Helix-helix interactions in membrane domains of bitopic proteins: Specificity and role of lipid environment

    Helix-helix interactions in membrane domains of bitopic proteins: Specificity and role of lipid environment Eduard V. Bocharov, Konstantin S. Mineev, Konstantin V. Pavlov, Sergey A. Akimov, Andrey S. Kuznetsov, Roman G. Efremov, Alexander S. Arseniev PII: DOI: Reference:

S0005-2736(16)30370-4 doi:10.1016/j.bbamem.2016.10.024 BBAMEM 82356

To appear in:

BBA - Biomembranes

Received date: Revised date: Accepted date:

30 June 2016 18 September 2016 20 October 2016

Please cite this article as: Eduard V. Bocharov, Konstantin S. Mineev, Konstantin V. Pavlov, Sergey A. Akimov, Andrey S. Kuznetsov, Roman G. Efremov, Alexander S. Arseniev, Helix-helix interactions in membrane domains of bitopic proteins: Specificity and role of lipid environment, BBA - Biomembranes (2016), doi:10.1016/j.bbamem.2016.10.024

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ACCEPTED MANUSCRIPT Helix-helix interactions in membrane domains of bitopic proteins: specificity and role of lipid environment Eduard V. Bocharova,c*, Konstantin S. Mineeva, Konstantin V. Pavlovb, Sergey A. Akimovb,d, Andrey S. Kuznetsova, Roman G. Efremova,e and Alexander S. Arsenieva* a

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Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya ul. 16/10, Moscow, 117997, Russian Federation; b Frumkin Institute of Physical Chemistry and Electrochemistry RAS, Leninskiy prospect 31/5, Moscow, 119071, Russian Federation; c National Research Centre "Kurchatov Institute", Akad. Kurchatova pl. 1, Moscow, 123182, Russian Federation; d National University of Science and Technology “MISiS”, Leninskiy prospect 4, Moscow, 119049, Russian Federation; e Higher School of Economics, Myasnitskaya ul. 20, Moscow, 101000, Russian Federation. *

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Corresponding authors at: Dept. of Structural Biology, IBCh RAS, Str. Mikluho-Maklaya, 16/10, Moscow, Russian Federation, 117997. Tel.: +7(495)330-74-83. E-mail addresses: [email protected] (E.V. Bocharov), [email protected] (A.S. Arseniev).

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ABSTRACT

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Interaction between transmembrane helices often determines biological activity of membrane proteins. Bitopic proteins, a broad subclass of membrane proteins, form dimers containing two membrane-spanning helices. Some aspects of their structure-function relationship cannot be fully understood without considering the protein-lipid interaction, which can determine the protein conformational ensemble. Experimental and computer modeling data concerning transmembrane parts of bitopic proteins are reviewed in the present paper. They highlight the importance of lipid-protein interactions and resolve certain paradoxes in the behavior of such proteins. Besides, some properties of membrane organization provided a clue to understanding of allosteric interactions between distant parts of proteins. Interactions of these kinds appear to underlie a signaling mechanism, which could be widely employed in the functioning of many membrane proteins. Treatment of membrane proteins as parts of integrated fine-tuned proteolipid system promises new insights into biological function mechanisms and approaches to drug design. Highlights 1. Activation of bitopic proteins is described with a focus on the membrane domain role 2. Structural data on the dimeric membrane domains of bitopic proteins is reviewed 3. Effects of membrane environment on the activation of bitopic proteins are discussed 4. Bitopic protein signaling and allostery can be modulated by protein-lipid interaction Keywords: bitopic membrane protein; receptor tyrosine kinase; transmembrane domain; protein-lipid and protein-protein interactions; lipid density fluctuations; signal transduction. Abbreviations: BP, bitopic protein; RTK, receptor tyrosine kinase; TMD, transmembrane domain; JM, juxtamembrane; ECD, extracellular domain; ICD, intracellular domain; NMR, nuclear magnetic resonance; MD, molecular dynamics; LID ―ligand-induced dimerization‖ mechanism; LIR, ―ligand-induced rotation‖ mechanism.

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ACCEPTED MANUSCRIPT 1. Plasma membrane as a venue for membrane-protein mediated functions

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Biological membranes form the boundaries of living cells and organelles therein, performing a broad range of vital functions, providing compartmentalization and serving as a venue for multiple enzymatic reaction and electrochemical processes. The fluid-mosaic model of the membrane originally proposed by Singer and Nicolson back in early 1970-s [1,2], though still universally recognized at the conceptual level, has since then been substantially refined and augmented. Multiple lines of experimental evidence revealed that different levels of structural asymmetry and inhomogeneity exist in the membrane, including lipid distribution between leaflets, lateral lipid distribution and organization, and interactions between lipidic and non-lipidic components. Besides the liquid-crystalline lamellar phase, lipid molecules in the membrane form other phases, coexisting in dynamic equilibrium under physiologically relevant conditions. These phases are characterized by different degree of ordering of the lipid hydrophobic tails, membrane thickness, elastic moduli and other parameters. This provides the necessary diversity of environments needed for proper functioning of a large array of membrane proteins coexisting in the same cell to maintain its homeostasis. The hypothesis that the lipid subsystem of the plasma membrane may be phase-separated under physiological conditions was explicitly formulated in 1997 by Simons and Ikonen [3]. It was assumed that microdomains enriched with saturated tail lipids and cholesterol can coexist with the phase rich in unsaturated lipids. The hypothesis is supported by experimental observations that macroscopic domains can be induced by temperature drop in model membranes with the composition resembling that of the outer leaflet of the plasma membranes [4–6]. In experiments with model systems, the domains were found to be in a liquid state, which is however more ordered than the state of the surrounding membrane, and is therefore referred to as ―liquid-ordered‖ state. Existence of the domains at physiological temperatures is generally recognized, though the details are still debated. In the experiments with dequenching of fluorescently labeled lipids it was shown that domains of the size comparable to the Foerster radius (5 nm) started to form exactly at T  37 C [7]. Nevertheless, according to [8], the critical temperature of the plasma membrane is about 20 C, and hence no phase separation appears to be possible at physiological temperatures. However, in [8] the domains were visualized optically, i.e. the resolution is determined by light diffraction limit and the irregularities of the size smaller than hundreds of nanometers could not be detected. Besides, even if the physiological temperature is too high for complete phase separation of the lipid subsystem, microdomains of the ordered phase can still form locally around membrane proteins by means of a phenomena known as wetting [9,10]. Moreover, the membrane proteins constitute a substantial part of the membrane surface and the membrane structure itself is a dynamic product of concerted function of proteins. However, the role of the membrane lipids with respect to membrane proteins is far more complex than merely providing adaptable environment that enables their functioning. In fact, the diversity of lipid species is at least comparable with the diversity of membrane proteins. The Lipid Maps Structure Database (LMSD, http://lipidmaps.org/data/structure/) of biologically relevant lipids contains an astounding number of over 40,000+ unique lipid structures, which greatly exceeds the number of known membrane proteins (see below). Lipids are known to specifically bind membrane proteins; according to a recent review on non-covalent interaction of membrane proteins with lipids [11], over 100 of the currently available structures of membrane proteins contain electronic densities attributable to bound lipid molecules. A proteome-wide analysis of cholesterol-binding proteins [12] revealed 250 proteins binding cholesterol, including various enzymes, channels and receptors that bind the lipid via their transmembrane domains (TMD). A far-reaching potential functional implication of such binding is prompted by the fact that in many cases (e.g., for amyloid precursor protein and protein receptor kinases [13–15]) it occurs via the so-called GxxxG motif (see below) often involved in helix-helix oligomerization, with which cholesterol binding can compete in a concentration-dependent manner. Specific binding of lipids is often implicated in allosteric regulation, and is sometimes a prerequisite for proper function of the protein. Another aspect of plasma membrane organization, understanding of which evolved notably based on the accumulated information is the amount of restraints imposed on the membrane constituents by interactions with membrane proteins, cytoskeleton or extracellular matrix. The lipid coat of membrane

