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1 Departments of Chemistry & Biochemistry, 2 Department of Medicine and Biomedical Sciences Graduate Program, and 3 Howard Hughes Medical Institute, University of California at San Diego, La Jolla, California 92093, USA
4 Sierra Analytics, Modesto, California 95356, USA
Reprint requests to: Virgil L. Woods Jr., Department of Medicine, University of California at San Diego, Department 0656, 9500 Gilman Drive, La Jolla, CA 92093-0656, USA; e-mail: vwoods{at}ucsd.edu; fax: (858) 534-2606.
(RECEIVED July 7, 2005; FINAL REVISION September 8, 2005; ACCEPTED September 12, 2005)
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Abstract |
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) isoform of protein kinase A (PKA) has been well characterized, but there currently is no detailed structural information of an AKAP docked to the type I (RI) isoform. Dual-specific AKAP2 (D-AKAP2) binds in the nanomolar range to both isoforms and provided us with an opportunity to characterize the isoform-selective nature of AKAP binding using a common docked ligand. Hydrogen/deuterium (H/D) exchange combined with mass spectrometry (DXMS) was used to probe backbone structural changes of an -helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RI and RII D/D domains. The region of protection upon complex formation and the magnitude of protection from H/D exchange were determined for both interacting partners in each complex. The backbone of the AKB ligand was more protected when bound to RI compared to RII, suggesting an increased helical stabilization of the docked AKB ligand. This combined with a broader region of backbone protection induced by the AKAP on the docking surface of RI indicated that there were more binding constraints for the AKB ligand when bound to RI. This was in contrast to RII, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RI AKAP-docking surface. DXMS provides valuable structural information for understanding binding specificity in the absence of a high-resolution structure, and can readily be applied to other protein–ligand and protein–protein interactions. Keywords: DXMS; PKA; D-AKAP2; isoform diversity; hydrogen/deuterium exchange; mass spectrometry
Abbreviations: PKA, protein kinase A • AKAP, A kinase anchoring protein • DAKAP, dual specific A kinase anchoring protein • AKB, A kinase binding • R, regulatory subunit of protein kinase A • D/D, dimerization/docking domain of regulatory subunit • RGS, regulators of G-protein signaling • DXMS, deuterium exchange mass spectrometry • GdnHCl, guanidine hydrochloride • TFA, trifluoracidic acid
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051687305.
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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cAMP-dependent protein kinase (PKA) phosphorylates a diverse set of proteins involved in numerous biological signaling pathways. A major way of achieving signal specificity for PKA is through subcellular localization via A-kinase anchoring proteins (AKAPs), a family of proteins which binds to PKA and targets it to various intracellular compartments (Colledge and Scott 1999; Edwards and Scott 2000). AKAPs tether PKA through a critical interaction motif, which we have termed the A-kinase binding (AKB) domain. For most AKAPs this consists of a 15–20 amino acid amphipathic, helical motif that binds to the dimerization/docking (D/D) domainofthe regulatorysub-unit homodimer. This interaction is essential for intracellular compartmentation of PKA and for integration of PKA signaling in the cell.
The regulatory (R) subunit homodimer of PKA is a modular and highly dynamic protein, Each R subunit monomer consists of an N-terminal D/D domain, two cAMP binding domains, and a disordered interconnecting hinge region (Taylor et al. 1990). It regulates the catalytic activity of the kinase directly through cAMP binding and indirectly through subcellular localization by AKAPs. The D/D domain is responsible for homodimerization and, once dimerized, provides a binding surface for the AKB helix. Four regulatory subunit isoforms of PKA (RI, RI
, RII, and RII) serve to diversify cAMP-mediated activation of the kinase (Skalhegg and Tasken 2000; Feliciello et al. 2001). These isoforms have a similar domain organization but differ in tissue distribution, subcellular localization, and cAMP sensitivity. In general, PKA-RI isoforms bind fewer AKAPs than PKA-RII and have a reduced affinity. Another important difference in the biology of these isoforms is their sensitivity to cAMP activation. PKA-RI isoforms are more sensitive to cAMP concentrations compared with PKA-RII, and this can result in a differential cAMP response (Doskeland et al. 1993; Skalhegg and Tasken 2000; Feliciello et al. 2001). Localization through AKAPs and cAMP sensitivity are two functions of the regulatory subunit that seem to have evolved to diversify PKA signaling.
