Binding Proteins and Complexes via Ultraviolet Photodissociation Mass Spectrometry John P. O’Brien,† Laura K. Mayberry,†,‡ Patricia A. Murphy,†,‡ Karen S. Browning,†,‡ and Jennifer S. Brodbelt*,† †

Department of Chemistry and Biochemistry and ‡Institute for Cell and Molecular Biology, The University of Texas at Austin, 1 University Station A5300, Austin, Texas 78712, United States S Supporting Information *

ABSTRACT: We report the structural analysis of cap-binding proteins using a chemical probe/ultraviolet photodissociation (UVPD) mass spectrometry strategy for evaluating solvent accessibility of proteins. Our methodology utilized a chromogenic probe (NN) to probe the exposed amine residues of wheat eukaryotic translation initiation factor 4E (eIF4E), eIF4E in complex with a fragment of eIF4G (“minieIF4F”), eIF4E in complex with full length eIF4G, and the plant specific cap-binding protein, eIFiso4E. Structural changes of eIF4E in the absence and presence of excess dithiothreitol and in complex with a fragment of eIF4G or full-length eIF4G are mapped. The results indicate that there are particular lysine residues whose environment changes in the presence of dithiothreitol or eIF4G, suggesting that changes in the structure of eIF4E are occurring. On the basis of the crystal structure of wheat eIF4E and a constructed homology model of the structure for eIFiso4E, the reactivities of lysines in each protein are rationalized. Our results suggest that chemical probe/UVPD mass spectrometry can successfully predict dynamic structural changes in solution that are consistent with known crystal structures. Our findings reveal that the binding of m7GTP to eIF4E and eIFiso4E appears to be dependent on the redox state of a pair of cysteines near the m7GTP binding site. In addition, tertiary structural changes of eIF4E initiated by the formation of a complex containing a fragment of eIF4G and eIF4E were observed. KEYWORDS: chromogenic probe, wheat eukaryotic translation initiation factor, chemical probe, eIF4E, eIF4G



protein translation,2 and misregulation of its expression has been linked to various cancers.5 The functional role of eIF4E has sparked the development of anticancer therapeutics that inhibit eIF4E binding to the cap group.6,7 The structures of human, yeast, mouse, and wheat eIF4E have been solved using X-ray crystallography or NMR methods.1,8−13 These solved structures have included eIF4E with bound cap analog,1 eIF4E without cap analog,1,8,9 the complex of eIF4E with eIF4G fragments or eIF4E binding protein (4EBP) peptides with bound cap analog,10−12 and more recently an intermediate state between bound and unbound cap analog to the complex of eIF4E and eIF4G peptide.13 There are few examples of eIF4E without the m7GTP ligand for comparison.9 Plant eIF4E has a unique disulfide bond (colored in red font in Figure 1), identified by X-ray crystallography, near the binding pocket that may play a redox-mediated regulatory role in cap binding.1 In addition, plants express different isoforms of

INTRODUCTION

Protein translation initiation in eukaryotes begins with the binding of the 7-methylguanosine (m7G) or “cap” group at the 5′ end of mRNAs to eukaryotic initiation factor 4E (eIF4E).1,2 eIF4E, the cap-binding protein (∼24 kDa), is one of the key constituents of the larger eIF4F complex, which also includes eIF4G (∼162 kDa) and eIF4A (∼50 kDa), the latter of which also copurifies depending upon the purification methods.2 The interactions of eIF4E with eIF4G and eIF4E with the cap are separate but related binding events, and it is believed that the presence of eIF4G influences eIF4E/m7G cap binding, as evidenced by the 10-fold greater binding affinity of the protein complex eIF4F for the 7-methylguanosine triphosphate (m7GTP) cap analog than eIF4E alone.2−4 The complex of eIF4F along with other initiation factors facilitates the binding of the mRNA to the 40S ribosome. The 40S ribosome and associated factors scan to find the correct initiation codon on the mRNA, and the 60S ribosome binds to complete the 80S ribosome and elongation of the polypeptide begins. eIF4E has been the subject of great interest because of its importance in © 2013 American Chemical Society

Received: August 24, 2013 Published: November 7, 2013 5867

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Figure 1. Structures of truncated wheat eIF4E (A) in the absence of m7GTP and (B) with m7GTP bound. The m7GTP cap analog is shown in yellow; the tryptophan residues that hold the cap analog are shown in orange. The blue box highlights the C113 and C151 cysteines, which are accompanied by a zoomed-in image (below each structure in the lower left corner) to focus on the residues.

spectrometry in combination with chemical probe methods offers a new frontier for evaluation of protein conformations, determination of solvent accessibility, examination of protein− ligand interactions, and mapping of protein−protein interfaces.27−31 In particular, developments in ion mobility, hydrogen−deuterium exchange, and covalent labeling techniques have provided compelling advances.26,32−35 A number of these methods rely on monitoring the differential reactivity of targeted residues that reflect specific regions or surfaces of proteins, typically based on tracking modified residues in peptides after proteolytic digestion of the proteins of interest. Several labeling-based strategies have been developed, including hydrogen−deuterium exchange (HDX),36−45 chemical cross-linking,31,46−53 noncovalent labeling,54−56 and the use of chemical probes,28,27,57−70 to characterize protein structures. Chemical probe strategies,28,27,58−70 typically involve incubating a protein or protein complex with a reactive agent that covalently modifies exposed residues, thus allowing the development of a semiquantitative correlation between sitespecific reactivity and solvent accessibility. It is assumed that the more accessible residues are more reactive, whereas the inaccessible residues are less likely to be modified. Other factors, such as pKa values of amino acid side chains and participation in hydrogen bonds and salt bridges, also modulate the reactivity. After the labeling protocol, the proteins are typically processed via a traditional bottom-up mass spectrometric workflow with determination of the extent of reaction of individual amino acids based on the abundances of modified and unmodified peptides that contain those residues. The modified peptides must be pinpointed from complex proteolytic digests, and methods that enrich the modified peptides or uniquely target them offer substantial analytical advantages. This issue has been addressed by developing chemical probes and cross-linkers that are selectively cleavable or have recognition moieties for enrichment, thus streamlining the identification of the modified peptides.28,49−53,65−70 Protein homology modeling has played an important role in the interpretation of solvent accessibility and cross-linking results.58,71−77 Homology modeling typically entails the superimposition of a primary sequence of an unknown structure onto a known template structure. Homology models

the eIF4E and eIF4G proteins that form the eIFiso4F complex.14,15 The roles of the isomeric forms of the plant eIF4F proteins are still unknown. In plants, eIF4F and eIFiso4F both initiate translation,15 but there is evidence that the two isoforms mediate translation of mRNA differently and are expressed at different developmental points.16−18 eIF4E and eIFiso4E have different amino acid sequences (sharing only ∼50% sequence identity), and it is postulated that isoformspecific residues influence mRNA cap binding. Moreover, plant eIF4E and eIFiso4E exhibit different translational activities toward different levels of structure in viral and capped mRNAs.16 The genes for the subunits of both eIF4F and eIFiso4F have been shown to be natural virus resistance genes, suggesting that these isoforms could be an evolutionary feature in plants to mediate viral attacks.17,19−21 These intriguing hypotheses create an urgency to better understand the structural interactions and mechanisms of cap binding in eIF4F and eIFiso4F to potentially support the pursuit of genetic modification methods to enhance virus resistance in food crops.22 Given the numerous unresolved questions about the function and mechanistic details of the eIF4E proteins and their role in viral infection, they remain a compelling target for structural studies that can provide new insights. The determination of protein and macromolecular structures has primarily been undertaken by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, but the underlying difficulty of producing crystals (for the former) and the molecular size and sensitivity limitations of NMR have created a bottleneck in the growing field of structural biology. In fact, only a small fraction of known protein sequences have published structures listed in the protein database, and the increasingly sophisticated computational methods like ITASSER23 and ROSETTA24,25 for construction of structural models have begun to outpace some of the experimental methods. In this context, the development of new mass spectrometric approaches has gained popularity for the evaluation of higher order structures26 and in fact may offer one of the few methods for generating structural information for large proteins or protein complexes or for those that cannot be crystallized. Although well-established as a means to determine protein sequences via top-down or bottom-up strategies, tandem mass 5868