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proteins includes a belt of annular lipids defining intimate environment of the membrane proteins, and nonannular lipids interacting with them in a more specific manner [15]. According to the current knowledge of the share of non-lipidic components in the membrane composition, only a small fraction of lipid in the membrane is unaffected by some kind of interaction with proteins or polysaccharides. It is convincingly illustrated by the example of a synaptic vesicle, protein and lipid composition of which was studied in [16], that with the transmembrane helix-to-lipid ratio of about 20 the TMDs occupy over a quarter of the membrane surface area, and with some lipids being tightly bound to the protein surface little is left of the concept of a two-dimensional fluid of lipids, which are free to diffuse and dissolve other membrane components. The role of lipids in large-scale spatial organization of proteins in the membrane is illustrated by a recent example of syntaxin 1A clustering induced by phosphatidylinositol-triphosphate (PIP3) [17], the effect being abolished by mutation of two neighboring lysine residues. On a broader scale, lateral inhomogeneity of lipid distribution and membrane properties, and in particular – formation of nanoscopic domains of liquid-ordered phase, has been demonstrated to be vital for many protein functions. For proteins involved in signaling [18], apoptosis [19] and endocytosis [3], collective recruitment of all the key players of the process into small platforms is a common mechanism. Proper folding and functioning of transmembrane proteins are affected by a large number of membrane parameters, such as thickness, fluidity, elasticity moduli, spontaneous curvature of the constituent lipids, lateral tension/pressure effects, water permeation, packing density, electrical charge density distribution and dielectric permittivity. At the present post-genomic stage of development of structural biology, spatial structures of various proteins are being accumulated in an attempt to find generic and specific structure-function interrelations. Protein Data Bank (PDB) presently contains more than 100 000 structures of soluble proteins, and modern force fields allow almost exact reproduction of not only spatial structure but also dynamic behavior of soluble proteins. On the contrary, membrane proteins are not studied that thoroughly. There are only ~3000 structures in PDB, which are annotated as membrane proteins, and a large fraction of them are actually the structures of separate water-soluble domains of membrane proteins. The capabilities of molecular dynamics (MD) with respect to the membrane proteins are also reduced. Therefore, structural data on the architecture of the membrane domains of integral membrane proteins is of the great interest and importance. The functional and structural importance of lipid interactions with large polytopic proteins is widely recognized and extensively discussed in many publications. In contrast, specific binding of lipids by individual transmembrane helices of bitopic proteins (BP) and regulation of conformational and monomer-oligomer equilibrium by lipid environment are relatively new concepts that are only beginning to find experimental and theoretical bases. This is attributable distinct properties of these proteins that limit applicability of crystallographic methods to their structure investigation. These proteins have large extramembrane domains, and only a single transmembrane α-helical segment. A vast majority of BPs are cell receptors, in which the information on the state of the system outside the cell is transferred inside via a very small TMD. The specified architecture of BPs complicates their crystallization: large extra- and intracellular domains and presence of flexible domain-connecting regions do not allow tight and stable crystallographic contacts to be formed. Another distinctive feature of a majority of BPs is weakness of self-association and interaction with other membrane proteins. For these reasons, spatial structures of only separate domains of BPs were described until recently, and the full-size receptors were studied only by the cryo-electron microscopy with resolution as low as c.a. 3 nm [20], which is definitely not enough to study the structural basis of the activity of BPs in atomistic details. Most of the available structural information on these proteins is obtained with the aid of NMR and computer modeling, with the exception of the recently obtained crystal dimeric structure of the isolated TMD of glycophorin A (GpA) [21], which is distinguished by exceptionally strong dimerization. Moreover, the role of TMDs and juxtamembrane (JM) regions in the protein function had been largely underestimated until the last decade, probably for the same reasons. It is becoming increasingly clear that despite apparent flexibility of the JM regions, they often deterministically respond to conformational transitions in the transmembrane part, thus mediating the protein function. Such a behavior cannot be reasonably explained exclusively by protein-protein interactions without careful consideration of interplay between protein and lipids on different levels. This is not limited to specific binding of lipids, but also

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ACCEPTED MANUSCRIPT includes spatially and/or temporally localized changes of physico-chemical parameters and membrane composition. Such interactions stabilize functionally relevant protein conformations, modulate dimerization or oligomerization modes, and cause functionally related proteins to cluster. Disregarding these types of interactions appears to make the existing information about structure and function of certain proteins look very counterintuitive, if not paradoxical.

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2. Bitopic membrane proteins as cell receptors

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A large class of typical representatives of BPs consists of cell receptors, which are extensively studied as potential therapeutic targets. They recognize hormones, growth factors, cytokines and various pathogens participating in regulation of proliferation, differentiation, migration, survival, death and metabolic activity of eukariotic cells. Almost all BPs are not functional in the monomeric form, requiring formation of homomeric or heteromeric complexes [22–27]. Intracellular domains (ICDs) of BPs typically respond to ligand recognition by catalytic activity or bind effector proteins that launch downstream signaling cascades. Most common types of ICDs are tyrosine kinases (in receptor tyrosine kinases, RTKs), serine/threonine kinases (receptors for the transforming growth factor β superfamily, RSTKs), tollinterleukine like domains (TIR, in toll-like receptors, TLRs), death domains (tumor necrosis factor receptor superfamily members, TNFRs), guanylyl cyclases (e.g. Natriuretic peptide receptor A), intrinsically disordered polypeptide regions associated with cytoplasmic Janus kinases (hormone receptors, e.g. erythropoietin and growth hormone receptors). Extracellular domains (ECDs) are responsible for recognition and binding of the ligands. Based on the type of ligands, BPs are divided into subfamilies, such as HER (human epidermal growth factor receptors), FGFR (fibroblast growth factor receptors), PDGFR (plateletderived growth factor receptors), VEGFR (vascular endothelium growth factor receptors), EphR (ephrin receptors) and many others. There are also other BPs, which do not fall into this classification. Some do not have any structured ECD, such as the subunits of T-cell and B-cell receptors or other co-receptors like NRADD; and others do not have any globular ICD, e.g. GpA, integrins or Amyloid precursor protein. Among other BPs, RSTKs and RTKs, and especially the members of HER [28,29], FGFR, EphR and VEGFR families, are the most studied, because they are involved in a number of socially important diseases, including cancer. The first model proposed for activation of BP cell receptors was the so-called ―ligand-induced dimerization‖ (LID) mechanism [30–32]. According to this model, proteins exist as monomers on the cell surface and dimerize upon ligand binding. However, a growing number of examples of BPs that can form dimers in the absence of ligands has been appearing lately [24,33,34]. These ligand-free dimers may be inactive and not lead to the signaling, wherefore they are referred to as pre-dimers. For example, p75NTR and insulin receptor (INSR) are covalent dimers formed via a disulfide bond [24,35]. Cross-linking [36] and fluorescence [37–41] studies revealed that EGFR and HER2 receptors are dimeric in the absence of bound ligand at physiological expression levels, and this is the cases for the majority if not all members of the HER family [33]. Mutagenesis data show that the dimerization of Neu receptors is not enough for the activation; specific orientation of TMDs is required [42]. Furthermore, single molecule observations also reveal presence of EGFR dimers in the living cells in the absence of EGF [43], and, moreover, according to luciferase fragment complementation assays, the dimer fraction is not increased upon addition of EGF, implying that in the absence of a ligand the majority of EGFR already exist in the form of pre-dimers [44,45]. However, some controversy still exists on the subject matter, e.g., a recent work by Yamashita et al. claims that only a small fraction of the total EGFR population in cell is in the dimeric state and EGF binding substantially increases this ratio [46]. Existence of inactive pre-dimers was also observed for some other RTKs and JAK2 kinase associated receptors, such as FGFRs [47], Trk [48,49], VEGFR2 [50], GHR [51], and EpoR [52]. For that reason, an alternative mechanism was suggested for the BPs of the RTK family, which is usually referred to as ―ligand-induced rotation‖ (LIR) [36,53,54] or ―pre-dimerization‖ mechanism. According to this model, receptors exist in the monomer-dimer equilibrium in the absence of ligand. Ligand binding causes interaction of ECDs in the dimeric complex and rearrangement of the TMDs and ICDs, which eventually triggers trans-phophorylation and launches the downstream cascade. On the other hand, it is

