Finding a Needle in a Haystack: The Role of Electrostatics in Target Lipid Recognition by PH Domains

Interactions between protein domains and lipid molecules play key roles in controlling cell membrane signalling and trafficking. The pleckstrin homology (PH) domain is one of the most widespread, binding specifically to phosphatidylinositol phosphates (PIPs) in cell membranes. PH domains must locate specific PIPs in the presence of a background of approximately 20% anionic lipids within the cytoplasmic leaflet of the plasma membrane. We investigate the mechanism of such recognition via a multiscale procedure combining Brownian dynamics (BD) and molecular dynamics (MD) simulations of the GRP1 PH domain interacting with phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)). The interaction of GRP1-PH with PI(3,4,5)P(3) in a zwitterionic bilayer is compared with the interaction in bilayers containing different levels of anionic 'decoy' lipids. BD simulations reveal both translational and orientational electrostatic steering of the PH domain towards the PI(3,4,5)P(3)-containing anionic bilayer surface. There is a payoff between non-PIP anionic lipids attracting the PH domain to the bilayer surface in a favourable orientation and their role as 'decoys', disrupting the interaction of GRP1-PH with the PI(3,4,5)P(3) molecule. Significantly, approximately 20% anionic lipid in the cytoplasmic leaflet of the bilayer is nearly optimal to both enhance orientational steering and to localise GRP1-PH proximal to the surface of the membrane without sacrificing its ability to locate PI(3,4,5)P(3) within the bilayer plane. Subsequent MD simulations reveal binding to PI(3,4,5)P(3), forming protein-phosphate contacts comparable to those in X-ray structures. These studies demonstrate a computational framework which addresses lipid recognition within a cell membrane environment, offering a link between structural and cell biological characterisation.     

Molecular mechanism of membrane targeting by the GRP1 PH domain

The general receptor for phosphoinositides isoform 1 (GRP1) is recruited to the plasma membrane in response to activation of phosphoinositide 3-kinases and accumulation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. GRP1's pleckstrin homology (PH) domain recognizes PtdIns(3,4,5)P(3) with high specificity and affinity, however, the precise mechanism of its association with membranes remains unclear. Here, we detail the molecular basis of membrane anchoring by the GRP1 PH domain. Our data reveal a multivalent membrane docking involving PtdIns(3,4,5)P(3) binding, regulated by pH and facilitated by electrostatic interactions with other anionic lipids. The specific recognition of PtdIns(3,4,5)P(3) triggers insertion of the GRP1 PH domain into membranes. An acidic environment enhances PtdIns(3,4,5)P(3) binding and increases membrane penetration as demonstrated by NMR and monolayer surface tension and surface plasmon resonance experiments. The GRP1 PH domain displays a 28 nM affinity for POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/PtdIns(3,4,5)P(3) vesicles at pH 6.0, but binds 22-fold weaker at pH 8.0. The pH sensitivity is attributed in part to the His355 residue, protonation of which is required for the robust interaction with PtdIns(3,4,5)P(3) and significant membrane penetration, as illustrated by mutagenesis data. The binding affinity of the GRP1 PH domain for PtdIns(3,4,5)P(3)-containing vesicles is further amplified (by approximately 6-fold) by nonspecific electrostatic interactions with phosphatidylserine/phosphatidylinositol. Together, our results provide new insight into the multivalent mechanism of the membrane targeting and regulation of the GRP1 PH domain.     

Steered molecular dynamics simulations reveal important mechanisms in reversible monoamine oxidase B inhibition

The monotopic membrane protein monoamine oxidase B (MAO B) is an important drug target for Parkinson's disease. In order to design more specific, and thereby more effective, inhibitors for this enzyme, it is necessary to determine what factors govern inhibitor specificity and the inhibitor binding process, including the roles of the lipid bilayer, the active site loop, and several key residues within the binding pocket. Atomistic molecular dynamics simulations of MAO B either embedded in a lipid bilayer or free in solution have been performed. The simulations suggest that the bilayer controls the availability of the active site cavity by regulating the degree of fluctuation in two key loops that form the greater part of the active site entrance (residues 85-110 and 155-165). In turn, the enzyme itself causes local thinning and a decrease in area per lipid of the surrounding bilayer environment. Additional MD simulations of MAO B in complex with seven different reversible inhibitors followed by nonequilibrium steered MD simulations of the inhibitor unbinding have also been performed. The simulations demonstrate that the average energy of interaction between inhibitor and MAO B residues during inhibitor egress is an effective indicator of inhibitor strength and is also useful for identifying key residues that govern inhibitor specificity. These data provide researchers with valuable tools for designing effective MAO B inhibitors as well as outline a method that can be translated to the study of other enzyme-inhibitor complexes.     

