272 Mini-Reviews in Medicinal Chemistry, 2011, 11, 272-282 Attachment of Rod-Like (BAR) Proteins and Membrane Shape D. Kabaso*,1,E. Gongadze2,P. Elter3, U. van Rienen2,J. Gimsa4,V. Kralj-Igli(cid:7)5and A. Igli(cid:7)1 1Laboratory of Physics, Faculty of Electrical Engineering, University of Ljubljana, Tr(cid:5)a(cid:4)ka 25, SI-1000 Ljubljana, Slovenia 2Institute of General Electrical Engineering, University of Rostock, Albert-Einstein-Straße 2, 18051 Rostock, Germany 3Department for Interface Science, Institute for Electronic Appliances and Circuits, University of Rostock, Albert- Einstein-Str. 2, D-18059, Rostock, Germany 4Department of Biology, Chair of Biophysics, Institute for Biosciences, University of Rostock, Gertrudenstr.11A, D-18057, Rostock, Germany 5Laboratory of Clinical Biophysics, Faculty of Medicine, Lipiceva 2, SI-1000 Ljubljana, Slovenia Abstract: Previous studies have shown that cellular function depends on rod-like membrane proteins, among them Bin/Amphiphysin/Rvs (BAR) proteins may curve the membrane leading to physiologically important membrane invaginations and membrane protrusions. The membrane shaping induced by BAR proteins has a major role in various biological processes such as cell motility and cell growth. Different models of binding of BAR domains to the lipid bilayer are described. The binding includes hydrophobic insertion loops and electrostatic interactions between basic amino acids at the concave region of the BAR domain and negatively charged lipids. To shed light on the elusive binding dynamics, a novel experiment is proposed to expand the technique of single-molecule AFM for the traction of binding energy of a single BAR domain. Keywords: AFM, attachment dynamics, curvature membrane proteins, membrane shape. INTRODUCTION charged lipids along the inner leaflet of the membrane [13, 19]. In addition, two hydrophobic insertion loops, one of The membrane curvature induced by BAR proteins has a each monomer, were suggested to penetrate the inner major role in various biological processes such as cell membrane leaflet and to increase its surface area and shaping, cell growth, cell motility, receptor-ligand curvature [20]. Substitution assays and mutagenesis studies interactions, adhesion to the extracellular matrix, and further demonstrated that the replacement of either basic intracellular signalling [1-7]. Thus, it is not surprising that amino acids at the concave side or hydrophobic amino acids the number of human diseases implicated with dysfunctional at the insertion loops with neutral amino acids would reduce BAR proteins is growing. For example, the gene encoding the BAR domain capacity to bend the membrane [18]. one of the BAR proteins in humans has been shown to be fused to mixed lineage leukaemia (MLL) observed in In the first section of this review, we describe a model for patients suffering from acute myelogenous leukaemia [8]. the binding of a BAR domain to the inner leaflet of a lipid Furthermore, some dysfunctional mammalian BAR proteins bilayer. In the second section, the binding dynamics is were observed in renal cancer [9], in Huntington disease [10- presented while taking into account the hydrophobic and 11],and in mental retardation [12].The waiting questions are electrostatic interactions. In the third section, we calculate whether the BAR domain binding and bending energies can the electric field in the vicinity of a BAR domain. In the be altered and what are the functional consequences of such fourth section, the theory of flexible or semi-flexible rod-like alterations in human diseases. proteins is presented. In the fifth section, the case of rigid (stiff) rod-like proteins is discussed, where the effects of BAR domains are rod-like membrane proteins which can BAR domain orientations are taken into account. In the sixth sense or induce a curvature to the membrane [13-15]. By section, the theory is further employed to determine the mapping their X-ray crystallography structures, it is evident effects of BAR domain density. Finally, in the discussion that BAR domains are homodimers of crescent-like shapes section, we propose an experiment by which it may be that are rich with basic amino-acids at the concave side possible to measure the binding energy of a BAR domain to [13,16-18] (Fig. 1). Thus, it was suggested that the basic a lipid bilayer/cell membrane, as well as possible amino acids at the concave side interact with negatively applications of the outlined experiments and theory. A MODEL FOR THE BINDING OF BAR DOMAINS *Address correspondence to this author at the Laboratory of Physics, Faculty of Electrical Engineering, University of Ljubljana, Tr(cid:6)a(cid:5)ka 25, SI- In the present review, we mainly concentrate on one ty pe 1000 Ljubljana, Slovenia; Tel: +386147688 25; Fax: +38614768850; of BAR domains, which is pacsin2 EFC/F-BAR [7,21]. Its E-mail: [email protected] three-dimensional structure was already revealed using 1389-5575/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd. Attachment Dynamics of BAR Proteins Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 4 273 Fig. (1). The molecular structure and a schematic diagram of pacsin2 EFC/F-BAR domain binding to a cell membrane. (a) Side view of the electric charge surface of pacsin2 dimer. Note that the molecule is positively charged (dark grey) at the concave, whereas the convex side is more negatively charged (light grey) (adapted from [18]). (b) Schematic diagram of pacsin2 EFC/F-BAR domain bound to the membrane. Note that the inner side of the membrane is negatively charged, while the domain contact interface is positively charged. In addition, two hydrophobic protrusions that belong to the BAR domain are