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important to note that the LIR mechanism is not the paradigm for all BPs. For instance, the interferon receptors (IFNAR) are known to act via the LID pathway [55]. In any case, for many RTKs the existence of another, non-active dimeric state has been positively established. Therefore, the information about ligand binding needs to somehow be transferred across the membrane to induce a change of conformation of the dimeric ICDs. It appears that there is only a small two-helical (in dimer) TMD available for achieving this purpose, and the TMD is usually connected to the intracellular and extracellular domains via rather flexible JM segments, the length of which can be varied to some extent without loss of the signaling function [56,57]. Transmembrane signal transduction in such a system can seem especially paradoxical taking into account the apparently loose coupling between the sequence of TM domains and RTK activation, structural heterogeneity of the pre-formed ECD dimers, and general lack of correlation between ligand binding and stabilization of the EGFR dimers [58–60]. However, there is yet another potential player in the signal transduction process that has been neglected so far – the lipid bilayer itself, a multi-component, dynamic and spatially inhomogeneous system that can undergo local structural rearrangements in response to external impacts, i.e. effectively transduce signals on its own. It therefore appears especially important to take into account all the aspects of subtle bilateral interplay between the components of the cellular membrane and the TMDs and JM regions of BPs.

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3. Involvement of the membrane regions in the function of bitopic proteins

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For the past 20 years, the question whether the TMD is a simple anchor necessary for correct positioning of a BP on the cell surface or an active player in the BP signaling had been in the focus of many investigations. In the context of the LID mechanism, TMDs are needed only to provide the necessary environment for the other domains of a BP: the dimerization of ECDs should automatically trigger the activation of ICDs regardless the conformation of the transmembrane part of the protein. On the contrary, if the LIR concept is accepted, TMDs should have at least two stable dimeric conformations, and it is the switch between the conformations that alters the functional state of the ICD complex. Nowadays, there is a vast amount of data proving significance of the TMDs for the signaling of BPs of various classes. First of all, isolated TMDs of most BPs reveal the propensity to self-associate in model membranes and in-vivo. It was shown by a variety of techniques including ultracentrifugation in detergents [61,62], solution NMR [63], FRET in detergents and liposomes [64–69], FRET in mammalian membranes [70–72] and by TOXCAT/TOXRED assays in bacterial membranes for all members of RTK [73] and TLR families [74]. Furthermore, addition of the TMD to the kinases of EGFR dramatically enhances their activity [75] and isolated TMDs can inhibit the full-size receptors [76]. This was ascribed to the ability of the TMDs to interact specifically with the TMD of the full-size receptor, which hinders the formation of both the active and the inactive dimers, if the latter do exist. Involvement of TMD in the function of BPs is also illustrated by a number of activating or inhibiting mutations, both naturally occurring (usually pathogenic) and artificial, in the transmembrane sequences of HER [77–81], EphR [79], FGFR [82] and VEGFR [83] members. Most of these mutations consist of substitutions of hydrophobic or weakly polar residues (Gly, Val, Ile) to the residues with ionogenic or highly polar sidechains (Glu, Gln, Arg). The mutations were shown not to affect the orientation of the isolated TMDs [72,78,84], moreover, some of them were shown to have no influence on the free energy of the transmembrane helix-helix interaction, but rather change the mutual arrangement of the TMD in a dimer [72,83,85]. Additionally, several polymorphisms in the TMDs of RTK members that increase the risk of cancer or other pathologies are documented [80,86]. Some recent works utilized the scanning mutagenesis approach to demonstrate that X/Glu substitutions at certain positions of the VEGFR2 TMDs or artificial TMDs in the context of wild-type receptor can cause the spontaneous activation of the protein with deleted ECD. The same work revealed that the introduction of two Glu sidechains separated by 7-8 residues on the certain face of the TMD helix of VEGFR2 results in the intrinsically active full-size receptors in the absence of the ligand [83,87]. These studies demonstrate clearly, that some BPs have at least one helix-helix interaction mode that favors activation of the ICDs. Conversely, the existence of inhibitory TMD mutation of EGFR implies that there is an interaction mode precluding ICD activation.

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Aside from the TMDs, JM regions of some BPs were also shown to be important for their function. Deletion of the short cytoplasmic hydrophobic region of TLR4 receptor alters substantially its ability to form high-order oligomers and prevents its activation [88]. ―Chopper‖ domain, which is a cytoplasmic juxtamembrane region of p75NTR was proved to be highly important for the receptor signaling [89,90]. A number of recent studies [75,91] revealed the significance of the cytoplasmic juxtamembrane-A (JM-A) regions of EGFR. These regions form short amphiphilic helices, which are thought to reside on the surface of the cell membrane [92]. The presence of JM-A enhanced the catalytic activity of the isolated kinase domains of EGFR [91], whereas substitutions of arginine residues in JM-A of EGFR/HER1 caused substantial reduction in receptor activity [75]. Analysis of the JM-A sequence suggests that the small domains form antiparallel dimer in the active state of the receptor. Later these assumptions were supported by direct structural data obtained for TMDs of EGFR and HER2 in junctions with their JM-A regions in model membrane-like systems and by computer simulations [57,93,94]. These facts suggest that the role played by the TMDs in the functioning of BPs has to be analyzed on the structural level taking into account the presence of the JM regions. Indeed, the JM region conformation and its interaction with the membrane was shown to be coupled with the TMD helix orientation in the dimer [95,96]. Moreover, transitions between the alternative modes of interaction of the JM regions with the membrane appear to be the key events in activation of many receptor BPs [57,97]. This interaction can be readily modulated by the lipid composition and physicochemical properties of the membrane.

4. Characteristic amino acid sequence determinants of helix-helix interactions in membranes

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Since early 1990s, it was known that certain specific patterns in the amino acid sequence of transmembrane α-helices can enhance their ability to form homodimers in model systems. The first helixhelix dimerization motif was found in GpA, a strongly dimerizing glycoprotein, and is commonly designated as GxxxG, where x stands for an arbitrary hydrophobic residue [98]. Works on the artificial transmembrane peptides revealed that GxxxG, or, more generally, SmxxxSm (Sm is an aminoacid with a small and slightly polar sidechain: G, A, S, or T), usually induce an increased propensity for TMD dimerization s [99]. This motif and its structural analogues are abundant in membrane-spanning proteins, being present in more than half of the single-pass transmembrane helices [100]. The importance of the specified motif for the dimerization of many BPs was confirmed by mutagenesis [98,101,102], and later supported by NMR and crystal structures of GpA [21,103–105], BniP3 [106,107], HER2 [108], HER4 [109], EGFR/HER2 heterodimer [110], EphA1 [111] and αIIb/β3 integrin complex [112,113]. Initially it was thought that dimerization via the GxxxG-like motifs is favorable due to the good steric match of the interacting surfaces: glycines form a ―groove‖ on the surface of the TMD helix, into which the bulky sidechains of hydrophobic residues of the counterpart TMD are tightly packed [103]. In addition, it is believed that polar Gly residues form unfavorable contacts with lipid molecules, and dimerization via the GxxxG motif protects the TMD helix from these interactions, thus decreasing the free energy of the system [114]. This hypothesis is based on the so-called ―hydrophobicity scale‖, which reflects the energy penalty for slightly polar residues inside the membrane, compared to the aqueous environment [115]. On the other hand, one can reason that while in the aqueous solution polar moieties of Gly, Ser, Ala and Thr can form hydrogen bonds with the solvent, there is no evidence that protein-lipid contacts by these groups result in the energetic penalty. In other words, ―hydrophobicity scale‖ describes the free energy losses of the system upon insertion of a TMD into a spatially homogeneous lipid bilayer, but can say nothing on the thermodynamics of protein-lipid interactions. Direct structural information reveals that a significant contribution to the energy of the GxxxG-driven dimerization is made by specific non-canonical hydrogen bonds [116,117]. According to the quantum chemical calculations, Cα-H bond is polarized in proteins, and can form favorable contacts with carbonyl or hydroxyl oxygens of protein backbone and sidechains [118,119]. Moreover, such contacts may be deemed hydrogen bonds, because a shared electron density is formed, resulting in a measurable Cα-Co J-couplings in NMR spectra [120]. The energy of such non-canonic contacts can reach 3 kcal/M, which is comparable to classical NH-CO hydrogen bonding [118]. Thus, many agree that GxxxG-like motifs allow the correct packing of the TMDs in a dimer that is accompanied by formation of a net of the non-canonical hydrogen

