Characterizing protein-ligand interactions is crucial to understanding cellular metabolism and guiding drug discovery and development. Herein, we explore complementing hydrogen/deuterium exchange mass spectrometry (HDX-MS) with a recently developed Fenton chemistry-based approach to protein oxidative footprinting mass spectrometry (OX-MS) to discriminate the binding of small-molecule therapeutics. Using drug-dependent perturbation as the experimental report, this combination of techniques more clearly differentiates the in-solution binding profiles of Venetoclax (ABT-199, GDC-0199-AbbVie and Genentech) and a drug candidate S55746 (Servier) to the apoptotic regulatory protein Bcl-2 than either technique alone. These results highlight the value of combining these methods to compare compounds in drug discovery and development. To better understand the structural context of the HDX-MS and OX-MS drug-dependent perturbations, we mapped these data on Bcl-2-Venetoclax and Bcl-2-S55746 cocrystal structures and compared these results with the structure of apo Bcl-2. HDX-MS shows that Venetoclax more strongly impacts the protein backbone compared to S55746. OX-MS reveals oxidation perturbations rationalized by direct side-chain protection as well as by crystallographically observed drug-induced protein restructuring. Both methods report the perturbation of some, but not all, residues mapped within 4 Å of the bound drugs in the crystal structures. Concordant characterization of backbone and side-chain accessibility will enhance our understanding of in-solution protein structure dynamics and protein-ligand interactions during drug discovery, development, and characterization, particularly when high-resolution structures are lacking.

Data collection and refinement statistics

Abstract KRAS is the most frequently mutated oncogene. The incidence of specific KRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specific mutant KRAS proteins. We used a cross-disciplinary approach to compare KRASG12D, a common mutant form, and KRASA146T, a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRASA146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factor–induced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRASG12D and KRASA146T exhibit distinct tissue-specific effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specific phenotypes result from allele-specific signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. Significance: Although epidemiologic and clinical studies have suggested allele-specific behaviors for KRAS, experimental evidence for allele-specific biological properties is limited. We combined structural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specific KRAS mutants in human cancers from different tissues is due to their distinct signaling properties. See related commentary by Hobbs and Der, p. 696. This article is highlighted in the In This Issue feature, p. 681

GSEA analysis of pancreata expressing WT or mutant K-Ras.

Proteomics data from mouse tissues.

GSEA analysis of spleens expressing WT or mutant K-Ras.

GSEA analysis of colons expressing WT or mutant K-Ras.

Supplementary Figures S1-S18

Proteins often undergo large conformational changes when binding small molecules, but atomic-level descriptions of such events have been elusive. Here, we report unguided molecular dynamics simulations of Abl kinase binding to the cancer drug imatinib. In the simulations, imatinib first selectively engages Abl kinase in its autoinhibitory conformation. Consistent with inferences drawn from previous experimental studies, imatinib then induces a large conformational change of the protein to reach a bound complex that closely resembles published crystal structures. Moreover, the simulations reveal a surprising local structural instability in the C-terminal lobe of Abl kinase during binding. The unstable region includes a number of residues that, when mutated, confer imatinib resistance by an unknown mechanism. Based on the simulations, NMR spectra, hydrogen-deuterium exchange measurements, and thermostability measurements and estimates, we suggest that these mutations confer imatinib resistance by exacerbating structural instability in the C-terminal lobe, rendering the imatinib-bound state energetically unfavorable.

Abstract KRAS is the most frequently mutated oncogene. The incidence of specific KRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specific mutant KRAS proteins. We used a cross-disciplinary approach to compare KRASG12D, a common mutant form, and KRASA146T, a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRASA146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factor–induced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRASG12D and KRASA146T exhibit distinct tissue-specific effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specific phenotypes result from allele-specific signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. Significance: Although epidemiologic and clinical studies have suggested allele-specific behaviors for KRAS, experimental evidence for allele-specific biological properties is limited. We combined structural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specific KRAS mutants in human cancers from different tissues is due to their distinct signaling properties. See related commentary by Hobbs and Der, p. 696. This article is highlighted in the In This Issue feature, p. 681

Proteomics data from human cell lines.

Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.