The GRP1 PH domain, like the AKT1 PH domain, possesses a sentry glutamate residue essential for specific targeting to plasma membrane PI(3,4,5)P(3)

During the appearance of the signaling lipid PI(3,4,5)P(3), an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P(3)-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P(2) and bind the rare PI(3,4,5)P(3) target lipid with sufficiently high affinity. Our previous study of the E17K mutant of the protein kinase B (AKT1) PH domain, together with evidence from Carpten et al. [Carpten, J. D., et al. (2007) Nature 448, 439-444], revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P(2), thereby playing an essential role in specific PI(3,4,5)P(3) targeting [Landgraf, K. E., et al. (2008) Biochemistry 47, 12260-12269]. The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P(3)-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P(2) affinity and constitutive plasma membrane targeting. To test this hypothesis, we investigated the E345 residue, a putative sentry glutamate, of the general receptor for phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into the GRP1 PH domain enhances PI(4,5)P(2) affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in the AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P(2) releases the E345K GRP1 PH domain into the cytoplasm, and the efficiency of this release increases when Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K [Carpten, J. D., et al. (2007) Nature 448, 439-444; Lindhurst, M. J., et al. (2011) N. Engl. J. Med. 365, 611-619]. Analysis of available PH domain structures suggests that a lone glutamate residue (or, in some cases, an aspartate) is a common, perhaps ubiquitous, feature of PI(3,4,5)P(3)-specific binding pockets that functions to lower PI(4,5)P(2) affinity.     

GRP1 pleckstrin homology domain: activation parameters and novel search mechanism for rare target lipid

Pleckstrin homology (PH) domains play a central role in a wide array of signaling pathways by binding second messenger lipids of the phosphatidylinositol phosphate (PIP) lipid family. A given type of PIP lipid is formed in a specific cellular membrane where it is generally a minor component of the bulk lipid mixture. For example, the signaling lipid PI(3,4,5)P(3) (or PIP(3)) is generated primarily in the inner leaflet of the plasma membrane where it is believed to never exceed 0.02% of the bulk lipid. The present study focuses on the PH domain of the general receptor for phosphoinositides, isoform 1 (GRP1), which regulates the actin cytoskeleton in response to PIP(3) signals at the plasma membrane surface. The study systematically analyzes both the equilibrium and kinetic features of GRP1-PH domain binding to its PIP lipid target on a bilayer surface. Equilibrium binding measurements utilizing protein-to-membrane fluorescence resonance energy transfer (FRET) to detect GRP1-PH domain docking to membrane-bound PIP lipids confirm specific binding to PIP(3). A novel FRET competitive binding measurement developed to quantitate docking affinity yields a K(D) of 50 +/- 10 nM for GRP1-PH domain binding to membrane-bound PIP(3) in a physiological lipid mixture approximating the composition of the plasma membrane inner leaflet. This observed K(D) lies in a suitable range for regulation by physiological PIP(3) signals. Interestingly, the affinity of the interaction decreases at least 12-fold when the background anionic lipids phosphatidylserine (PS) and phosphatidylinositol (PI) are removed from the lipid mixture. Stopped-flow kinetic studies using protein-to-membrane FRET to monitor association and dissociation time courses reveal that this affinity decrease arises from a corresponding decrease in the on-rate for GRP1-PH domain docking with little or no change in the off-rate for domain dissociation from membrane-bound PIP(3). Overall, these findings indicate that the PH domain interacts not only with its target lipid, but also with other features of the membrane surface. The results are consistent with a previously undescribed type of two-step search mechanism for lipid binding domains in which weak, nonspecific electrostatic interactions between the PH domain and background anionic lipids facilitate searching of the membrane surface for PIP(3) headgroups, thereby speeding the high-affinity, specific docking of the domain to its rare target lipid.     