Recent studies have advanced our understanding of the molecular basis for the R–AKAP interaction (Angelo and Rubin 1998, 2000; Banky et al. 1998, 2000, 2003; Miki and Eddy 1998, 1999; Newlon et al. 1999; Herberg et al. 2000; Newlon et al. 2001; Burns et al. 2003). Despite a similar X-type four-helix bundle comprising the D/D domain of these isoforms, there are distinct structural differences (Banky et al. 2003). The RI D/D domain contains an extended N-terminal helix, helix N-1, which gives a more rugged surface topology to the AKAP binding groove. A small hinge segment allows for significant variation in the positioning of the N-1 helix. The RII D/D domain contains a shorter, -strand-like N termini, which creates a more accessible AKAP binding surface (Newlon et al. 1999). The charge distribution is also very different for these isoforms. The RI D/D domain contains more acidic and basic residues that line the surface of the AKAP binding groove, whereas the AKAP binding surface of RII contains primarily hydrophobic residues (Newlon et al. 1999, 2001; Banky et al. 2000, 2003; Burns et al. 2003).
Dual-specific AKAPs (D-AKAPs) can bind to both RI and RII, and represent an interesting subfamily of AKAPs, which can potentially recruit both PKA-RI and PKA-RII to a given intracellular location. D-AKAP2 is a multisubunit protein containing two putative RGS domains and a 40 amino acid C-terminal domain containing the AKB helix and a PDZ binding motif. To better understand how the differences in surface topology contributes to isoform specific binding, we have examined the binding of a peptide corresponding to the AKB domain of D-AKAP2 to the D/D domains of both RI and RII using amide hydrogen/deuterium (H/D) exchange combined with mass spectrometry (DXMS) (Woods and Hamuro 2001). Amide H/D exchange has proven to be an invaluable tool for studying protein structure (Zhang and Smith 1993; Resing et al. 1999; Hamuro et al. 2002a; Yan et al. 2002), protein dynamics (Neubert et al. 1997; Engen and Smith 2001; Hoofnagle et al. 2001), protein–ligand interactions (Engen et al. 1999; Andersen et al. 2001; Hamuro et al. 2002b), and protein–protein interactions (Ehring 1999; Mandell et al. 2001; Anand et al. 2002; Hamuro et al. 2003). Using this technique we have shown that the backbone of the AKB ligand was more stabilized when bound to RI, and that the peptide ligand induced a broader region of protection on the surface of RI. We suggest that these differences represent distinct modes of AKAP binding to the regulatory subunit isoforms, and may contribute to the more discriminating nature of the RI binding surface.
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Results |
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and RII D/D domains
). Nine peptides were of high enough quality to measure the extent of deuteration in both the free and RI and RII D/D bound states (solid line in Fig. 1A).
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H/D exchange of the AKB ligand in complex with the RI and RII D/D domain
H/D exchange experiments were performed with the AKB ligand in complex with either the RI D/D or RII D/D domain at pH 6.9. Using the dissociation constants, maximal complex formation was calculated using the concentrations of analyte and binding partner (see Table 1 and Material and Methods). Approximately 99% of the analyte was complexed for both RI and RII.