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Figure 2. UVPD mass spectra of (A) MVYSFHDDSR (3+) and (B) NN-modified WKEVIDYNDK (3+). The precursor ion is labeled with an asterisk. Δ indicates the retention of the NN modification. # indicates water loss.



have been used as a means to assess chemical labeling and cross-linking data to support or refute particular structures. For example, Wang and Chance demonstrated the confirmation of the structure of protein gp120-OD8 using oxidative footprinting and homology modeling, which accessed the structural differences within looped regions of the protein by monitoring oxidative reactivity.58 More recently, Levitt et al. have constructed a convincing model of the TRiC 16 unit subunit from a total of 40,320 potential structures using cross-linking and homology modeling.76 As we have recently reported, the NN chemical probe (named for the probe’s nitrogen−nitrogen hydrazone bond) reacts with primary amines and confers high UV absorption cross sections to any tagged species, thus allowing the NNmodified peptides to be readily pinpointed in complex mixtures based on selective UV photodissociation mass spectrometry.69,70 We assess the changes in the reactivities of lysine residues of wheat eIF4E upon complex formation with a fragment of eIF4G and full-length eIF4G. In addition, the reactivities of the lysine residues upon changes in redox state in eIF4E and eIFiso4E are monitored using the NN probe/ ultraviolet photodissociation (UVPD) mass spectrometry methodology. Structural analysis of the X-ray crystal structure of eIF4E and a homology model of eIFiso4E aided in the interpretation of the observed NN reactivates for all of the cap proteins. This method clearly shows that structural changes occur upon binding of eIF4G and that there are also changes in the eIF4E and eIFiso4E structures in the presence of high concentrations of dithiothreitol that are consistent with the reduction of a disulfide bond. We differentiate exposed reactive residues from unreactive residues within the 186 kDa eIF4F complex and compare the reactivity of eIF4E residues in the full protein complex with the unbound and “mini-eIF4F” states.

EXPERIMENTAL SECTION

Materials and Reagents

Proteomics grade trypsin, dithiothreitol (DTT), and PBS were purchased from Sigma Aldrich (St. Louis, MO). Recombinant eIF4E, eIFiso4E, and eIF4F were expressed and purified as described previously and were isolated using m7GTP sepharose affinity chromatography.78 The amine-reactive reagent, NN, was synthesized in house as previously reported.69 Expression and Purification of the eIF4E/eIF4G Mini-Complex

Limited proteolysis of the recombinant wheat eIF4F complex was carried out with thermolysin.79 The fragments of eIF4G that were still bound to eIF4E were recovered by chromatography on m7GTP sepharose. The most prominent peptide was identified by Edman degradation and mass spectrometry. The peptide (p27) contained amino acids 587−844 of wheat eIF4G (GenBank ABO15893), and the eIF4E binding site is roughly in the middle of this eIF4G peptide. Oliogonucleotides were made to amplify this region, and the DNA product was cloned into a vector (pET15b) containing truncated eIF4E (p26T) to form a discistronic expression construct (p26Tp27). The truncated eIF4E was the N-terminal deleted version previously used for structure determination.1 Four 1.2 L cultures of p26Tp27 (“mini-eIF4F complex”) in BL21(DE3) were grown to an OD600 of 0.5 and induced with 0.5 mM IPTG for 2 h prior to being harvested by centrifugation. The pellets were stored at −80 °C. The mini-eIF4F complex was then purified similarly to wheat eIF4F, and the purest fractions from the m7GTP sepharose column, as visualized using SDS-PAGE, were pooled and concentrated using an Amicon Ultra centrifugal filter.78 LC−MS/MS Analysis

The NN-chemical probe reactions of the various proteins, their proteolytic digestion and cleanup, and the LC−MS/MS analysis, subsequent data workup, and calculation of primary 5869

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Table 1. Predicted eIF4E and eIFiso4E Solvent Accessibilities (SAs) and Experimental NN Percent Reactivitiesa elF4E residue

predicted 4E SAb

4E−DTTc

4E+DTTd

+mini4G

+elF4G

mini4F−DTTc

4F−DTTc

N-term









18%

Ala39



0%

0%

0%



Lys56

36%

31 ± 2%

27 ± 7%

34 ± 4%

1%

Lys90 Lys101 Lys103 Lys107 Lys118

66% 7% 81% 16% 16%

ND 0% 91 ± 7% 0% ND

0% 0% 84 ± 5% 0% 0%

ND 0% 87 ± 5% 0% 0%

0% 0% 70% 0% 12%

Lys127 Lys160 Lys169 Lys182

66% 78% 19% 57%

0% 100% 4 ± 1% 67 ± 3%

0% 100% 0% 59 ± 4%

0% 100% 7 ± 2% 77 ± 2%

0% 100% 0% 15%

Lys185 Lys191 Lys203 Lys207 Lys210

11% 83% 39% 63% 69%

0% 0% 27 ± 7% 98 ± 1% 0%

0% 0% 4 ± 0% 97 ± 3% 0%

0% 0% 2 ± 1% 100% 0%

0% 2% 1% 100% 39%

elFiso4E residue

predicted iso4E SAe

iso4E−DTTc

iso4E+DTTd

Ala1



8 ± 1%

2 ± 0%

Lys29 Lys 33 Lys47/Lys49 Lys59 Lys60 Lys82 Lys93

− 66% 81%/64% 62% 51% 73% 6%

1 ± 0% 41 ± 4% 70% ± 10 0% 0% 0% 2 ± 0%

0% 45 ± 7% 81 ± 3% 0% 0% 0% 3 ± 2%

Lys99 Lys110 Lys118 Lys156

17% 9% 78% 30%

70 ± 7% 32 ± 4% 0% 57 ± 9%

60 ± 9% 5 ± 2% 0% 21 ± 6%

Lys162 Lys175 Lys176 Lys178 Lys186

25% 55% 20% 10% 65%

1 ± 0% 14 ± 1% 10 ± 2% 23 ± 4% 16 ± 4%

1 ± 0% 4 ± 1% 3 ± 1% 15 ± 3% 4 ± 2%

Lys199

86%

63 ± 6%

49 ± 7%

All reactions were carried out in the presence of 10 μM m7GTP. ND indicates that the peptide was not detected. A value of 0% indicates that the residue was identified but no corresponding NN-modified peptides were detected, and a value of 100% indicates that only NN-modified peptides were identified but not any unmodified peptides. All values are based on triplicate measurements except the full eIF4F percent reactivities, which are based on a single analysis. The entries displayed in bold indicate lysine residues that are conserved between both eIF4E and eIFiso4E. “−” indicates that the residue SA calculation or the NN reactivity was not calculated because of its absence for that particular protein or protein model. bPredicted solvent accessibilities (SAs) were calculated using the eIF4E structure in the presence of m7GTP ligand. cReactions were performed without excess DTT (0.1 mM). dReactions were carried out in excess DTT (1 mM). ePredicted SAs were calculated using the eIFiso4E homology model structure in the presence of m7GTP ligand. a

solvent accessibilities of m7GTP-free eIF4E and eIFiso4E would be inappropriate. Therefore, all solvent accessibilities were calculated using the models derived from the PDB 2IDV eIF4E X-ray crystal structure. The NMR structures of yeast eIF4F complex was used to model wheat eIF4E bound to the eIF4G peptide residues 393−490 (PDB 1RF8).11

amine reactivities were undertaken in a manner described previously.70 All reactions were in the presence of 10 μM m7GTP and either 0.1 mM DTT (−DTT) or 1 mM DTT (+DTT). Extensive details and protocols are included in the Supporting Information.