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Aside from the weakly polar GxxxG-like motifs, there are many other factors in the TMD sequences that can enhance the helix-helix interactions. The presence of highly polar sidechains such as Asn, Asp, His, Glu, Gln or Arg in the TMD may result in intermolecular hydrogen bonding and favor its dimerization. For example, highly polar His and Asn residues are found on the dimerization interfaces of BNiP3 [106,107] and TLR3 TMDs [121]. Bacterial aspartate receptors employ the Gln sidechain to form a homodimeric complex. A number of genetic assays, such as TOXCAT [122] reveal that polar sidechains may often enhance the helix-helix interactions in TMDs, both artificial and native ones [123]. The ability of polar residues to increase the dimerization propensity of the transmembrane helices was the base of the scanning Glu mutagenesis approach, applied to study the functional interfaces in the TMDs of VEGFR-2 receptor tyrosine kinase [83,87]. The dimer-stabilizing effect was ascribed to the famous Neu (Val/Glu) mutation [77] that causes cancer in rats. However, a single polar aminoacid does not necessarily enhance the dimerization. Polar sidechains need to be on the proper side of the transmembrane helix to act in resonance with other factors, favorable for helix-helix interactions, e.g. GxxxG-like motifs [83]. When hydrophobic residue and existing dimerization motif are on the opposite faces of the transmembrane helix, this may lead to competition of the two interfaces rather than a decrease in the overall dimerization free energy. This can explain the fact that in many documented cases hydrophobic/polar mutations in the TMDs do not enhance dimerization, but lead to rearrangements of the TMD dimer [72,83,85]. Sequence-dependent parameters of helix-helix interaction can also be analyzed based on the known structures of multi-helical membrane domains. Two similar studies of that kind were undertaken by William DeGrado in 2006 and 2015 [124,125]. The authors took all the spatial structures of transmembrane and soluble helical proteins known to date and investigated the geometric parameters and sequences of helixhelix pairs. As a result, DeGrado and colleagues identified several most frequent conformations of helixhelix pairs. The most abundant were parallel and antiparallel pairs with a right-handed twist, small interhelical distance and large helix-crossing angle ( -30-40 º). The probability of simultaneous occurrence of Gly/Ser/Ala in positions i and i+4 was higher than for other residues, which corresponds to the GxxxG-like motif. In other words, it was shown that dimerization via the GxxxG or similar motif results in a specific mutual arrangement of transmembrane helices in dimer. In addition, it was revealed that almost 40% of helical pairs form left-handed parallel or antiparallel dimers with intermediate interhelical distance and small helix crossing angle (10-20º). This geometry was accompanied with the increased probability of Gly/Ser/Ala at positions i and i+7, which is referred to as ―heptad repeat‖ motif. All clusters were characterized by different patterns of hydrogen bonding, and for each cluster there was an increased chance to meet a certain aminoacid at certain positions of the dimerization interface. These studies clearly demonstrate that there is a correlation between the amino acid sequence of a transmembrane helix and both the geometry and the free energy of the helix-helix interaction. Once the interface is known, one can assume the structure of the dimer. E.g., if the protein is dimerized via the GxxxG-like motif, the right-handed structure with ca. 35 º helix crossing angle is most likely formed. However, the straightforward analysis of TMD sequence does not allow exactly predicting the dimer structure and interaction energy. The structure is a result of interplay of many factors, including stacking of aromatic rings, presence of polar residues, quality of packing on different interfaces, possibilities for formation of canonical and non-canonical hydrogen bonds. This statement is supported by the recently obtained NMR structures of EphA2 [126], HER2 [93], VEGFR2 [83], PDGFR [127], FGFR3 [128], ζζ [129], DAP12 [130] homodimers and the crystal structure of the Sx1a/Syb2 heterodimer of the SNARE complex [131]. These TMDs contain at least one and sometimes several GxxxGlike motifs, but none of them was employed for dimerization in the structures obtained under the given experimental conditions (in model systems, such as detergent micelles, bicelles and crystals, and in the absence of certain domains of the protein, which can affect the mode of the TMD interaction). This does not exclude the possibility of biological relevance of the non-employed motifs (including the GxxxG-like motifs), which can provide alternative dimerization modes, as was demonstrated for some receptors [50,79,132,133].

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ACCEPTED MANUSCRIPT 5. Spatial structures of dimeric transmembrane domains of bitopic proteins

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The identified common motifs of helix-helix interactions can be used for analyzing and classifying the structures of the dimeric TMDs of BPs known to date. PDB database contains 27 spatial structures of the TMD helical dimers. The first such structure was resolved in 1997 for GpA [107], and the field was then abandoned for 10 years. Since 2007, with the growth of interest in the signaling of RTKs and other BPs, many structures of helical TMD dimers were published by several groups. Out of 27 dimers, 25 were solved by solution NMR spectroscopy and two were determined using the X-ray crystallography. Fourteen structures are of the proteins from the RTK superfamily EGFR [57,134], HER2 [93,108], HER3[135], HER4 [109], VEGFR2 [83], PDGFR [127], FGFR3 [128], EphA1[111], EphA2 [126]. Other structures are those of GpA (4 structures determined using various techniques and membrane mimetics) [21,103,104], TLR3 [121], p75NTR [136], alphaIIb/beta3 integrins [112,113], ζζ dimer from the T-cell receptor [129], amyloid precursor protein APP [137, 186], DAP12 complex from the natural killer cell activating receptor [130], proapoptotic mitochodrial protein BNip3 [106,107]. Based on the known dimerization motifs, obtained structures can be clustered into several groups: (1) right-handed dimers with the strong and weak interactions formed via the weakly polar tandem GxxxG-like motifs providing high helix crossing-angle, (2) right- and left-handed structures formed via the extended dimeric interface abundant in hydrophobic residues with bulk sidechains (usually resulting in low helix-crossing angle), (3) dimers with specific features such as partially folded extracellular or cytoplasmic JM regions, disulfide and salt-bridge cross-links between the TMDs (Figure 1). There is a general correlation between the properties of lipid environments and dimerization mode, more specifically – in the bicelles forming a narrow rim of true bilayer around the protein there is a trend in favor of the N-terminal interaction via the weakly polar tandem GxxxG-like motif, whereas in the micelles where the lipid tails are in more disordered state and the hydrophobic core is more water permeable the helices trend to interact via more hydrophobic interfaces. These trends are predictably more pronounced for weakly dimerizing TMDs, e.g., of such BPs as RTK, whereas for such a strongly self-associated dimer as GpA the dimerization mode remained the same in both environments. A major question regarding the above mentioned structures is their biological relevance. They are obtained in artificial environments – detergent micelles and bicelles, and in the absence of ECDs and ICDs. In case of BPs that act via the LID mechanism, the obtained spatial structures would present a possible interface of helix-helix interaction in the TMD of the protein dimer, which can be verified using single-point mutagenesis. In the case of RTKs, which were shown to function through the LIR pathway, one needs to consider the known activating and inactivating mutations and take into account the possible mechanics that can cause the switch between two dimeric conformations of the TMD. This could help to find the correspondence between the functional state of the full-size receptor and the mode of TMD dimerization. In this respect, it is important that for three proteins, EGFR [57,134], VEGFR2 [83] and HER2 [93,108], alternative dimer structures were obtained in different membrane mimetics (having different degree of lipid ordering, dielectric properties, water penetration, hydrophobic thickness, etc.) and sequence context (presence/absence of juxtamembrane regions, activating mutations). It should be, however, understood that these alternative dimeric structures do not necessarily represent the difference between the active and inactive states of the full-length receptors, as the functional states can be more diverse, including multiple activated states caused by interaction with alternative ligands [132], or certain transient states [138].