docked into the hydrophobic part of the inner membrane leaflet. The binding of BAR domains of positive intrinsic curvature (e.g. pacsin2) to both membrane invaginations and protrusions. (cid:1) is the rotation angle of the molecule on the membrane surface. In high concentrations of BAR domains, a spiral aggregate can be formed stabilizing the formation of a membrane invagination. X-ray crystallography and molecular mapping techniques. It tubules with a curvature comparable to the intrinsic has been demonstrated that pacsin2 is a homo dimer forming curvature of pacsin2 EFC/F-BAR domain, and that the a crescent-like shape, where the concave side is rich with distribution of pacsin2 was not only at membrane basic amino acids (Fig. 1). Since the membrane surface on invaginations, but also at the base of membrane protrusions, the cytoplasmic side is negatively charged, it was proposed which could be due to similar curvatures [18](Fig. 2). that the concave side of BAR protein binds the negatively THE BINDING DYNAMICS OF BAR DOMAINS TO A charged parts of the membrane. In addition, two hydrophobic MEMBRANE protrusions, one on each side of a BAR domain could be inserted into the hydrophobic lipid layer. It has been shown On the outer lipid leaflet of a cell membrane, epithelia that over expression of pacsin2 EFC/F-BAR domain in and other cells produce an extracellular polymeric layer phosphatidylserine rich liposomes deform the membrane to (glycoproteins) called glycocalyx [22], which is negatively 274 Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 4 Kabaso et al. charged mainly due to sialic acids. The binding between the inner leaflet of a membrane, which lacks glycoproteins and outer surface of the cell membrane and a substrate may glycolipids but has some negatively charged lipid molecules. include the membrane bending energy, short-range ligand Therefore, the energy landscape of the interaction is receptor attraction, and long-range glycocalyx repulsion [23- expected to be different from the adhesion of a cell 24].This binding leads to the formation of a double well in membrane to a substrate. It has been demonstrated using the distance energy curve [23]. The long distance well is due molecular dynamics simulations that both the positively to repulsive forces of protruding hydrophilic sugar chains of charged amino acids at the interface of a BAR domain and glycolipids and glycoproteins [25]. Normally, these repulsive the hydrophobic insertion loops near its tips could facilitate forces are stronger than the attractive van der Waals the bending of a membrane according to the intrinsic interactions, preventing the adhesion of neutral membrane curvature of a BAR domain [13,30]. To summarize, we surfaces. In contrast, short-range attractive forces due to propose that the dynamics of a BAR domain (e.g. pacsin2 ligand receptor interactions, which are balanced by repulsive EFC/F-BAR) binding to a lipid bilayer may include three forces due to the ordering of water molecules, give rise to a steps: a) the two tips of a BAR domain are attracted to the short distance well [24,26-28] at distances comparable with membrane by strong electrostatic interactions towards the ligand size [29]. The attractive short range interactions negatively charged lipids (Fig. 2a); b) the two hydrophobic and the repulsive long range interactions creates a high loops are inserted into the hydrophobic layer of the inner energy barrier at a distance close to the membrane. leaflet (Fig. 2b). Due to the resulted local area difference between the two leaflets, the membrane will be slightly bent In contrary to the adhesion dynamics between a cell and into the convexity of both insertion points and to concavity a substrate, the binding of BAR domains is mostly to the Fig. (2). Schematic diagram of the binding dynamics of a BAR domain to a lipid bilayer. The adhesion process is divided into three steps. The first step (a) is the electrostatic attraction between the positively charged tips of a BAR domain and negatively charged lipids attracted to both tips. The second step (b) is the insertion of hydrophobic protrusions into the inner leaflet of a lipid bilayer, which generates a slight curvature to the membrane. The third step (c) maybe electrostatic attraction between positively charged amino acids of the BAR domain to the lipid bilayer, thereby bending the membrane to match the intrinsic curvature of the BAR domain. Attachment Dynamics of BAR Proteins Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 4 275 in between; c) the membrane is attracted also to the middle Here e is the elementary charge, p is the magnitude of part of the protein thereby closing the gap between the BAR 0 0 the dipole moment of water (or small cluster of water domain and the lipid bilayer. As a result, the curvature of the membrane becomes similar to the intrinsic curvature of the molecules), (cid:1) is the permittivity the free space, E = |(cid:1)(cid:1) | 0 BAR domain (Fig. 2c). It is here proposed that since the is the magnitude of electric field strength, n is the unit electric field strength of a BAR protein at the membrane normal vector in direction of (cid:1)(cid:1)(r), (cid:6) = 1/kT, kT is thermal surface increases with increasing curvature radius R (Fig. 4), energy and n = n (cid:3)2n is the number density of water the attraction is stronger for BAR proteins with lower 0w s 0 molecules in the bulk, n is the number density of lattice intrinsic curvature (i.e. larger radius of curvature). We also s note that in steps (a) and (b) there might be more negatively sites, and n0 the bulk number density of monovalent salt charged lipids near both ends of a BAR domain because of anions and