Structural Modeling of Wheat eIF4E, eIF4F, and eIFiso4E

Several known X-ray structures of mammalian eIF4E and eIF4E in complex with eIF4G fragments were used to model the conformational changes incurred by wheat eIF4E in the absence and presence of the m7GTP cap analog and upon subsequent binding of eIF4E to the eIF4G peptide. The wheat homology models were constructed based on using the SWISSMODEL (http://swissmodel.expasy.org/) program with the solved wheat eIF4E X-ray crystal structures. The structural models of wheat eIF4E and eIFiso4E in the absence of the cap analog were generated from the wheat eIF4E C113S mutant (bound with m7GTP) X-ray crystal structure (PDB 2IDR).1 Additional models were produced to visualize the structures of eIF4E and eIFiso4E without m7GTP based on the X-ray crystal structure of the apo-eIF4E dimer (PDB 2IDR). The solvent accessibilities of the lysines and N-termini of the m7GTP-free eIF4E and eIFiso4E models were not calculated because the eIF4E dimer structure is believed to be artifactually produced during protein crystallization. eIF4E does not naturally form a dimer, and thus using the PDB 2IDR structure to calculate the

RESULTS

Properties of the Chemical Probe

The chemical probe NN (Supplementary Figure 1 in the Supporting Information) was designed to react with primary amines, such as those found on the side-chain of lysine and the N-terminus. Each resulting NN-modified residue has a trackable mass shift (+223 Da) and is endowed with a UV chromophore at 351 nm. Upon proteolysis of the NN-modified protein or protein complex and LCMS analysis, those peptides that contain the NN modification are readily detected and differentiated from the large number of highly abundant nonmodified peptides based on the production of characteristic fragment ions upon UVPD. Peptides without the NN chromophore do not undergo UVPD (i.e., no fragmentation pattern), as demonstrated in Figure 2A for the eIFiso4E peptide MVYSFHDDSR. However, peptides that contain the NN-tag, such as WKNNEVIDYNDK (where the subscripted NN indicates the site of NN modification) in Figure 2B, undergo 5870

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mass shift of the NN-modified lysine-containing b and y sequence ions. Every peptide was subjected to UVPD to readily differentiate the modified peptides from the unmodified peptides (and facilitate tabulation of the modified peptides), and CID was employed to identify the unmodified and modified peptides. After identification of all peptides upon LCMS/MS of each tryptic digest, their abundances were quantified by manual integration of peak areas in the total ion current (TIC) profiles and used to calculate percent reactivities based on the equation shown in the Experimental Section. The percent reactivities shown in Table 1 under various conditions reflect the accessibilities of each amine site to the NN probe. The entries for those lysines that are conserved are displayed in bold in Table 1 to help facilitate the comparison between the reactivities and lysine residues of eIF4E, eIF4E in complex with eIF4G or the truncated version of eIF4G, and eIFiso4E. The data in Table 1 are also shown as histograms in Supplemental Figure 2 in the Supporting Information to assist in visualization of the trends in the NN reactivities of the various amines.

photodissociation upon exposure to 351 nm photons and produce diagnostic b and y ions that allow the probe-modified peptides to be sequenced. It is this striking difference in UVPD response that allows the probe-modified and unmodified peptides to be readily differentiated. This NN chemical probe/UVPD workflow provides a robust method to pinpoint the probe-modified peptides with great selectivity in complex proteolytic mixtures containing many unmodified peptides. NN/UVPD Strategy for eIF4E and eIFiso4E

In addition to the N-terminus, eIF4E has 15 lysines and eIFiso4E has 17 lysines. which are all potential reaction sites upon exposure to the NN chemical probe. (See Table 1 and Figure 3.) In general, chemical probes interact with a protein

Probing the Primary Amine Reactivities and Solvent Accessibilities of eIF4E and eIFiso4E

Alignment of the amino acid sequences of wheat eIF4E and eIFiso4E shows that there are 11 lysine residues that are conserved exactly (seven residues, highlighted in blue in Figure 3) or within 1 or 2 amino acids (four residues highlighted in orange in Figure 3). Given the high degree of similarity between these two proteins, it is expected that the environments of the lysine residues would be very similar; however, of the 11 lysine residues that are conserved, 7 show significant differences in reactivity with the chemical probe (Table 1). Specifically, the eIF4E lysine residues K56, K107, K118, K185, and K191 demonstrate higher reactivity in eIFiso4E, whereas K182 and K207 show lower reactivity in eIFiso4E. The crystal structure for eIF4E (PDB 2IDV) and predicted structure for eIFiso4E (Figure 1B and Supplemental Figure 3B in the Supporting Information) provide explanations for these differences in reactivity based on the ability of the lysine residue to form hydrogen bonds or ion pairs (see Supplementary Figure 4 in the Supporting Information). This analysis suggests that although the protein structures are highly conserved there are subtle differences that could influence their ability to interact with mRNA or other proteins during initiation of translation. The present work shows that variable protein environments may be successfully sampled using chemical probe/UVPD mass spectrometry and that the results are consistent with known or modeled structures. To further assess the utility of this method to identify dynamic structural changes, a potential disulfide bond in eIF4E and eIFiso4E was reduced in the presence of 1 mM dithiothreitiol, and any structural changes were assessed using chemical probe/UVPD mass spectrometry. As shown in Table 1, only K203 in eIF4E showed a significant change (∼7-fold) in reactivity in the presence of excess dithiothreitol, whereas three residues in eIFiso4E (K110, K156, K186) showed ∼2−6 fold changes in reactivity. In each case where there was a change in reactivity, the structure of eIF4E or model of eIFiso4E indicated that these residues were in regions that may be affected by disulfide bond formation. Thus the use of the chemical probe/UVPD-MS strategy was able to indicate structural changes due to the reduction of a predicted disulfide bond.

Figure 3. CLUSTALW2 protein sequence alignment between wheat eIF4E and eIFiso4E. Conserved tryptophan and cysteine residues important for m7GTP binding or disulfide bond formation are highlighted with red and green bands, respectively. Perfectly conserved lysine residues are highlighted with blue bands or orange for those lysine positions off by one or two amino acids. The location of the eIF4E truncation is shown with an arrow.

during a time period in which the protein dynamically transitions among various states, and thus the measured reactivities of particular sites reflect average conformations of the protein or its complexes in solution. The corresponding calculations of solvent accessibility are based on static conformations (often derived from X-ray crystal structures). Both the reaction time and the molar ratio of NN to protein influence the number of sites modified such that longer reaction periods and higher relative concentrations of NN lead to more extensive protein modification. For the present study, a reaction time of 30 min and an NN:protein molar ratio of 20:1 was used to afford a balance between minimizing the reaction time and obtaining a comprehensive map of primary amine reactivity. eIF4E demonstrated some reactivity at seven sites, and eIFiso4E showed reactivity at 12 sites with the NN probe based on UVPD/CID analysis of the modified and unmodified peptides produced after tryptic digestion of the proteins. 351 nm UVPD-MS was performed to pinpoint the elution of the low-abundance NN-modified peptides among the complex digests. The NN-modified peptides produce characteristic b/y fragment ion maps and also yield two reporter ions, m/z 130 and m/z 224, which are indicative of the presence of the NN tag. In the same context, CID provides facile sequencing of both modified and unmodified peptides, and the NN modification is not labile so it is tracked via the consistent 5871

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Figure 4. Modeled structures of (A) truncated eIF4E and (B) eIFiso4E with NN-modified lysine residues shown. These structures were produced based on the PDB 2IDR crystal structure and in the absence of m7GTP.



