Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants.

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras-GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility-mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOScat) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRas. We also find KRasG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its activity. A structure of the KRasG13D•SOScat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRasG13D-GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas-GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRasG13D•SOScat complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.

Selective regulation of human TRAAK channels by biologically active phospholipids.

TRAAK is an ion channel from the two-pore domain potassium (K2P) channel family with roles in maintaining the resting membrane potential and fast action potential conduction. Regulated by a wide range of physical and chemical stimuli, the affinity and selectivity of K2P4.1 toward lipids remains poorly understood. Here we show the two isoforms of K2P4.1 have distinct binding preferences for lipids dependent on acyl chain length and position on the glycerol backbone. The channel can also discriminate the fatty acid linkage at the SN1 position. Of the 33 lipids interrogated using native mass spectrometry, phosphatidic acid had the lowest equilibrium dissociation constants for both isoforms of K2P4.1. Liposome potassium flux assays with K2P4.1 reconstituted in defined lipid environments show that those containing phosphatidic acid activate the channel in a dose-dependent fashion. Our results begin to define the molecular requirements for the specific binding of lipids to K2P4.1.

Variation of CI-2 Conformers upon Addition of Methanol to Water: An IMS-MS-Based Thermodynamic Analysis.

Chymotrypsin inhibitor 2 (CI-2) is a well-studied, textbook example of a cooperative, two-state, native ↔ denatured folding transition. A recent hybrid ion mobility spectrometry (IMS)/mass spectrometry (MS) thermal denaturation study of CI-2 (the well-studied truncated 64-residue model) in water reported evidence that this two-state transition involves numerous (∼41) unique native and non-native (denatured) solution conformations. The characterization of so many, often low-abundance, states is possible because of the very high dynamic range of IMS-MS measurements of ionic species that are produced upon electrospraying CI-2 solutions from a variable temperature electrospray ionization source. A thermodynamic analysis of these states revealed large changes in enthalpy (ΔH) and entropy (ΔS) at different temperatures, and it was suggested that such variation might arise because of temperature-dependent conformational changes of the protein in response to changes in the conformational entropy and the dielectric permeability of water, which drops from a value of ε ∼ 79 at 24 °C to ∼ 60 at 82 °C. Herein, we examine how adding methanol to water influences the distributions of CI-2 conformers and their ensuing stabilities. The dielectric constant of a 60:40 water:methanol (MeOH) drops from ε ∼ 60 at 24 °C to ∼ 51 at 64 °C. Although the same set of conformers observed in water appears to be present in 60:40 water:MeOH, the abundance of each is substantially altered by the presence of methanol. Relative free energy values (ΔG) and thermodynamic values [ΔH and ΔS and heat capacities (ΔCp)] are derived from a Gibbs-Helmholtz analysis. A comparison of these data from water and water:MeOH systems allows rare insight into how variations in solvation and temperature affect many-state protein equilibria. While these studies confirm that variations in solvent dielectric constant with temperature affect the distributions of conformers that are observed, our findings suggest that other solvent differences may also affect abundances.

Cupric Ions Selectively Modulate TRAAK-Phosphatidylserine Interactions.

TRAAK and TREK2 are two-pore domain K+ (K2P) channels and are modulated by diverse factors including temperature, membrane stretching, and lipids, such as phosphatidic acid. In addition, copper and zinc, both of which are essential for life, are known to regulate TREK2 and a number of other ion channels. However, the role of ions in the association of lipids with integral membrane proteins is poorly understood. Here, we discover cupric ions selectively modulate the binding of phosphatidylserine (PS) to TRAAK but not TREK2. Other divalent cations (Ca2+, Mg2+, and Zn2+) bind both channels but have no impact on binding PS and other lipids. Additionally, TRAAK binds more avidly to Cu2+ and Zn2+ than TREK2. In the presence of Cu2+, TRAAK binds similarly to PS with different acyl chains, indicating a crucial role of the serine headgroup in coordinating Cu2+. High-resolution native mass spectrometry (MS) enables the determination of equilibrium binding constants for distinct Cu2+-bound stoichiometries and uncovered the highest coupling factor corresponds to a 1:1 PS-to-Cu2+ ratio. Interestingly, the next three highest coupling factors had a ∼1.5:1 PS-to-Cu2+ ratio. Our findings bring forth the role of cupric ions as an essential cofactor in selective TRAAK-PS interactions.

Insight into the Phospholipid-Binding Preferences of Kir3.4.