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D/D and RII D/D domains to the AKB ligand resulted in a significant slowing of deuteron incorporation into the backbone amides of the AKB ligand. However, the extent of the deuterium incorporation was different for the two isoforms. Figure 2 illustrates an example of a peptide isotopic envelope from the AKB ligand (residue 14–19, indicated by asterisk in Fig. 1A
) in the nondeuterated state (Fig. 2A), after 300 sec on-exchange when complexed with RI D/D (Fig. 2B), with RII D/D (Fig. 2C) or in the free state (Fig. 2D). Fewer deuterons were incorporated into this region of the AKB ligand when bound to RI with an isotopic envelope that more closely resembles the non-deuterated state. In contrast, the isotopic envelope for RII was shifted further to the right indicating more deuteron uptake and less protection in this region when RII was bound.
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and RII-bound states (Fig. 3). The data is illustrated in two different formats. In Figure 3A, a color-coded grid makes it easy to qualitatively visualize the regions in the AKB ligand that are protected in the complexes. Regions highlighted under the amino acid sequence represent the peptide analyzed minus the first two residues of the peptide. This designation was used since it has been shown that the N-terminal amino group of the peptide and the amide hydrogen of the second amino acid exchange too rapidly to retain deuterons during the experiment (Bai et al. 1993). For example, exchange data for residues 14–19 corresponded to peptide 12–19. Residues 12–25 showed varying degrees of protection from exchange in both the RI and RII complexes, surrounded by unprotected regions (Fig. 3A). This 14 amino acid stretch when modeled using a helical wheel projection (data not shown) includes four turns of an amphipathic helix that place hydrophobic residues (underlined in Fig. 3A) on one face of the helix. These residues have previously been shown to be essential for high affinity binding to the D/D domain (Burns-Hamuro et al. 2003). The magnitude of RI-induced protection was focused over the length of the helix (12–25), while that of RII showed greater protection at the C terminus of the helix (Fig. 3A).
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). There was almost no protection in the AKB ligand for either complex at the far N- and C-terminal [see Fig. 3B, residues VQGNTDE (1–7) and QQAHHDQPLEKSTKL (26–40)]. Beginning with the residues "LA" (12–13), the AKB ligand was more protected when bound to RI. Since this difference was nearly 2 deuterons at 10 sec, this suggested that both of these residues are protected to a greater extent in the RI complex. It was clear from the data that RI induced more protection throughout this region (residues 14–25) in the AKB ligand. The residues "SD" (22–23) were protected for both the RI and RII complex. Since these residues map to the opposite side of the interacting amphipathic helix, this suggested to us that protection along the length of the helix was most likely due to a direct stabilization of the backbone of the helix rather than specific contacting sites along the helix.
H/D exchange of the D/D domain isoforms in the free and bound state
Digestion of RI and RII D/D domains
The same digestion and analysis method used for the AKB domain digestion above identified 15 and 17 peptides for the RI and RII D/D domains, respectively (Fig. 1B,C). After complexation and deuteration, a subset of these peptides was used to monitor amide H/D exchange patterns (highlighted bold in Fig. 1B,C).
H/D exchange of RI and RII D/D domains in the free state
RI and RII D/D domains were incubated in 75% deuterated water at pH 6.9 for various time periods in the absence of the AKB domain (10–10,000 sec = ~2.8 h for RI and 10–300,000 sec = ~83 h for RII). The extent of deuteration of nine pepsin-generated peptides from the RI D/D domain and 10 peptides from the RII D/D domain was evaluated (Fig. 4 [RII] and Fig. 5 [RI]). The slow exchanged regions of the free state for both RI and RII D/D domains were centered around helix I and helix II, and were consistent with secondary structure assignments by NMR as well as H/D exchange by NMR (Newlon et al. 1997, 1999; Banky et al. 2000, 2003; Fayos et al. 2003).