6. Alternative conformational states as an evidence of the conformational switching in the dimeric transmembrane domains of RTKs The first proposed mechanical interpretation of the LIR signaling of RTKs was based on the sequence motif analysis of the TMDs of HER members [36,108,139]. Almost all HER proteins have two GxxxG-like domains in their TMD helix, suggesting that the activation might proceed via switching of the TMD dimerization mode between the N- and C-terminal GxxxG-like motifs. It was tempting to assume that in the inactive state ECDs pull the N-termini of the TMD helices apart, stabilizing the C-terminal motif, while in the active state ECDs pull the N-termini closer, which results in the N-terminal dimerization and large spacing of the C-termini of the TMDs. This model implies that the distance between the N- and C-

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termini of the TMD helices is important for the switching between the active and inactive states of the ICDs. The hypothesis was first supported by the structural data: NMR structures of the HER2 [108], HER4 [109], EGFR/HER2 TMD dimers [110] were characterized by the N-terminal interaction through the weakly polar tandem GxxxG-like motifs (cluster 1 of the previous section, see Figure 1A), and were assigned by the authors to the receptor active state. Recent structural and biochemical data on the EGFR receptor also reveal that a large spacing is needed between the C-termini of the TMDs to allow the antiparallel interaction of cytoplasmic JM-A helices [75,91], which is clearly seen in the NMR-derived model (Figure 1C) of the EGFR TMD and JM-A dimer [57]. Although the credibility of this latter structure was recently argued [94], all other data, including the position of the activating Neu mutation in the TMD of HER2 [78], mutagenesis of JM-A [75,91], etc., suggests that the specified morphology of TMD helix-helix dimer corresponds to the active state of HER proteins, and, probably, of other RTKs. The situation with the inactive state of the RTK TMD dimer is much less straightforward. Until very recently, when the structure of EGFR TMD in detergent micelles was published [134], no structure of the TMD dimer with C-terminal interaction utilizing the GxxxG-like motif was known to exist. On the other hand, five structures of the RTK TMD dimers belong to the cluster 2 as defined in the previous section: left-handed dimers with low helix crossing angle and long non-polar dimerization motif (Figure 1B) (also including the right-handed structure of the HER2 TMD dimer obtained in the presence of JM-A regions [93], Figure 1C). Moreover, the analysis of mutations affecting functioning and dimerization of full-length receptors, in particular FGFR3 [72,85,128] and VEGFR2 [50], implies that the observed dimer conformation may indeed correspond to the receptor inactive state or another active state (for recognition of a different ligand [132]). Thus, while the structure of the receptor TMD in the active state fits well the proposed mechanistic interpretation of the LIR pathway, the conformation of the TMD in the inactive state does not. This raises two major issues regarding the switching between the two states of the RTK TMDs. Firstly, is it the helix-crossing angle or rotation of helices rather than the spacing between the termini of TMD helices that is critical for activation of ICDs. Secondly, another source of energy aside from the repulsion of unbound ECDs is needed to explain the transition between the two conformations of TMD dimer. The concept that mutual rotation of helices is responsible for the activation of the kinase domains is quintessential for the LIR mechanism. It was supported by the experiments on chemical crosslinking of the EGFR receptors [36], by the experiments with artificial sequences with Neu receptor [42], and later confirmed by Glu scanning mutagenesis of the VEGFR2 TMDs [83,87]. The inactive TMD conformation can be stabilized by the interactions of juxtamembrane regions and kinase domains of the receptor, and the dimerization of ECDs could shift the equilibrium of the system towards the formation of the active dimer. However, it is quite difficult to imagine how the interaction of ECDs can change the structure of the TMDs, if both active and inactive conformations are characterized by the same distance between the N-termini of the TMD helices, and according to the X-ray structures of ECDs the connection between the ECDs and TMDs is flexible enough to allow almost arbitrary rotation of the TMD helices. The length of this link varies notably between the representatives of the same subfamily, moreover, it took insertion of as many as 20-40 additional residues into the sequence to cause constitutive (ligand-independent) activation, similarly to oncogenic ECD-truncated receptors [56,57]. Inapplicability of the LIR mechanism is especially evident in the case of the p75 neurotrophin receptor, which is a disulfide-linked dimer in both ligand-bound and ligandfree states [24]. The disulfide bond is located in the first N-terminal turn of the TMD α-helix, and allows neither change of the distance between the N-termini of TMDs nor the rotation of the transmembrane segments upon activation. The motions of the ICDs were shown to be uncoupled from the conformation of the TMD [140]. Altogether, it implies that the inhibition of constitutive signaling by unliganded ECD is achieved by other mechanism than simple mechanical traction of TMD helix N-termini with extracellular JM regions. Furthermore, recent studies suggest that interaction between the TMDs in the active state of RTK can be non-specific or weakly specific [58]. For example, substitutions of the first ten residues of EGFR TMD to leucines did not cause any substantial effect on the receptor activity [58], which indicates that the presence of the N-terminal dimerization motif is not necessary or not enough for the receptor activation. Therefore, other driving factors of the conformational switch in the dimeric TMDs of RTK superfamily proteins associated with receptor activation need to be considered, and, as we are going to show below, lipids

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7. Physics beyond the membrane protein interactions: the role of lipid environment.

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Free energies of the TMD helix-helix interactions of BPs were measured actively since the beginning of 2000-ies using sedimentation equilibrium [62], FRET [68] and NMR [63] in detergent micelles, bicelles, liposomes and even on the living cell surface [46]. These studies mainly focused on the effects of aminoacid substitutions on the dimerization of TMDs themselves and in the context of larger constructs, containing various extramembrane domains of the BPs. They revealed that in many cases mutations causing the activation of the full-size receptor do not enhance the dimerization propensity of its TMD, but rather change the conformation of a TMD dimer [72,83,85]. However, the effect of lipids was usually neglected. On the other hand, many studies reveal that the properties of lipid bilayer may be important for the thermodynamics of the membrane protein dimerization. It was shown that the phase of bilayer has an impact on the free energy of the helix-helix interaction: dimerization of the model TMD helices was enhanced in the gel phase, in comparison to the liquid-crystalline membrane [141]. An obvious contributor to the driving force of enhanced dimerization in the more ordered gel phase is higher energy cost of incorporation of a protein helix into it, making dimeric state with smaller surface area more preferable. A similar effect was shown for the presence of cholesterol: addition of this lipid to the bilayers substantially increased the propensity of the transmembrane segments to interact with each other [142,143]. NMR studies of the thermodynamics of the HER4 TMD dimerization in bicelles revealed that the process is entropy driven [109], and is accompanied by increase of heat capacity. It was attributable to the observed increased degree of ordering of lipids around the transmembrane protein through formation of a larger ordered lipid coat, which is in many aspects analogous to the hydrophobic effect for the soluble membrane proteins in aqueous environment. Such changes in the local ordering of lipids around the proteins upon the dimerization were observed in MD simulations [144– 146]. Apart from the phase behavior, hydrophobic thickness of the membrane is also believed to affect the energy of interaction between the TMDs and structure of TMD dimers, as was shown in a number of computational works [147,148]. Both the free energy of the dimerization and spatial structure of TLR3 TMD dimer were altered, when the length of fatty tail was varied for the micelle-forming detergent [121]. Looking at the spatial structures of the TMD dimers known to the date, one could also notice that the conformation of the dimer correlates with the type of selected membrane mimetic. The dimers solved in the environment of bicelles are likely to be formed via the GxxxG-like motif and have large helix-crossing angle, while in the micelles TMDs usually form left-handed dimers with a small helix-crossing angle and extensive non-polar interface. This is, however, not the case for the GpA [104] and BNiP3 [107], which form strong dimers through the GxxxG-like motifs in all kinds of membrane mimetics, while weakly interacting HER2 TMDs dimerize differently in bicelles and micelles [93,108]. Additional effects are observed for the type of membrane-like environment with respect to the juxtamembrane regions. Short cytoplasmic juxtamembrane regions of EGFR were shown to be helical and membrane-bound in micellar solution and disordered and water-dissolved in bicelles [93,94]. Therefore, we can assume that local properties of the membrane environment, including the state of the lipids, are essential and sometimes can determine both the energy and conformation of the TMD dimer. Consequently, if the ligand binding to the BP can change the properties of the cell membrane around the protein, it can trigger the conformational changes in the TMD dimer and juxtamembrane regions and thus transfer the information on the ligand binding across the plasma membrane. Though complex interplay between protein, carbohydrate and lipid components of plasma membranes cannot be fully controlled in the current experimental systems, it is tempting to evaluate the role of each component individually. Clearly, even local lipid composition of biological membranes containing multitude of lipid species presently can be neither reproduced in the model system, nor directly measured in cells. Thus, experimental evaluation of the role of lipid-protein interplay for the signal transduction in the systems allowing direct control of local composition of the membrane realistically representing biological conditions is yet to be achieved. For the time being, computer modeling methods proved to be a reasonable alternative to biophysical experiments, allowing evaluating many characteristics of the membrane that are not accessible for direct