Membrane docking geometry of GRP1 PH domain bound to a target lipid bilayer: an EPR site-directed spin-labeling and relaxation study

The second messenger lipid PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) is generated by the lipid kinase PI3K (phosphoinositide-3-kinase) in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP(3)-specific pleckstrin homology (PH) domains to the membrane surface. Despite the broad importance of PIP(3)-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP(3) lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP(3). The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i) PIP(3) target lipid that provides specificity and affinity, and (ii) PS facilitator lipid that enhances the PIP(3) on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP(3) headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP(3) headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP(3) headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral diffusion observed for PIP(3)-bound GRP1 PH domain on supported lipid bilayers.     

One membrane protein,two structures and six environments: a comparative molecular dynamics simulation study of the bacterial outer membrane protein PagP

PagP is a bacterial outer membrane protein consisting of an 8 stranded transmembrane β-barrel and an N-terminal α-helix. It is an enzyme which catalyses transfer of a palmitoyl chain from a phospholipid to lipid A. Molecular dynamics simulations have been used to compare the dynamic behaviour in simulations starting from two different structures (X-ray vs. NMR) and in six different environments (detergent micelles formed by dodecyl phosphocholine and by octyl glucoside, vs. four species of phospholipid bilayer). Analysis of interactions between the protein and its environment reveals the role played by the N-terminal α-helix, which interacts with the lipid headgroups to lock the PagP molecule into the bilayer. The PagP β-barrel adopts a tilted orientation in lipid bilayers, facilitating access of lipid tails into the mouth of the central binding pocket. In simulations starting from the X-ray structure in lipid bilayer, the L1 and L2 loops move towards one another, leading to the formation of a putative active site by residues H33, D76 and S77 coming closer together.     

Identification of a functional phosphatidylinositol 3,4,5-trisphosphate binding site in the epithelial Na+ channel

Membrane phospholipids, such as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)), are signaling molecules that can directly modulate the activity of ion channels, including the epithelial Na(+) channel (ENaC). Whereas PI(3,4,5)P(3) directly activates ENaC, its binding site within the channel has not been identified. We identify here a region of gamma-mENaC just following the second trans-membrane domain (residues 569-583) important to PI(3,4,5)P(3) binding and regulation. Deletion of this track decreases activity of ENaC heterologously expressed in Chinese hamster ovary cells. K-Ras and its first effector phosphoinositide 3-OH kinase (PI3-K), as well as RhoA and its effector phosphatidylinositol 4-phosphate 5-kinase increase ENaC activity. Whereas the former, via generation of PI(3,4,5)P(3), increases ENaC open probability, the latter increases activity by increasing membrane levels of the channel. Deletion of the region just distal to the second trans-membrane domain disrupted regulation by K-Ras and PI3-K but not RhoA and phosphatidylinositol 4-phosphate 5-kinase. Moreover, PI(3,4,5)P(3) binds ENaC with deletion of the region following the second transmembrane domain disrupting this interaction and disrupting direct activation of the channel by PI(3,4,5)P(3). Mutation analysis revealed the importance of conserved positive and negative charged residues as well as bulky amino acids within this region to modulation of ENaC by PI3-K. The current results identify the region just distal to the second trans-membrane domain within gamma-mENaC as being part of a functional PI(3,4,5)P(3) binding site that directly impacts ENaC activity. Phospholipid binding to this site is probably mediated by the positively charged amino acids within this track, with negatively charged and bulky residues also influencing specificity of interactions.     

NMR-Based Simulation Studies of Pf1 Coat Protein in Explicit Membranes

As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.     

NMR-Based Simulation Studies of Pf1 Coat Protein in Explicit Membranes

As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.     

