Nucleotide-binding State Research Articles

Native kinesin-1 does not bind preferentially to GTP-tubulin-rich microtubules in vitro.

Molecular motors such as kinesin-1 work in small teams to actively shuttle cargos in cells, for example in polarized transport in axons. Here, we examined the potential regulatory role of the nucleotide state of tubulin on the run length of cargos carried by multiple kinesin motors, using an optical trapping-based in vitro assay. Based on a previous report that kinesin binds preferentially to GTP-tubulin-rich microtubules, we anticipated that multiple-kinesin cargos would run substantially greater distances along GMPCPP microtubules than along GDP microtubules. Surprisingly, we did not uncover any significant differences in run length between microtubule types. A combination of single-molecule experiments, comparison with previous theory, and classic microtubule affinity pulldown assays revealed that native kinesin-1 does not bind preferentially to GTP-tubulin-rich microtubules. The apparent discrepancy between our observations and the previous report likely reflects differences in post-translational modifications between the native motors used here and the recombinant motors examined previously. Future investigations will help shed light on the interplay between the motor's post-translational modification and the microtubule's nucleotide-binding state for transport regulation in vivo.

Structural intermediates and directionality of the swiveling motion of Pyruvate Phosphate Dikinase

Pyruvate phosphate dikinase (PPDK) is a vital enzyme in cellular energy metabolism catalyzing the ATP- and P i -dependent formation of phosphoenolpyruvate from pyruvate in C4 -plants, but the reverse reaction forming ATP in bacteria and protozoa. The multi-domain enzyme is considered an efficient molecular machine that performs one of the largest single domain movements in proteins. However, a comprehensive understanding of the proposed swiveling domain motion has been limited by not knowing structural intermediates or molecular dynamics of the catalytic process. Here, we present crystal structures of PPDKs from Flaveria, a model genus for studying the evolution of C4 -enzymes from phylogenetic ancestors. These structures resolve yet unknown conformational intermediates and provide the first detailed view on the large conformational transitions of the protein in the catalytic cycle. Independently performed unrestrained MD simulations and configurational free energy calculations also identified these intermediates. In all, our experimental and computational data reveal strict coupling of the CD swiveling motion to the conformational state of the NBD. Moreover, structural asymmetries and nucleotide binding states in the PPDK dimer support an alternate binding change mechanism for this intriguing bioenergetic enzyme.

The HOPS/Class C Vps Complex Tethers Membranes by Binding to a Rab Gtpase in one Membrane and Directly to a Second Membrane via a Curvature-Sensing Motif

Many Rab GTPase effectors are membrane tethering factors, that is, they physically link two apposed membranes prior to intracellular membrane fusion. Here, we investigate the distinct binding factors needed on apposed membranes for Rab effector-dependent tethering. We show that the homotypic fusion and protein sorting/Class C vacuole protein sorting (HOPS/Class C Vps) complex can tether low-curvature membranes, i.e. liposomes with a diameter of ∼100 nm, only when the yeast vacuolar Rab GTPase Ypt7p is present in both tethered membranes. When HOPS is phosphorylated by the vacuolar casein kinase I, Yck3p, tethering only takes place when GTP-bound Ypt7p is present in both tethered membranes. When HOPS is not phosphorylated, however, its tethering activity shows little specificity for the nucleotide-binding state of Ypt7p. These results suggest a model for HOPS-mediated tethering in which HOPS tethers membranes by binding to Ypt7p in each of the two tethered membranes. Moreover, because vacuole-associated HOPS is presumably phosphorylated by Yck3p, our results suggest that nucleotide exchange of Ypt7p on multivesicular bodies (MVB's)/late endosomes must take place before HOPS can mediate tethering at vacuoles.

The HOPS/class C Vps complex tethers membranes by binding to one Rab GTPase in each apposed membrane.

Many Rab GTPase effectors are membrane-tethering factors, that is, they physically link two apposed membranes before intracellular membrane fusion. In this study, we investigate the distinct binding factors needed on apposed membranes for Rab effector-dependent tethering. We show that the homotypic fusion and protein-sorting/class C vacuole protein-sorting (HOPS/class C Vps) complex can tether low-curvature membranes, that is, liposomes with a diameter of ∼100 nm, only when the yeast vacuolar Rab GTPase Ypt7p is present in both tethered membranes. When HOPS is phosphorylated by the vacuolar casein kinase I, Yck3p, tethering only takes place when GTP-bound Ypt7p is present in both tethered membranes. When HOPS is not phosphorylated, however, its tethering activity shows little specificity for the nucleotide-binding state of Ypt7p. These results suggest a model for HOPS-mediated tethering in which HOPS tethers membranes by binding to Ypt7p in each of the two tethered membranes. Moreover, because vacuole-associated HOPS is presumably phosphorylated by Yck3p, our results suggest that nucleotide exchange of Ypt7p on multivesicular bodies (MVBs)/late endosomes must take place before HOPS can mediate tethering at vacuoles.

