Revealing KRas4b topology on the membrane surface.

KRas4b is a membrane-bound regulatory protein belonging to the family of small GTPases that function as a molecular switch, facilitating signal transduction from activated membrane receptors to intracellular pathways controlling cell growth and proliferation. Oncogenic mutations locking KRas4b in the active GTP state are responsible for nearly 85% of all Ras-driven cancers. Understanding the membrane-bound state of KRas4b is crucial for designing new therapeutic approaches targeting oncogenic KRas-driven signaling pathways. Extensive research demonstrates the significant involvement of the membrane bilayer in Ras-effector interactions, with anionic lipids playing a critical role in determining protein conformations The preferred topology of KRas4b for interacting with signaling partners has been a long-time question. Computational studies suggest a membrane-proximal conformation, while other biophysical methods like neutron reflectivity propose a membrane-distal conformation. To address these gaps, we employed FRET measurements to investigate the conformation of KRas4b. Using fully post-translationally modified KRas4b, we designed a Nanodisc based FRET assay to study KRas4b-membrane interactions. We suggest an extended conformation of KRas4b relative to the membrane surface. Measurement of FRET donor - acceptor distances reveal that a negatively charged membrane surface weakly favors closer association with the membrane surface. Our findings provide insights into the role of anionic lipids in determining the dynamic conformations of KRas4b and shed light on the predominant conformation of its topology on lipid headgroups.

Engineering C-C Bond Cleavage Activity into a P450 Monooxygenase Enzyme.

The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon-carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon-carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C-C bond cleavage toward one of the α-hydroxy ketones. This C-C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.

Solvent isotope effects in the catalytic cycle of P450 CYP17A1: Computational modeling of the hydroxylation and lyase reactions.

The catalytic cycle of the cytochromes P450 (CYP) requires two electrons from a protein redox partner and two protons from water to generate the main catalytic intermediate, a ferryl-oxo complex with π-cation on the heme porphyrin ring, termed Compound 1. The protonation steps are at least partially rate-limiting, therefore the steady-state rates of P450 catalysis are usually slower in deuterated solvent (D2O) by a factor of 1.5-3. However, in several P450 systems a pronounced inverse kinetic solvent isotope effect (KSIE ∼0.4-0.7) is observed, where the reaction is faster in D2O. This raises an important mechanistic question: Is this inverse solvent isotope effect compatible with Compound 1 catalyzed reactions, or is it indicative of another catalytic intermediate being involved? In this communication we use exhaustive numerical modeling of the P450 steady-state kinetics to demonstrate that a significant inverse KSIE cannot be obtained for a pure Compound 1 driven catalytic cycle of P450. Rather, an alternative, protonation independent, catalytic intermediate needs to be introduced. This result is applicable to the broad spectrum of P450s in nature, but as an example we use the extensively documented inverse isotope effect in the human steroid biosynthetic P450 CYP17A1 where the involvement of a heme peroxo anion intermediate has been characterized. Based on this analysis, we show that the observation of an inverse KSIE can be used as a general mechanistic probe for reaction cycle intermediates in the cytochromes P450.

Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4.

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F' loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation.

Importance of Asparagine 202 in Manipulating Active Site Structure and Substrate Preference for Human CYP17A1.

The multifunctional cytochrome P450 17A1 (CYP17A1) plays a crucial role in human steroid hormone synthesis (UniProtKB─P05093). It first carries out standard monooxygenase chemistry, converting pregnenolone (PREG) and progesterone (PROG) into 17OH-PREG and 17OH-PROG, utilizing a "Compound I" to initiate hydrogen abstraction and radical recombination in the classic "oxygen rebound" mechanism. Additionally, these hydroxylated products also serve as substrates in a second oxidative cycle which cleaves the 17-20 carbon-carbon bond to form dehydroepiandrosterone and androstenedione, which are key precursors in the generation of powerful androgens and estrogens. Interestingly, in humans, with 17OH-PREG, this so-called lyase reaction is more efficient than with 17OH-PROG, based on Kcat/Km values. In the present work, the asparagine residue at 202 position was replaced by serine, an alteration which can affect substrate orientation and control substrate preference for the lyase reaction. First, we report studies of solvent isotope effects for the N202S CYP17A1 mutant in the presence of 17OH-PREG and 17OH-PROG, which suggest that the ferric peroxo species is the predominant catalytically active intermediate in the lyase step. This conclusion is further supported by employing a combination of cryoradiolysis and resonance Raman techniques to successfully trap and structurally characterize the key reaction intermediates, including the peroxo, the hydroperoxo, and the crucial peroxo-hemiketal intermediate. Collectively, these studies show that the mutation causes active site structural changes that alter the H-bonding interactions with the key Fe-O-O fragment and the degree of protonation of the reactive ferric peroxo intermediate, thereby impacting lyase efficiency.