cations. Eq. (1) describes the electrostatics of a demixing induced by the charged tips of a BAR domain [31- charged surface in contact with an electrolyte solution, 33]. The electrostatic attraction may then start from the taking into account the finite size of ions and spatial edges spreading to the middle in concomitant to the bending variation of the permittivity near the charged surface. The of the membrane. equation has two boundary conditions. The first one states that the electric field in the bulk solution is zero: THE ELECTRIC FIELD IN THE VICINITY OF BAR PROTEINS (cid:4)(cid:1)(r(cid:3)(cid:2))=0 . (7) To understand the influence of the intrinsic curvature of a The second boundary condition is [34]: BAR domain to the electric field strength at the membrane surface (Fig. 4), we constructed a simple electrostatic model (cid:4)n of a BAR domain using Finite Element Method (FEM) in (cid:5)(cid:3)(r=r )=(cid:1) , (8) Comsol Multiphysics 3.5a®. The inner positively charged surf (cid:2)(cid:2) (r=r ) 0 r surf surface of a BAR domain with a negative concave curvature is presented as an arch of curvature radius R (Fig. 4). Based where (cid:1) (r) is defined by Eq. (3). r on experimental data, the concave surface of the BAR domain is assumed to have a surface charge density In order to determine the spatial dependency of (cid:1) (r), (cid:5) = 0.2 As/m2. The electric field at point 1 (shown in Fig. 4) r we first solve Eq. (1) in a planar geometry within the is calculated as described below. program package Comsol Multiphysics 3.5a Software. In The spatial dependency of electric potential (cid:1)(r) (which this procedure the space dependency of (cid:1) (r) (Eq. (3)) is r enables us to determine the electric field strength E=(cid:3)(cid:1)(cid:1) at taken into account in an iterative procedure, where the initial point 1 in Fig. (4) is calculated using the Langevin-Bikerman value of (cid:1) (r) is a constant equal to the permittivity of the r equation [28,34] rewritten in the form appropriate for FEM bulk solution. Fig. (3) shows the calculated spatial Comsol Multiphysics program package [34]: dependency of (cid:1)(r) in the vicinity of a charged planar r (cid:5)·((cid:2)(cid:2)(r)(cid:5)(cid:3)(r))=(cid:1)(cid:4) (r), surface. The predicted decrease of the permittivity relative to 0 r free (1) its bulk value is the consequence of the orientational where (cid:1) (r) is the macroscopic (net) volume charge ordering of water dipoles in the vicinity of the charged free surface and the depletion of water dipoles at the charged density of coions and counterions in contact with the BAR surface [28,34-35]. domain concave charged surface [34]: Next, we calculated the electric potential and electric (cid:3) (r)=(cid:1)2enn sinh(e(0(cid:2)/k)T) (2) field strength at point 1 at a certain distance from the curved free 0 s 0 H (cid:2),E inner charged surface of BAR domain as shown in Fig. (4). We solved numerically the Langevin-Bikerman equation and (cid:1)(r) is the relative permittivity of the electrolyte r (Eq. (1)) using the program package Comsol Multiphysics solution in contact with the BAR domain [34]: 3.5a Software by taking into account the boundary conditions in Eqs. (7) and (8). However, unlike the planar (cid:2)(r)=1+ nn p0 F(p(0E(cid:1))) , (3) case, to avoid the numerical problems, we expand Eq. (2), r s 0w(cid:2) EH (cid:3),E 0 while the relative permittivity (cid:1)(r) (defined by Eq. (3)) is r where approximated by a step function with the value (cid:4) in the ord F(p E(cid:1)) = L(p E(cid:1))sinh (p0E(cid:1)), (4) region rsurf (cid:1) r (cid:1) ( rsurf + a), where the value (cid:4)ord = 54.5 is 0 0 p E(cid:1) taken from Fig. (3). Here a is the thickness of the thin layer 0 near the charged concave surface of BAR protein with a L(p0E(cid:1)) = [coth(p0E(cid:1))-1/(p0E(cid:1))] , (5) strong preferential orientation of water molecules and accumulated counterions. In the region r (cid:2) (r + a), we ( ) n surf H (cid:2),E =2n0 cosh(e0(cid:2)(cid:1))+p0E0w(cid:1) sinh (p0E(cid:1)) . (6) assume the bulk value of permittivity, i.e. (cid:4)r( r)= (cid:4)b= 78.5. 276 Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 4 Kabaso et al. Fig. (3). The relative permittivity (cid:1) as a function of the distance r from the planar charged surface x calculated within the Langevin- Bikerman model of the electric double layer [28,34-35]for surface charge density (cid:2) = 0.2 As/m2. Eqs.(1)-(8) were solved numerically for planar geometry using Finite Element Method as described in the text. Dipole moment of water p0 = 4.79 D, bulk concentration of salt n /N = 0.15 mol/l, bulk concentration of water n /N = 55 0 A 0w A mol/l. It can be seen in Fig. (4) that the electric field strength at the point 1 strongly increases with the increased curvature radius of the BAR domain, which means that the probability that the BAR domain would be fully electrostatically attached to the underlying membrane surface strongly increases with increasing intrinsic curvature radius R of the BAR domain. Fig. (4). Calculated electric field of a BAR protein at the point 1 as FLEXIBLE OR SEMI-FLEXIBLE ROD-LIKE a function of its curvature radius R. The surface charge density of PROTEINS the inner concave BAR domain (cid:2) = 0.2 As/m2 is considered As every member in the family of BAR proteins is a uniform. The specific parameters of the model are chosen as (cid:1) = ord homodimer, it is quite probable that the interaction between 54.5, a= 0.32 nm, bulk concentration of salt n /N = 0.15 mol/l, 0 A the monomers would determine the rigidity of the BAR bulk concentration of water n /N = 55 mol/l. Note that the total 0w A domain. At the interface between the two monomers, there length