Variations in the binding pocket of an inhibitor of the bacterial division protein FtsZ across genotypes and species.

The recent increase in antibiotic resistance in pathogenic bacteria calls for new approaches to drug-target selection and drug development. Targeting the mechanisms of action of proteins involved in bacterial cell division bypasses problems associated with increasingly ineffective variants of older antibiotics; to this end, the essential bacterial cytoskeletal protein FtsZ is a promising target. Recent work on its allosteric inhibitor, PC190723, revealed in vitro activity on Staphylococcus aureus FtsZ and in vivo antimicrobial activities. However, the mechanism of drug action and its effect on FtsZ in other bacterial species are unclear. Here, we examine the structural environment of the PC190723 binding pocket using PocketFEATURE, a statistical method that scores the similarity between pairs of small-molecule binding sites based on 3D structure information about the local microenvironment, and molecular dynamics (MD) simulations. We observed that species and nucleotide-binding state have significant impacts on the structural properties of the binding site, with substantially disparate microenvironments for bacterial species not from the Staphylococcus genus. Based on PocketFEATURE analysis of MD simulations of S. aureus FtsZ bound to GTP or with mutations that are known to confer PC190723 resistance, we predict that PC190723 strongly prefers to bind Staphylococcus FtsZ in the nucleotide-bound state. Furthermore, MD simulations of an FtsZ dimer indicated that polymerization may enhance PC190723 binding. Taken together, our results demonstrate that a drug-binding pocket can vary significantly across species, genetic perturbations, and in different polymerization states, yielding important information for the further development of FtsZ inhibitors.

Structural Basis for Dodecameric Assembly States and Conformational Plasticity of the Full-Length AAA+ ATPases Rvb1·Rvb2

As building blocks of diverse macromolecular complexes, the AAA+ ATPases Rvb1 and Rvb2 are crucial for many cellular activities including cancer-related processes. Their oligomeric structure and function remain unclear. We report the crystal structures of full-length heteromeric Rvb1·Rvb2 complexes in distinct nucleotide binding states. Chaetomium thermophilum Rvb1·Rvb2 assemble into hexameric rings of alternating molecules and into stable dodecamers. Intriguingly, the characteristic oligonucleotide-binding (OB) fold domains (DIIs) of Rvb1 and Rvb2 occupy unequal places relative to the compact AAA+ core ring. While Rvb1's DII forms contacts between hexamers, Rvb2's DII is rotated 100° outward, occupying lateral positions. ATP was retained bound to Rvb1 but not Rvb2 throughout purification, suggesting nonconcerted ATPase activities and nucleotide binding. Significant conformational differences between nucleotide-free and ATP-/ADP-bound states in the crystal structures and in solution suggest that the functional role of Rvb1·Rvb2 is mediated by highly interconnected structural switches. Our structures provide an atomic framework for dodecameric states and Rvb1·Rvb2's conformational plasticity.

Nucleotides Linkage Analysis of RecBCD DNA Helicase

RecBCD is a bacterial enzyme complex that possesses both DNA helicase and nuclease activities necessary for its biological function. RecBCD is both a continuous and a processive DNA helicase, with an unwinding rate of ∼ 1,600 bp/s. Research on RecBCD function and mechanism in last decades had provided wealth of information on its biochemistry, structure and unwinding mechanism. Still, very little is known about the biochemical transitions, and the nucleotide linkage between RecBCD and DNA during its ATPase cycle. In this work we present thermodynamic linkage analysis using multiple approaches of different nucleotides states of RecBCD free and bound to ssDNA or dsDNA. Primarily, we have purified RecBCD, free of nucleic acids contaminates, which limits such measurements. We show that a nucleotide state mimicking ATP hydrolysis binds with the tightest affinity in comparison to the ADP or nucleotide free binding states in the presence of either ssDNA or dsDNA. Furthermore, by utilizing transient kinetics approach, we have measured dissociation rate constants of ssDNA and dsDNA from RecBCD with different nucleotides states. We demonstrate that ADP state increases the dissociation rate constant of ssDNA in comparison to all these states: dsDNA-bound, nucleotide free or with the binding of ATP states. Collectively, our results identified a strong nucleotide binding state of the RecBCD complex and a second nucleotide state, which is most likely to dissociate from the DNA track during the course of unwinding and translocation reaction. These results will allow us to construct a model of the different biochemical intermediates during a single ATPase cycle and reveal the allostery that may exist between RecBCD subunits during its unwinding reaction.