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measurement and performing parametric studies under a broader spectrum of conditions than is experimentally attainable. Although like the current experimental techniques, the computation methods cannot presently capture the details of biological environment of the plasma membrane, some generic mechanisms can be elucidated with the aid of simplified, substantially reduced models. Computer simulations also allow directly evaluating the effects of macroscopic physico-chemical parameters, such as membrane potential, dielectric properties, membrane asymmetry etc. The timescale of computer simulations allows resolving fast processes that are often difficult to observe experimentally due to time averaging, though it is bound on the upper end due to computer power limitations and accumulation of the simulation uncertainties with time. Thus, experimental measurements and computer modeling essentially complement each other, providing useful crosschecking opportunities. In the most simplistic computational models, the membrane is represented implicitly or imposed as a set of constraints, allowing to evaluate the effects of global membrane properties, such as hydrophobic thickness, lateral pressure, dielectric permittivity profile, charge distribution [149,150]. At this level of discretization, the protein properties are conveniently described by the molecular hydrophobicity potential [151]. Such an approach yields a broad spectrum of possible models of interactions for the given amino acid sequence, so it can be effectively employed in combination with MD simulations in explicit membrane allowing assessment stability of the models, some of which can represent alternative functional states of the protein. This level of details allows detecting basic effects, such as hydrophobic mismatch [152,153], disregarding specific protein-lipid interactions and reciprocal influence between the protein and the state of the membrane in its vicinity. The last couple of decades have seen many examples of vital functional importance of specific interactions of BPs with particular lipid species (e.g., cholesterol, gangliosides, sphyngomielins, phosphatidylinositol bi- and thriphosphate, cardiolipines, or phosphatidylserine). Thus, computer modeling revealed the details of cholesterol binding occurring in different parts of BPs, both near the membrane surface and deep in the hydrophobic core [14,154], however the functional implications have so far been only speculated. For many RTKs it was shown that their juxtamembrane segments strongly interact with anionic lipids [155]. These interactions involve polar lipid headgroups. The corresponding free energy estimated using molecular modeling techniques was shown to range from -40 to -4 kJ/mol for EGFR interacting with glycolipid GM3 and phosphoinositide PIP2, respectively [11]. This energy is comparable to the estimates of GpA TMD helices dimerization strength (-40 to -60 kJ/mol) that was calculated using different approaches [147,148]. Moreover, systematic analysis of all 58 representatives of RTK family confirmed that the PIP2 interaction site is conserved throughout the family [155]. These conserved interactions were implicated in inducing local bilayer reorganization and anionic lipid clustering. This behavior, which can extend to a broader range of objects, is of special interest for RTK since PIP2 have been reported to modulate activity and cellular distribution of several members of the family [11,155]. Activated EphA2 is known to preferentially localize in lipid nanodomains [156], and requires presence of negatively charged lipids for its functions [157,158]. Similarly to EGFR [159], such EphA2 interaction with specific anionic lipids was suggested as a mechanism triggering coalescence of lipid nanodomains into more extended signaling platforms. Non-specific lipid interactions are also common in membranes. For example, dimerization of human GpA, an archetypal dimerizing membrane protein, is strongly modulated by lipid properties [147,160]. GpA dimer prefers the neighborhood of lipids with unsaturated acyl chains and promotes formation of raft-like microdomains [161]. Nature of the polar heads of surrounding lipids also affects GpA dimerization: anionic lipids make the dimer less stable [162]. Such interactions can be viewed dualistically: either as GpA dimerization causing the protein to adapt the lipid environment to its preferences, or as certain lipid environments favoring GpA dimerization. Strictly speaking, these viewpoints are complementary to each other, both effects taking place in the same time. It can result in precipitation of proteolipid clusters of specific composition, in other words, the membrane in this case can act as a medium broadcasting to large distances the perturbation caused by the presence of a protein in certain functional state and guiding the necessary counterparts (proteins or lipids) towards it. According to computer modeling results, insertion of a single transmembrane helix is capable of inducing strong perturbation of the hydrophobic core of the bilayer enabling ―communication‖ between transmembrane helices at the distances of 2-3 nm [144]. Calculated lipid

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density was also shown to increase in the vicinity of transmembrane helices, the effect being almost negligible for poly-Leu sequence, but becoming substantial for a native sequence containing GxxxG-like motifs and notably increasing with dimerization in both cases (Figures 2 and 3). The density distribution is quasi-periodic on the scale of 1 Ǻ, indicative of the existence of certain vibrational normal modes of the protein-lipid system, which can play a role in formation of the clusters and defects. This point of view can be supported by recent experimental results from phonon spectroscopy of biomembranes [163]. Even more interesting effects come up when both lateral and transversal asymmetry of the membrane is taken into account. Certain interaction appears to exist between lipid microdomains in the opposing leaflets of the membrane. In model bilayer membranes the domains are always bilayer, i.e. monolayer domains in different leaflets of the membrane are always in register [4–6]. Moreover, in the work [164] it is claimed that phase separation in one leaflet can induce phase separation in an opposing leaflet that would not otherwise take place. Recently, the energy gain due to in-registry domain configuration has been experimentally estimated as 0.016 kBT/nm2 at T  300 K [165], which is non-negligeable even for as small microdomain characteristic size as 5 nm. Such a tendency of the lipid domains located in different leaflets to couple into bilayer microdomains can be reflected in an ability of a protein on one side of the membrane to respond to changes of state of another protein (or part of the same protein) located on the other side of the membrane, i.e. this property of lipids can render the proteins able to communicate across the membrane. In the simplest case, formation of the liquid-ordered patch under an adsorbed protein (e.g., ECD, ICD, or JM region of a receptor) can induce liquid-ordered state of the membrane patch in the opposite leaflet. This allows transmitting across the plasma membrane the information about the protein conformational changes on the extracellular side resulting from ligand binding. There is no consensual description of the physics behind correlation between phase states of the membrane leaflets, but it is clear that the coupling does not require any specific lipids or cholesterol [166– 169]. Most probably, it is only sensitive to the relative degree of ordering of lipid in the membrane domains, while the exact reasons of the ordering are immaterial. For example, in [167,169] different elastic rigidities and thicknesses of monolayer patches are induced by electrostatic adsorption of polycation molecules, without global phase separation in the membranes. Hence, elastic deformations are thought to arise at the boundary between the lipid domains with the surrounding membrane, and the energy of such deformations was found to be minimal when the boundaries of domains in the opposing leaflets are shifted relative to each other a by small distance of 2-4 nm [170]. Such a small shift of boundaries at equilibrium was also observed in several MD studies [169,171,172]. Thus, elastic membrane deformations can contribute to the domain coupling, especially since the energy of single monolayer domain is higher than the energy of coupled bilayer domain of the same radius with the optimal shift between the monolayers [173,174]. Another promising hypothesis on possible mechanism of coupling between the domains is based on the difference between the elastic properties of the ordered and disordered membrane phases [167]. The ordered domains were experimentally shown to possess a 2-5 times higher bending modulus than the surrounding membrane [175–177]. This should result in suppression of thermal fluctuations (undulations) of the membrane surface in regions of more ordered phase (Figure 4). Coupling of lateral positions of domains between the opposite leaflets allows maximizing the membrane area, which is free to fluctuate, thus leading to favorable entropy contribution [167]. Both mechanisms — elastic energy minimization and maximization of the undulating membrane area — only require certain patches of the opposing leaflets to have properties differing from those of the surrounding membrane: monolayer thickness or elastic rigidity. The sign of the difference is not essential, a local thinning, loosening or other form of defect in the membrane can equally serve the purpose. Provided this difference, the mechanisms are able to drive the patches into the register. They are quite universal in the sense that they do not impose any limitations on membrane composition. The mechanisms even do not require the phase separation to take place in the membrane: these different properties of monolayer patches compared to the surrounding membrane may originate from any physical reason. Moreover, both mechanisms can contribute into indirect protein-protein interaction across the membrane, allowing a protein on one side of the membrane to be affected by conformational transition of a protein on the opposite side through the induced change of membrane properties. For the proteins with transmembrane