L of BAR domain remains fixed during variation of 0 are hydrophobic amino acids from six alpha helices, three curvature radius R. from each monomer. The BAR domain could be flexible or rigid behaving as a worm-like polymer or a stiff rod. two principal curvatures of the lipid bilayer. The Membrane-attached proteins can be less rigid or of th e same contribution of membrane protein orientation to the bending order of magnitude as lipid bilayer membranes [36].In this energy was investigated in a recent study by Perutková et al. section the membrane-attached proteins are considered as (2010) for the case of protein and membrane rigidities of the flexible elongated curved rod-like proteins having similar same order of magnitude [40]. It was indicated that rigidity as a membrane bilayer. The limit of strong adhesion accumulation of anisotropic curved rod-like membrane is assumed. In this limit, the protein should adapt its proteins can stabilize highly curved membrane regions (Fig. curvature to the curvature of the membrane. 1) while overcoming the decrease in the configurational entropy during the process of lateral sorting of membrane The bending energy of flexible membrane attached BAR proteins [40]. However, in the case of isotropic membrane domain (Ep) can be calculated as follows [20,37-39]: proteins, substantial sorting of membrane proteins is not K L ( ) possible without strong enough interactions between Ep = 2p 0 C(cid:1)Cp 2, (9) proteins. The local membrane curvature seen by the rod-like BAR protein for a given rotation of the protein [38] where C is the membrane curvature, Kp is the flexural described by the angle (cid:3) between the normal plane in which rigidity, L0 is the length of the protein, and Cp is the intrinsic the protein is lying and the plane of the first principal curvature of the BAR protein. curvature (C = 1/R ) is : 1 1 Since BAR proteins have a rod-like shape, it is very C= H+Dcos(2(cid:1)) , (10) likely that the induced curvature is not symmetric along the Attachment Dynamics of BAR Proteins Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 4 277 Fig. (5). Schematic diagram of a flexible rod-like protein strongly attached to the inner side of a cylindrical membrane surface having C = 1 1/R and C = 0, i.e. H = D= 1/R . At a given value of the protein orientation angle (cid:3) the protein senses the curvature C=(C +C )/2+ ((C (cid:1) 1 2 1 1 2 1 C )/2)cos(2(cid:3)) (adapted from [38]). 2 where D= |C (cid:1)C |/2 and H =(C +C )/2 are the curvature their strong adhesion, the nanotube diameter is bent to fit the 1 2 1 2 deviator and the mean curvature at the given location on the intrinsic curvature of (cid:2)2-GPI. membrane surface, respectively, and C and C are the two 1 2 principal curvatures (Fig. 5). By inserting Eq. (9) into Eq. (10), we get : E = KpL0(H(cid:1)C +Dcos(2(cid:2)))2. (11) p 2 p The energy of symmetric bilayer membranes is [41-42] : W =k /2 (cid:2)(2H)2dA+k (cid:2)CCdA(cid:1) 2mkT(cid:2)ln(2cosh(D D))dA, (12) b c G 1 2 0 eff where k and k are the membrane local bending constant c G and the Gaussian saddle-splay constant, respectively, D is eff the effective intrinsic curvature deviator of lipid molecules, and m is the area density of the lipid molecules. If the 0 flexural rigidity of the protein and the membrane are of the same order of magnitude, the interplay between the bending energy of a membrane (Eq. (12)) and the bending energy of a protein (Eq. (9)) determines the curvature of the membrane. At this point it should be stressed that neglecting the Fig. (6). Network of thin nanotubular connections (indicated by deviatoric term in Eq. (11) may considerably reduce the black arrows) between negatively charged POPC-cholesterol- depth of the free energy minima. This indicates that the cardiolipin giant unilamellar vesicles (GUVs) in the presence of (cid:2)2- decrease of isotropic curvature energy of the BAR domains GPI (100 mg/L) and serum IgG antibodies (75 mg/mL) from an in the region of membrane protrusions is usually not large antiphospholipid syndrome patient, containing antibodies against enough for substantial protein sorting and consequent (cid:2)2-GPI. GUVs were observed under Zeiss Axiovert 200 Phase stabilization of the membrane protrusion [40,43-44]. In this Contrast Microscope (Zeiss, Germany), magnification 1000 x in 0.2 case only the decrease of the deviatoric part of the bending mol/l sucrose/glucose/ PBS solution; pH 7.4; T =37oC; ionic v energy of the attached rod-like proteins and their direct strength 10 mmol/l. POPC:cholesterol: cardiolipin mass propor- interaction energy may overcome the increase of the free tionin GUVs = 7:2:1. (adapted from [38]). energy due to decrease of the configurational (mixing) entropy, upon the lateral sorting of curved rod-like BAR RIGID (STIFF) ROD-LIKE PROTEINS domains, thereby stabilizing the nanotubular membrane protrusion [40,43-46]. An experimental evidence for In the limit of large bending modulus, the membrane or membrane proteins that can stabilize and bend highly curved part of the membrane should adapt its curvature to the membrane regions is the appearance of a network of thin intrinsic curvature of the attached rigid rod-like proteins (Cp) nanotubular connections [38,47-49]. These thin nanotubular [4,19-20]. For a special case of a tubular shaped membrane: connections can be seen using a phase contrast microscope C = H+ Dcos(2(cid:3)) , (13) upon the addition of (cid:2)2-GPI molecules [47-49]. One possible p explanation to the observed increase in nanotube diameter where the two principal curvatures of the tube are C = 1/R 1 1 (Fig. 6) is that (cid:2)2-GPI molecules have a smaller intrinsic and C = 0, while (cid:3) describes the