Mechanism of the Clinically Relevant E305G Mutation in Human P450 CYP17A1.

Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon-carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C17-C20 bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon-carbon bond scission.

A Pathfinder in High-Pressure Bioscience: In Memoriam of Gaston Hui Bon Hoa.

On 26 July 2020, our colleague and friend Dr [...].

Midazolam as a Probe for Drug-Drug Interactions Mediated by CYP3A4: Homotropic Allosteric Mechanism of Site-Specific Hydroxylation.

We developed an efficient and sensitive probe for drug-drug interactions mediated by human CYP3A4 by using midazolam (MDZ) as a probe substrate. Using global analysis of four parameters over several experimental data sets, we demonstrate that the first MDZ molecule (MDZ1) binds with high affinity at the productive site near the heme iron and gives only hydroxylation at the 1 position (1OH). The second midazolam molecule (MDZ2) binds at an allosteric site at the membrane surface and perturbs the position and mobility of MDZ1 such that the minor hydroxylation product at the 4 position (4OH) is formed in a 1:2 ratio (35%). No increase in catalytic rate is observed after the second MDZ binding. Hence, the site of the 1OH:4OH metabolism ratio is a sensitive probe for drugs, such as progesterone, that bind with high affinity to the allosteric site and serve as effectors. We observe similar changes in the MDZ 1OH:4OH ratio in the presence of progesterone (PGS), suggesting a direct communication between the active and allosteric sites. Mutations introduced into the F-F' loop indicate that residues F213 and D214 are directly involved in allosteric interactions leading to MDZ homotropic cooperativity, and these same residues, together with L211, are involved in heterotropic allosteric interactions in which PGS is the effector and MDZ the substrate. Molecular dynamics simulations provide a mechanistic picture of the origin of this cooperativity. These results show that the midazolam can be used as a sensitive probe for drug-drug interactions in human P450 CYP3A4.

Substrate-Specific Allosteric Effects on the Enhancement of CYP17A1 Lyase Efficiency by Cytochrome b5.

CYP17A1 is an essential human steroidogenic enzyme, which catalyzes two sequential reactions leading to the formation of androstenedione from progesterone and dehydroepiandrosterone from pregnenolone. The second reaction is the C17-C20 bond scission, which is strongly dependent on the presence of cytochrome b5 and displays a heretofore unexplained more pronounced acceleration when 17OH-progesteone (17OH-PROG) is a substrate. The origin of the stimulating effect of cytochrome b5 on C-C bond scission catalyzed by CYP17A1 is still debated as mostly due to either the acceleration of the electron transfer to the P450 oxy complex or allosteric effects of cytochrome b5 favoring active site conformations that promote lyase activity. Using resonance Raman spectroscopy, we compared the effect of Mn-substituted cytochrome b5 (Mn-Cytb5) on the oxy complex of CYP17A1 with both proteins co-incorporated in lipid nanodiscs. For CYP17A1 with 17OH-PROG, a characteristic shift of the Fe-O mode is observed in the presence of Mn-b5, indicating reorientation of a hydrogen bond between the 17OH group of the substrate from the terminal to the proximal oxygen atom of the Fe-O-O moiety, a configuration favorable for the lyase catalysis. For 17OH-pregnenolone, no such shift is observed, the favorable H-bonding orientation being present even without Mn-Cytb5. These new data provide a precise allosteric interpretation for the more pronounced acceleration seen for the 17OH-PROG substrate.

Nanodiscs: A toolkit for membrane protein science.

Membrane proteins are involved in numerous vital biological processes, including transport, signal transduction and the enzymes in a variety of metabolic pathways. Integral membrane proteins account for up to 30% of the human proteome and they make up more than half of all currently marketed therapeutic targets. Unfortunately, membrane proteins are inherently recalcitrant to study using the normal toolkit available to scientists, and one is most often left with the challenge of finding inhibitors, activators and specific antibodies using a denatured or detergent solubilized aggregate. The Nanodisc platform circumvents these challenges by providing a self-assembled system that renders typically insoluble, yet biologically and pharmacologically significant, targets such as receptors, transporters, enzymes, and viral antigens soluble in aqueous media in a native-like bilayer environment that maintain a target's functional activity. By providing a bilayer surface of defined composition and structure, Nanodiscs have found great utility in the study of cellular signaling complexes that assemble on a membrane surface. Nanodiscs provide a nanometer scale vehicle for the in vivo delivery of amphipathic drugs, therapeutic lipids, tethered nucleic acids, imaging agents and active protein complexes. This means for generating nanoscale lipid bilayers has spawned the successful use of numerous other polymer and peptide amphipathic systems. This review, in celebration of the Anfinsen Award, summarizes some recent results and provides an inroad into the current and historical literature.

