The design of potent RAS inhibitors benefits from a molecular understanding of the dynamics in KRAS and NRAS and their oncogenic mutants. Here we characterize switch-1 dynamics in GTP-state KRAS and NRAS by P-31 NMR, by N-15 relaxation dispersion NMR, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and molecular dynamics simulations. In GMPPNP-bound KRAS and NRAS, we see the co-existence of two conformational states, corresponding to an "inactive" state-1 and an "active" state-2, as previously reported. The KRAS oncogenic mutations G12D, G12C and G12V only slightly affect this equilibrium towards the "inactive" state-1, with rank order wt < G12C < G12D < G12V. In contrast, the NRAS Q61R oncogenic mutation shifts the equilibrium fully towards the "active" state-2. Our molecular dynamics simulations explain this by the observation of a transient hydrogen bond between the Arg61 side chain and the Thr35 backbone carbonyl oxygen. NMR relaxation dispersion experiments with GTP-bound KRAS Q61R confirm a drastic decrease in the population of state-1, but still detect a small residual population (1.8%) of this conformer. HDX-MS indicates that higher populations of state-1 correspond to increased hydrogen-deuterium exchange rates in some regions and increased flexibility, whereas low state-1 populations are associated with KRAS rigidification. We elucidated the mechanism of action of a potent KRAS G12D inhibitor, MRTX1133. Binding of this inhibitor to the switch-2 pocket causes a complete shift of KRAS G12D towards the "inactive" conformation and prevents binding of effector RAS-binding domain (RBD) at physiological concentrations, by signaling through an allosteric network.

An inherent strength of hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is its ability to detect the presence of multiple conformational states of a protein, which often manifest as multimodal isotopic envelopes. However, the statistical considerations for accurate analysis of multimodal spectra have yet to be established. Here we outline an unrestrained binomial distribution fitting approach with the corresponding statistical tests to accurately detect and, when possible, deconvolute isotopic distributions that contain multiple subpopulations. The algorithms have been incorporated into an updated version of the freely available software, HX-Express, and validated using known mixtures of peptides deuterated to varying degrees. This approach presents a readily accessible tool to fit and interpret bimodal and trimodal behavior in HDX-MS data for mixed populations, EX1 kinetics, and pulse labeling data.

The cellular environment is critical for efficient protein maturation, but how proteins fold during biogenesis remains poorly understood. We used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. On the ribosome, the nascent polypeptide folds via structured intermediates not populated during refolding from denaturant. Association with the ribosome allows these intermediates to form, as otherwise destabilizing C-terminal sequences remain confined in the ribosome exit tunnel. We find that partially-folded nascent chains recruit the chaperone Trigger factor, which uses a large composite hydrophobic/hydrophilic interface to engage folding intermediates without disrupting their structure. In addition, we comprehensively mapped dynamic interactions between the nascent chain and ribosomal proteins, tracing the path of the emerging polypeptide during synthesis. Our work provides a high-resolution description of de novo protein folding dynamics, thereby revealing new mechanisms by which cellular factors shape the conformational search for the native state.

Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.

AbstractProtein complexes are essential for proteins' folding and biological function. Currently, native analysis of large multimeric protein complexes remains challenging. Structural biology techniques are time‐consuming and often cannot monitor the proteins' dynamics in solution. Here, a capillary electrophoresis‐mass spectrometry (CE–MS) method is reported to characterize, under near‐physiological conditions, the conformational rearrangements of ∽1 MDa GroEL upon complexation with binding partners involved in a protein folding cycle. The developed CE–MS method is fast (30 min per run), highly sensitive (low‐amol level), and requires ∽10 000‐fold fewer samples compared to biochemical/biophysical techniques. The method successfully separates GroEL14 (∽800 kDa), GroEL7 (∽400 kDa), GroES7 (∽73 kDa), and NanA4 (∽130 kDa) oligomers. The non‐covalent binding of natural substrate proteins with GroEL14 can be detected and quantified. The technique allows monitoring of GroEL14 conformational changes upon complexation with (ATPγS)4–14 and GroES7 (∽876 kDa). Native CE‐pseudo‐MS3 analyses of wild‐type (WT) GroEL and two GroEL mutants result in up to 60% sequence coverage and highlight subtle structural differences between WT and mutated GroEL. The presented results demonstrate the superior CE–MS performance for multimeric complexes' characterization versus direct infusion ESI–MS. This study shows the CE–MS potential to provide information on binding stoichiometry and kinetics for various protein complexes.