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in complex with the AKB ligand; Material and Methods). Figure 4 illustrates the color-coded data format and plots of individual segments for the RII-AKB complex. Helix I and helix II showed the greatest protection by the AKB ligand. AKB-induced protection in helix I was consistent with this region being the direct interaction site of the AKB ligand as seen by NMR (Newlon et al. 2001). Since there was no evidence by NMR that helix II directly interacts with the ligand, protection in this region was likely due to indirect effects propagated through the binding surface. The AKB ligand induced little if any changes at the N terminus, the turn between helix I and II and the C terminus (Fig. 4).
H/D exchange of RI in complex with the AKB ligand
The AKB ligand induced similar regions of protection in RI with some notable differences (Fig. 5). Helix I and Helix II were protected, suggesting that the docking surface for RI was the same as for RII. However, the AKB-induced protection of RI extended more into the N terminus when compared with RII [Fig. 5B, see segment QKHNIQAL (21–28)]. At least four deuterons were protected from exchange after 10 sec on-exchange. This was in contrast to RII, in which little if any AKB-induced protection was observed in the region leading into helix I [Fig. 4, see segment GLTEL (12–16)].
To compare the differences in AKB-induced protection between RI and RII, the percent deuteration difference between free and bound states was calculated and plotted versus the sequence for each isoform (Fig. 6). In the bar-graph format it was clear that the region leading into helix I of RI was more protected by the AKB ligand compared with a similar region in RII. When the protected regions were mapped onto the structures of RI and RII, the enhanced protection leading into helix I of RI translated into a broader protected region on the AKAP docking surface (Fig. 7).
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Discussion |
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Backbone of the AKB ligand is stabilized to a greater extent when bound to RI
The AKB ligand spanning residues 12–25 was protected upon binding RI and RII. The more localized region of AKB protection when bound to RII was consistent with recent peptide array analysis in which each residue in the AKB peptide was replaced with the other 19 amino acids and binding to both RI and RII characterized (Burns-Hamuro et al. 2003). The critical contact areas mapped out by the peptide substitution array extended the length of the AKB helix for RI binding (residues 12–25), but were more localized to the C-terminal residues of the AKB helix for RII binding (residues 16–25) (Burns-Hamuro et al. 2003).
Interestingly, the magnitude of protection for the AKAP peptide was different when bound to each isoform, suggesting differences in helical stabilization of the docked AKAP. The binding affinity for the AKAP peptide is 25-fold weaker for RI than RII (Burns et al. 2003), yet was protected to a greater extent when docked to the RI isoform, suggesting an increase in helical stabilization of the AKB ligand. This at first interpretation seemed counterintuitive, but H/D exchange measures only backbone contributions to the free energy change for an EX2 mechanism (Englander and Kallenbach 1984) and solvent effects and side-chain contributions to the binding energy are not measured directly by this technique. Likewise, binding affinities that are dominated by side chain or solvent effects may have lower than expected protection levels. Weaker protection of the RII-bound AKB ligand suggested that the ligand backbone was less stabilized and perhaps more dynamic when bound, even though the overall affinity is higher relative to RI. Support of this interpretation came from NMR experiments, where modest H/D exchange protection factors were found for residues contacting the AKAP along with increases in backbone flexibility for selected regions of the RII docking surface when the AKAP is bound (Fayos et al. 2003). This type of observation may be a consequence of the predominately, hydrophobic binding surface of RII and increased backbone dynamics may further contribute to a favorable binding entropy, leading to enhanced AKAP binding affinity for this isoform. Further experiments that characterize the thermodynamic contributions to binding of each isoform will be important to verify this hypothesis.
Similar regions of exchange for both RI and RII isoforms in the unbound state
The H/D exchange patterns for the isolated D/D domains are similar for the two isoforms, reflecting the fact that both domains have similar four helix bundle structures. Concentrated regions of protection map to helix I and helix II for both of these isoforms. Helix I and helix I' form the AKAP docking surface and helix II and helix II' form the primary dimerization contacts for this domain. The slow exchanged regions of the native state were consistent with previous H/D exchange experiments by NMR, which map slowly, exchanging amide hydrogens to similar regions (Newlon et al. 1997; Banky et al. 2000).
