Richard Y.-C. Huang

Hydrogen/Deuterium Exchange and Electron-Transfer Dissociation Mass Spectrometry Determine the Interface and Dynamics of Apolipoprotein E Oligomerization

Apolipoprotein E, a 34 kDa protein, plays a key role in triglyceride and cholesterol metabolism. Of the three common isoforms (ApoE2, -3, and -4), only ApoE4 is a risk factor for Alzheimers disease. All three isoforms of wild-type ApoE self-associate to form oligomers, a process that may have functional consequences. Although the C-terminal domain, residues 216-299, of ApoE is believed to mediate self-association, the specific residues involved in this process are not known. Here we report the use of hydrogen/deuterium exchange (H/DX) coupled with enzymatic digestion to identify those regions in the sequence of full-length apoE involved in oligomerization. For this determination, we compared the results of H/DX of the wild-type proteins and those of monomeric forms obtained by modifying four residues in the C-terminal domain. The three wild-type and mutant isoforms show similar structures based on their similar H/DX kinetics and extents of exchange. Regions of the C-terminus (residues 230-270) of the ApoE isoforms show significant differences of deuterium uptake between oligomeric and monomeric forms, confirming that oligomerization occurs at these regions. To achieve single amino acid resolution, we examined the extents of H/DX by using electron transfer dissociation (ETD) fragmentation of peptides representing selected regions of both the monomeric and the oligomeric forms of ApoE4. From these experiments, we could identify the specific residues involved in ApoE oligomerization. In addition, our results verify that ApoE4 is composed of a compact structure at its N-terminal domain. Regions of C-terminal domain, however, appear to lack defined structure.

Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

AbstractEpitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin (10Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein–protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods. Figureᅟ

Psb27, a transiently associated protein, binds to the chlorophyll binding protein CP43 in photosystem II assembly intermediates

Photosystem II (PSII), a large multisubunit pigment–protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, mediates light-driven evolution of oxygen from water. Recently, a high-resolution X-ray structure of the mature PSII complex has become available. Two PSII polypeptides, D1 and CP43, provide many of the ligands to an inorganic Mn4Ca center that is essential for water oxidation. Because of its unusual redox chemistry, PSII often undergoes degradation followed by stepwise assembly. Psb27, a small luminal polypeptide, functions as an important accessory factor in this elaborate assembly pathway. However, the structural location of Psb27 within PSII assembly intermediates has remained elusive. Here we report that Psb27 binds to CP43 in such assembly intermediates. We treated purified genetically tagged PSII assembly intermediate complexes from the cyanobacterium Synechocystis 6803 with chemical cross-linkers to examine intermolecular interactions between Psb27 and various PSII proteins. First, the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to cross-link proteins with complementary charged groups in close association to one another. In the His27△ctpAPSII preparation, a 58-kDa cross-linked species containing Psb27 and CP43 was identified. This species was not formed in the HT3△ctpA△psb27PSII complex in which Psb27 was absent. Second, the homobifunctional thiol-cleavable cross-linker 3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used to reversibly cross-link Psb27 to CP43 in His27△ctpAPSII preparations, which allowed the use of liquid chromatography/tandem MS to map the cross-linking sites as Psb27K63↔CP43D321 (trypsin) and CP43K215↔Psb27D58AGGLK63↔CP43D321 (chymotrypsin), respectively. Our data suggest that Psb27 acts as an important regulatory protein during PSII assembly through specific interactions with the luminal domain of CP43.

Higher order structure characterization of protein therapeutics by hydrogen/deuterium exchange mass spectrometry

AbstractCharacterization of therapeutic drugs is a crucial step in drug development in the biopharmaceutical industry. Analysis of protein therapeutics is a challenging task because of the complexities associated with large molecular size and 3D structures. Recent advances in hydrogen/deuterium-exchange mass spectrometry (HDX-MS) have provided a means to assess higher-order structure of protein therapeutics in solution. In this review, the principles and procedures of HDX-MS for protein therapeutics characterization are presented, focusing on specific applications of epitope mapping for protein–protein interactions and higher-order structure comparison studies for conformational dynamics of protein therapeutics. FigureHDX of protein backbone amide hydrogen

Hydrogen-deuterium exchange mass spectrometry reveals the interaction of Fenna-Matthews-Olson protein and chlorosome CsmA protein.

In green-sulfur bacterial photosynthesis, excitation energy absorbed by a peripheral antenna structure known as the chlorosome is sequentially transferred through a baseplate protein to the Fenna-Matthews-Olson (FMO) antenna protein and into the reaction center, which is embedded in the cytoplasmic membrane. The molecular details of the optimized photosystem architecture required for efficient energy transfer are only partially understood. We address here the question of how the baseplate interacts with the FMO protein by applying hydrogen/deuterium exchange coupled with enzymatic digestion and mass spectrometry analysis to reveal the binding interface of the FMO antenna protein and the CsmA baseplate protein. Several regions on the FMO protein, represented by peptides consisting of 123-129, 140-149, 150-162, 191-208, and 224-232, show significant decreases of deuterium uptake after CsmA binding. The results indicate that the CsmA protein interacts with the Bchl a #1 side of the FMO protein. A global picture including peptide-level details for the architecture of the photosystem from green-sulfur bacteria can now be drawn.

Characterization of antibody-drug conjugates by mass spectrometry: advances and future trends.

Antibody-drug conjugates (ADCs) are emerging modalities in the pharmaceutical industry. The unique target-specific binding of antibody allows targeted delivery of cytotoxic small molecules to cancer cells, and thus expands the therapeutic window. However, in-depth characterization of ADCs is complex because it involves the characterization of antibody, conjugated molecules and antibody conjugates as a whole. In this review, we describe the practical use of mass spectrometry for ADC characterization including qualitative and quantitative analysis. Technical advances, limitations and future trends will also be discussed.

