3166-Pos Board B27 The Autotransporter b Domain: Insights into Structure and Function through Molecular Dynamics Simulations Daniel A. Holdbrook, Thomas J. Piggot, Syma Khalid. University of Southampton, Southampton, United Kingdom. The autotransporters are a family of bacterial outer membrane proteins that are characterised by three functional domains: an N-terminal signal sequence, a central passenger domain, and a C-terminal transmembrane b-barrel domain. Using molecular dynamics simulations we explore the structural dynamics and membrane interactions of the b-barrel domains from six autotransporters: BrkA, NalP, EspP, EstA, Hia and Hbp. The b-barrel domain has been proposed to aid in the translocation of the passenger domain across the outer membrane. Extended timescale simulations are performed to investigate the flexibility of the b-barrel domains from the six autotransporters in lipid bilayers designed to capture the complexity of the bacterial outer membrane. We propose a model of the missing pore a-helix from the crystal structure of the Bordetella pertussis autotransporter, BrkA, and we explore the dynamics of the ubiquitous hydrophobic cavity that is required for translocation of the passenger domain. Furthermore, we explore the structure-function relationship of EstA from Pseudomonas aeruginosa by simulating the translocator domain and the associated passenger domain. Thus our simulations provide unprecedented details regarding the membrane interactions and dynamics of the autotransporter proteins we have studied.

Anionic phosphotidylinositol 4,5-bisphosphate (PIP2) occupies only 2∼3% of the phospholipids in the inner leaflet of the plasma membrane, but participates in numerous cellular processes. Concentrated pools of PIP2 on the membrane surface appear to be the source for such PIP2-involved cellular processes, and recent experimental observations indicate that a "protein fence" prevents PIP2 from diffusing out of the pool. Here the necessary properties of the protein fence are examined using Langevin dynamics simulation with effective potential maps. PIP2 molecules are modeled as explicitly diffusing spheres, and the rest of the system represented by steric and electrostatic potentials. Simple models of charged or porous rods, as well as rigid proteins are considered. Retardation by electrostatic forces from a rod is only significant when the charges are highly concentrated and close to the diffusion plane; charge densities and geometries of proteins are insufficient to effectively block diffusion. Hence, fencing appears to be dominated by steric effects, though even a small (> 1%) opening in a porous rod in contact with the surface results in substantial leakage. The three protein fence candidates, actin, human septin, and yeast septin, are known to self-assemble into filaments on the cell surface and to interact with PIP2. Simulations reveal that actin is a poor fence for all depths and orientations simulated because its arch-type shape leaves passageways for diffusion. In contrast, the septins effectively block PIP2 diffusion when buried in the membrane to 10∼15 Å from the lipid surface. Free energy calculations using an implicit membrane model support the possibility of burial of septins at these depths. It is also possible that the septins are not buried as deeply, but associate with other proteins or peptides to make a fence.

To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP2), several investigators have proposed that there are separate pools of PIP2 in the plasma membrane. Recent experiments show the surface concentration of PIP2 is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP2 are also concentrated in these regions. However, how is the PIP2 produced by these kinases prevented from diffusing rapidly away? First, proteins could act as “fences” around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP2 within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP2. FCS measurements show that PIP2 diffuses rapidly (D ∼ 1 μm2/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP2 does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (∼10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane–anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP2 into and out of forming phagosomes.

Understanding the activation mechanism of the epidermal growth factor receptor (EGFR) is of fundamental importance in anticancer drug development. Crystal structures establish the two kinase domains in the receptor dimer bind to and activate each other by an allosteric mechanism. However, information about the conformation of the juxtamembrane (JM) region and the proximity of kinase domains to the membrane in live cells is lacking. Electrostatic potential calculations suggest that both the positively charged N-terminal JM region and a positive face of the kinase domain could bind to the negatively charged inner leaflet of the plasma membrane. The objective of this report is to investigate the proximity of the kinase domain of EGFR to the plasma membrane. We truncated the EGFR after the kinase domain and genetically tagged it with the monomeric yellow fluorescent protein (mYFP). We measured FRET between a plasma membrane target cyan fluorescent protein (PMT-CFP) and the truncated EGFR- mYFP (tEGFR-mYFP). The observed FRET is consistent with the hypothesis that the kinase domain is located close to the membrane in the absence of ligand. Addition of epidermal growth factor (EGF) produced a decrease in FRET, which indicates the kinase domain may dissociate from the membrane during activation.

