Hydrogen exchange coupled to mass spectrometry (MS) has become a valuable analytical tool for the study of protein dynamics. By combining information about protein dynamics with more classical functional data, a more thorough understanding of protein function can be obtained. In many cases, protein dynamics are directly related to specific protein functions such as conformational changes during enzyme activation or protein movements during binding. The method is made possible because labile backbone hydrogens in a protein will exchange with deuterium atoms when the protein is placed in a D2O solution. The subsequent increase in protein mass over time is measured with high-resolution MS. The location of the deuterium incorporation is determined by monitoring deuterium incorporation in peptic fragments that are produced after the labeling reaction. In this review, we will summarize the general principles of the method, discuss the latest variations on the experimental protocol that probe different types of protein movements, and review other recent work and improvements in the field.

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR mutant non-small cell lung cancer (NSCLC) is limited by the development of drug resistance mutations, including the gatekeeper T790M mutation1-3. Strategies aimed at targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild type EGFR4,5. All current EGFR inhibitors possess a structurally related quinazoline based core scaffold and were identified as ATP-competitive inhibitors of wild type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30-100 fold more potent against EGFR T790M, and up to 100 fold less potent against wild type EGFR, than quinazoline based EGFR inhibitors in vitro and are effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant selective kinase inhibitors.

SUMMARY In an effort to find new pharmacological modalities to overcome resistance to ATP-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry we demonstrate that GNF-2 binds to the myristate binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analog of GNF-2 having improved pharmacokinetic properties, when utilized in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I Bcr-Abl and displayed in vivo efficacy against the recalcitrant T315I Bcr-Abl mutant in a murine bone-marrow transplantation model. These results demonstrate that therapeutically relevant inhibition of Bcr-Abl activity can be achieved using inhibitors that bind to the myristate binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Biologics such as monoclonal antibodies are much more complex than small-molecule drugs, which raises challenging questions for the development and regulatory evaluation of follow-on versions of such biopharmaceutical products (also known as biosimilars) and their clinical use once patent protection for the pioneering biologic has expired. With the recent introduction of regulatory pathways for follow-on versions of complex biologics, the role of analytical technologies in comparing biosimilars with the corresponding reference product is attracting substantial interest in establishing the development requirements for biosimilars. Here, we discuss the current state of the art in analytical technologies to assess three characteristics of protein biopharmaceuticals that regulatory authorities have identified as being important in development strategies for biosimilars: post-translational modifications, three-dimensional structures and protein aggregation.

Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin ␥1 (IgG1) antibody. IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs both in vivo and in vitro. For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response. We investigated the impact of differential galactosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the Fc␥RIIIa receptor. The results showed that methionine oxidation and galactosylation both impact IgG1 conformation, whereas fucosylation appears to have little or no impact to the conformation. Fc␥RIIIa binding was strongly influenced by both the glycan structure/composition (namely galactose and fucose) and conformational changes that were induced by some of the modifications. Molecular & Cellular Proteomics 9:1716 -1728, 2010.

The Cdc48 adenosine triphosphatase (ATPase) (p97 or valosin-containing protein in mammals) and its cofactor Ufd1/Npl4 extract polyubiquitinated proteins from membranes or macromolecular complexes for subsequent degradation by the proteasome. How Cdc48 processes its diverse and often well-folded substrates is unclear. Here, we report cryo–electron microscopy structures of the Cdc48 ATPase in complex with Ufd1/Npl4 and polyubiquitinated substrate. The structures show that the Cdc48 complex initiates substrate processing by unfolding a ubiquitin molecule. The unfolded ubiquitin molecule binds to Npl4 and projects its N-terminal segment through both hexameric ATPase rings. Pore loops of the second ring form a staircase that acts as a conveyer belt to move the polypeptide through the central pore. Inducing the unfolding of ubiquitin allows the Cdc48 ATPase complex to process a broad range of substrates.

Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced, multipoint binding of ubiquitin chains. The structures of the T1 site in its free state and complexed with monoubiquitin or K48-linked diubiquitin were solved, revealing that three neighboring outer helices from the T1 toroid engage two ubiquitins. This binding mode leads to a preference for certain ubiquitin chain types, especially K6- and K48-linked chains, in a distinct configuration that can position substrates close to the entry port of the proteasome. The fate of proteasome-docked ubiquitin conjugates is determined by a competition between deubiquitination and substrate degradation. We find that proximal to the T1 site within the Rpn1 toroid is a second UBL-binding site, T2, that does not assist in ubiquitin chain recognition, but rather in chain disassembly, by binding to the UBL domain of deubiquitinating enzyme Ubp6. Importantly, the UBL interactors at T1 and T2 are distinct, assigning substrate localization to T1 and substrate deubiquitination to T2. CONCLUSION A ligand-binding hotspot was identified in the Rpn1 toroid, consisting of two adj...