The D/D domain is quite stable for its size, and can function as an isolated unit as indicated by similar AKAP binding affinities for this domain compared with full-length protein (Newlon et al. 1999; Burns et al. 2003). Previous studies have shown that the RI D/D domain is highly thermostable with interchain disulfide bonds that are resistant to high concentrations of reducing agent (Leon et al. 1997). The disulfide bonds, although not necessary for dimerization, are unusually stabilized by the X-type helical motif (Banky et al. 2003).
Backbone perturbations in the N-1 helix upon AKAP-binding to the RI isoform
Two primary regions of the D/D domain were protected in the AKAP–R complex for each isoform: helix I and helix I' and helix II and helix II'. Segments covering 51% of the backbone hydrogens of RI were protected significantly from exchange upon AKAP binding, compared with only 29% for RII. The additional protected residues in RI primarily map to the N-1 helix. Although there is no structural information for an AKAP docked to the RI D/D domain, the data presented here predicts that the N-1 helical region undergoes backbone structural rearrangements to accommodate the AKAP. It cannot be distinguished by this technique alone if the N-1 helix is directly contacting the peptide, but mutagenesis experiments have shown that residues within this helix are important for AKAP binding. Mutating tyrosine at position 19 with an alanine significantly interferes with binding of the D-AKAP2, AKB peptide to RI (L.L. Burns-Hamuro, unpubl.). In addition, valine at position 20 is also important for binding the AKB domain from D-AKAP1 (Banky et al. 1998).
The N-1 helical extensions are the least well defined in the ensemble of NMR structures of the free RI D/D domain due to the apparent rotation of these helices with respect to the core of the domain (Banky et al. 2003). Residues His23 and Asn24 have been proposed as a hinge, allowing the N-1 helix to adopt multiple conformations (Banky et al. 2003). These residues are contained within the AKB-induced protected region, and consistent with the model that the N-1 helices rearrange to accommodate the AKB ligand (Banky et al. 2003).
The increased protection of the AKB peptide backbone when bound to RI relative to when bound to RII, suggested that the AKB peptide formed a more stabilized hydrogen-bonded network with RI. The N-1 helices may wrap around the docked peptide, extending the interaction surface and further stabilizing the hydrogen-bonding network of the bound peptide. Whether this is through direct interaction of the AKB ligand with the N-1 helical region or through an indirect effect cannot be determined by H/D exchange. Further structural characterization will answer this question.
Intradomain communication within the X-type four-helix bundle motif
Helix II and helix II', which directly oppose the AKAP binding surface, were also protected when the AKB peptide was bound to each isoform (Fig. 6). The magnitude of protection was similar for both AKB–R complexes, suggesting a similar level of backbone communication in this region. This area of protection was consistent with recent H/D exchange experiments by NMR which showed a similar protection pattern for helix II and helix II' when the AKAP peptide, HT31, was bound to RII, indicating that protection in helix II is not specific to a given AKAP ligand (Fayos et al. 2003). Protection in these helices was presumably due to longer-range effects that were propagated through the helix I and II interface, as there was no evidence to suggest from mutagenesis or structural experiments that helix II interacts directly with the AKAP. These longer-range effects potentially highlight communication networks within the domain. Propagation of a signal through the domain upon AKAP binding may be important for signal transmittance to other regions of the multidomain regulatory subunit. Future experiments with a full-length regulatory subunit will determine if there are long-range protections induced by the AKB ligand in other domains of the regulatory subunit.