Experiments described by Golebiewska et al. at this meeting suggest the existence of a 'fence'that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate (PIP2) into and out of nascent phagosomes in macrophages. Although the nature of the fence remains an enigma, actin filaments are plausible components. They are highly negatively charged (as is PIP2), are swept away from the central region and are concentrated at the perimeter of the forming phagosome. To explore the actin fence hypothesis, we have used (1) Poisson-Boltzmann continuum electrostatics and a grid-based repulsive potential to describe a fence model made of a single layer of actin filaments, and (2) Brownian dynamics to describe the diffusion of PIP2 molecules modeled as single spheres. The simulations with actin filaments positioned parallel to the membrane indicate that a single filament without attached proteins does not significantly impede the diffusion of PIP2. A helical stripe of basic residues on the acidic actin filament provides a hole in the putative fence through which PIP2 can diffuse, no matter how close the filament is positioned to the membrane. Results from simulations of PIP2 diffusion out of corrals formed of multiple layers of actin filaments, and mazes of non-electrostatic barriers will also be presented.

To account for the many roles that phosphatidylinositol 4,5-bisphosphate (PIP2) plays (e.g., in phagocytosis, exocytosis, activation of ion channels) a number of investigators have suggested there are separate pools of PIP2 in the plasma membrane. Recent experiments show that the free concentration of PIP2 is indeed enhanced in nascent phagosomes, syntaxin clusters, and the furrows of dividing cells. Kinases that produce PIP2 (PIPkins) are also concentrated in these regions. But how is the PIP2 produced by these PIPkins prevented from diffusing rapidly away? First, proteins could act as ‘fences/corrals’around the perimeter of these regions. Second, some factor (e.g., a protein that acted as a PIP2 buffer) could decrease significantly the diffusion coefficient, D, of PIP2 within these regions. We used FCS and FRAP to investigate these two possibilities in the nascent phagosomes of J774 macrophages injected with fluorescent PIP2. FCS measurements show PIP2 diffuses with similar fast diffusion coefficients in the nascent phagosomes and in the bulk (unengaged) plasma membrane: D = 0.6 ± 0.3 μm2/s and 0.8 ± 0.2 μm2/s, respectively. FRAP measurements show the fluorescence from PIP2 recovers slowly (>100 s) after photobleaching the entire nascent phagosome but recovers rapidly (<10 s) in a comparable area of the plasma membrane outside the cup. These results support the first hypothesis: a ‘fence’impedes the diffusion of PIP2 into and out of nascent phagosomes. The nature of the PIP2 fence remains an enigma: although actin filaments are concentrated at the perimeter of the forming phagosomes, electrostatic and Brownian Dynamics calculations suggest individual negatively charged actin filaments near the membrane do not significantly impede the diffusion of PIP2 into or out of the cup.

The juxtamembrane domain of vesicle-associated membrane protein (VAMP) 2 (also known as synaptobrevin2) contains a conserved cluster of basic/hydrophobic residues that may play an important role in membrane fusion. Our measurements on peptides corresponding to this domain determine the electrostatic and hydrophobic energies by which this domain of VAMP2 could bind to the adjacent lipid bilayer in an insulin granule or other transport vesicle. Mutation of residues within the juxtamembrane domain that reduce the VAMP2 net positive charge, and thus its interaction with membranes, inhibits secretion of insulin granules in beta cells. Increasing salt concentration in permeabilized cells, which reduces electrostatic interactions, also results in an inhibition of insulin secretion. Similarly, amphipathic weak bases (e.g., sphingosine) that reverse the negative electrostatic surface potential of a bilayer reverse membrane binding of the positively charged juxtamembrane domain of a reconstituted VAMP2 protein and inhibit membrane fusion. We propose a model in which the positively charged VAMP and syntaxin juxtamembrane regions facilitate fusion by bridging the negatively charged vesicle and plasma membrane leaflets.