Nucleotide bound to rab11a controls localization in rod cells but not interaction with rhodopsin.

Precise vectorial transport of rhodopsin is essential for rod photoreceptor health and function. Mutations that truncate or extend the C terminus of rhodopsin disrupt this transport, and lead to retinal degeneration and blindness in human patients and in mouse models. Here we show that such mutations disrupt the binding of rhodopsin to the small GTPase rab11a. The rhodopsin-rab11a interaction is a direct binding interaction that does not depend on the nucleotide binding state of rab11a. Expression of EGFP-rab11a fusion proteins in Xenopus laevis photoreceptors revealed that the nucleotide binding status of rab11a affects its subcellular localization, with GTP-locked mutants concentrated in the inner segment and GDP-locked mutants concentrated in the outer segment. shRNA-mediated knockdown of rab11a in rods led to shortened outer segments and retinal degeneration. Together, our results show the critical importance of direct rhodopsin-rab11a interactions for the formation and maintenance of vertebrate photoreceptors.

Reciprocal conversion of Gtr1 and Gtr2 nucleotide-binding states by Npr2-Npr3 inactivates TORC1 and induces autophagy

Autophagy is an intracellular degradation process that delivers cytosolic material to lysosomes and vacuoles. To investigate the mechanisms that regulate autophagy, we performed a genome-wide screen using a yeast deletion-mutant collection, and found that Npr2 and Npr3 mutants were defective in autophagy. Their mammalian homologs, NPRL2 and NPRL3, were also involved in regulation of autophagy. Npr2-Npr3 function upstream of Gtr1-Gtr2, homologs of the mammalian RRAG GTPase complex, which is crucial for TORC1 regulation. Both npr2∆ mutants and a GTP-bound Gtr1 mutant suppressed autophagy and increased Tor1 vacuole localization. Furthermore, Gtr2 binds to the TORC1 subunit Kog1. A GDP-bound Gtr1 mutant induced autophagy even under nutrient-rich conditions, and this effect was dependent on the direct binding of Gtr2 to Kog1. These results revealed that 2 molecular mechanisms, Npr2-Npr3-dependent GTP hydrolysis of Gtr1 and direct binding of Gtr2 to Kog1, are involved in TORC1 inactivation and autophagic induction.

Npr2 regulates autophagy via Gtr1‐Gtr2‐dependent target of rapamycin complex 1 signaling (539.11)

Autophagy is an intracellular degradation process that delivers cytosolic material to lysosomes and vacuoles. To understand the mechanisms that regulate autophagy, we performed a genome‐wide screen using a yeast deletion mutant collection, and identified Npr2 and Npr3. Mutation of Npr2 and Npr3 disrupted autophagy upstream of Target of Rapamycin Complex 1 (TORC1). Additionally, we found that the Npr2‐Npr3 complexes function upstream of Gtr1‐Gtr2, a GTPase complex that is homologous to Rag. A constitutively active GTP‐bound Gtr1 mutant suppressed autophagy by activating TORC1, whereas a constitutively GDP‐bound Gtr1 mutant induced autophagy. The autophagic defects observed in ∆npr2 cells and cells expressing the GTP‐bound Gtr1 mutant were quite comparable, but different from Gtr1‐GDP expressing cell. We hypothesized that the autophagic defect in the ∆npr2 cells resulted from a defect in the switching of the nucleotide‐binding state of Gtr1. Thus, Npr2‐Npr3 appears to regulate GTP hydrolysis in Gtr1, which affects TORC1 activity and autophagy.

Intersubunit communications within KaiC hexamers contribute the robust rhythmicity of the cyanobacterial circadian clock.