The G-protein-gated inwardly rectifying potassium channel 4 (Kir3.4) subunit forms functional tetramers. Previous studies have established that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is required for Kir3.4 function. However, the binding preferences of Kir3.4 for the headgroup and acyl chains of phosphorylated phosphatidylinositides (PIPs) and other lipids are not well understood. Here, the interactions between full-length, human Kir3.4 and lipids are characterized using native mass spectrometry (MS) in conjunction with a soluble fluorescent lipid-binding assay. Kir3.4 displays binding preferences for PIPs, and, in some cases, the degree of binding is influenced by the type of acyl chains. The interactions between Kir3.4 and PIPs are weaker in comparison to full-length, human Kir3.2. The binding of PI(4,5)P2 modified with a fluorophore to Kir3.2 can be enhanced by other lipids, such as phosphatidylcholine. Introduction of S143T, a mutation that enhances Kir3.4 activity, results in an overall reduction in the channel binding PIPs. In contrast, the D223N mutant of Kir3.4 that mimics the sodium-bound state exhibited stronger binding for PI(4,5)P2, particularly for those with 18:0-20:4 acyl chains. Taken together, these results provide additional insight into the interaction between Kir3.4 and lipids that are important for channel function.

Discovery of Potent Charge-Reducing Molecules for Native Ion Mobility Mass Spectrometry Studies.

There is growing interest in the characterization of protein complexes and their interactions with ligands using native ion mobility mass spectrometry. A particular challenge, especially for membrane proteins, is preserving noncovalent interactions and maintaining native-like structures. Different approaches have been developed to minimize activation of protein complexes by manipulating charge on protein complexes in solution and the gas-phase. Here, we report the utility of polyamines that have exceptionally high charge-reducing potencies with some molecules requiring 5-fold less than trimethylamine oxide to elicit the same effect. The charge-reducing molecules do not adduct to membrane protein complexes and are also compatible with ion-mobility mass spectrometry, paving the way for improved methods of charge reduction.

TREK2 Lipid Binding Preferences Revealed by Native Mass Spectrometry.

TREK2, a two-pore domain potassium channel, is recognized for its regulation by various stimuli, including lipids. While previous members of the TREK subfamily, TREK1 and TRAAK, have been investigated to elucidate their lipid affinity and selectivity, TREK2 has not been similarly studied in this regard. Our findings indicate that while TRAAK and TREK2 exhibit similarities in terms of electrostatics and share an overall structural resemblance, there are notable distinctions in their interaction with lipids. Specifically, SAPI(4,5)P2,1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate) exhibits a strong affinity for TREK2, surpassing that of dOPI(4,5)P2,1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate), which differs in its acyl chains. TREK2 displays lipid binding preferences not only for the headgroup of lipids but also toward the acyl chains. Functional studies draw a correlation for lipid binding affinity and activity of the channel. These findings provide important insight into elucidating the molecular prerequisites for specific lipid binding to TREK2 important for function.

Grafting the ALFA tag for structural studies of aquaporin Z.

Aquaporin Z (AqpZ), a bacterial water channel, forms a tetrameric complex and, like many other membrane proteins, activity is regulated by lipids. Various methods have been developed to facilitate structure determination of membrane proteins, such as the use of antibodies. Here, we graft onto AqpZ the ALFA tag (AqpZ-ALFA), an alpha helical epitope, to make use of the high-affinity anti-ALFA nanobody (nB). Native mass spectrometry reveals the AqpZ-ALFA fusion forms a stable, 1:1 complex with nB. Single-particle cryogenic electron microscopy studies reveal the octameric (AqpZ-ALFA)4(nB)4 complex forms a dimeric assembly and the structure was determined to 1.9 Å resolution. Dimerization of the octamer is mediated through stacking of the symmetrically bound nBs. Tube-like density is also observed, revealing a potential cardiolipin binding site. Grafting of the ALFA tag, or other epitope, along with binding and association of nBs to promote larger complexes will have applications in structural studies and protein engineering.

Combining native mass spectrometry and lipidomics to uncover specific membrane protein-lipid interactions from natural lipid sources.

While it is known that lipids play an essential role in regulating membrane protein structure and function, it remains challenging to identify specific protein-lipid interactions. Here, we present an innovative approach that combines native mass spectrometry (MS) and lipidomics to identify lipids retained by membrane proteins from natural lipid extracts. Our results reveal that the bacterial ammonia channel (AmtB) enriches specific cardiolipin (CDL) and phosphatidylethanolamine (PE) from natural headgroup extracts. When the two extracts are mixed, AmtB retains more species, wherein selectivity is tuned to bias headgroup selection. Using a series of natural headgroup extracts, we show TRAAK, a two-pore domain K+ channel (K2P), retains specific acyl chains that is independent of the headgroup. A brain polar lipid extract was then combined with the K2Ps, TRAAK and TREK2, to understand lipid specificity. More than a hundred lipids demonstrated affinity for each protein, and both channels were found to retain specific fatty acids and lysophospholipids known to stimulate channel activity, even after several column washes. Natural lipid extracts provide the unique opportunity to not only present natural lipid diversity to purified membrane proteins but also identify lipids that may be important for membrane protein structure and function.