Calcium/calmodulin (Ca/CaM) binds to the intracellular juxtamembrane domain (JMD) of the epidermal growth factor receptor (EGFR). The basic JMD also binds to acidic lipids in the inner leaflet of the plasma membrane, and this interaction may contribute an extra level of autoinhibition to the receptor. Binding of a ligand to the EGFR produces a rapid increase in intracellular calcium, [Ca2+]i, and thus Ca/CaM. How does Ca/CaM compete with the plasma membrane for the JMD? Does Ca/CaM directly pull the JMD off the membrane or does Ca/CaM only bind to the JMD after it has dissociated spontaneously from the bilayer? To answer this question, we studied the effect of Ca/CaM on the rate of dissociation of fluorescent JMD peptides from phospholipid vesicles by making kinetic stop-flow measurements. Ca/CaM increases the rate of dissociation: an analysis of the differential equations that describe the dissociation shows that Ca/CaM must directly pull the basic JMD peptide off the membrane surface. These measurements lead to a detailed atomic-level mechanism for EGFR activation that reconciles the existence of preformed EGFR dimers/oligomers with the Kuriyan allosteric model for activation of the EGFR kinase domains.

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) controls a surprisingly large number of processes in cells. Thus, many investigators have suggested that there might be different pools of PIP(2) on the inner leaflet of the plasma membrane. If a significant fraction of PIP(2) is bound electrostatically to unstructured clusters of basic residues on membrane proteins, the PIP(2) diffusion constant, D, should be reduced. We microinjected micelles of Bodipy TMR-PIP(2) into cells, and we measured D on the inner leaflet of fibroblasts and epithelial cells by using fluorescence correlation spectroscopy. The average +/- SD value from all cell types was D = 0.8 +/- 0.2 microm(2)/s (n = 218; 25 degrees C). This is threefold lower than the D in blebs formed on Rat1 cells, D = 2.5 +/- 0.8 microm(2)/s (n = 26). It is also significantly lower than the D in the outer leaflet or in giant unilamellar vesicles and the diffusion coefficient for other lipids on the inner leaflet of these cell membranes. The simplest interpretation is that approximately two thirds of the PIP(2) on inner leaflet of these plasma membranes is bound reversibly.

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca2+/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca2+/CaM, but the latter results could be due to binding of W-13 to the plasma membrane.

Phospholipase C- zeta ( PLC- zeta) is a sperm- specific enzyme that initiates the Ca2+ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-delta 1, but lacks the PH domain that anchors PLC-delta 1 to phosphatidylinositol 4,5-bisphosphate, PIP2. Thus it is unclear how PLC-zeta interacts with membranes. The linker region between the X and Y catalytic domains of PLC-zeta, however, contains a cluster of basic residues not present in PLC-zeta 1. Application of electrostatic theory to a homology model of PLC-zeta suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC-zeta to phospholipid vesicles: for 2: 1 phosphatidylcholine/phosphatidylserine (PC/ PS) vesicles, the molar partition coefficient, K, is too weak to be of physiological significance. Incorporating 1% PIP2 into the 2: 1 PC/ PS vesicles increases K about 10-fold, to 5 X 103 M-1, a biologically relevant value. Expressed fragments corresponding to the PLC- zeta X-Y linker region also bind with higher affinity to polyvalent than monovalent phosphoinositides on nitrocellulose filters. A peptide corresponding to the basic cluster (charge = +7) within the linker region, PLC-zeta-( 374-385), binds to PC/ PS vesicles with higher affinity than PLC-zeta, but its binding is less sensitive to incorporating PIP2. The acidic residues flanking this basic cluster in PLC-zeta may account for both these phenomena. FRET experiments suggest the basic cluster could not only anchor the protein to the membrane, but also enhance the local concentration of PIP2 adjacent to the catalytic domain.

