Elizaveta A. Kovrigina

Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the off and on allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.

The Ras G Domain Lacks the Intrinsic Propensity to Form Dimers

Ras GTPase is a molecular switch controlling a number of cellular pathways including growth, proliferation, differentiation, and apoptosis. Recent reports indicated that Ras undergoes dimerization at the membrane surface through protein-protein interactions. If firmly established this property of Ras would require profound reassessment of a large amount of published data and modification of the Ras signaling paradigm. One proposed mechanism of dimerization involves formation of salt bridges between the two GTPase domains (G domains) leading to formation of a compact dimer as observed in Ras crystal structures. In this work, we interrogated the intrinsic ability of Ras to self-associate in solution by creating conditions of high local concentration through irreversibly tethering the two G domains together at their unstructured C-terminal tails. We evaluated possible self-association in this inverted tandem conjugate via analysis of the time-domain fluorescence anisotropy and NMR chemical shift perturbations. We did not observe the increased rotational correlation time expected for the G domain dimer. Variation of the ionic strength (to modulate stability of the salt bridges) did not affect the rotational correlation time in the tandem further supporting independent rotational diffusion of two G domains. In a parallel line of experiments to detect and map weak self-association of the G domains, we analyzed NMR chemical shifts perturbations at a number of sites near the crystallographic dimer interface. The nearly complete lack of chemical shift perturbations in the tandem construct supported a simple model with the independent G domains repelled from each other by their overall negative charge. These results lead us to the conclusion that self-association of the G domains cannot be responsible for homodimerization of Ras reported in the literature.

Application of methyl-TROSY to a large paramagnetic membrane protein without perdeuteration: 13 C-MMTS-labeled NADPH-cytochrome P450 oxidoreductase

NMR spectroscopy of membrane proteins involved in electron transport is difficult due to the presence of both the lipids and paramagnetic centers. Here we report the solution NMR study of the NADPH-cytochrome P450 oxidoreductase (POR) in its reduced and oxidized states. We interrogate POR, first, in its truncated soluble form (70xa0kDa), which is followed by experiments with the full-length protein incorporated in a lipid nanodisc (240xa0kDa). To overcome paramagnetic relaxation in the reduced state of POR as well as the signal broadening due to its high molecular weight, we utilized the methyl-TROSY approach. Extrinsic 13C-methyl groups were introduced by modifying the engineered surface-exposed cysteines with methyl-methanethiosulfonate. Chemical shift dispersion of the resonances from different sites in POR was sufficient to monitor differential effects of the reduction–oxidation process and conformation changes in the POR structure related to its function. Despite the high molecular weight of the POR-nanodisc complex, the surface-localized 13C-methyl probes were sufficiently mobile to allow for signal detection at 600xa0MHz without perdeuteration. This work demonstrates a potential of the solution methyl-TROSY in analysis of structure, dynamics, and function of POR, which may also be applicable to similar paramagnetic and flexible membrane proteins.

Extraction of a recombinant full-length NADPH-cytochrome P450 oxidoreductase from bacterial membranes: effect of detergents and additives

NADPH-cytochrome P450 oxidoreductase (POR) is a membrane protein in the endoplasmic reticulum of eukaryotic cells. POR is as a key reducing partner for a number of cytochrome P450 proteins involved in different metabolic degradation and signaling pathways. Preparation of the full-length recombinant POR expressed in bacteria has been reported and, typically, involved the use of Triton X-100 detergent for extraction of the overexpressed POR from bacterial membranes. However, extraction efficiency is always relatively low hindering structural studies, particularly—the NMR spectroscopy requiring isotopic enrichment. In this paper, we assessed the effect of a variety of detergents and additives on the efficiency of the membrane-extraction step in POR preparation protocol. We evaluated non-ionic detergents with the variable hydrophobicity (Triton X-100, X-114, and X-405) and structure (Triton X-100, TWEEN-20, Brij-35), a zwitterionic/non-ionic detergent combination (Triton X-100 and CHAPS), as well as a range of alkylamines and polyamines as additives to the conventional extraction buffer containing Triton X-100. None of the detergents or detergent-additive combinations yielded better extraction efficiency than the conventional protocol with the Triton X-100. Lack of variation of the extraction yield allows to hypothesize that the conventional protocol extracts all of the available natively-folded monomeric POR while the remaining fraction is possibly an unfolded aggregated POR, which did not insert in the membranes during expression. We propose that the yield of soluble POR may be increased by a careful optimization of expression conditions while monitoring the distribution of POR between soluble and insoluble fractions in the detergent extraction step.

Study of Förster Resonance Energy Transfer to Lipid Domain Markers Ascertains Partitioning of Semisynthetic Lipidated N-Ras in Lipid Raft Nanodomains

Cellular membranes are heterogeneous planar lipid bilayers displaying lateral phase separation with the nanometer-scale liquid-ordered phase (also known as lipid rafts) surrounded by the liquid-disordered phase. Many membrane-associated proteins were found to permanently integrate into the lipid rafts, which is critical for their biological function. Isoforms H and N of Ras GTPase possess a unique ability to switch their lipid domain preference depending on the type of bound guanine nucleotide (GDP or GTP). This behavior, however, has never been demonstrated in vitro in model bilayers with recombinant proteins and therefore has been attributed to the action of binding of Ras to other proteins at the membrane surface. In this paper, we report the observation of the nucleotide-dependent switch of lipid domain preferences of the semisynthetic lipidated N-Ras in lipid raft vesicles in the absence of additional proteins. To detect segregation of Ras molecules in raft and disordered lipid domains, we measured Förster resonance energy transfer between the donor fluorophore, mant, attached to the protein-bound guanine nucleotides, and the acceptor, rhodamine-conjugated lipid, localized into the liquid-disordered domains. Herein, we established that N-Ras preferentially populated raft domains when bound to mant-GDP, while losing its preference for rafts when it was associated with a GTP mimic, mant-GppNHp. At the same time, the isolated lipidated C-terminal peptide of N-Ras was found to be localized outside of the liquid-ordered rafts, most likely in the bulk-disordered lipid. Substitution of the N-terminal G domain of N-Ras with a homologous G domain of H-Ras disrupted the nucleotide-dependent lipid domain switch.