Circadian rhythms, endogenous oscillations with periods of ~24 h, are found in many organisms, and they enhance fitness in alternating day/night environments. In cyanobacteria, three clock proteins, KaiA, KaiB, and KaiC, control the timekeeping mechanism. KaiC, the central component of the system, is a hexameric ATPase that also has autokinase and autophosphatase activities. It has been assumed that KaiC’s hexameric structure was critical for regulation of the circadian clock; however, the underlying molecular mechanism of such regulation has remained unclear. Recently, we elucidated the regulation of KaiC’s activities by its phosphorylation state, in the context of its hexameric structure. We found that local interactions at subunit interfaces regulate KaiC’s activities by coupling the nucleotide-binding states. We also discovered the mechanism of regulation by intersubunit communication in KaiC hexamers. Our observations suggest that intersubunit communication precisely synchronizes KaiC subunits to avoid dephasing, and contributes to the robustness of circadian rhythms in cyanobacteria [Kitayama, Y. et al. Nat. Commun. 4:2897 doi: 10.1038/ncomms3897 (2013)].

Variation in the Binding Pocket of an Inhibitor of the Bacterial Division Protein FtsZ Across Genotypes, Nucleotide States, and Species

The recent increase in antibiotic resistance in disease-causing bacteria calls for new approaches to drug target selection and drug development. Targeting proteins involved in the bacterial cell division process provides a novel mechanism of action that bypasses problems associated with the increasingly ineffective variants of older antibiotics. One potentially powerful target for inhibition is the bacterial cytoskeletal protein FtsZ, a GTPase homolog of eukaryotic tubulin. This highly conserved and essential protein acts as a scaffold to recruit other cell division machinery and can exert mechanical forces that may be responsible for constriction of the division site. Recent work on an allosteric inhibitor (PC190723) has shown promising in vitro and in vivo antimicrobial activities on Staphylococcus aureus, however the mechanism of drug action and its effect on FtsZ of other bacterial species is unclear. Here, we examine the structural environment of the FtsZ-PC190723 binding pocket using Molecular Dynamics simulations and PocketFEATURE, a statistical method that performs pairwise comparisons of small molecule binding sites based on 3D structure information. By comparing all currently existing FtsZ crystal structures to the crystalized FtsZ-PC190723 complex, we observe that species and nucleotide binding state have significant impact on the structural properties of the binding site, with a GDP-bound Staph-FtsZ displaying the highest similarity to the inhibitor complex. However, during extended Molecular Dynamics simulations, this similarity is negated upon introduction of point mutations known to nullify the inhibitor's antimicrobial affect. Together, these results give insight into the mechanism and specificity of PC190723 interaction with various bacterial FtsZ.

KaiC intersubunit communication facilitates robustness of circadian rhythms in cyanobacteria

The cyanobacterial circadian clock is the only model clock to have been reconstituted in vitro. KaiC, the central clock component, is a homohexameric ATPase with autokinase and autophosphatase activities. Changes in phosphorylation state have been proposed to switch KaiC’s activity between autokinase and autophosphatase. Here we analyse the molecular mechanism underlying the regulation of KaiC’s activity, in the context of its hexameric structure. We reconstitute KaiC hexamers containing different variant protomers, and measure their autophosphatase and autokinase activities. We identify two types of regulatory mechanisms with distinct functions. First, local interactions between adjacent phosphorylation sites regulate KaiC’s activities, coupling the ATPase and nucleotide-binding states at subunit interfaces of the CII domain. Second, the phosphorylation states of the protomers affect the overall activity of KaiC hexamers via intersubunit communication. Our findings indicate that intra-hexameric interactions play an important role in sustaining robust circadian rhythmicity.

Altered Intersubunit Communication Is the Molecular Basis for Functional Defects of Pathogenic p97 Mutants

The human AAA ATPase p97 is a molecular chaperone essential in cellular proteostasis. Single amino acid substitutions in p97 have been linked to a clinical multiple-disorder condition known as inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia. How the mutations affect the molecular mechanism that governs the function of p97 remains unclear. Here, we show that within the hexameric ring of a mutant p97, D1 domains fail to regulate their respective nucleotide-binding states, as evidenced by the lower amount of prebound ADP, weaker ADP binding affinity, full occupancy of adenosine-5'-O-(3-thiotriphosphate) binding, and elevated overall ATPase activity, indicating a loss of communication among subunits. Defective communication between subunits is further illustrated by altered conformation in the side chain of residue Phe-360 that probes into the nucleotide-binding pocket from a neighboring subunit. Consequently, conformations of N domains in a hexameric ring of a mutant p97 become uncoordinated, thus impacting its ability to process substrate.