Distinct modes of AKAP interaction with the regulatory subunit isoforms
The RII isoform can bind many different amphipathic AKAP binding motifs, which contain primarily branched chain amino acids along one face of the AKAP helix. The RI surface is more discriminating, and has only been shown to bind a few AKAPs (Huang et al. 1997a,b; Angelo and Rubin 1998; Miki and Eddy 1998; Li et al. 2001). The structural features that may contribute to reduced AKAP binding affinity and higher binding selectivity of the RI surface has recently been revealed with the solution NMR structure of the RI D/D domain. Although, the overall structures of the RI and RII D/D domains are similar, there are distinct differences in the surfaces that they present to the AKAP (Banky et al. 2003). RI has a central, deep cleft created by the isoform-specific N-terminal extensions (helix N-1). These helical extensions are tethered through a disulfide bridge between C16 (helix N-1) and C37 (helix 1) of the adjacent protomer, which most likely place conformational restrictions on this segment of the docking surface. The RII surface is more extended and preformed for AKAP binding (Fig. 6). The N-terminal extensions in RI also contain several charged residues that most likely play a role in AKAP binding, whereas the surface of RII is primarily hydrophobic (Burns et al. 2003).
The H/D exchange data presented here expand our current understanding of the isoform-selective nature to AKAP binding. The broader region of backbone protection induced by the AKAP on the RI surface and the more enhanced backbone stabilization of the AKAP helix when bound to RI versus RII suggest that the AKAP helix forms a more intimate contact with the hydrogen-bonded network of the RI core. In this model for AKAP binding to RI, the AKAP preferentially interacts with an N-1 helical conformation on the RI surface that allows for optimized helical stabilization of the AKAP backbone. Molecular details of this complex await structural determination of the RI–AKAP complex, but this model is in stark contrast to the RII D/D surface, which presents as a preformed docking surface with only minor changes in conformation of the N-terminal region between the free and bound forms (Newlon et al. 2001). The data presented here also suggests that the higher affinity AKAP docking surface of RII is more localized than RI. The AKAP induced a narrower region of backbone protection on the surface of RII and contained decreased backbone stabilization relative to when docked to RI. The localized, pre-formed and primarily hydrophobic binding surface of RII may enable this surface to recognize a multitude of binding partners with fewer sequence constraints on AKAP binding. Presumably, water exclusion from the interacting hydrophobic surfaces and the preformed nature of the RII binding surface contribute to the enhanced affinity of this isoform for AKAPs. Although in general the RI surface binds AKAPs weaker than RII, AKAP peptides that contain appropriately spaced aromatic groups (Phe or Trp) dramatically enhance the affinity and confer selective binding to RI (Angelo and Rubin 2000; Burns-Hamuro et al. 2003). This demonstrates that while the RI surface is selective in binding, it can accommodate the appropriate side chains for high affinity binding, perhaps by filling the deep cleft on the docking surface. A future challenge will be to design cell-permeable, isoform-specific AKAP disruptors of PKA localization to enable a better understand of the functional importance of isoform-selective anchoring of PKA in cells.
We have used hydrogen/deuterium exchange mass spectrometry to map the binding of an AKAP helical peptide to the D/D domain of RI and RII subunits of PKA. The results reveal a common docking surface but show differences in ligand stabilization and surface interactions, which have enabled us to propose distinct interaction modes for the docked ligand. This technique is applicable to any set of binding partners, and can be used to map both local and long-range perturbations in the backbone upon complex formation.
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Materials and methods |
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D/D and RI D/D was previously described (Banky et al. 2000; Burns et al. 2003). The protein concentrations were determined at OD280 using an extinction coefficient of 5499 M–1 cm–1, 5960 M–1 cm–1, and 6210 M–1 cm–1 for the AKB domain, RII D/D and RI D/D, respectively (Pace et al. 1995; Burns et al. 2003).