Changes in the structure of eIF4E were also evaluated when its protein binding partner eIF4G was present. First, a tryptic peptide fragment (∼27 kDa) of eIF4G containing the eIF4E binding site was identified. The fragment was cloned and coexpressed with eIF4E to form the eIF4F “mini-complex”. The “mini-complex” was evaluated using chemical probe/UVPDmass spectrometry, and the reactivity of one lysine residue of eIF4E (K203) was found to change significantly (Table 1). Interestingly, this is the same residue for which its reactivity changed in the presence of excess dithiothreitol. This suggests that the binding of eIF4G to eIF4E may induce changes in the cap-binding pocket similar to reduction of the disulfide bond that has been shown in vitro to modestly increase the binding affinity for m7GTP.1 Interestingly, the complex of eIF4E with full-length eIF4G shows even greater changes in lysine reactivity. The reactivities of lysines 56, 182, and 203 decrease in the complex, whereas the reactivities of lysines 118 and 210 increase. Given that the size of the eIF4G subunit is ∼180 kDa compared with ∼27 kDa for the eIF4G fragment, it is not surprising that the eIF4G engages in additional contacts with eIF4E that likely change the solvent accessibility (and reactivity) of the lysine residues either by masking the lysines or changing their local environments to make them more exposed. In the absence of a crystal structure for eIF4G in complex with eIF4E, chemical probe studies such as these help to provide clues about the dynamic nature of these proteins. The modeled structures based on X-ray data for eIF4E in both states (with and without m7GTP cap) are illustrated in Figure 1 and provide a visual guide of eIF4E cap binding. The cap-binding residues (amino acids W62 and W108) are highlighted in orange in the structure of eIF4E in the absence of ligand shown in Figure 1A and are in fact themselves highly accessible residues. Upon m7GTP cap analog binding, oxidized eIF4E exhibits the formation of a hydrophobic core around the cap analog. (See Figure 1B.) The tryptophan residues that were highly accessible in the unbound state interact significantly with the m7GTP cap analog, thus creating a far less solvent accessible region for all residues near the binding site, including the highlighted K203 site. The equilibrium established between m7GTP bound and unbound eIF4E creates a mixture of bound and unbound structures in solution, both of which would be sampled in the reactions with the NN chemical probe consistent with the observed results. The predicted solvent accessibilities of the N-terminal and lysine residues using the m7GTP bound eIF4E model from Figure 1B are listed in Table 1

DISCUSSION

Primary Amine Reactivities and Solvent Accessibilities of eIF4E

For uncomplexed eIF4E, the primary amines that exhibited NN reactivities included K56, K103, K160, K169, K182, K203, and K207 (highlighted with arrows in Figure 4A) and are summarized in Table 1 (calculated using the data shown in Supplemental Tables 1−6 in the Supporting Information). The least reactive and presumably the less accessible or least nucleophilic residues were determined to be the N-terminus (A39), K90, K101, K107, K118, K127, K185, K191, and K210 (Table 1, in the column 4E−DTT column). Among the lysines, K103, K160, K182, and K207 were the most highly reactive sites, thus correlating well with their predicted higher solvent accessibilities in both the cap analog bound and unbound forms of eIF4E. The residues K56 and K203 showed evidence of moderate reactivity toward NN, and this behavior can be rationalized by inspection of the structures in Figure 1. Both K203 and K56 convert from exposed to less accessible positions upon binding the cap analog, consistent with their overall moderate reactivities and suggesting that both structures in Figure 1 are sampled in the chemical probe experiment. As illustrated in Table 1 (in the 4E−DTT column), the majority of residues that exhibited no NN reactivity were predicted to have very low solvent accessibilities. However, the calculated solvent accessibilities of K90, K127, K191, and K210 were rather high (ranging from 66 to 83%), whereas the experimentally measured reactivities were zero. One rationalization for this discrepancy is that all of these residues are likely to engage in polar contacts (hydrogen bonds, salt bridges) with surrounding atoms (Supplemental Figure 4 in the Supporting Information), not only altering the exposure of the amines but also modulating their nucleophilicities and pKa values. Any changes in the pKa values of the lysines will alter the equilibrium distribution of deprotonated and protonated forms, with the latter being unreactive with the probe. Other factors such as the hydrophobicity and size of the NN probe may affect its interaction with the various amine sites. These factors, which are not accounted for in the solvent accessibility calculations, likely contribute to the low reactivities of K90, K127, K191, and K210. This conglomeration of factors likewise increases the sensitivity of the NN probe to subtle structural differences that may occur when a protein is probed in different environments, making comparative measurements under varying conditions highly relevant. This method may be highly useful when 5872

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analyzing small molecule effectors for changes in structure at or near active sites. There was little significant change in NN reactivity for K56, K160, and K207 when the reactions were undertaken in a reducing buffer to promote cleavage of any disulfide bonds in eIF4E, thus suggesting little change in environment (Table 1, column 4E +DTT). However, K203 displayed a substantial decrease in NN reactivity and parallels an expected decrease in solvent accessibility for residues localized around the m7GTP cap-binding site. Previous studies have suggested that eIF4E when in a reducing environment1 or when bound to eIF4G should favor the m7GTP cap bound structure.3,4 Our results suggest that the reduction of the disulfide bond of eIF4E or interaction with the binding partner eIF4G likely enhances m7GTP cap binding. Given this information, the change in the NN reactivity of K203 implies that the m7GTP cap binding may be favored in a reducing environment, as reflected by the lower accessibility of K203 as the neighboring hydrophobic pocket forms around the m7GTP cap analog (Figure 1B). Interestingly K103, K169, and K182 all showed a slight yet reproducible reduction in NN reactivity in the reducing environment. This suggests that these residues, upon binding to m7GTP in a reduced environment, may be shifted toward the less accessible hydrophobic core of the protein during stabilization of the protein upon m7GTP cap binding.

Figure 5. Homology model of truncated wheat eIF4E as it would likely appear in the complex eIF4F with m7GTP. Models were based on the yeast eIF4F structure PDB 1RF8. eIF4E is shown in green, the eIF4G peptide is shown in magenta, and the cap structure is shown in yellow.

Primary Amine Reactivities and Solvent Accessibilities of eIF4E in Complex with eIF4G Peptide (Mini-eIF4F Complex)

complex was incubated with NN at a 30:1 molar ratio prior to proteolytic digestion and analysis. The congestion of the base peak chromatogram of one of the digests is exemplified in Figure 6A, resulting in over 15,000 spectra, most of them from

The NN reactivity of eIF4E while in complex with the eIF4G peptide (i.e., formation of the eIF4F mini-complex, see Table 1 in the column +mini4G) follows a similar NN reactivity trend as observed for free eIF4E in its reduced state (Table 1, in the column 4E−DDT), with two notable exceptions, residues K182 and K203. The similarities in the NN reactivities of most of the lysine residues mirror the structural similarities between the modeled eIF4E structure in complex with eIF4G and m7GTP (Figure 5) and the wheat eIF4E models (Figure 1). These similarities suggest that eIF4E has similar solvent accessibility and thus should exhibit similar NN reactivity regardless of eIF4G binding state. However, there are noticeable differences in NN reactivity for residues K182 and K203. The experimentally determined NN reactivity of K203 while bound in the eIF4F mini-complex decreased relative to the NN reactivity of K203 in free eIF4E. This result suggests that m7GTP may bind more tightly to the eIF4F mini-complex relative to the binding of m7GTP to free eIF4E, thus significantly reducing the accessibility of K203. A similar trend is observed for eIF4E bound to the full eIF4G protein and is discussed in more detail below. In contrast, K182 displays an increase in reactivity in the eIF4F mini-complex, revealing that the interaction of eIF4E in the mini-eIF4F complex appears to favor the cap analog bound structure of eIF4E and increases the accessibility of the K182 site. The full eIF4F complex (180 kDa; composed of the 24 kDa eIF4E and the full length 162 kDa eIF4G proteins) contains 120 lysine residues and two N-terminal primary amines, all with the potential to react with the NN probe. Proteolytic digest of the eIF4F complex produces hundreds of peptides, creating a significant analytical challenge when trying to identify the low abundance NN-modified peptides that reveal relative reactivity/ solvent accessibility information. To evaluate the utility of the UVPD-MS strategy to facilitate the identification of probemodified peptides in a very complex mixture, the eIF4F

Figure 6. LC-UVPD-MS results showing (A) base peak chromatogram and (B) extracted ion chromatogram of NN reporter ion (m/z 130) of the tryptic digest of NN-modified protein complex eIF4F.