Polyamine detergents tailored for native mass spectrometry studies of membrane proteins.

Native mass spectrometry (MS) is a powerful technique for interrogating membrane protein complexes and their interactions with other molecules. A key aspect of the technique is the ability to preserve native-like structures and noncovalent interactions, which can be challenging depending on the choice of detergent. Different strategies have been employed to reduce charge on protein complexes to minimize activation and preserve non-covalent interactions. Here, we report the synthesis of a class of polyamine detergents tailored for native MS studies of membrane proteins. These detergents, a series of spermine covalently attached to various alkyl tails, are exceptional charge-reducing molecules, exhibiting a ten-fold enhanced potency over spermine. Addition of polyamine detergents to proteins solubilized in maltoside detergents results in improved, charge-reduced native mass spectra and reduced dissociation of subunits. Polyamine detergents open new opportunities to investigate membrane proteins in different detergent environments that have thwarted previous native MS studies.

Structural basis for lipid and copper regulation of the ABC transporter MsbA.

A critical step in lipopolysaccharide (LPS) biogenesis involves flipping lipooligosaccharide, an LPS precursor, from the cytoplasmic to the periplasmic leaflet of the inner membrane, an operation carried out by the ATP-binding cassette transporter MsbA. Although LPS binding to the inner cavity of MsbA is well established, the selectivity of MsbA-lipid interactions at other site(s) remains poorly understood. Here we use native mass spectrometry (MS) to characterize MsbA-lipid interactions and guide structural studies. We show the transporter co-purifies with copper(II) and metal binding modulates protein-lipid interactions. A 2.15 Å resolution structure of an N-terminal region of MsbA in complex with copper(II) is presented, revealing a structure reminiscent of the GHK peptide, a high-affinity copper(II) chelator. Our results demonstrate conformation-dependent lipid binding affinities, particularly for the LPS-precursor, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL). We report a 3.6 Å-resolution structure of MsbA trapped in an open, outward-facing conformation with adenosine 5'-diphosphate and vanadate, revealing a distinct KDL binding site, wherein the lipid forms extensive interactions with the transporter. Additional studies provide evidence that the exterior KDL binding site is conserved and a positive allosteric modulator of ATPase activity, serving as a feedforward activation mechanism to couple transporter activity with LPS biosynthesis.

Entropy in the Molecular Recognition of Membrane Protein-Lipid Interactions.

Understanding the molecular driving forces that underlie membrane protein-lipid interactions requires the characterization of their binding thermodynamics. Here, we employ variable-temperature native mass spectrometry to determine the thermodynamics of lipid binding events to the human G-protein-gated inward rectifier potassium channel, Kir3.2. The channel displays distinct thermodynamic strategies to engage phosphatidylinositol (PI) and phosphorylated forms thereof. The addition of a 4'-phosphate to PI results in an increase in favorable entropy. PI with two or more phosphates exhibits more complex binding, where lipids appear to bind two nonidentical sites on Kir3.2. Remarkably, the interaction of 4,5-bisphosphate PI with Kir3.2 is solely driven by a large, favorable change in entropy. Installment of a 3'-phosphate to PI(4,5)P2 results in an altered thermodynamic strategy. The acyl chain of the lipid has a marked impact on binding thermodynamics and, in some cases, enthalpy becomes favorable.

Amphiphile self-assembly dynamics at the solution-solid interface reveal asymmetry in head/tail desorption.

Amphiphilic alkoxybenzonitriles (ABNs) of varying chain length are studied at the solution/graphite interface to analyze dynamics of assembly. Competitive self-assembly between ABNs and alkanoic acid solvent is shown by scanning tunneling microscopy (STM) to be controlled by concentration and molecular size. Molecular dynamics (MD) simulations reveal key roles of the sub-nanosecond fundamental steps of desorption, adsorption, and on-surface motion. We discovered asymmetry in desorption-adsorption steps. Desorption starting from alkyl chain detachment from the surface is favored due to dynamic occlusion by neighbouring chains. Even though the nitrile head has a strong solvent affinity, it more frequently re-adsorbs following a detachment event.