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8. Local lipid environment properties affect bitopic protein folding and can enable remote

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protein-protein interactions, providing a novel mechanism of cell signaling The most obvious possible way for signal transduction through plasma membrane is transmission of conformational change of receptor ectodomain resulting from ligand binding to TMD and finally to cytoplasmic part of the receptor. LIR and LID mechanisms are specific cases of this generic way. However, such mechanisms are not feasible if a receptor TMD is linked to the extracellular and intracellular parts by long flexible linkers, or for receptors lacking transmembrane segment, such as GPI-anchored proteins. In this case, lipid-protein interaction appears to be the key to understanding of the signaling mechanism. For lipid-mediated mechanism to work, the interaction of ECD with the external leaflet of the plasma membrane should be substantially different in free and ligand-bound states. The interaction can cause changes of electrostatic properties of the membrane, changes of lipid ordering due to partial insertion of the ectodomain hydrophobic parts into the outer leaflet, or induce binding of lipids into specific ―pockets‖ of the receptor, modify water permeability, or otherwise locally affect the physical state of the lipid monolayer in the vicinity of the ECD. There are experimental and computer modeling clues that interaction with proteins can cause the required local changes in the lipid environment. MD simulations demonstrated that electrostatic adsorption of polycations on the negatively charged lipid membrane results in substantial ordering of lipids, observed as increase of monolayer thickness and tighter packing of lipid tails [169]. Under similar experimental conditions, the adsorption resulted in a drop of the average diffusion coefficient of fluorescently labeled lipid, consistent with decreased mobility of lipid molecules [166]. Partial insertion of protein hydrophobic residues also leads to alteration of lipid chains order parameter as revealed by X-ray scattering for amphipathic α-helical peptides [176] . In a similar manner, surface active peptides strongly affect the lipid membrane state, locally increasing the depth of penetration of water and polar lipid moieties into the membrane, resulting in greater disorder and smaller density of lipid tails underneath, matched by complementary changes on the other side of the membrane [178–181]. As mentioned above, changes of properties of the lipid environment of opposite signs (e.g., membrane thickening/thinning, lipid ordering/disordering, dielectric permittivity increase/decrease etc.) are equally effective for signal transduction across the membrane, as well in lateral direction. Lateral interactions resulting in formation of signaling platforms are extensively discussed and reviewed elsewhere [182], whereas the less obvious and less widely recognized transversal signal propagation on the basis of such effects deserves a more detailed treatment. Figure 4 illustrates possible implications of lipid-mediated communication between proteins for the mechanisms of cell signaling by the receptors, whose intracellular and extracellular parts are not strongly connected by large and complex transmembrane protein complex, as is the case with GPCRs or ion channels. In principle, these effects can underlie the mechanism of signaling by GPI-anchored receptors, conditionally to certain additional assumptions needed to explain how the necessary cytoplasmic counterpart selectively recognizes the occurring change of the intracellular leaflet properties. It can require specific lipid recognition or formation of signaling platforms, in which all the necessary participants of the signaling process colocalize. However, any transmembrane link resolves this problem, and even a single-span receptor can transduce signals via this mechanism effectively (Figure 4). It might appear that in a plasma membrane abundant in various lipid species and non-lipidic components the signal transduced via such mechanism will be lost in the noise of stochastic interactions, and the mechanism will therefor lack specificity. However, the changes of the lipid state induced by ligand binding by the receptor concentrate in the immediate vicinity of the receptor, which limits the response of other membrane proteins that are more than ~2-3 nm (the characteristic length at which individual TM helices can sense each other [144]) away from the receptor. Certain level of interplay between the closest neighbors (within this distance) is possible, but it should not be construed as compromising the required signaling specificity since such close proximity in cellular membrane is usually not random and the known phenomenon of cross-talk between different receptors can

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occur through lipid-mediated interactions in addition to direct protein-protein contacts. A dimer formed by such receptors provides extra opportunities for signal modulation and possibility for alternative modes of activation (e.g., in response to different ligands), making the signaling process more deterministic and more complex in the same time, which is especially essential for multi-cellular organisms (Figure 5). This does not exclude LIR, LID, or other signaling mechanisms suggested in the past (such as ―string puppet‖, ―flexible rotation‖, or ―electrostatic engine‖ models [36,42,128,183]), rather complementing them and extending the spectrum of conditions, under which they can occur by adding alternative allosteric pathways. The TMD role is thus more complex than that of a passive anchor ensuring that the cytoplasmic counterpart of signaling is there to respond; the TMD dimer rearrangements in response to a change of environment due to lipid interaction with extracellular protein components can play active role in signal transduction and amplification. Change of TMD conformation, in its turn, can introduce further perturbations the state of the surrounding lipids throughout the entire surface of the dimer in its new state, thus making the protein together with its lipid environment a self-consistent signal transduction system. It can be partly responsible for pathogenic mutations known to exist in the transmembrane part of RTK, since they can prevent the change of dimer conformation, contribution of which into adaptive rearrangement of the cytoplasmic leaflet of the membrane might be necessary for effective signaling. This mechanism appears to be perfectly exemplified by one of the representatives of BP receptors, the human epidermal growth factor receptor (EGFR), also known as HER1 or ErbB1, which has for many years served as an excellent model RTK to illustrate how ligand-induced conformational rearrangements and specific dimerization of ECDs lead to allosteric activation of the ICD kinase, resulting in signal propagation across the membrane [60,184,185]. Recently, the EGFR TMD dimerization through alternative interfaces was experimentally proved to occur in alternative membrane-mimetic systems differing by the degree of ordering of lipid tails, elastic moduli and polarizability [57,93,108,134]. A similar case of alternative dimerization mode in a different membrane mimetic was also recently observed for HER2, another representative of ErbB/HER family member [93]. As discussed in the reference [134], the lipid-mediated signaling mechanism, illustrated for this dimeric receptor by the Figure 6 explains the available functional and structural information and reconciles certain apparent contradictions between experimental observations. Although such alternative dimerization in a different membrane mimetic does not constitute a lipid-induced conformational transition, it clearly illustrates the influence of local lipid environment on the protein configuration (and on the TMD dimerization mode). The influence of certain lipid species on BP function, and in particular inhibition of the EGFR activity by GM3 [187] also implies strong modulation of the receptor structure by the neighboring lipids. In this respect, the notion of the plasma membrane being a highly inhomogeneous glyco-proteo-lipidic system is fully consistent with the hypothesis of lipid-mediated receptor function, especially taking into account the concept of signaling platforms consisting of specific combinations of lipids and membrane proteins. Lipid-protein interactions within such spatially localized platforms can contribute to the cross-talk between different membrane protein species and enable their complex concerted responses to different combination of stimuli, providing the diversity of biological responses required for the observed complexity of behavior patterns of cells. The plasma membrane containing multiple lipid species and non-lipidic components might allow cells to form adjustable local environment most suitable for supporting receptor function. Thus, the suggested mechanism combines the necessary degree of specificity of response to ligand binding event with the possibility to adapt the response to the local conditions and the state of the immediate neighbors of the receptors. It means that the implications of lipid-protein interactions in plasma membrane are even more relevant to receptor functions requiring complexity of cell signaling responses.

