1H, 15N backbone assignment and comparative analysis of the wild type and G12C, G12D, G12V mutants of K-Ras bound to GDP at physiological pH

K-Ras protein is a membrane-bound small GTPase acting as a molecular switch. It plays a key role in many signal transduction pathways regulating cell proliferation, differentiation, survival, etc. It alternates between its GTP-bound active and the GDP-bound inactive conformers regulated by guanine nucleotide exchange factors and GTPase activating proteins. Its most frequent oncogenic mutants are G12C, G12D, and G12V that have impaired GTPase activity, thus induce malignant tumors. Here we report the resonance assignment of the backbone 1H and 15N nuclei of K-Ras wildtype, G12C, G12D and G12V proteins’ catalytic G domain (1–169 residues) in GDP-bound state, and 13C of backbone and side chains of G12C mutant at physiological pH 7.4. Triple resonance data were used to get secondary structure information and backbone dynamics of G12C, the best-known drug target among K-Ras mutants. Simultaneous investigation of G12C, G12D and G12V mutants, along with the wild type form at the very same conditions allowed us to perform a comprehensive analysis based on the combined chemical shifts to reveal the effect of mutation at G12 position on structure. Intriguingly, the G12C and G12V mutants found to be structurally very similar at the three most important regions of K-Ras (P-loop, Switch-I, Switch-II), while the G12D mutant significantly differs at P-loop and Switch-II from the wildtype as well as G12C and G12V mutants. However, in Switch-I it hardly deviates from the wildtype protein

Ezrin interacts with S100A4 via both its N- and C-terminal domains

Ezrin belongs to the ERM (ezrin, radixin, moesin) protein family that has a role in cell morphology changes, adhesion and migration as an organizer of the cortical cytoskeleton by linking actin filaments to the apical membrane of epithelial cells. It is highly expressed in a variety of human cancers and promotes metastasis. Members of the Ca2+-binding EF-hand containing S100 proteins have similar pathological properties; they are overexpressed in cancer cells and involved in metastatic processes. In this study, using tryptophan fluorescence and stopped-flow kinetics, we show that S100A4 binds to the N-terminal ERM domain (N-ERMAD) of ezrin with a micromolar affinity. The binding involves the F2 lobe of the N-ERMAD and follows an induced fit kinetic mechanism. Interestingly, S100A4 binds also to the unstructured C-terminal actin binding domain (C-ERMAD) with similar affinity. Using NMR spectroscopy, we characterized the complex of S100A4 with the C-ERMAD and demonstrate that no ternary complex is simultaneously formed with the two ezrin domains. Furthermore, we show that S100A4 co-localizes with ezrin in HEK-293T cells. However, S100A4 very weakly binds to full-length ezrin in vitro indicating that the interaction of S100A4 with ezrin requires other regulatory events such as protein phosphorylation and/or membrane binding, shifting the conformational equilibrium of ezrin towards the open state. As both proteins play an important role in promoting metastasis, the characterization of their interaction could shed more light on the molecular events contributing to this pathological process

NMR-Chemical-Shift-Driven Protocol Reveals the Cofactor-Bound, Complete Structure of Dynamic Intermediates of the Catalytic Cycle of Oncogenic KRAS G12C Protein and the Significance of the Mg²⁺ Ion

In this work, catalytically significant states of the oncogenic G12C variant of KRAS, those of Mg2+-free and Mg²⁺-bound GDP-loaded forms, have been determined using CS-Rosetta software and NMR-data-driven molecular dynamics simulations. There are several Mg²⁺-bound G12C KRAS/GDP structures deposited in the Protein Data Bank (PDB), so this system was used as a reference, while the structure of the Mg²⁺-free but GDP-bound state of the RAS cycle has not been determined previously. Due to the high flexibility of the Switch-I and Switch-II regions, which also happen to be the catalytically most significant segments, only chemical shift information could be collected for the most important regions of both systems. CS-Rosetta was used to derive an “NMR ensemble” based on the measured chemical shifts, which, however, did not contain the nonprotein components of the complex. We developed a torsional restraint set for backbone torsions based on the CS-Rosetta ensembles for MD simulations, overriding the force-field-based parametrization in the presence of the reinserted cofactors. This protocol (csdMD) resulted in complete models for both systems that also retained the structural features and heterogeneity defined by the measured chemical shifts and allowed a detailed comparison of the Mg²⁺-bound and Mg²⁺-free states of G12C KRAS/GDP.ISSN:1422-006

The Importance of Mg2 + -Free State in Nucleotide Exchange of Oncogenic K-Ras Mutants

For efficient targeting of oncogenic K-Ras interaction sites, a mechanistic picture of the Ras-cycle is necessary. Herein, we used NMR relaxation techniques and molecular dynamics simulations to decipher the role of slow dynamics in wild-type and three oncogenic P-loop mutants of K-Ras. Our measurements reveal a dominant two-state conformational exchange on the ms timescale in both GDP- and GTP-bound K-Ras. The identified low-populated higher energy state in GDP-loaded K-Ras has a conformation reminiscent of a nucleotide-bound/Mg2+ -free state characterized by shortened β2/β3-strands and a partially released switch-I region preparing K-Ras for the interaction with the incoming nucleotide exchange factor and subsequent reactivation. By providing insight into mutation-specific differences in K-Ras structural dynamics, our systematic analysis improves our understanding of prolonged K-Ras signaling and may aid the development of allosteric inhibitors targeting nucleotide exchange in K-Ras