orientation of proteins. It 2 curvature than the membrane nanotube curvature, and due to follows from Eq. (13) that 278 Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 4 Kabaso et al. (cid:2)1 1 ( )(cid:5) decreasing the tube diameter. The direct attractive Cp =C1(cid:4)(cid:3)2+2cos 2(cid:1) (cid:7)(cid:6) =C1cos2(cid:1), (14) interactions between BAR domain ends, contributing to the negative interaction energy, would compensate the increase to yield of configurational entropy (Fig. 8). In large concentrations of BAR domains, the self-assembly into a spiral aggregate (Fig. C = Cp , (15) 8) may not only minimize the energy of direct attractive 1 cos2(cid:1) interactions between BAR domains but also minimize the local membrane deformation in the vicinity of attached BAR demonstrating that the principal curvature of the tube is domains. According to Eq. (13), for a rotation angle of (cid:1) = determined by the intrinsic curvature of the attached protein (cid:2)/4, the contribution of the deviatoric term is zero, and C = 1 (Cp) and its orientation angle ((cid:1)). Thus, the principal 2Cp. The possible increase in the membrane bending energy curvature of the tube is not constant but is changing along (Eq. (12)) due to larger C can be counterbalanced by a 1 with the orientation of the BAR domain. For (cid:1)=0 (Fig. 7a), negative interaction energy due to electrostatic attraction the value of C1 is minimal, and the membrane bending between neighboring BAR domains. In intermediate energy is minimal as well. The BAR domain will fit concentrations of BAR domains, the overlap between perfectly to the tube surface at (cid:1)=0. The BAR domain can be neighboring BAR domains in the possible formation of ring rotated at an angle (cid:1)> 0 in order to maximize the aggregates is partial, compensating the smaller loss of electrostatic and hydrophobic interaction with the target configurational entropy. Finally, for the case of low BAR membrane (Fig. 7b). The BAR domain preferred orientation domain concentrations, the system energy is at minimum can be also binding from the top side (convex) rather than when the BAR domains are randomly dispersed over the the concave side of the molecule, lying down on the tube tube surface contributing large configurational entropy to the surface. To conclude, the preferred orientation of the BAR system free energy. To summarize, high concentrations of domain and the tube diameter could be according to the BAR proteins not only increase the tube curvature but also maximum binding strength and the BAR domain intrinsic affect the aggregate shape of BAR domains. curvature (Eq. (15)), respectively. The possible reason for (cid:1)(cid:2)0 (or R (cid:2)R ) could be also the direct interaction between DISCUSSION 1 p BAR proteins, requiring (cid:1)(cid:2)0 as is discussed in the next In the present review, we describe how positively section. charged amino acids and the two hydrophobic insertion THE EFFECTS OF BAR DOMAIN DENSITY AND loops of BAR domains (e.g. pacsin2 EFC/F-BAR) at the DIRECT ATTRACTIVE INTERACTIONS membrane contact interface (Fig. 1), may affect their binding dynamics to a lipid bilayer (Fig. 2). Electric field In the literature, there is inconsistency with respect to the calculations reveal that by varying the BAR domain intrinsic effect of BAR domain density on the induced curvature of a radius of curvature the electric field of a BAR protein close membrane. In a recent study [18], it was demonstrated that at to the membrane is increased with the radius of curvature high expression levels of EFC/F-BAR proteins the diameters (Fig. 4). The effects of rod-like BAR domain orientation on of the formed tubes fit the intrinsic curv ature of the BAR the bending energy are discussed. The limits of BAR domain domains. Whereas, a different study [30]has shown that the rigidity and adhesion strengths are considered. It is formed tube curvatures are smaller than the BAR domain demonstrated that in the limit of a rigid BAR domain, the intrinsic curvature. We propose that the density of BAR tube diameter depends on the orientation of the BAR domain domains affects the formation of spiral domains, thereby on the tube surface (Fig. 7). The interplay between Fig. (7). Schematic diagram for different stiff BAR domain tilt and orientation on the interaction with the membrane. The intrinsic curvature radius of the domain can be equal (a), or larger (b) than the tube radius. The protein can be rotated ((cid:1)(cid:2)0) to optimize the area of contact, which minimizes the binding energy. Attachment Dynamics of BAR Proteins Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 4 279 configurational entropy, membrane bending, protein binding, In a similar manner, we propose an AFM experiment for and domain-domain interactions as a function of the BAR the retraction of a BAR domain from the interface of a lipid domain density are shown to affect the self-assembly of bilayer in order to determine the contributions of BAR domain aggregates (Fig. 8). hydrophobic and electrostatic interactions to the binding energy landscape. The soft or rigid BAR domain could be reconstituted into artificial membranes that are rich in negatively charged phosphatidylserine. The crystallization of the lipid bilayer is an important step, since it prevents thermal undulations from unbinding of the attached AFM stylus from the BAR domain. On the other hand, it introduces a limitation not revealing the protein-induced membrane deformation energy. Future experiments may circumvent this limitation by adding a strong cross linker to the AFM stylus, increasing the life span of the BAR domain on the AFM stylus. Fig. (9) shows a schematic of the possible F-D