unmodified peptides. The extracted ion chromatogram for the NN reporter ion (m/z 130) generated by UVPD yields a subset of spectra that readily earmark the modified peptide candidates (Figure 6B). This type of selective reporter ion filtering significantly streamlines the initial identification of the modified peptides required for large proteomic workflows. For the peptides highlighted in the tryptic digest shown in Figure 6B, 70 modified sites were identified. The affiliated NN-modified tryptic peptides are summarized in Supplemental Table 7 in the 5873

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Supporting Information for each of the two interacting proteins (with the corresponding lists generated for the chymotrypsin and GluC digests provided in Supplemental Table 8 in the Supporting Information). CID-MS was then employed to monitor and calculate the changes of reactivity of the eIF4E in the untruncated eIF4F complex relative to the truncated versions of eIF4E in its monomeric and complexed states. It was determined that the N-terms, K56, K103, K118, K160, K182, K191, K203, and K207, exhibited at least some reactivity under all reaction conditions. Additionally, no NN modifications of K90, K101, K107, K127, K169, and K185 were found. Several trends emerge for the lysine reactivity of eIF4E in the full eIF4F complex (Table 1, in the column +eIF4G), truncated eIF4E (Table 1, in the column 4E−DTT), and within the mini eIF4F complex (Table 1, in the column +mini4G). In particular, for all reaction conditions, the most reactive residues were K103, K160, and K207, further suggesting that these residues are the most exposed lysine residues for eIF4E in the protein’s native state. The most unreactive residues were K90, K101, K107, K127, and K185. Importantly, K203, located near the cap-binding site, demonstrated a decrease in NN reactivity (Table 1, in the column +eIF4G), suggesting that K203 is exposed in the unbound ligand or oxidized state (Figure 1B) and parallels the observed reactivity changes for eIF4E within the mini-eIF4F complex (Table 1, in the column +mini4G). There are several additional modified sites (N-term, K118, K191, and K210) and also reactivity changes noted for eIF4E in the full eIF4F complex that were not observed for any of the previous reaction conditions. The N-terminus, a residue not present in the truncated form of the eIF4E protein, exhibits moderate reactivity toward NN. This result suggests that the Nterminus interacts with the eIF4G protein and is inaccessible for the majority of the time in solution, but the moderate reactivity suggests that the N-terminus has some time to react with the NN probe. This outcome was expected as the Nterminal region of eIF4E is known to be flexible in its monomeric state but is stabilized upon interaction with the eIF4G protein. K56 and K182 of eIF4E each showed significant decreases in NN reactivity within the full version of the eIF4F complex (Table 1, in the column 4F−DTT) but not in the mini-eIF4F complex (Table 1, in the column +mini4G), an outcome that may be directly tied to the difference in size of the two protein partners (27 kDa for mini-eIF4G versus 162 kDa for eIF4G).

predicted to be very solvent exposed, yet no reactivity with the NN probe was observed. The sequence alignment of eIF4E and eIFiso4E (Figure 3) shows that K82 in eIFiso4E and K90 in eIF4E (as well as an analogous correlation for K118 in eIFiso4E and K127 in eIF4E) are found in similar locations as these residues overlap in the primary structures. Furthermore, neither of these residues in either protein proved to be reactive, suggesting that other factors, such as low nucleophilicity or participation in polar or ionic contacts, influenced the reactivities of these amines. The reactions of eIFiso4E with the NN probe were also undertaken in a reducing environment in an identical manner to eIF4E to chemically disrupt the predicted C105−C145 disulfide bond (Supplemental Figure 3A in the Supporting Information) and favor the m 7 GTP bound structure (Supplemental Figure 3B in the Supporting Information). Relatively minor or no changes in reactivity were observed for K33, K93, and K162 (see Table 1, in the column iso4E+DTT), an outcome that parallels the predicted solvent accessibilities of eIFiso4E (Supplemental Table 9 in the Supporting Information, in the column predicted iso4E SA). Overall eIFiso4E, like eIF4E, exhibits structural changes dependent on the C105− C145 oxidation states (Supplemental Figure 3 in the Supporting Information). In particular an 11% increase in NN reactivity occurred for K47/K49 on going to the reducing environment (Table 1, in the columns iso4E−DTT and iso4E +DTT), and this result matches the average increase in solvent accessibility for those lysine sites for the unbound versus bound states. Unfortunately, K203 that showed the most change in eIF4E is not a conserved lysine in eIFiso4E to provide information about changes in the cap-binding pocket. However, K99, K110, K156, K175, K176, K178, K186, and K199 all showed a reduction in NN reactivity in the reducing environment (Table 1, in the column iso4E +DTT). The residues K99, K110, and K156 all lie within the hydrophobic pocket, and a decrease in reactivity is expected as the model for reduced eIFiso4E favors the more rigid m7GTP-bound structure (Supplemental Figure 3B in the Supporting Information), which should reduce the accessibilities of those lysine residues. K199 demonstrated a reduction in reactivity, whereas solvent-accessibility calculations predicted greater exposure, suggesting that K199 may play a role in cap binding that does not directly correlate with accessibility. K175, K176, K178, and K186, as accessible residues, are not found within the cap-binding hydrophobic pocket but did exhibit a reduction in reactivity in the reducing environment, which implies that these residues are affected by cap-binding. The corresponding NN reactivities reflected this prediction for these four residues. Additional discussion of the NN reactivities and structural differences between eIF4E and eIFiso4E is included as Supporting Information.

Primary Amine Reactivities and Solvent Accessibilities of eIFiso4E

A total of 15 of 19 lysine amines plus the N-terminus of eIFiso4E displayed some reactivity toward the NN probe, as summarized in Table 1 (in the column iso4E−DTT) and visualized in Figure 4B. Four lysine amines, K59, K60, K82, and K118 exhibited no reactivity toward the NN probe for any of the reaction conditions. The amine sites A1 (N-term), K29, K93, K162, K175, K176, and K186, exhibited modest reactivity ( K199 ≈ K156 > K33 > K110 > K178 ≈ K186 > K175 ≈ K 176 > Ala1 > K29 ≈ K93 ≈ K162 > K59, K60, K82, and K118. The most notable discrepancy between the calculated solvent accessibilities and NN probe reactivities arose for K82 and K118 because these residues were



CONCLUSIONS The reported chemical probe/UVPD-MS approach allowed the examination of a number of structural details of eIF4E and the isoform protein eIFiso4E by targeting local reactivities of lysine residues. Subtle changes in the reactivities of lysine residues of eIF4E were observed upon the formation of a complex with an eIF4G peptide or upon reduction of its disulfide bonds, both of which are consistent with the ability of eIF4E to bind the m7GTP cap analog. The NN chemical probe methodology in conjunction with homology modeling also facilitated the elucidation of structural details of eIFiso4E and the 5874