Single-Molecule Fluorescence Studies of a PH Domain: New Insights into the Membrane Docking Reaction

Proteins containing membrane targeting domains play essential roles in many cellular signaling pathways. However, important features of the membrane-bound state are invisible to bulk methods, thereby hindering mechanistic analysis of membrane targeting reactions. Here we use total internal reflection fluorescence microscopy (TIRFM), combined with single particle tracking, to probe the membrane docking mechanism of a representative pleckstrin homology (PH) domain isolated from the general receptor for phosphoinositides, isoform 1 (GRP1). The findings show three previously undescribed features of GRP1 PH domain docking to membranes containing its rare target lipid, phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P₃]. First, analysis of surface diffusion kinetics on supported lipid bilayers shows that in the absence of other anionic lipids, the PI(3,4,5)P₃-bound protein exhibits the same diffusion constant as a single lipid molecule. Second, the binding of the anionic lipid phosphatidylserine to a previously unidentified secondary binding site slows both diffusion and dissociation kinetics. Third, TIRFM enables direct observation of rare events in which dissociation from the membrane surface is followed by transient diffusion through solution and rapid rebinding to a nearby, membrane-associated target lipid. Overall, this study shows that in vitro single-molecule TIRFM provides a new window into the molecular mechanisms of membrane docking reactions.     

Interaction of cardiotoxin A5 with a membrane: role of conformational heterogeneity and hydrophilic properties

The hypothesis that local conformational differences of snake venom cardiotoxins (cytotoxins, CTs) may play a significant role in their interaction with membrane was tested by molecular modeling of the behavior of the CT A5 from the venom of Naja atra in water and at the water-membrane interface. Two models of the CT A5 spatial structure are known: the first was obtained by X-ray analysis and the second, by NMR studies in solution. A molecular dynamics (MD) analysis demonstrated that loop II of the toxin has a fixed omega-like shape in water, which does not depend on its initial structure. Interaction of the experimentally derived (X-ray and NMR) conformations and MD-simulated conformations of CT A5 with the lipid bilayer was studied by the Monte Carlo method using the previously developed model of the implicit membrane. The following was found: (1) Unlike the previously studied CT2 from the venom of cobra Naja oxiana, CT A5 has only loops I and II bound to the membrane, with the involvement of a lesser number of hydrophobic residues. (2) A long hydrophobic area is formed on the surface of CT A5 due to the omega-like shape of loop II and the arrangement of loop I in proximity to loop II. This hydrophobic area favors the toxin embedding into the lipid bilayer. (3) The toxin retains its conformation upon interaction with the membrane. (4). The CT A5 molecule has close values of the potential energy in the membrane and in an aqueous environment, which suggests a dynamic character of the binding. The results of the molecular modeling indicate a definite configuration of loops I and II and, consequently, a specific character of distribution of polar and apolar properties on the toxin surface, which turns out to be the most energetically favorable. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Creating stable stem regions for loop elongation in Fcabs — Insights from combining yeast surface display,in silico loop reconstruction and molecular dynamics simulations

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules.     

In meso crystal structure and docking simulations suggest an alternative proteoglycan binding site in the OpcA outer membrane adhesin

OpcA is an integral outer membrane adhesin protein from Neisseria meningitidis, the causative agent of meningococcal meningitis and septicemia. It binds to sialic acid (SA)-containing polysaccharides on the surface of epithelial cells. The crystal structure of OpcA showed that the protein adopts a 10-stranded beta-barrel structure, with five extensive loop regions on the extracellular side of the membrane. These form a crevice structure, lined with basic residues, which was hypothesized to act as the binding site for polysaccharide ligands. In the current study, a distinctly different OpcA structure has been obtained using crystals grown from a lipidic mesophase. Comparison of the two structures shows that the largest loop (L2), which closes over the end of the beta-barrel in the original crystal form, adopts a much more extended structure by reaching outward and away from the protein. The difference in conformation may be attributable to the absence of zinc ions from the crystallization conditions for the in meso crystal form: in the original structure, two zinc ions were bound to the external loops. Molecular dynamics (MD) simulations performed on the two OpcA models in a lipid bilayer environment demonstrated pronounced loop mobility. These observations support the view that the loop regions of OpcA are capable of a high degree of conformational flexibility. The original binding site for polysaccharide is not present in the in meso crystal form, and is disrupted during MD simulations. Docking analysis suggests a putative alternative location for the SA ligand in the new crystal form of OpcA.     