Phospholipase C-zeta (PLC-zeta) is a sperm-specific enzyme that initiates the Ca2+ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-delta1, but lacks the PH domain that anchors PLC-delta1 to phosphatidylinositol 4,5-bisphosphate, PIP2. Thus it is unclear how PLC-zeta interacts with membranes. The linker region between the X and Y catalytic domains of PLC-zeta, however, contains a cluster of basic residues not present in PLC-delta1. Application of electrostatic theory to a homology model of PLC-zeta suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC-zeta to phospholipid vesicles: for 2:1 phosphatidylcholine/phosphatidylserine (PC/PS) vesicles, the molar partition coefficient, K, is too weak to be of physiological significance. Incorporating 1% PIP2 into the 2:1 PC/PS vesicles increases K about 10-fold, to 5x10(3) M-1, a biologically relevant value. Expressed fragments corresponding to the PLC-zeta X-Y linker region also bind with higher affinity to polyvalent than monovalent phosphoinositides on nitrocellulose filters. A peptide corresponding to the basic cluster (charge=+7) within the linker region, PLC-zeta-(374-385), binds to PC/PS vesicles with higher affinity than PLC-zeta, but its binding is less sensitive to incorporating PIP2. The acidic residues flanking this basic cluster in PLC-zeta may account for both these phenomena. FRET experiments suggest the basic cluster could not only anchor the protein to the membrane, but also enhance the local concentration of PIP2 adjacent to the catalytic domain.

The transmembrane (TM) and juxtamembrane (JM) regions of the epidermal growth factor receptor (EGFR) couple ligand binding in the extracellular domain to activation of the kinase domain. Solid-state NMR and polarized FTIR measurements of peptides corresponding to the TM plus JM regions of EGFR (residues 622-660) reconstituted in model phospholipid membranes are presented to address the role of the short cytoplasmic JM sequence (residues 645-660) in regulating EGFR activity. We show that the TM domain is helical with a transition to non-helical structure at the TM-JM boundary. Fluorescence measurements indicate that the JM region of EGFR(622-660) binds to the membrane surface and that binding can be reversed by the addition of the complex of Ca2+ and calmodulin. Together these data support models suggesting the cytoplasmic JM region of EGFR plays an active role in regulating receptor activity.

The AKAP gravin is a scaffold for protein kinases, phosphatases, and adaptor molecules obligate for resensitization and recycling of β2-adrenergic receptors. Gravin binds to the receptor through well characterized protein-protein interactions. These interactions are facilitated ∼1000-fold when gravin is anchored to the cytoplasmic leaflet of the plasma membrane. Although the N-terminal region (∼550 residues) is highly negatively charged and probably natively unfolded, it could anchor gravin to the inner leaflet through hydrophobic insertion of its N-terminal myristate and electrostatic binding of three short positively charged domains (PCDs). Loss of the site of N-myristoylation was found to affect neither AKAP macro-scopic localization nor AKAP function. Synthetic peptides corresponding to PCD1-3 bound in vitro to unilamellar phospholipid vesicles with high affinity, a binding reversed by calmodulin in the presence of Ca2+. In vivo gravin localization is regulated by intracellular Ca2+, a function mapping to the N terminus of the protein harboring PCD1, PCD2, and PCD3. Mutation of any two PCDs eliminates membrane association of the non-myristoylated gravin, the sensitivity to Ca2+/calmodulin, and the ability of this scaffold to catalyze receptor resensitization and recycling.

Several biologically important peripheral (e.g., myristoylated alanine-rich C kinase substrate) and integral (e.g., the epidermal growth factor receptor) membrane proteins contain clusters of basic residues that interact with acidic lipids in the plasma membrane. Previous measurements demonstrate that the polyvalent acidic lipid phosphatidylinositol 4,5-bisphosphate is bound electrostatically (i.e., sequestered) by membrane-adsorbed basic peptides corresponding to these clusters. We report here three experimental observations that suggest monovalent acidic lipids are not sequestered by membrane-bound basic peptides. 1), Binding of basic peptides to vesicles does not decrease when the temperature is lowered below the fluid-to-gel phase transition. 2), The binding energy of Lys-13 to lipid vesicles increases linearly with the fraction of monovalent acidic lipids. 3), Binding of basic peptides to vesicles produces no self-quenching of fluorescent monovalent acidic lipids. One potential explanation for these results is that membrane-bound basic peptides diffuse too rapidly for the monovalent lipids to be sequestered. Indeed, our fluorescence correlation spectroscopy measurements show basic peptides bound to phosphatidylcholine/phosphatidylserine membranes have a diffusion coefficient approximately twofold higher than that of lipids, and those bound to phosphatidylcholine/phosphatidylinositol 4,5-bisphosphate membranes have a diffusion coefficient comparable to that of lipids.