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Characterization of mRNA Discriminatory Initiation Factors. Proc. Natl. Acad. Sci. U. S. A. 1983, 80, 663−667. (5) Furic, L.; Rong, L.; Larsson, O.; Koumakpayi, I. H.; Yoshida, K.; Brueschke, A.; Petroulakis, E.; Robichaud, N.; Pollak, M.; Gaboury, L. A.; et al. eIF4E Phosphorylation Promotes Tumorigenesis and Is Associated with Prostate Cancer Progression. Proc. Natl. Acad. Sci. 2010, 107, 14134−14139. (6) Chen, X.; Kopecky, D. J.; Mihalic, J.; Jeffries, S.; Min, X.; Heath, J.; Deignan, J.; Lai, S.; Fu, Z.; Guimaraes, C.; et al. Structure-Guided Design, Synthesis, and Evaluation of Guanine-Derived Inhibitors of the eIF4E mRNA−Cap Interaction. J. Med. Chem. 2012, 55, 3837− 3851. (7) Graff, J. R.; Konicek, B. W.; Vincent, T. M.; Lynch, R. L.; Monteith, D.; Weir, S. N.; Schwier, P.; Capen, A.; Goode, R. L.; Dowless, M. S.; et al. Therapeutic Suppression of Translation Initiation Factor eIF4E Expression Reduces Tumor Growth Without Toxicity. J. Clin. Invest. 2007, 117, 2638−2648. (8) Volpon, L.; Osborne, M. J.; Topisirovic, I.; Siddiqui, N.; Borden, K. L. Cap-free Structure of eIF4E Suggests a Basis for Conformational Regulation by Its Ligands. EMBO J. 2006, 25, 5138−5149. (9) Siddiqui, N.; Tempel, W.; Nedyalkova, L.; Volpon, L.; Wernimont, A. K.; Osborne, M. J.; Park, H.-W.; Borden, K. L. B. Structural Insights into the Allosteric Effects of 4EBP1 on the Eukaryotic Translation Initiation Factor eIF4E. J. Mol. Biol. 2012, 415, 781−792. (10) Marcotrigiano, J.; Gingras, A. C.; Sonenberg, N.; Burley, S. K. Cap-dependent Translation Initiation in Eukaryotes Is Regulated by a Molecular Mimic of eIF4G. Mol. Cell 1999, 3, 707−716. (11) Gross, J. D.; Moerke, N. J.; von der Haar, T.; Lugovskoy, A. A.; Sachs, A. B.; McCarthy, J. E. G.; Wagner, G. Ribosome Loading onto the mRNA Cap Is Driven by Conformational Coupling Between eIF4G and eIF4E. Cell 2003, 115, 739−750. (12) Matsuo, H.; Li, H.; McGuire, A. M.; Fletcher, C. M.; Gingras, A. C.; Sonenberg, N.; Wagner, G. Structure of Translation Factor eIF4E Bound to m7GDP and Interaction with 4E-binding Protein. Nat. Struct. Biol. 1997, 4, 717−724. (13) Brown, C. J.; Verma, C. S.; Walkinshaw, M. D.; Lane, D. P. Crystallization of eIF4E Complexed with eIF4GI Peptide and Glycerol Reveals Distinct Structural Differences Around the Cap-binding Site. Cell Cycle 2009, 8, 1905−1911. (14) Browning, K. S. The Plant Translational Apparatus. Plant Mol. Biol. 1996, 32, 107−144. (15) Browning, K. S.; Webster, C.; Roberts, J. K.; Ravel, J. M. Identification of an Isozyme Form of Protein Synthesis Initiation Factor 4F in Plants. J. Biol. Chem. 1992, 267, 10096−10100. (16) Gallie, D. R.; Browning, K. S. eIF4G Functionally Differs from eIFiso4G in Promoting Internal Initiation, Cap-Independent Translation, and Translation of Structured mRNAs. J. Biol. Chem. 2001, 276, 36951−36960. (17) Duprat, A.; Caranta, C.; Revers, F.; Menand, B.; Browning, K. S.; Robaglia, C. The Arabidopsis Eukaryotic Initiation Factor (iso)4E Is Dispensable for Plant Growth but Required for Susceptibility to Potyviruses. Plant J. 2002, 32, 927−934. (18) Rodriguez, C. M.; Freire, M. A.; Camilleri, C.; Robaglia, C. The Arabidopsis Thaliana cDNAs Coding for eIF4E and eIF(iso)4E Are Not Functionally Equivalent for Yeast Complementation and Are Differentially Expressed During Plant Development. Plant J. 1998, 13, 465−473. (19) Robaglia, C.; Caranta, C. Translation Initiation Factors: a Weak Link in Plant RNA Virus Infection. Trends Plant Sci. 2006, 11, 40−45. (20) Song, A.; Lou, W.; Jiang, J.; Chen, S.; Sun, Z.; Guan, Z.; Fang, W.; Teng, N.; Chen, F. An Isoform of Eukaryotic Initiation Factor 4E from Chrysanthemum Morifolium Interacts with Chrysanthemum Virus B Coat Protein. PLoS ONE 2013, 8, e57229. (21) Gallois, J.-L.; Charron, C.; Sanchez, F.; Pagny, G.; Houvenaghel, M.-C.; Moretti, A.; Ponz, F.; Revers, F.; Caranta, C.; German-Retana, S. Single Amino Acid Changes in the Turnip Mosaic Virus Viral Genome-linked Protein (VPg) Confer Virulence Towards Arabidopsis

confirmation of redox-mediated cap-binding behavior. The probe/UVPD-MS approach highlighted the ability to selectively track modified lysine-containing peptides within the complex digest of the 186 kDa eIF4F protein complex. Overall, several conclusions can be drawn from monitoring lysine reactivity based on the NN chemical probe/UVPD-MS strategy and supplemented by homology modeling. eIF4E and eIFiso4E both exhibit structural changes that are dependent on the oxidation state of regulatory disulfide bonds. This is most evident from the reduction of lysine NN reactivity for residues associated with the m7GTP cap-binding region. Similarly, both eIF4G and mini-eIF4G alter the structure of eIF4E within the eIF4F or mini-eIF4F complexes. eIF4E and eIFiso4E both have similar structures yet retain subtle differences in their tertiary and secondary structures. The lysine amines of eIF4E showed an overall lower reactivity toward the NN probe relative to the amines of eIFiso4E. eIF4E reacted at a total of 7 out of a possible 16 amine sites, and eIFiso4E reacted at a total of 14 out of a total of 19 possible reactive sites, many of which are within the cap-binding site. We anticipate that this NN-probe/ UVPD methodology can be extended for examination of other protein−protein and protein−ligand complexes, where protein structural data are absent, such as the eIFiso4E, mini-eIF4F, and eIF4F protein systems described in this report.



ASSOCIATED CONTENT

S Supporting Information *

Details of mass spectrometry, database searching, derivatization and sample preparation, circular dichroism, determination of primary amine reactivities, primary amine reactivities and solvent accessibilities of eIF4E in complex with eIF4G peptide (mini-eIF4F complex), homology model of eIFisoE, and NN reactivities and structural comparison of eIF4E and eIFiso4E. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Funding from the NSF (CHE-1012622 to J.S.B. and MCB1052530 and Arabidopsis 2010 S-0000335 to K.S.B.) and the Welch Foundation (F1155 to J.S.B) is gratefully acknowledged. We thank Art Monzingo for thoughtful and insightful discussions.



REFERENCES

(1) Monzingo, A. F.; Dhaliwal, S.; Dutt-Chaudhuri, A.; Lyon, A.; Sadow, J. H.; Hoffman, D. W.; Robertus, J. D.; Browning, K. S. The Structure of Eukaryotic Translation Initiation Factor-4E from Wheat Reveals a Novel Disulfide Bond. Plant Physiol. 2007, 143, 1504−1518. (2) Prévôt, D.; Darlix, J.; Ohlmann, T. Conducting the Initiation of Protein Synthesis: The Role of eIF4G. Biol. Cell 2003, 95, 141−156. (3) Haghighat, A.; Sonenberg, N. eIF4G Dramatically Enhances the Binding of eIF4E to the mRNA 5′-Cap Structure. J. Biol. Chem. 1997, 272, 21677−21680. (4) Ray, B. K.; Brendler, T. G.; Adya, S.; Daniels-McQueen, S.; Miller, J. K.; Hershey, J. W.; Grifo, J. A.; Merrick, W. C.; Thach, R. E. Role of mRNA Competition in Regulating Translation: Further 5875