Mass Spectrometry-based Footprinting Reveals Structural Dynamics of Loop E of the Chlorophyll-binding Protein CP43 during Photosystem II Assembly in the Cyanobacterium Synechocystis 6803

Background: The mechanism for association and dissociation of Psb27 and CP43 is poorly understood. Results: Loop E of CP43 undergoes significant conformational change upon D1 processing. Conclusion: D1 processing initiates the dissociation of Psb27 from CP43. Significance: The structural dynamics of the lumenal domain of CP43 plays a critical role in the assembly of functional Photosystem II centers. The PSII repair cycle is required for sustainable photosynthesis in oxygenic photosynthetic organisms. In cyanobacteria and higher plants, proteolysis of the precursor D1 protein (pD1) to expose a C-terminal carboxylate group is an essential step leading to coordination of the Mn4CaO5 cluster, the site of water oxidation. Psb27 appears to associate with both pD1- and D1-containing PSII assembly intermediates by closely interacting with CP43. Here, we report that reduced binding affinity between CP43 and Psb27 is triggered by the removal of the C-terminal extension of the pD1 protein. A mass spectrometry-based footprinting strategy was adopted to probe solvent-exposed aspartic and glutamic acid residues on the CP43 protein. By comparing the extent of footprinting between HT3ΔctpAΔ27PSII and HT3ΔctpAPSII, two genetically modified PSII assembly complexes, we found that Psb27 binds to CP43 on the side of Loop E distal to the pseudo-symmetrical D1-D2 axis. By comparing a second pair of PSII assembly complexes, we discovered that Loop E of CP43 undergoes a significant conformational rearrangement due to the removal of the pD1 C-terminal extension, altering the Psb27-CP43 binding interface. The significance of this conformational rearrangement is discussed in the context of recruitment of the PSII lumenal extrinsic proteins and Mn4CaO5 cluster assembly. In addition to CP43s previously known function as one of the core PSII antenna proteins, this work demonstrates that Loop E of CP43 plays an important role in the functional assembly of the Water Oxidizing Center (WOC) during PSII biogenesis.

Improved Mass Spectrometric Characterization of Protein Glycosylation Reveals Unusual Glycosylation of Maize-Derived Bovine Trypsin

Although bottom-up proteomics using tryptic digests is widely used to locate post-translational modifications (PTM) in proteins, there are cases where the protein has several potential modification sites within a tryptic fragment and MS(2) strategies fail to pinpoint the location. We report here a method using two proteolytic enzymes, trypsin and pepsin, in combination followed by tandem mass spectrometric analysis to provide fragments that allow one to locate the modification sites. We used this strategy to find a glycosylation site on bovine trypsin expressed in maize (TrypZean). Several glycans are present, and all are attached to a nonconsensus N-glycosylation site on the protein.

Effects of desialylation on human α1-acid glycoprotein-ligand interactions.

Human α1-acid glycoprotein (AGP), an acute-phase glycoprotein, exists predominantly in blood. With its ability to bind basic, lipophilic, and acidic drugs, AGP has served as a drug carrier. It has been shown that the carbohydrate composition of AGP changes in response to tissue injury, inflammation, or infection and can have a great impact on AGPs drug binding activities. The molecular-level details of the effects of desialylation on the AGP conformation and AGP-ligand interactions, however, are unknown. Here we report the use of hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) to reveal the changes in AGP conformational dynamics induced by the removal of terminal sialic acid. HDX-MS also reveals the changes in the conformational dynamics of sialylated and unsialylated AGP upon formation of complexes of holo-AGP with progesterone or propranolol. Our HDX-MS results demonstrate that desialylation stabilizes two loop regions that are exterior to the β-sheet barrel in AGP, and this stabilization minimizes the conformational changes of AGP upon binding with progesterone or propranolol.

Mapping the Energetic Epitope of an Antibody/Interleukin-23 Interaction with Hydrogen/Deuterium Exchange, Fast Photochemical Oxidation of Proteins Mass Spectrometry, and Alanine Shave Mutagenesis

Epitope mapping the specific residues of an antibody/antigen interaction can be used to support mechanistic interpretation, antibody optimization, and epitope novelty assessment. Thus, there is a strong need for mapping methods, particularly integrative ones. Here, we report the identification of an energetic epitope by determining the interfacial hot-spot that dominates the binding affinity for an anti-interleukin-23 (anti-IL-23) antibody by using the complementary approaches of hydrogen/deuterium exchange mass spectrometry (HDX-MS), fast photochemical oxidation of proteins (FPOP), alanine shave mutagenesis, and binding analytics. Five peptide regions on IL-23 with reduced backbone amide solvent accessibility upon antibody binding were identified by HDX-MS, and five different peptides over the same three regions were identified by FPOP. In addition, FPOP analysis at the residue level reveals potentially key interacting residues. Mutants with 3-5 residues changed to alanine have no measurable differences from wild-type IL-23 except for binding of and signaling blockade by the 7B7 anti-IL-23 antibody. The M5 IL-23 mutant differs from wild-type by five alanine substitutions and represents the dominant energetic epitope of 7B7. M5 shows a dramatic decrease in binding to BMS-986010 (which contains the 7B7 Fab, where Fab is fragment antigen-binding region of an antibody), yet it maintains functional activity, binding to p40 and p19 specific reagents, and maintains biophysical properties similar to wild-type IL-23 (monomeric state, thermal stability, and secondary structural features).