The 33 members of the transforming growth factor beta (TGF-β) family are fundamentally important for organismal development and homeostasis. Family members are synthesized and secreted as pro-complexes of prodomains that are non-covalently bound to the growth factor (GF). The pro-complexes of some members are latent and require activation steps to release the GF for signaling. Why some members are latent while others are non-latent is incompletely understood, but crystal structures and hydrogen-deuterium exchange (HDX) of four family members have begun to unravel how latency is regulated. Here, we extend this understanding by comparing pro-complex conformation in negative stain EM (nsEM) and HDX of ActA, BMP7, BMP9, BMP10, GDF8, TGF-β1, and TGF-β2. nsEM revealed that family members varied in either adopting cross-armed, open-armed, or V-armed configurations. Latency was achieved in both cross-armed and V-armed but not open-armed conformations. HDX revealed remarkably varying patterns of exchange between family members, consistent with large prodomain sequence divergence. We observed a strong correlation between latency and protection of the prodomain α1-helix from exchange, which in latent members coincided with greater buried surface area of the α1-helix and more hydrogen and cation-pi bonds from the prodomain fastener and GF to the α1-helix. Strong sequence conservation of the α1-helix and fastener only in latent members suggests that similar interactions are conserved and sufficient to confer latency. Moreover, most members exhibited rapid exchange in the unstructured “association region” at the prodomain N-terminus, highlighting their availability for interacting with factors that may regulate latency and extracellular storage.

Integrin α5β1 mediates cell adhesion to the extracellular matrix by binding fibronectin (Fn). Selectivity for Fn by α5β1 is achieved through recognition of an RGD motif in the 10th type III Fn domain (Fn10) and the synergy site in the ninth type III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5β1-binding affinities and stabilities. We also interrogated binding of the α5β1 ectodomain headpiece fragment to Fn using hydrogen-deuterium exchange (HDX) mass spectrometry to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two β-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit β-propeller domain for the Fn9 synergy site and in the β1-subunit βI domain for Fn10 based on decreases in α5β1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5β1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5β1 binding to Fn and dynamics associated with this interaction.

Integrin alpha 5 beta 1 mediates cell adhesion to the extracellular matrix by binding fibronectin (Fn). Selectivity for Fn by alpha 5 beta 1 is achieved through recognition of an RGD motif in the 10th type III Fn domain (Fn10) and the synergy site in the ninth type III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their alpha 5 beta 1-binding affinities and stabilities. We also interrogated binding of the alpha 5 beta 1 ectodomain headpiece fragment to Fn using hydrogen-deuterium exchange (HDX) mass spectrometry to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two beta-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the alpha 5-subunit beta-propeller domain for the Fn9 synergy site and in the beta 1-subunit beta I domain for Fn10 based on decreases in alpha 5 beta 1 HDX. Interestingly, the dominant effect of Fn binding was an increase in alpha 5 beta 1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for alpha 5 beta 1 binding to Fn and dynamics associated with this interaction.

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multi-substrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structure-guided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of ~500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A.

The higher-order structure of a protein defines its function, and protein structural dynamics are often essential for protein binding and enzyme catalysis. Methods for protein characterization in solution are continuously being developed to understand and explore protein conformational changes with regards to function and activity. The goal of this study was to survey the use of combining HDX-MS global conformational screening with in silico modeling and continuous labeling peptide-level HDX-MS as an approach to highlight regions of interest within an enzyme required for biocatalytic processes. We surveyed in silico modeling correlated with peptide level HDX-MS experiments to characterize and localize transaminase enzyme structural dynamics at different conditions. This approach was orthogonally correlated with a global Size-Exclusion-HDX (SEC-HDX) screen for global conformational comparison and global alpha-helical content measurements by circular dichroism. Enzymatic activity and stereo-selectivity of transaminases were compared at different reaction-solution conditions that forced protein conformational changes by increasing acetonitrile concentration. The experimental peptide-level HDX-MS results demonstrated similar trends to the modeling data showing that certain regions remained folded in transaminases ATA-036 and ATA-303 with increasing acetonitrile concentration, which is also associated with shifting stereoselectivity. HDX modeling, SEC-HDX and CD experimental data showed that transaminase ATA-234 had the highest level of global unfolding with increasing acetonitrile concentration compared to the other two enzymes, which correlated with drastically reduced product conversion in transamination reaction. The combined HDX modeling/experimental workflow, based on enzymatic reactions studied at different conditions to induce changes in enzyme conformation, could be used as a tool to guide directed evolution efforts by identifying and focusing on the regions of an enzyme required for reaction product conversion and stereoselectivity.

Integrin αVβ8, which like αVβ6 functions to activate TGF-βs, is atypical. Its β8 subunit binds to a distinctive cytoskeleton adaptor and does not exhibit large changes in conformation upon binding to ligand. Here, crystal structures, hydrogen-deuterium exchange dynamics, and affinity measurements on mutants are used to compare αVβ8 and αVβ6. Lack of a binding site for one of three βI domain divalent cations and a unique β6-α7 loop conformation in β8 facilitate movements of the α1 and α1' helices at the ligand binding pocket toward the high affinity state, without coupling to β6-α7 loop reshaping and α7-helix pistoning that drive large changes in βI domain-hybrid domain orientation seen in other integrins. Reciprocal swaps between β6 and β8 βI domains increase affinity of αVβ6 and decrease affinity of αVβ8 and define features that regulate affinity of the βI domain and its coupling to the hybrid domain.

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.