dx.doi.org/10.1021/pr400869u | J. Proteome Res. 2013, 12, 5867−5877

Journal of Proteome Research

Article

Thaliana Mutants Knocked Out for Eukaryotic Initiation Factors eIF(iso)4E and eIF(iso)4G. J. Gen. Virol. 2009, 91, 288−293. (22) Woloshen, V.; Huang, S.; Li, X. RNA-Binding Proteins in Plant Immunity. J. Pathog. 2011, 2011, 1−11. (23) Roy, A.; Kucukural, A.; Zhang, Y. I-TASSER: a Unified Platform for Automated Protein Structure and Function Prediction. Nat. Protoc. 2010, 5, 725−738. (24) Simons, K. T.; Bonneau, R.; Ruczinski, I.; Baker, D. Ab Initio Protein Structure Prediction of CASP III Targets Using ROSETTA. Proteins: Struct., Funct., Bioinf. 1999, 37, 171−176. (25) Kuhlman, B.; Dantas, G.; Ireton, G. C.; Varani, G.; Stoddard, B. L.; Baker, D. Design of a Novel Globular Protein Fold with AtomicLevel Accuracy. Science 2003, 302, 1364−1368. (26) Benesch, J. L. P.; Ruotolo, B. T. Mass Spectrometry: Come of Age for Structural and Dynamical Biology. Curr. Opin. Struct. Biol. 2011, 21, 641−649. (27) Mendoza, V. L.; Vachet, R. W. Probing Protein Structure by Amino Acid-specific Covalent Labeling and Mass Spectrometry. Mass Spectrom. Rev. 2009, 28, 785−815. (28) Zhou, X.; Lu, Y.; Wang, W.; Borhan, B.; Reid, G. Fixed Charge” Chemical Derivatization and Data Dependant Multistage Tandem Mass Spectrometry for Mapping Protein Surface Residue Accessibility. J. Am. Soc. Mass Spectrom. 2010, 21, 1339−1351. (29) Zhang, H.; Gau, B. C.; Jones, L. M.; Vidavsky, I.; Gross, M. L. Fast Photochemical Oxidation of Proteins for Comparing Structures of Protein-ligand Complexes: The Calmodulin-peptide Model System. Anal. Chem. 2011, 83, 311−318. (30) Hambly, D. M.; Gross, M. L. Laser Flash Photolysis of Hydrogen Peroxide to Oxidize Protein Solvent-accessible Residues on the Microsecond Timescale. J. Am. Soc. Mass Spectrom. 2005, 16, 2057−2063. (31) Lauber, M. A.; Reilly, J. P. Novel Amidinating Cross-Linker for Facilitating Analyses of Protein Structures and Interactions. Anal. Chem. 2010, 82, 7736−7743. (32) Bornschein, R.; Hyung, S.-J.; Ruotolo, B. Ion Mobility-Mass Spectrometry Reveals Conformational Changes in Charge Reduced Multiprotein Complexes. J. Am. Soc. Mass Spectrom. 2011, 22, 1690− 1698. (33) Ruotolo, B. T.; Benesch, J. L. P.; Sandercock, A. M.; Hyung, S.J.; Robinson, C. V. Ion Mobility|[ndash]|mass Spectrometry Analysis of Large Protein Complexes. Nat. Protoc. 2008, 3, 1139−1152. (34) Hilton, G. R.; Benesch, J. L. P. Two Decades of Studying NonCovalent Biomolecular Assemblies by Means of Electrospray Ionization Mass Spectrometry. J. R. Soc. Interface 2012, 9, 801−816. (35) Loo, J. A. Studying Noncovalent Protein Complexes by Electrospray Ionization Mass Spectrometry. Mass Spectrom. Rev. 1998, 16, 1−23. (36) Abzalimov, R. R.; Kaplan, D. A.; Easterling, M. L.; Kaltashov, I. A. Protein Conformations Can Be Probed in Top-Down HDX MS Experiments Utilizing Electron Transfer Dissociation of Protein Ions Without Hydrogen Scrambling. J. Am. Soc. Mass Spectrom. 2009, 20, 1514−1517. (37) Pan, Y.; Piyadasa, H.; O’Neil, J. D.; Konermann, L. Conformational Dynamics of a Membrane Transport Protein Probed by H/D Exchange and Covalent Labeling: The Glycerol Facilitator. J. Mol. Biol. 2012, 416, 400−413. (38) Zhang, H.; Yu, X.; Greig, M. J.; Gajiwala, K. S.; Wu, J. C.; Diehl, W.; Lunney, E. A.; Emmett, M. R.; Marshall, A. G. Drug Binding and Resistance Mechanism of KIT Tyrosine Kinase Revealed by Hydrogen/deuterium Exchange FTICR Mass Spectrometry. Protein Sci. 2010, 19, 703−715. (39) Frantom, P. A.; Zhang, H.-M.; Emmett, M. R.; Marshall, A. G.; Blanchard, J. S. Mapping of the Allosteric Network in the Regulation of α-Isopropylmalate Synthase from Mycobacterium Tuberculosis by the Feedback Inhibitor l-Leucine: Solution-Phase H/D Exchange Monitored by FT-ICR Mass Spectrometry. Biochemistry 2009, 48, 7457−7464.

(40) Katta, V.; Chait, B. T.; Carr, S. Conformational Changes in Proteins Probed by Hydrogen-exchange Electrospray-ionization Mass Spectrometry. Rapid Commun. Mass Spectrom. 2005, 5, 214−217. (41) Niedzwiecka, A.; Marcotrigiano, J.; Stepinski, J.; JankowskaAnyszka, M.; Wyslouch-Cieszynska, A.; Dadlez, M.; Gingras, A.-C.; Mak, P.; Darzynkiewicz, E.; Sonenberg, N.; et al. Biophysical Studies of eIF4E Cap-binding Protein: Recognition of mRNA 5′ Cap Structure and Synthetic Fragments of eIF4G and 4E-BP1 Proteins. J. Mol. Biol. 2002, 319, 615−635. (42) Rozbesky, D.; Man, P.; Kavan, D.; Chmelik, J.; Cerny, J.; Bezouska, K.; Novak, P. Chemical Cross-Linking and H/D Exchange for Fast Refinement of Protein Crystal Structure. Anal. Chem. 2011, 84, 867−870. (43) Zhang, Z.; Smith, D. L. Determination of Amide Hydrogen Exchange by Mass Spectrometry: A New Tool for Protein Structure Elucidation. Protein Sci. 2008, 2, 522−531. (44) Kaltashov, I. A.; Bobst, C. E.; Abzalimov, R. R. H/D Exchange and Mass Spectrometry in the Studies of Protein Conformation and Dynamics: Is There a Need for a Top-down Approach? Anal. Chem. 2009, 81, 7892−7899. (45) Valeja, S.; Emmett, M.; Marshall, A. Polar Aprotic Modifiers for Chromatographic Separation and Back-Exchange Reduction for Protein Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2012, 23, 699−707. (46) Collins, C. J.; Schilling, B.; Young, M.; Dollinger, G.; Guy, R. K. Isotopically Labeled Crosslinking Reagents: Resolution of Mass Degeneracy in the Identification of Crosslinked Peptides. Bioorg. Med. Chem. Lett. 2003, 13, 4023−4026. (47) Dimova, K.; Kalkhof, S.; Pottratz, I.; Ihling, C.; RodriguezCastaneda, F.; Liepold, T.; Griesinger, C.; Brose, N.; Sinz, A.; Jahn, O. Structural Insights into the Calmodulin−Munc13 Interaction Obtained by Cross-Linking and Mass Spectrometry. Biochemistry 2009, 48, 5908−5921. (48) Fujii, N.; Jacobsen, R. B.; Wood, N. L.; Schoeniger, J. S.; Guy, R. K. A Novel Protein Crosslinking Reagent for the Determination of Moderate Resolution Protein Structures by Mass Spectrometry (MS3D). Bioorg. Med. Chem. Lett. 2004, 14, 427−429. (49) Gardner, M. W.; Vasicek, L. A.; Shabbir, S.; Anslyn, E. V.; Brodbelt, J. S. Chromogenic Cross-Linker for the Characterization of Protein Structure by Infrared Multiphoton Dissociation Mass Spectrometry. Anal. Chem. 2008, 80, 4807−4819. (50) Gardner, M. W.; Brodbelt, J. S. Preferential Cleavage of N−N Hydrazone Bonds for Sequencing Bis-arylhydrazone Conjugated Peptides by Electron Transfer Dissociation. Anal. Chem. 2010, 82, 5751−5759. (51) Gomes, A. F.; Gozzo, F. C. Chemical Cross-linking with a Diazirine Photoactivatable Cross-linker Investigated by MALDI- and ESI-MS/MS. J. Mass Spectrom. 2010, 45, 892−899. (52) Lu, Y.; Tanasova, M.; Borhan, B.; Reid, G. E. Ionic Reagent for Controlling the Gas-phase Fragmentation Reactions of Cross-linked Peptides. Anal. Chem. 2008, 80, 9279−9287. (53) Yang, L.; Zheng, C.; Weisbrod, C. R.; Tang, X.; Munske, G. R.; Hoopmann, M. R.; Eng, J. K.; Bruce, J. E. In Vivo Application of Photocleavable Protein Interaction Reporter Technology. J. Proteome Res. 2012, 11, 1027−1041. (54) Liu, Z.; Cheng, S.; Gallie, D. R.; Julian, R. R. Exploring the Mechanism of Selective Noncovalent Adduct Protein Probing Mass Spectrometry Utilizing Site-directed Mutagenesis to Examine Ubiquitin. Anal. Chem. 2008, 80, 3846−3852. (55) Ly, T.; Julian, R. R. Using ESI-MS to Probe Protein Structure by Site-Specific Noncovalent Attachment of 18-Crown-6. J. Am. Soc. Mass Spectrom. 2006, 17, 1209−1215. (56) Tao, Y.; Julian, R. R. Examining Protein Surface Structure in Highly Conserved Sequence Variants with Mass Spectrometry. Biochemistry 2012, 51, 1796−1802. (57) Zhou, Y.; Vachet, R. Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass 5876