Amide hydrogen/deuterium exchange analysis
General operation procedure
A 20-µL hydrogen-exchanged protein solution was quenched by shifting to pH 2.2–2.5, 0°C with 30 µL of 0.8% formic acid with various concentrations of GdnHCl (final pH was measured on a nondeuterated mock solution at room temperature using a Model 250 pH meter; Denver Instrument Co.). At 0°C, the quenched solution was immediately passed over a column (66 µL bed volume; Upchurch Scientific) filled with porcine pepsin (Sigma) immobilized on Poros 20 AL media at 30 mg/mL per the manufacturer’s instructions, with 0.05% TFA (200 µL/min) for 2 min with contemporaneous collection of proteolytic products using a C18 column (Vydac). Inline filters (Upchurch) were placed on each side of the pepsin column, and just before the C18 column (Vydac prefilter) to minimize column fouling. Subsequently, the C18 column was eluted with a linear gradient of 10% to 50% solvent B over 10 min (solvent A was 0.05% TFA in water, and solvent B was 80% acetonitrile, 20% water, 0.01% TFA). Mass spectrometric analysis was carried out with a Finnigan LCQ mass spectrometer with a capillary temperature of 200°C.
Sequence identification of pepsin-generated peptides
To identify pepsin-generated peptides for each digestion condition employed, spectral data was acquired in "data-dependent MS/MS" mode. The "data-dependent MS/MS" data set was then analyzed using Sequest (Finnigan, Inc.) to identify the sequence of the dynamically selected parent peptide ions.
Hydrogen/deuterium exchange experiments
Deuterated samples were prepared by diluting 5 µL of protein solution (either analyte alone or with excess binding partner) with 15 µL of deuterated buffer, followed by "on-exchange" incubation at room temperature (23 ± 1°C) for varying times (10–300,000 sec) prior to quenching in 30 µL of 0.8% formic acid with GdnHCl at 0°C. For pH 7 experiments, the deuterated buffer was 10 mM HEPES, 150 mM NaCl, pHread = 6.90, and the pHread of exchange solution was 6.88 ± 0.01. These functionally deuterated samples were subjected to DXMS processing as described above, along with control samples of nondeuterated and fully deuterated protein. The centroids of probe peptide isotopic envelopes were measured using the DXMS software provided by Sierra Analytics. The corrections for back-exchange were made employing the methods of Zhang and Smith (1993),
 | (1) |
 | (2) |
where m(P), m(N), and m(F) are the centroid value of partially deuterated peptide, nondeuterated peptide, and fully deuterated peptide, respectively. MaxD is the maximum deuterium incorporation calculated by subtracting the first two residues of the peptide, which have been shown to be fully exchanged due to end effects (Bai et al. 1993) and by subtracting the number of prolines from the total number of amide hydrogens in the peptide. The experimentally determined deuteron recovery of the fully deuterated sample was on average 90% (i.e., [m(F) – m(N)]/MaxD/0.75, 0.75 is the deuteron content in the exchange buffer).
Sublocalization of deuteriums
The deuteration levels of the peptides were further sublocalized using overlapping peptides. For example, the deuterium incorporation of segment 10–11 was obtained by the subtraction of the deuterium incorporation of 3–9 from that of 3–11 (for more details, see Supplemental Material).
Complex formation
The proteins were mixed in the ratios indicated (Table 1) and the fraction of analyte bound was calculated using the following equations (Mandell et al. 2001):
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 | (4) |
where A0 is the original concentration of the analyte in the exchange reaction, B0 is that of the binding partner, and KD is the dissociation constant.
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Footnotes |
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5 These authors contributed equally to this work. 
6 Present addresses: L.L. Burns-Hamuro, Provid Pharmaceuticals, 671 US Route 1 South, North Brunswick, NJ 08902, USA;
7 Y. Hamuro, ExSAR Corporation, 11 Deer Park Drive, Suite 103, Monmouth Junction, NJ 08852, USA.
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Acknowledgments |
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Conflict of interest
V.L.W., D.D.S., and Y.H. have financial interests in ExSAR Corporation.
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References |
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), RII
), or RI
). It is clear from the plots that the backbone of the AKB ligand is protected to a greater extent from amide hydrogen exchange when bound to RI