Mechanistic basis of differential cellular responses of phosphatidylinositol 3,4-bisphosphate- and phosphatidylinositol 3,4,5-trisphosphate-binding pleckstrin homology domains

Phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) are lipid second messengers that regulate various cellular processes by recruiting a wide range of downstream effector proteins to membranes. Several pleckstrin homology (PH) domains have been reported to interact with PtdIns(3,4)P2 and PtdIns(3,4,5)P3. To understand how these PH domains differentially respond to PtdIns(3,4)P2 and PtdIns(3,4,5)P3 signals, we quantitatively determined the PtdIns(3,4)P2 and PtdIns(3,4,5)P3 binding properties of several PH domains, including Akt, ARNO, Btk, DAPP1, Grp1, and C-terminal TAPP1 PH domains by surface plasmon resonance and monolayer penetration analyses. The measurements revealed that these PH domains have significant different phosphoinositide specificities and affinities. Btk-PH and TAPP1-PH showed genuine PtdIns(3,4,5)P3 and PtdIns(3,4)P2 specificities, respectively, whereas other PH domains exhibited less pronounced specificities. Also, the PH domains showed different degrees of membrane penetration, which greatly affected the kinetics of their membrane dissociation. Mutational studies showed that the presence of two proximal hydrophobic residues on the membrane-binding surface of the PH domain is important for membrane penetration and sustained membrane residence. When NIH 3T3 cells were stimulated with platelet-derived growth factor to generate PtdIns(3,4,5)P3, reversible translocation of Btk-PH, Grp1-PH, ARNO-PH, DAPP1-PH, and its L177A mutant to the plasma membrane was consistent with their in vitro membrane binding properties. Collectively, these studies provide new insight into how various PH domains would differentially respond to cellular PtdIns(3,4)P2 and PtdIns(3,4,5)P3 signals.     

Characterization of PROPPIN-Phosphoinositide Binding and Role of Loop 6CD in PROPPIN-Membrane Binding

PROPPINs (β-propellers that bind polyphosphoinositides) are a family of PtdIns3P- and PtdIns(3,5)P₂-binding proteins that play an important role in autophagy. We analyzed PROPPIN-membrane binding through isothermal titration calorimetry (ITC), stopped-flow measurements, mutagenesis studies, and molecular dynamics (MD) simulations. ITC measurements showed that the yeast PROPPIN family members Atg18, Atg21, and Hsv2 bind PtdIns3P and PtdIns(3,5)P₂ with high affinities in the nanomolar to low-micromolar range and have two phosphoinositide (PIP)-binding sites. Single PIP-binding site mutants have a 15- to 30-fold reduced affinity, which explains the requirement of two PIP-binding sites in PROPPINs. Hsv2 bound small unilamellar vesicles with a higher affinity than it bound large unilamellar vesicles in stopped-flow measurements. Thus, we conclude that PROPPIN membrane binding is curvature dependent. MD simulations revealed that loop 6CD is an anchor for membrane binding, as it is the region of the protein that inserts most deeply into the lipid bilayer. Mutagenesis studies showed that both hydrophobic and electrostatic interactions are required for membrane insertion of loop 6CD. We propose a model for PROPPIN-membrane binding in which PROPPINs are initially targeted to membranes through nonspecific electrostatic interactions and are then retained at the membrane through PIP binding.     

Interaction of Cardiotoxin A5 with Membrane: Role of Conformational Heterogeneity and Hydrophobic Properties

The hypothesis that local conformational differences of the snake venom cardiotoxins (cytotoxins, CT) may play a significant role in their interaction with membrane was tested by molecular modeling of the behavior of the CT A5 from the venom of Naja atra in water and at the water–membrane interface. Two models of the CT A5 spatial structure are known: the first was obtained by X-ray analysis and the second, by NMR studies in solution. A molecular dynamics (MD) analysis demonstrated that loop II of the toxin has a fixed -like shape in water, which does not depend on its initial structure. An interaction of the experimentally derived (X-ray and NMR) conformations and MD simulated conformations of CT A5 with the lipid bilayer was studied by the Monte Carlo method using the previously developed model of the implicit membrane. It is found that: (1) unlike the previously studied CT2 from the venom of cobra Naja oxiana, CT A5 has only loops I and II bound to the membrane with the involvement of a lesser number of hydrophobic residues. (2) A long hydrophobic area is formed on the surface of CT A5 due to the -like shape of loop II and the arrangement of loop I in proximity to loop II. This hydrophobic area favors the toxin embedding into the lipid bilayer. (3) The toxin retains its conformation upon interaction with the membrane. (4) The CT A5 molecule has close values of the potential energy in the membrane and in aqueous environment, which suggests dynamic character of the binding. The results of the molecular modeling indicate a definite configuration of loops I and II and, consequently, a specific character of distribution of polar and apolar properties on the toxin surface, which turns out to be the most energetically favorable.     