Structural Dynamics of the MecA-ClpC Complex: A TYPE II AAA+ PROTEIN UNFOLDING MACHINE

The MecA-ClpC complex is a bacterial type II AAA(+) molecular machine responsible for regulated unfolding of substrates, such as transcription factors ComK and ComS, and targeting them to ClpP for degradation. The six subunits of the MecA-ClpC complex form a closed barrel-like structure, featured with three stacked rings and a hollow passage, where substrates are threaded and translocated through successive pores. Although the general concepts of how polypeptides are unfolded and translocated by internal pore loops of AAA(+) proteins have long been conceived, the detailed mechanistic model remains elusive. With cryoelectron microscopy, we captured four different structures of the MecA-ClpC complexes. These complexes differ in the nucleotide binding states of the two AAA(+) rings and therefore might presumably reflect distinctive, representative snapshots from a dynamic unfolding cycle of this hexameric complex. Structural analysis reveals that nucleotide binding and hydrolysis modulate the hexameric complex in a number of ways, including the opening of the N-terminal ring, the axial and radial positions of pore loops, the compactness of the C-terminal ring, as well as the relative rotation between the two nucleotide-binding domain rings. More importantly, our structural and biochemical data indicate there is an active allosteric communication between the two AAA(+) rings and suggest that concerted actions of the two AAA(+) rings are required for the efficiency of the substrate unfolding and translocation. These findings provide important mechanistic insights into the dynamic cycle of the MecA-ClpC unfoldase and especially lay a foundation toward the complete understanding of the structural dynamics of the general type II AAA(+) hexamers.

DNTP-dependent Conformational Transitions in the Fingers Subdomain of Klentaq1 DNA Polymerase: INSIGHTS INTO THE ROLE OF THE “NUCLEOTIDE-BINDING” STATE

Conformational selection plays a key role in the polymerase cycle. Klentaq1 exists in conformational equilibrium between three states (open, closed, and “nucleotide-binding”) whose level of occupancy is determined by the bound substrate. The “nucleotide-binding” state plays a pivotal role in the reaction pathway. Direct evidence is provided for the role of a conformationally distinct “nucleotide-binding” state during dNTP incorporation. DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single-molecule, and x-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high precision FRET measurements, we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP, it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed the "nucleotide-binding" state predominates. Photon distribution and hidden Markov modeling revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the nucleotide-binding state. After nucleotide binding, correct dNTPs are transported to the closed state, whereas incorrect dNTPs are delivered to the open state.

Conformational dynamics of full-length inducible human Hsp70 derived from microsecond molecular dynamics simulations in explicit solvent

Human 70 kDa heat shock protein (hHsp70) is an ATP-dependent chaperone and is currently an important target for developing new drugs in cancer therapy. Knowledge of the conformations of hHsp70 is central to understand the interactions between its nucleotide-binding domain (NBD) and substrate-binding domain (SBD) and is a prerequisite to design inhibitors. The conformations of ADP-bound (or nucleotide-free) hHsp70 and ATP-bound hHsp70 was investigated by using unbiased all-atom molecular dynamics (MD) simulations of homology models of hHsp70 in explicit solvent on a timescale of .5 and 2.7 μs, respectively. The conformational heterogeneity of hHsp70 was analyzed by computing effective free-energy landscapes (FELs) and distance distribution between selected pair of residues. These theoretical data were compared with those extracted from single-molecule Förster resonance energy transfer (FRET) experiments and to small-angle X-ray scattering (SAXS) data of Hsp70 homologs. The distance between a pair of residues in FRET is systematically larger than the distance computed in MD which is interpreted as an effect of the size and of the dynamics of the fluorescent probes. The origin of the conformational heterogeneity of hHsp70 in the ATP-bound state is due to different binding modes of the helix B of the SBD onto the NBD. In the ADP-bound (or nucleotide-free) state, it arises from the different closed conformations of the SBD and from the different positions of the SBD relative to the NBD. In each nucleotide-binding state, Hsp70 is better represented by an ensemble of conformations on a μs timescale corresponding to different local minima of the FEL. An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:30

Nucleotide-dependent conformations of FtsZ dimers and force generation observed through molecular dynamics simulations