Effect of ligands on stability of H-Ras GTPase

The G domain of a small monomeric GTPase Ras contains a nucleotide-binding pocket and a magnesium-binding site essential for the Ras function in cellular signaling. The G domain also has another (allosteric) ion-binding site on the rear surface of the G domain, which function is still unknown. In this paper, we detailed the effect of calcium and magnesium ions on stability of Ras bound to GDP, GTP, and GTP-mimic GppNHp. We revealed that the remote allosteric ion-binding site contributes very significantly to stability of Ras in the GDP-bound conformation, but nearly not at all—when Ras is bound to a GTP mimic. These findings highlight that further studies of the remote ion-binding site are warranted to reveal its role in the Ras function.

Preparation of H-Ras GTPase conjugated to lipid nanodiscs for NMR spectroscopy

Ras GTPase is a peripheral membrane protein central to cellular signaling of growth and proliferation. Membrane attachment is critical for a range of Ras activities, therefore, ability to make faithful in-vitro samples of a mem-brane-bound Ras for detailed biophysical studies is a highly desirable goal. In this manuscript, we are describing preparation of a large-scale sample of isotopically labeled H-Ras conjugated to lipid nanodiscs. We demonstrate that the Ras-nanodisc sample is fairly stable to allow for a range of Nuclear Magnetic Resonance (NMR) and other biophysical measurements. The need to achieve a homogeneous protein-nanodisc ratio is also emphasized.

Detection of domain motion in NADPH-cytochrome P450 oxidoreductase through polarization anisotropy measurements

Conformational transitions between closed and open states in the NADPH-cytochrome P450 oxidoreductase (POR) play a critical role in its electron-transport function. In this study, we determined rotational diffusion coefficients of the EDANS fluorophore attached to the cytosolic POR construct lacking the N-terminal transmembrane region. We identified two dynamic modes, slow and fast, which are interpreted as the rotational diffusion of POR as a whole and the local domain motion, respectively. Timescale of the local rotational diffusion component suggests that it may correspond to the transient opening of the fully oxidized POR structure.

Partitioning of semisynthetic lipidated N-Ras in lipid raft nanodomains determined by FRET to lipid domain markers

Cellular membranes are heterogeneous planar lipid bilayers displaying lateral phase separation with the nanometer-scale liquid-ordered phase (aka “lipid rafts” or Lo) surrounded by the liquid-disordered phase (Ld). Many membrane-associated proteins were found to stably integrate in the rafts, which is critical for their biological function. Isoforms H and N of Ras GTPase possess a unique ability to switch their lipid domain preference depending on the type of bound guanine nucleotide (GDP or GTP). This behavior, however, has never been reproduced in vitro in model bilayers with recombinant proteins, and therefore has been attributed to action of other proteins binding Ras at the membrane surface. In this paper, we report the observation of the nucleotide-dependent switch of lipid domain preferences of the semisynthetic lipidated N-Ras in raft lipid vesicles in the absence of other proteins. To detect segregation of Ras molecules in raft and disordered lipid domains, we measured Förster Resonance Energy Transfer (FRET) between the donor fluorophore, mant, attached to the protein-bound guanine nucleotides, and the acceptor, rhodamine-conjugated lipid, localized to the liquid-disordered domains. We demonstrated that N-Ras preferentially populated raft domains when bound to mant-GDP, while losing preference for rafts when it was associated with a GTP mimic, mant-GppNHp. At the same time, the isolated lipidated C-terminal peptide of N-Ras was found localized outside of the liquid-ordered rafts, most likely—in the bulk disordered lipid.

Fluorescence of Supported Phospholipid Bilayers Recorded in a Conventional Horizontal-Beam Spectrofluorometer

Supported phospholipid bilayers are a convenient model of cellular membranes in studies of membrane biophysics and protein-lipid interactions. Traditionally, supported lipid bilayers are formed on a flat surface of a glass slide to be observed through fluorescence microscopes. This paper describes a method to enable fluorescence detection from the supported lipid bilayers using standard horizontal-beam spectrofluorometers instead of the microscopes. In the proposed approach, the supported lipid bilayers are formed on the inner optical surfaces of the standard fluorescence microcell. To enable observation of the bilayer absorbed on the cell wall, the microcell is placed in a standard fluorometer cell holder and specifically oriented to expose the inner cell walls to both excitation and emission channels with a help of the custom cell adaptor. The signal intensity from supported bilayers doped with 1xa0% (mol) of rhodamine-labeled lipid in the standard 3-mm optical microcell was equivalent to fluorescence of the 70–80xa0nM reference solution of rhodamine recorded in a commercial microcell adaptor. Because no modifications to the instruments are required in this method, a variety of steady-state and time-domain fluorescence measurements of the supported phospholipid bilayers may be performed with the spectral resolution using standard horizontal-beam spectrofluorometers.