Characterization of the outer membrane protein OprF of Pseudomonas aeruginosa in a lipopolysaccharide membrane by computer simulation

The N-terminal domain of outer membrane protein OprF of Pseudomonas aeruginosa forms a membrane spanning eight-stranded antiparallel beta-barrel domain that folds into a membrane channel with low conductance. The structure of this protein has been modeled after the crystal structure of the homologous protein OmpA of Escherichia coli. A number of molecular dynamics simulations have been carried out for the homology modeled structure of OprF in an explicit molecular model for the rough lipopolysaccharide (LPS) outer membrane of P. aeruginosa. The structural stability of the outer membrane model as a result of the strong electrostatic interactions compared with simple lipid bilayers is restricting both the conformational flexibility and the lateral diffusion of the porin in the membrane. Constricting side-chain interactions within the pore are similar to those found in reported simulations of the protein in a solvated lipid bilayer membrane. Because of the strong interactions between the loop regions of OprF and functional groups in the saccharide core of the LPS, the entrance to the channel from the extracellular space is widened compared with the lipid bilayer simulations in which the loops are extruding in the solvent. The specific electrostatic signature of the LPS membrane, which results in a net intrinsic dipole across the membrane, is found to be altered by the presence of OprF, resulting in a small electrically positive patch at the position of the channel.     

Structure and dynamics of the phospholipase C-delta1 pleckstrin homology domain located at the lipid bilayer surface

Despite the importance of signal transduction pathways at membrane surfaces, there have been few means of investigating their molecular mechanisms based on the structural information of membrane-bound proteins. We applied solid state NMR as a novel method to obtain structural information about the phospholipase C-delta1 (PLC-delta1) pleckstrin homology (PH) domain at the lipid bilayer surface. NMR spectra of the alanine residues in the vicinity of the beta5/beta6 loop in the PH domain revealed changes in local conformations due to the membrane localization of the protein. We propose that these conformational changes originate from a hydrophobic interaction between the amphipathic alpha-helix located in the beta5/beta6 loop and the hydrophobic layer of the membrane and contribute to the membrane binding affinity, interdomain interactions and intermolecular interactions of PLC-delta1.     

Identification of membrane-contacting loops of the catalytic domain of cytochrome P450 2C2 by tryptophan fluorescence scanning

The catalytic domain of cytochrome P450 is thought to contact the lipid core of the endoplasmic reticulum membrane based on antibody epitope accessibility, protease susceptibility, and hydrophobic surfaces present on P450 structures of solubilized forms of the proteins. Quenching by nitroxide spin label-modified phospholipids of the fluorescence of tryptophan residues substituted into cytochrome P450 2C2, modified to contain tryptophan only at position 120, was used to identify regions of P450 inserted into the lipid core and to estimate the depth of penetration. Consistent with the proposed models of cytochrome P450-membrane interaction, the fluorescence of tryptophans inserted at residues 36 and 69 in the two segments of P450 2C2 flanking the A-helix and at residue 380 in the beta2-2 strand was quenched by nitroxide spin labels on carbon 5 of the fatty acid tails of the phospholipids within the lipid bilayer. The fluorescence of tryptophan at 380 was also strongly quenched by a spin label on carbon 12 of the fatty acids suggesting it was deepest in the membrane. However, fluorescence of tryptophan substituted at residue 225 in the F-G loop, which was predicted to be in the lipid bilayer, was not quenched by the spin labels at carbons 5 and 12 of the fatty acids. The pattern of quenching of fluorescence for tryptophans at the other positions tested, 80, 189, 239, and 347, was similar to the parent protein indicating they were not inserted into the lipid bilayer as expected. The results are consistent with an orientation of cytochrome P450 2C2 in the membrane in which positions 36, 69, and 380 are inserted into the lipid bilayer and residues 80 and 225 are near or within the phospholipid headgroup region. In this orientation, the F-G loop, which contains residue 225, could form a dimerization interface as was observed in the P450 2C8 crystal structure (Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497).