Dark, Ultra-Dark and Ultra-Bright Nanodiscs for membrane protein investigations.

We describe the construction, expression and purification of three new membrane scaffold proteins (MSP) for use in assembling Nanodiscs. These new MSPs have a variety of luminescent properties for use in combination with several analytical methods. "Dark" MSP has no tryptophan residues, "Ultra-Dark" replaces both tryptophan and tyrosine with non-fluorescent side chains, and "Ultra-Bright" adds additional tryptophans to the parent membrane scaffold protein to provide a dramatic increase in native tryptophan fluorescence. All MSPs were used to successfully assemble Nanodiscs nominally 10 nm in diameter, and the resultant bilayer structure was characterized. An example of the usefulness of these new scaffold proteins is provided.

P450 CYP17A1 Variant with a Disordered Proton Shuttle Assembly Retains Peroxo-Mediated Lyase Efficiency.

Human cytochrome P450 CYP17A1 first catalyzes hydroxylation at the C17 position of either pregnenolone (PREG) or progesterone (PROG), and a subsequent C17 -C20 bond scission to produce dehydroepiandrosterone (DHEA) or androstenedione (AD). In the T306A mutant, replacement of the Threonine 306 alcohol functionality, essential for efficient proton delivery in the hydroxylase reaction, has only a small effect on the lyase activity. In this work, resonance Raman spectroscopy is employed to provide crucial structural insight, confirming that this mutant, with its disordered proton shuttle, fails to generate essential hydroxylase pathway intermediates, accounting for the loss in hydroxylase efficiency. Significantly, a corresponding spectroscopic study with the susceptible lyase substrate, 17-OH PREG, not only reveals an initially trapped peroxo-iron intermediate experiencing an H-bond interaction of the 17-OH group with the proximal oxygen of the Fe-Op -Ot fragment, facilitating peroxo- attack on the C20 carbon, but also unequivocally shows the presence of the subsequent hemiketal intermediate of the lyase reaction.

Nanodisc self-assembly is thermodynamically reversible and controllable.

Many highly ordered complex systems form by the spontaneous self-assembly of simpler subunits. An important biophysical tool that relies on self-assembly is the Nanodisc system, which finds extensive use as native-like environments for studying membrane proteins. Nanodiscs are self-assembled from detergent-solubilized mixtures of phospholipids and engineered helical proteins called membrane scaffold proteins (MSPs). Detergent removal results in the formation of nanoscale bilayers stabilized by two MSP "belts." Despite their numerous applications in biology, and contributions from many laboratories world-wide, little is known about the self-assembly process such as when the bilayer forms or when the MSP associates with lipids. We use fluorescence and optical spectroscopy to probe self-assembly at various equilibria defined by the detergent concentration. We show that the bilayer begins forming below the critical micellar concentration of the detergent (10 mM), and the association of MSP and lipids begins at lower detergent levels, showing a dependence on the concentrations of MSP and lipids. Following the dissolution process by adding detergent to purified Nanodiscs demonstrates that the self-assembly is reversible. Our data demonstrate that Nanodisc self-assembly is experimentally accessible, and that controlling the detergent concentration allows exquisite control over the self-assembly reaction. This improved understanding of self-assembly could lead to better functional incorporation of hitherto intractable membrane target proteins.

Molecular Orientation Determination in Nanodiscs at the Single-Molecule Level.

The function of membrane-bound proteins often depends on their interactions with the lipid bilayer. Bulk absorption-based linear dichroism has been historically used to investigate molecular orientations in the phospholipid bilayer but cannot resolve the actual distribution of molecules embedded in the membrane and is often limited by a poor signal-to-noise ratio. Here, we present single-molecule orientation determination by fluorescence-detected linear dichroism visualization in Nanodisc grids or SOLVING, to determine the molecular orientation of molecules assembled into nanoscale lipid bilayers. We provide a proof-of-concept by using SOLVING to quantitate the orientation distribution of two commonly used fluorescent dyes, DiO and BODIPY, in 10 nm Nanodiscs. Besides confirming the mean orientation determined by bulk absorption measurement, SOLVING provides the actual distribution of orientations and promises to provide key molecular insights into the topology and interactions of multiprotein complexes, such as those observed in intracellular signal transduction.

PIP2 Influences the Conformational Dynamics of Membrane-Bound KRAS4b.