9. Concluding remarks and prospective directions for future development The suggested mediation of signal transduction by protein-induced targeted changes of lipid properties is complementary to other mechanisms hypothesized for RTK in the past, revealing a new tier of the underlying mechanism. Introduction of this additional tier makes the system more resistant to polymorphism and a wide range of mutations in the TMD segment, in the same time enabling another level

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of fine-tuning of receptor behavior by the membrane composition. In fact, signaling in this model is mediated by an integrated heterogeneous system and depends on the properties of every part thereof. This concept suggests a new possibility for the search of therapeutic agents targeting BP based on modulation of protein-lipid interaction in addition to the traditional protein functional determinant targets. Such compounds can combine selectivity with respect to the affected proteins and efficiency against a broad spectrum of deviations from their normal function and regulation. On a broader scale, the recent trend of shift of focus of attention from the lipid membrane properties towards structural aspects of proteins functioning in the membrane appears somewhat premature. In many cases, membrane protein functional mechanisms cannot be adequately described without consideration of the protein in an integrated, self-consistent, and evolutionary optimized complex with its dynamically changing lipid environment, and possibly – some other membrane proteins inalienable from the system. The roles of specific interaction with various lipid species, influence of diverse defects and peculiarities of membrane structure and variable parameters of the membrane upon the interplay of protein functions appear to form promising areas for both experimental and computer modeling investigations. Moreover, as evidenced by not yet very numerous recent examples of successful elucidation of some aspects of such complex interactions, the problem can be effectively tackled at the present level of development of experimental and computing tools and techniques.

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Acknowledgments This work was supported by Russian Science Foundation (project #14-14-00573), excluding the review analysis of lipid distribution around protein (Section 7) sponsored by Russian Science Foundation (project #14-14-00871). S.A.A. was supported by the Ministry of Education and Science of the Russian Federation in the framework of Increase of Competitiveness Program of ―MISiS‖. Support from the RAS Programme "Molecular and Cellular Biology" is greatly appreciated. The authors express their sincere thanks to Dr. K.A. Beirit for helpful discussions.

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FIGURES

Figure 1. Spatial structures of homo- and heterodimeric TMDs of BPs obtained by now. (A) Righthanded dimers with the strong and weak interactions formed via the weakly polar tandem GxxxG-like motifs providing high helix-crossing angle. (B) Right- and left-handed structures via the extended dimeric interface abundant in hydrophobic residues with bulk sidechains (usually resulting in low helix-crossing angle). (C) Dimers with specific features such as partially folded extracellular or cytoplasmic JM regions, disulfide and salt-bridge cross-links between the TMDs. Ribbon structure and heavy atom bonds are shown. Hydrogen atoms are depicted only for polar groups.

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Figure 2. Lipid density increases in proximity to the TMDs of BPs. Ribbon representation of monomeric (A) and dimeric (D) TMD of human glycophorin A (GpA) in POPC bilayer from MD simulations [144]. High density regions (average density > 1500 g/l) around monomeric and dimeric TMDs of GpA (B, E) and poly-leucine (C, F). Averaging time was 200 ns. Peptides are colored by the nature of amino acids: blue and red - positively and negatively charged, green - polar, yellow - aliphatic, magenta - aromatic. POPC molecules are colored by the element type with gray carbons.

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Figure 3. Lipid density perturbations around the TMDs of BPs. Average density representations in slices of the lipid bilayer in "pure" POPC (A) and around monomeric (B) and dimeric (C) TMD of GpA [144,148]. Darker areas correspond to higher density. Averaging time was 200 ns. Peptide representations are the same as on Figure 2. (D) Time evolution of the radial distribution of density in central slice of the POPC bilayer (corresponding to acyl chain tails) containing one GpA TMD. Density is plotted on Z axis and colored blue to red (low to high), R is the distance from the peptide helix, t - simulation time.

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Figure 4. Possible pathway of the signal transduction through the plasma membrane via the lipidprotein interactions of monomeric receptor having a single-span TMD flanked by the flexible JM regions. (A) Free receptor. (B) Ligand-bound receptor. (C) Adsorption of ectodomain to the outer leaflet of the plasma membrane, and formation of lipid cluster. (D) Formation of the lipid cluster at the inner leaflet of the plasma membrane. (E) Adsorption of cytoplasmic part of the BP receptor to the lipid cluster at the inner leaflet of the plasma membrane. Inset: two possible mechanisms of induction of lipid cluster at the inner leaflet by the cluster at the outer leaflet: i) maximization of membrane area, which is free to thermally undulate; ii) minimization of elastic energy arising from compensation of the thickness mismatch between lipid clusters and the surrounding membrane.

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Figure 5. Possible pathway of the signal transduction through the plasma membrane via the lipidprotein and protein-protein interactions of the dimeric receptor having a single-span TMD flanked by the flexible JM regions. (A) Receptor dimer in the inactive state. Ectodomain and cytoplasmic part are adsorbed to the plasma membrane, inducing lipid disorder in the center of the signaling platform. (B) Ligand-bound receptor. (C) Ectodomains detach from the membrane, thus inducing the formation of liquid-ordered cluster in the outer leaflet. (D) The newly formed ordered patch in the outer leaflet induces formations of the ordered patch at the inner leaflet of the plasma membrane at the same transmembrane position, close to the receptor dimer. TMDs change their orientation relative to the surrounding lipid (follow the positions of yellow circles). This is a key stage in the signal transduction through the plasma membrane. (E) Cytoplasmic parts of the dimeric BP receptor detach from the inner leaflet of the plasma membrane due to the unfavorable ordered lipid environment, thus finishing the signal transduction process.

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Figure 6. Alternative TMD packing and pathway of the signal transduction through plasma membrane proposed for human epidermal growth factor receptor. (A) Ribbon structures of the dimeric EGFR TMD (TMD) formed via the alternative C-terminal (single GxxxG-like A661xxxG665 surrounded by the bulky sidechains of hydrophobic residues) and more polar N-terminal (tandem GxxxG-like motif S645xxT648G649xxG652A653) interfaces, obtained in DPC micelles (PDB ID: 2M0B [134]) and DMPC/DHPC bicelles (PDB ID: 2M20 [57]; similar to other HER/ErbB TMD homo- and heterodimers, PDB ID: 2JWA [108], 2LCX [109], 2KS1 [110]), respectively. Different dimer subunits are colored in magenta and cyan. Approximate position of the membrane boundaries is highlighted by green and yellow strips for the dimer structures obtained in the micelles and bicelles, respectively. (B) Possible sequence of structural rearrangements of EGFR and associated perturbations of the lipidic bilayer in the course of ligand-induced receptor activation. According to mechanism described in [134], prior to ligand binding EGFR pre-dimers exist in the equilibrium with monomeric receptors. In this inactive state the extracellular domain (ECD) of EGFR is in unliganded ―tethered‖ conformation. TMD helices are dimerized via the C-terminal motifs (yellow ovals), whereas more polar N-terminal motifs (green ovals) face the lipid bilayer locally perturbed by tethered ECD, making it favorable for cytoplasmic juxtamembrane-A regions (JM-A) to be buried near the lipid polar head plane. Consequently, the intracellular kinase domains (ICD) have no access to phosphorylation sites (open orange circles). Lipid heads in the perturbed membrane area with a slightly increased dielectric permittivity are schematically shown in green. Ligand binding to EGFR causes ECD rearrangement to the ―extended‖ state (at right). In this state, the ECD interaction with the lipid in the vicinity of TMD is weakened, the lipid returns to its ―original‖, unperturbed state, and TMD dimerization switches to the polar N-terminal motifs, whereas the C-terminal motifs get exposed to the unperturbed (more hydrophobic) lipid environment. All these changes trigger the release from the membrane and subsequent antiparallel dimerization of the JM-A helices, ultimately allowing formation of the asymmetric ICD dimer and phosphorylation of the target tyrosine residues followed by the stimulation of the downstream signaling cascades.

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