The bacterial cytoskeletal protein FtsZ is a GTPase that is thought to provide mechanical constriction force via an unidentified mechanism. Purified FtsZ polymerizes into filaments with varying structures in vitro: while GTP-bound FtsZ assembles into straight or gently curved filaments, GDP-bound FtsZ forms highly curved filaments, prompting the hypothesis that a difference in the inherent curvature of FtsZ filaments provides mechanical force. However, no nucleotide-dependent structural transition of FtsZ monomers has been observed to support this force generation model. Here, we present a series of all-atom molecular dynamics simulations probing the effects of nucleotide binding on the structure of an FtsZ dimer. We found that the FtsZ-dimer structure is dependent on nucleotide-binding state. While a GTP-bound FtsZ dimer retained a firm monomer-monomer contact, a GDP-bound FtsZ dimer lost some of the monomer-monomer association, leading to a "hinge-opening" event that resulted in a more bent dimer, while leaving each monomer structure largely unaffected. We constructed models of FtsZ filaments and found that a GDP-FtsZ filament is much more curved than a GTP-FtsZ filament, with the degree of curvature matching prior experimental data. FtsZ dynamics were used to estimate the amount of force an FtsZ filament could exert when hydrolysis occurs (20-30 pN per monomer). This magnitude of force is sufficient to direct inward cell-wall growth during division, and to produce the observed degree of membrane pinching in liposomes. Taken together, our data provide molecular-scale insight on the origin of FtsZ-based constriction force, and the mechanism underlying prokaryotic cell division.

Using Pressure Perturbation for Studying Conformational and Functional Substates of Proteins

Regulation of protein function is often linked to a conformational switch triggered by chemical or physical signals. To evaluate such conformational changes and to elucidate the underlying molecular mechanisms of subsequent protein function, experimental identification of conformational substates and characterization of conformational equilibria are mandatory. We applied pressure modulation in combination with spectroscopic and scattering techniques to reveal equilibria between substates of various proteins. Pressure has the advantage that its thermodynamic conjugate is volume, a parameter that is directly related to structure. The conformational dynamics of a signaling protein, Ras, in the absence and presence of a model biomembrane was probed by pressure perturbation. We show that not only the nucleotide binding state, but also the presence of the lipid bilayer membrane has a drastic effect on the conformational dynamics and selection of conformational substates of the protein. These results provide also insights into the influence of pressure on Ras-controlled signaling events in organisms living under extreme environmental conditions as they are met in the deep sea where pressures reach the kbar range. Furthermore, the pressure perturbation approach has been used to disentangle aggregation pathways of amyloidogenic proteins, such as insulin, IAPP, and the prion protein. This approach reveals new insights into the pre-aggregated regime as well as mechanistic details about concurrent aggregation pathways and the differential stability of the protein aggregates formed.

Revealing Conformational Substates of Lipidated N-Ras Protein by Pressure Modulation

Signaling networks maintain their spatiality by localization of their protein constituents to distinct regions of the membrane, and the different Ras guanine nucleotide binding proteins are a paradigm example of this. Despite being highly homologous, they exhibit isoform specific diversity in generating explicit signal outputs governed by, but not limited to, their hyper variable region responsible for targeting them to particular membrane microdomains (1). In addition, Ras proteins are known to sample multiple conformations which exhibit varying affinities towards their interaction partners. To fully explore the conformational space exhibited by Ras, experimental identification of conformational substates and characterization of conformational equilibria are mandatory. We applied pressure modulation in combination with FT-IR spectroscopy to reveal equilibria between spectroscopically resolved, otherwise low lying, substates of the lipidated signaling protein N-Ras in its different nucleotide binding states and in the absence and presence of a model biomembrane. Not only the nucleotide binding, but also the presence of the membrane has a drastic effect on the conformational dynamics and selection of conformational substates of the protein, and a new substate appearing upon membrane binding could be uncovered. Population of this new substate is accompanied by structural reorientations of the G-domain involving α-helix-membrane interactions. These findings thus illustrate that the membrane controls signaling conformations by acting as an effective interaction partner which has consequences for the G-domain orientation of membrane-associated N-Ras which in turn is known to be critical for its effector and modulator interactions. Finally, these results provide first insights into the influence of pressure on Ras-controlled signaling events in organisms living under extreme environmental conditions as they are encountered in the deep sea.Reference1. Weise K, Kapoor S, ... Waldmann H, Winter R (2011) J Am Chem Soc 133:880-887.Table of Contents