KRAS4b is a small GTPase involved in cellular signaling through receptor tyrosine kinases. The activation of KRAS4b occurs only after recruitment of the regulatory proteins to the plasma membrane, thus making the role of the phospholipid bilayer an integral part of the signaling mechanism. Phospholipids, primarily with anionic headgroups, interact with both the membrane-anchoring hypervariable (HVR) region and the G-domain (catalytic domain) and influence the orientation of KRAS4b on the membrane surface, potentially playing a key role in the regulation of activation. Although there has been significant research focused on the role of the anionic phosphatidylserine, less effort has been spent on the role of the important signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Using instrumentation to measure the fluorescence anisotropy decay of site specifically labeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) Nanodiscs over a wide frequency range, we quantitate the binding of KRAS4b to Nanodiscs containing either 30% phosphatidylserine (PS) or 10% l-α-phosphatidylinositol 4,5-bisphosphate by measuring the rotational correlation time of the Nanodisc-KRAS4b complex. We find that KRAS4b binds significantly tighter to Nanodiscs containing PIP2 but that at any level of binding saturation of KRAS4b, both 30% PS and 10% PIP2 containing Nanodiscs display similar rotational correlation times. This shows that the overall hydrodynamic radii of the KRAS4b-Nanodisc complexes are similar regardless of the incorporated anionic lipid. Atomic force microscopy is used to visualize KRAS4b when bound to individual Nanodiscs. Clean images are observed with the PIP2-doped Nanodiscs, but significantly blurred images are obtained when the anionic lipid is PS. This suggests that KRAS4b is not only more tightly bound overall with PIP2 as the anionic lipid but also less mobile on the bilayer surface. Microsecond molecular dynamics simulations of KRAS4b on PS- and PIP2-containing membranes show that the dynamics of the G-domain at the bilayer surface are significantly altered in the presence of PIP2, due to the formation of long-lived salt bridges with basic residues on the G-domain. The orientation and dynamics of KRAS4b on the membrane are critical to understanding the mechanisms of oncoprotein signaling, and our results with the GDP-bound form show subtle differences from that published for GTP-KRAS4b.

Biotransformation of the Mycotoxin Enniatin B1 by CYP P450 3A4 and Potential for Drug-Drug Interactions.

Enniatins (ENNs) are fungal secondary metabolites that frequently occur in grain in temperate climates. Their toxic potency is connected to their ionophoric character and lipophilicity. The biotransformation of ENNs predominantly takes place via cytochrome P450 3A (CYP 3A)-dependent oxidation reactions. Possible interaction with ENNs is relevant since CYP3A4 is the main metabolic enzyme for numerous drugs and contaminants. In the present study, we have determined the kinetic characteristics and inhibitory potential of ENNB1 in human liver microsomes (HLM) and CYP3A4-containing nanodiscs (ND). We showed in both in vitro systems that ENNB1 is mainly metabolised by CYP3A4, producing at least eleven metabolites. Moreover, ENNB1 significantly decreased the hydroxylation rates of the typical CYP3A4-substrate midazolam (MDZ). Deoxynivalenol (DON), which is the most prevalent mycotoxin in grain and usually co-occurrs with the ENNs, was not metabolised by CYP3A4 or binding to its active site. Nevertheless, DON affected the efficiency of this biotransformation pathway both in HLM and ND. The metabolite formation rates of ENNB1 and the frequently used drugs progesterone (PGS) and atorvastatin (ARVS) lactone were noticeably reduced, which indicated a certain affinity of DON to the enzyme with subsequent conformational changes. Our results emphasise the importance of drug-drug interaction studies, also with regard to natural toxins.

Nanodiscs as a New Tool to Examine Lipid-Protein Interactions.

The interactions between lipids and proteins are one of the most fundamental processes in living organisms, responsible for critical cellular events ranging from replication, cell division, signaling, and movement. Enabling the central coupling responsible for maintaining the functionality of the breadth of proteins, receptors, and enzymes that find their natural home in biological membranes, the fundamental mechanisms of recognition of protein for lipid, and vice versa, have been a focal point of biochemical and biophysical investigations for many decades. Complexes of lipids and proteins, such as the various lipoprotein factions, play central roles in the trafficking of important proteins, small molecules and metabolites and are often implicated in disease states. Recently an engineered lipoprotein particle, termed the nanodisc, a modified form of the human high density lipoprotein fraction, has served as a membrane mimetic for the investigation of membrane proteins and studies of lipid-protein interactions. In this review, we summarize the current knowledge regarding this self-assembling lipid-protein complex and provide examples for its utility in the investigation of a large number of biological systems.