dx.doi.org/10.1021/pr400869u | J. Proteome Res. 2013, 12, 5867−5877

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Spectrometric Detection. J. Am. Soc. Mass Spectrom. 2012, 23, 708− 717. (58) Wang, L.; Chance, M. R. Structural Mass Spectrometry of Proteins Using Hydroxyl Radical Based Protein Footprinting. Anal. Chem. 2011, 83, 7234−7241. (59) Mendoza, V. L.; Barón-Rodríguez, M. A.; Blanco, C.; Vachet, R. W. Structural Insights into the Pre-Amyloid Tetramer of β-2Microglobulin from Covalent Labeling and Mass Spectrometry. Biochemistry 2011, 50, 6711−6722. (60) Zhou, Y.; Vachet, R. W. Diethylpyrocarbonate Labeling for the Structural Analysis of Proteins: Label Scrambling in Solution and How to Avoid It. J. Am. Soc. Mass Spectrom. 2012, 23, 899−907. (61) Janecki, D. J.; Beardsley, R. L.; Reilly, J. P. Probing Protein Tertiary Structure with Amidination. Anal. Chem. 2005, 77, 7274− 7281. (62) Lu, Y.; Zhou, X.; Stemmer, P. M.; Reid, G. E. Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization. J. Am. Soc. Mass Spectrom. 2011, 23, 577−593. (63) Reid, G. E.; Roberts, K. D.; Simpson, R. J.; O’Hair, R. A. J. Selective Identification and Quantitative Analysis of Methionine Containing Peptides by Charge Derivatization and Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2005, 16, 1131−1150. (64) Roberts, K. D.; Reid, G. E. Leaving Group Effects on the Selectivity of the Gas-phase Fragmentation Reactions of Side Chain Fixed-charge-containing Peptide Ions. J. Mass Spectrom. JMS 2007, 42, 187−198. (65) Petrotchenko, E. V.; Serpa, J. J.; Borchers, C. H. An Isotopically Coded CID-Cleavable Biotinylated Cross-Linker for Structural Proteomics. Mol. Cell. Proteomics 2011, 10, M110.001420. (66) Kao, A.; Chiu, C.-L.; Vellucci, D.; Yang, Y.; Patel, V. R.; Guan, S.; Randall, A.; Baldi, P.; Rychnovsky, S. D.; Huang, L. Development of a Novel Cross-Linking Strategy for Fast and Accurate Identification of Cross-Linked Peptides of Protein Complexes. Mol. Cell. Proteomics 2011, 10, M110.002212. (67) Müller, M. Q.; Dreiocker, F.; Ihling, C. H.; Schäfer, M.; Sinz, A. Cleavable Cross-Linker for Protein Structure Analysis: Reliable Identification of Cross-Linking Products by Tandem MS. Anal. Chem. 2010, 82, 6958−6968. (68) Yang, L.; Tang, X.; Weisbrod, C.; Munske, G.; Eng, J.; von Haller, P.; Kaiser, N.; Bruce, J. E. pcPIR, a Photocleavable and Mass Spectrometry Identifiable Cross-linker for Protein Interaction Studies. Anal. Chem. 2010, 82, 3556−3566. (69) Vasicek, L.; O’Brien, J. P.; Browning, K. S.; Tao, Z.; Liu, H.-W.; Brodbelt, J. S. Mapping Protein Surface Accessibility via an Electron Transfer Dissociation Selectively Cleavable Hydrazone Probe. Mol. Cell. Proteomics 2012, 11, O111.015826. (70) O’Brien, J. P.; Pruet, J. M.; Brodbelt, J. S. Chromogenic Chemical Probe for Protein Structural Characterization via Ultraviolet Photodissociation Mass Spectrometry. Anal. Chem. 2013, 85, 7391− 7397. (71) Li, D.; Harper, S. L.; Tang, H.-Y.; Maksimova, Y.; Gallagher, P. G.; Speicher, D. W. A Comprehensive Model of the Spectrin Divalent Tetramer Binding Region Deduced Using Homology Modeling and Chemical Cross-Linking of a Mini-Spectrin. J. Biol. Chem. 2010, 285, 29535−29545. (72) Fu, C.-Y.; Uetrecht, C.; Kang, S.; Morais, M. C.; Heck, A. J. R.; Walter, M. R.; Prevelige, P. E. A Docking Model Based on Mass Spectrometric and Biochemical Data Describes Phage Packaging Motor Incorporation. Mol. Cell. Proteomics 2010, 9, 1764−1773. (73) Young, M. M.; Tang, N.; Hempel, J. C.; Oshiro, C. M.; Taylor, E. W.; Kuntz, I. D.; Gibson, B. W.; Dollinger, G. High Throughput Protein Fold Identification by Using Experimental Constraints Derived from Intramolecular Cross-Links and Mass Spectrometry. Proc. Natl. Acad. Sci. 2000, 97, 5802−5806. (74) Dyksterhuis, L. B.; Weiss, A. S. Homology Models for Domains 21−23 of Human Tropoelastin Shed Light on Lysine Crosslinking. Biochem. Biophys. Res. Commun. 2010, 396, 870−873.

(75) Lacroix, M.; Rossi, V.; Gaboriaud, C.; Chevallier, S.; Jaquinod, M.; Thielens, N. M.; Gagnon, J.; Arlaud, G. J. Structure and Assembly of the Catalytic Region of Human Complement Protease C1r: A Three-Dimensional Model Based on Chemical Cross-Linking and Homology Modeling. Biochemistry 1997, 36, 6270−6282. (76) Kalisman, N.; Adams, C. M.; Levitt, M. Subunit Order of Eukaryotic TRiC/CCT Chaperonin by Cross-Linking, Mass Spectrometry, and Combinatorial Homology Modeling. Proc. Natl. Acad. Sci. 2012, 109, 2884−2889. (77) Rappsilber, J. The Beginning of a Beautiful Friendship: Crosslinking/mass Spectrometry and Modelling of Proteins and Multiprotein Complexes. J. Struct. Biol. 2011, 173, 530−540. (78) Mayberry, L. K.; Dennis, M. D.; Leah Allen, M.; Ruud Nitka, K.; Murphy, P. A.; Campbell, L.; Browning, K. S. Expression and Purification of Recombinant Wheat Translation Initiation Factors eIF1, eIF1A, eIF4A, eIF4B, eIF4F, eIF(iso)4F, and eIF5. Methods Enzymol. 2007, 430, 397−408. (79) Bantscheff, M.; Weiss, V.; Glocker, M. O. Identification of Linker Regions and Domain Borders of the Transcription Activator Protein NtrC from Escherichia Coli by Limited Proteolysis, In-gel Digestion, and Mass Spectrometry. Biochemistry 1999, 38, 11012− 11020.

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dx.doi.org/10.1021/pr400869u | J. Proteome Res. 2013, 12, 5867−5877