Data collection and refinement statistics

Immunoglobulin light chain (LC) amyloidosis is a life-threatening disease whose understanding and treatment is complicated by vast numbers of patient-specific mutations. To address molecular origins of the disease, we explored 14 patient-derived and engineered proteins related to κ1-family germline genes IGKVLD-33*01 and IGKVLD-39*01. Hydrogen-deuterium exchange mass spectrometry analysis of local conformational dynamics in full-length recombinant LCs and their fragments was integrated with studies of thermal stability, proteolytic susceptibility, amyloid formation, and amyloidogenic sequence propensities using spectroscopic, electron microscopic and bioinformatics tools. The results were mapped on the atomic structures of native and fibrillary proteins. Proteins from two κ1 subfamilies showed unexpected differences. Compared to their germline counterparts, amyloid LC related to IGKVLD-33*01 was less stable and formed amyloid faster, whereas amyloid LC related to IGKVLD-39*01 had similar stability and formed amyloid slower. These and other differences suggest different major factors influencing amyloid formation. In 33*01-related amyloid LC, these factors involved mutation-induced destabilization of the native structure and probable stabilization of amyloid. The atypical behaviour of 39*01-related amyloid LC tracked back to increased dynamics/exposure of amyloidogenic segments in βC' V and βE V that could initiate aggregation, combined with decreased dynamics/exposure near the Cys23-Cys88 disulfide whose rearrangement is rate-limiting to amyloidogenesis. The results suggest distinct amyloidogenic pathways for closely related LCs and point to the antigen-binding, complementarity-determining regions CDR1 and CDR3, which are linked via the conserved internal disulfide, as key factors in amyloid formation by various LCs.

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

Full-length BTK has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology (PHTH) domain and proline-rich regions (PRR) contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveals only the SH3-SH2-kinase core with no electron density visible for the PHTH-PRR segment. CryoEM data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH-PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3-SH2-kinase core. On the way to activation, disassembly of the SH3-SH2-kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with PIP3. Membrane-induced dimerizationactivates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to transautophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.

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

Reversed-phase peptide separation in hydrogen deuterium exchange (HDX) mass spectrometry (MS) must be done with conditions where the back exchange is the slowest possible, the so-called quench conditions of low pH and low temperature. To retain maximum deuterium, separation must also be done as quickly as possible. The low temperature (0 °C) of quench conditions complicates the separation and leads primarily to a reduction in separation quality and an increase in chromatographic backpressure. To improve the separation in HDX MS, one could use a longer gradient, smaller particles, a different separation mechanism (for example, capillary electrophoresis), or multi-dimensional separations such as combining ion mobility separation with reversed-phase separation. Another way to improve separations under HDX MS quench conditions is to use a higher flow rate where separation efficiency at 0 °C is more ideal. Higher flow rates, however, require chromatographic systems (both pumps and fittings) with higher backpressure limits. We tested what improvements could be realized with a commercial UPLC/UHPLC system capable of ~20,000 psi backpressure. We found that a maximum flow rate of 225 μL/min (using a 1×50mm column packed with 1.8 μm particles) was possible and that higher flow rate clearly led to higher peak capacity. HDX MS analysis of both simple and particularly complex samples improved, permitting both shorter separation time, if desired, and providing more deuterium recovery.

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

Proteomics data from mouse tissues.

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.

Exercise benefits the human body in many ways. Irisin is secreted by muscle, increased with exercise, and conveys physiological benefits, including improved cognition and resistance to neurodegeneration. Irisin acts via αV integrins; however, a mechanistic understanding of how small polypeptides like irisin can signal through integrins is poorly understood. Using mass spectrometry and cryo-EM, we demonstrate that the extracellular heat shock protein 90α (eHsp90α) is secreted by muscle with exercise and activates integrin αVβ5. This allows for high-affinity irisin binding and signaling through an Hsp90α/αV/β5 complex. By including hydrogen/deuterium exchange data, we generate and experimentally validate a 2.98 Å RMSD irisin/αVβ5 complex docking model. Irisin binds very tightly to an alternative interface on αVβ5 distinct from that used by known ligands. These data elucidate a non-canonical mechanism by which a small polypeptide hormone like irisin can function through an integrin receptor.