spectrum during the retraction of a bound BAR domain from a lipid bilayer, wherein the individual fluctuations in the F-D spectrum may reveal the binding strength of different sub-domains within the BAR domain. The integral of the force over the distance could give the binding energy. We note that the area under the F-D curve depends on the loading rate, thus the effect of time and rate constants during the rupture of particular intermolecular Fig. (8). The self-assembly of aggregates of attached BAR proteins bonds should be taken into account in a theoretical model into a spiral in large densities of BAR proteins. [57-58]. The BAR domain is of a crescent-shape dimer, where The proposed use of single molecule AFM technique to each monomer is composed of three alpha helices. The measure the energy landscape of binding BAR domains to a curvature of the BAR domain could be due to the kinks in lipid bilayer holds many advantages over other experimental alpha helices 2 and 3. It has been suggested that the large methods. The first advantage is the ability to determine the patches of basic amino acids on the concave side of the binding energy of a lipid bilayer and an individual BAR dimer are attracted to negatively charged cell membrane domain in their native state. The second advantage is that the lipids [13]. In agreement, an increase in the salt experimental set-up can be modified to investigate the concentration has been shown to screen the electrostatic consequences of changes in the lipid bilayer, such as the interactions with the membrane thereby decreasing the membrane curvature, and the lipid bilayer composition and binding rate of BAR domains [31]. On the other hand, shape. Other general features of the technique itself are the changes in the content of negatively charged lipids due to capacity to sense specific interactions, such as the lipid demixing may further increase the strength of contribution of hydrophobic insertion loops at the nano scale electrostatic interactions [31-33]. resolution of 10 nm, and the capacity to detect forces over a wide range from 5 pN to 100 nN. To reveal the strength of intermolecular bondings of an individual BAR domain with a lipid bilayer, we here propose In addition to rigid and flexible limits of rod-like a novel experiment that uses single molecule AFM proteins, the adhesion to the cell membrane could be strong techniques [50-55] to measure the force-distance (F-D) or weak. In the limit of strong adhesion, the mobility of the spectrum of a BAR domain bound to a lipid bilayer (Fig. 9). BAR domain could be low and its main function would be in The underlying assumption is that the unbinding dynamics of the stabilization of highly curved regions. Whereas, in the a BAR domain from the lipid bilayer is similar to the binding limit of weak adhesion, the mobility of the BAR domain dynamics but opposite in the temporal direction. Therefore, could be high facilitating intricate membrane dynamics such the force trace of the unbinding obtained in the following as vesiculation during endocytosis and exocytosis. While the experiment could unravel the interactions seen during the present review focused on BAR domains, the theory and binding of a BAR domain. The technique used in the experiments suggested herein would also apply for drug proposed experiment has been employed in adifferent study molecules which target the cell membrane. For example, the [56], in which the terminal end of a single transmembrane anisotropic nature of drug molecules that are incorporated protein (NhaA) was linked to a stylus bound to an AFM into the cell membrane would to some extent bend the cell cantilever. By pulling the stylus, the intra membrane membrane, which may cause their aggregation into domains of helical shapes are released one after the other. membrane protrusions or invaginations depending on their The F-D spectrum demonstrates that the pulling of each intra intrinsic curvature. Thus, the design of drug molecules with membrane domain out of the membrane would immediately specific intrinsic curvature may increase their specificity to unfold the helical structure causing a sharp decline in the targeted cell and organelle membranes of different pulling force and roughly no change in the pulling distance. curvatures and shapes. These fluctuations in the F-D spectrum may then reveal the To generate membrane bending, the binding energy interaction energies of intra-membrane domains. gained due to the electrostatic and hydrophobic interactions 280 Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 4 Kabaso et al. Fig. (9). Schematic diagram of a possible single molecule AFM experiment employed to measure the binding energy between a BAR domain and a negatively charged lipid bilayer. The stylus of an AFM setup is attached to the N-terminus of a BAR domain bound to a lipid bilayer (a). The AFM stylus is retracted from the lipid bilayer (b). The BAR domain is completely detached from the lipid bilayer (c). The obtained force distance (F-D) spectrum (hypothetical data) can be used to estimate the binding energy of a BAR domain (d). The possible fluctuations in the F-D spectrum could be due to hydrophobic and electrostatic interactions as described in Fig. (2) and corresponding text. should be larger than the energy costs required to bend the REFERENCES membrane [13,19]. Otherwise, the BAR domain could only [1] Farsad, K.; Ringstad, N.; Takei, K.; Floyd, S.; Rose, K.; Camilli, bind highly curved regions thereby functioning as a P.D. Generation of high curvature membranes mediated by direct membrane curvature sensor. An additional domain such as endophilin bilayer interactions. J. Cell Biol., 2001, 155, 193-200. PH (phosphoinositide-binding structural domain) or PX [2] Tarricone, C.; Xiao, B.; Justin, N.; Walker, P.; Rittinger, K.; (Pleckstrin homology domain) would confer the binding to Gamblin, S.; Smerdon, S. The structural basis of Arfaptin-mediated cross-talk between Rac and Arf signalling pathways. Nature, 2001, specific lipids in the cell membrane or involve in 411, 215-219. intracellular signalling, respectively. The possible coupling [3] Itoh, T.; Erdmann, K.S.; Roux, A.; Habermann, B.; Werner, H.; between membrane curvature and intracellular signaling Camilli, P.D. Dynamin and the actin cytoskeleton cooperatively could be taken into advantage for drug application, whereby regulate plasma membrane invagination by BAR and F-BAR proteins. Dev. Cell, 2005, 23, 791–804. the chimera of a BAR protein and a drug may increase the [4] Zimmerberg,Y.; Kozlov, M. How proteins produce cellular drug spatial specificity. curvature. Nat. Rev. Mol. Cell. Biol., 2006, 7, 9-19. [5] Veksler, A.; Gov, N.S. Phase transitions of the coupled membrane- Previous studies have shown that BAR protein cytoskeleton modify cellular shape. Biophys. J., 2007, 93, 3798– dysfunction may account for human diseases [8-12], while 3810. the underlying mechanisms are not clear. The suggested [6] Frost, A.; Unger, V.; Camilli, P.D. The BAR Domain Superfamily: novel AFM experiment could reveal how observed point Membrane-Molding Macromolecules. Cell, 2009, 137, 191-196. [7] Wang, Q.; Navarro, M.V.; Peng, G.; Molinelli, E.; Lin-Goh, S.; mutations affect the binding energy landscape and dynamics. Judson, B.L.; Rajashankar, K.R.; Sondermann, H. Molecular To conclude, we believe that the proposed experiments and mechanism of membrane constriction and tubulation mediated by discussed theory could help to prepare the ground for future the F-BAR protein Pacsin/Syndapin. Proc. Natl. Acad. Sci. USA, studies revealing the role of BAR proteins in the bending and 2009, 106, 12700–12705. curvature sensing of cell membranes. [8] Fuchs, U.; Rehkamp, G.; Haas, O.; Slany, R.; Konig, M.; Bojesen, S.; Bohlei, R.M.; Damm-Welk, C.; W. Ludwig; Harbott, J.; ACKNOWLEDGEMENTS Borkhardt, A. The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner This work was supported by ARRS grants J3-9219-0381, in acute myelogeneous leukemia. Proc. Natl. Acad. Sci. USA, 2001, P2-0232-1538, and by DFG for the project A3 in Research 98, 8756-8761. [9] Tsujia, E.; Tsujia, Y.; Fujiwarac, T.;Ogatad, S.; Tsukamotob, K.; Training group 1505/1 "welisa". A.I. is grateful to (cid:3)arka Sakub, K. Splicing variant of Cdc42 interacting protein-4 disrupts Perutkova for all the help and to Adi Pick from M. Kozlov beta-catenin-mediated cell cell adhesion: expression and function group for useful discussion. Attachment Dynamics of BAR Proteins Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 4 281 in renal cell carcinoma. Biochem. Biophys. Res. Commun. 2006, interaction: The BAR dimer adsorption. Biophys. J., 2009, 97, 339, 1083-1088. 1626–1635. [10] Modregger, J.; DiProspero,N.; Charles, V.; Tagle, D.; Plomann, M. [34] Gongadze, E.; van Rienen, U.; Kralj-Igli(cid:9), V., Igli(cid:9), A. Langevin PACSIN1 interacts with huntingtin and is absent from synaptic Poisson-Boltzmann equation. Gen. Physiol. Biophys., 2011, 30(2), varicosities in presymptomatic Huntington's disease brains. Hum. in print. Mol. Genet. 2002, 11, 2547-2558. [35] Gongadze, E.; Bohinc, K.; van Rienen, U.; Kralj-Igli(cid:9), A. Spatial [11] Holbert, S.; Dedeoglu, A.; Humbert, S.; Saudou, F.; Ferrante, R.; variation of permittivity near a charged membrane in contact with Neri, C. Cdc42-interacting protein 4 binds to huntingtin: electrolyte solution. In Advances in Planar Lipid Bilayers and neuropathologic and biological evidence for a role in Huntington's Liposomes (APLBL).; Elsevier, 2010; Vol. 11. disease. Proc. Natl. Acad. Sci. USA, 2003, 100, 2712-2717. [36] Nossal, R. Energetics of clathrin basket assembly. Traffic, 2001, 2, [12] Endris, V.; Wogatzky, B.; Leimer, U.; Bartsch, D.; Zatyka, M.; 138–147. Latif, F.; Maher, E. R.; Tariverdian, G.; Kirsch, S.; Karch, D.; [37] Landau, L.; Lifshitz, E. Theory of Elasticity; Butterworth- Rappold, G. The novel Rho-GTPase activating gene Heinemann, Oxford, 1996; Vol. 2nd ed., pp 67-70. MEGAP/srGAP3 has a putative role in severe mental retardation. [38] Perutková, S.; Kralj-Igli(cid:9), V.; Frank, M.; Igli(cid:9), A. Mechanical Proc. Natl. Acad. Sci. USA 2002, 99, 11754-11759. stability of membrane nanotubular protrusions influenced by [13] Peter, B. J.;Kent, H. M.; Mills, I. G.;Vallis,Y.; Butler,P. J.; attachment of flexible rod-like protein. J. Biomech., 2010, 43, Evans,P. R.; McMahon, H.T. BAR domains as sensors of 1612–1617. membrane curvature: the amphiphysin BAR structure. Science [39] Perutková, S.; Daniel, M.; Rappolt, M.; Pabst, G.; Dolinar, G.; 2004, 303, 495–499. Kralj-Igli(cid:9), V.; Igli(cid:9), A. Elastic deformations in hexagonal phases [14] Scita, G.;Confalonieri, S.; Lappalainen, P.; Suetsugu, S. IRSp53: studied by small angle x-ray diffraction and simulations. Phys. crossing the road of membrane and actin dynamics in the formation Chem., 2011, 13, 3100–3107. of membrane protrusions. Trends in Cell Biol., 2008, 18, 52–60. [40] Perutková, S.; Frank, M.; Bohinc, K.; Bobojevi(cid:9), V.; Rozman, B.; [15] Ayton, G.; Lyman, E.; Krishna, V.; Swenson, R.; Mim, C.; Kralj-Igli(cid:9), V.; Igli(cid:9), A. Interaction between equally charged Unger,V.; Voth, G. New insights into BAR domain-induced membrane surfaces mediated by positively and negatively charged membrane remodeling. Biophys. J., 2009, 97, 1616–1625. macroions. J. Membr. Biol., 2010, 236, 43–53. [16] Itoh, T.; Camilli, P.D. BAR, F-BAR (EFC) and ENTH/ANTH [41] Helfrich,W. Elastic propertiesoflipid bilayers: theoryand domains in the regulation of membrane-cytosol interfaces and possibleexperiments. Z. Naturforsch.C, 1973, 28, 693–703. membrane curvature. Biochim. Biophys. Acta, 2006, 1761, 897– [42] Kralj-Igli(cid:9), V.; Babnik, B.; Gauger, D.; May, S.; Igli(cid:9), A. 912. Quadrupolar ordering of phospholipid molecules in narrow necks [17] Heath, R. J.W.; Insall, R.H. F-BAR domains: multifunctional of phospholipid vesicles. J. Stat. Phys., 2006, 125, 727–752. regulators of membrane curvature. J. Cell Sci., .2008, 121, 1951– [43] Igli(cid:9), A.; Hägerstrand, H.; Verani(cid:9), P.; Plemenita(cid:6), A.; Kralj-Igli(cid:9), 1954. V.; Curvature induced accumulation of anisotropic membrane [18] Shimada, A.; Takano, K.; Shirouzu, M.; Hanawa-Suetsugu, K.; components and raft formation in cylindrical membrane Terada, T.; Toyooka, K.; Umehara, T.; Yamamoto, M.; Yokoyama, protrusions. J. Theor. Biol., 2006, 240, 368-373. S.; Suetsugu, S. Mapping of the basic amino-acid residues [44] Tian, A.; Baumgart, T. Sorting of Lipids and Proteins in Membrane responsible for tubulation and cellular protrusion by the EFC/F- Curvature Gradients. Biophys. J., 2009, 96, 2676–2688. BAR domain of pacsin2/syndapin ii. FEBS Lett, 2010, 584, 1111– [45] Kralj-Igli(cid:9), V.; Hägerstrand, H.; Verani(cid:9), P.; Jezernik, K.; Igli(cid:9), A. 1118. Amphiphile induced tubular budding of the bilayer membrane. [19] Zimmerberg, J.; McLaughlin, S. Membrane curvature: How BAR Europ. Biophys. J., 2005, 34, 1066–1070. domains bend bilayers. Curr. Biol., 2004, 14, 250-252. [46] Sorre, B.; Callan-Jones, A.; Manneville, J.; Nassoy, P.; Joanny, J.; [20] Igli(cid:9), A.; Slivnik, T.; Kralj-Igli(cid:9), V. Elastic properties of biological Prost, J.; Goud, B.; Bassereau, P. Curvature-driven lipid sorting membranes influenced by attached proteins. J. Biomech., 2007, 40, needs proximity to a demixing point and is aided by proteins. 2492–2500. PNAS, 2009, 106, 5622-5626. [21] Shimada, A.; Niwa, H.; Tsujita, K.; Suetsugu, S.; Nitta, K.; et al., [47] Ambro(cid:7)i(cid:9), A.; (cid:8)u(cid:9)nik, S.; Tom(cid:6)i(cid:9), N.; Urbanija, J.; Lokar, M.; Curved EFC/F-BAR domain dimers are joined end to end into a Babnik, B.; Rozman, B.; Igli(cid:9), A.; Kralj-Igli(cid:9), V. Interaction of filament for membrane invagination in endocytosis. Cell , 2007, giant phospholipid vesicles containing cardiolipin and cholesterol 129, 761–772. with (cid:10)2-glycoprotein-I and anti-(cid:10)2-glycoprotein-I antibodies. [22] Langer, G.A. The structure and function of the myocardial cell Autoimmun. Rev. 2006, 6, 10–15. surface. AJP -Heart and Circ. Phys., 1978, 235, 461–468. [48] Urbanija, J.; Tom(cid:6)i(cid:9), N.; Lokar, M.; Ambro(cid:7)i(cid:9), A.; (cid:8)u(cid:9)nik, S.; [23] Israelachvili, J. Intermolecular and Surface Forces,; Academic Rozman, B.; Kandu(cid:6)er, M.; Igli(cid:9), A.; Kralj- Igli(cid:9), V. Coalescence Press, San Diego., 1995. of phospholipid membranes as a possible origin of anticoagulant [24] Rand, R.; Parsegian, V. Hydration Forces Between Phospholipid effect of serum proteins. Chem. Phys. Lipids, 2007, 150, 49–57. Bilayers. Biochim. Biophys. Acta, 1989, 988, 351. [49] Lokar, M.; Urbanija, J.; Frank, M.; Hägerstrand, H.; Rozman, B.; [25] Leckband,D. Force as a probe of membrane protein structure and Bobrowska-Hägerstrand, M.; Igli(cid:9), A.; Kralj-Igli(cid:9), V. function. Curr. Opin. Colloid Interface Sci., 2001, 11, 433-439. Agglutination of like-charged red blood cells induced by binding of [26] Leikin, S.; Parsegian, V.; Rau, D.; Rand, R. Hydration forces. 2-glycoprotein I to outer cell surface. Bioelectrochemistry, 2008, Annu. Rev. Phys. Chem., 1993, 44, 369. 73, 110-116. [27] McIntosh,T.; Simon, S. Hydration and steric pressures between [50] Binnig, G.; Quate, C.; Gerber, C. Atomic force microscope. Phys. phospholipid bilayers. Biochemistry, 1993, 32, 8374. Rev. Lett., 1986, 56, 930-933. [28] Igli(cid:9), A.; Gongadze, E.; Bohinc, K. Excluded volume effect and [51] Lee, G.; Kidwell, D.; Colton, R. Sensing discrete streptavidin- orientation ordering near charged surface in solution of ions and biotin interactions with atomic force microscopy. Langmuir, 1994, langevin dipoles. Bioelectrochemistry, 2010, 79, 223–227. 10, 354-357. [29] Bruinsma, R.; Behrisch, A.; Sackmann, E. Adhesive switching of [52] Moy, V.; Florin, E.; Gaub, H. Intermolecular forces and energies membranes: experiment and theory. Phys. Rev.E, 2000, 61, 4253. between ligands and receptors. Science, 1994, 266, 257-259. [30] Blood, P.; Voth, G. Direct observation of bin/amphiphysin/rvs [53] Fritz, J.; Katopodis, A.; Kolbinger, F.; Anselmetti, D. Force- (BAR) domain-induced membrane curvature by means of mediated kinetics of single P selectin/ligand complexes observed molecular dynamics simulations. PNAS, 2006, 103, 15068– 15072. by atomic force microscopy. Proc. Natl. Acad. Sci. USA, 1998, 95, [31] Heimburg, T.; Angerstein, B.; Marsh, D. Binding of peripheral 12283-12288. proteins to mixed lipid membranes: effect of lipid demixing upon [54] Helenius, J.; Heisenberg, C.; Gaub, H.; Muller, D. Single-cell force binding. Biophys. J., 1999, 76, 2575–2586. spectroscopy. J. Cell Sci., 2008, 121, 1785-1791. [32] Fo(cid:6)nari(cid:9), A.; Igli(cid:9), A., Kroll, D.; May, S. Monte Carlo simulations [55] Neuman, K.; Nagy, A. Single-molecule force spectroscopy: optical of complex formation between a mixed fluid vesicle and a charged tweezers, magnetic tweezers and atomic force microscopy. Nat. colloid. J. Chem. Phys., 2009, 131, 105103. Methods, 2008, 5, 491-505. [33] Khelashvili, G.; Harries, D.; Weinstein, H. Modeling membrane deformations and lipid demixing upon protein-membrane