The architecture of transmembrane and cytoplasmic juxtamembrane regions of Toll-like receptors.

Toll-like receptors (TLRs) are the important participants of the innate immune response. Their spatial organization is well studied for the ligand-binding domains, while a lot of questions remain unanswered for the membrane and cytoplasmic regions of the proteins. Here we use solution NMR spectroscopy and computer simulations to investigate the spatial structures of transmembrane and cytoplasmic juxtamembrane regions of TLR2, TLR3, TLR5, and TLR9. According to our data, all the proteins reveal the presence of a previously unreported structural element, the cytoplasmic hydrophobic juxtamembrane α-helix. As indicated by the functional tests in living cells and bioinformatic analysis, this helix is important for receptor activation and plays a role, more complicated than a linker, connecting the transmembrane and cytoplasmic parts of the proteins.

Expression, purification and characterization of SORCS2 intracellular domain for structural studies.

Neurotrophin signaling pathways are one of the major cascades in neuronal development and involved in many key processes including proliferation, differentiation, apoptosis, synaptic plasticity, axonal growth. In addition to the main classes of neurotrophin receptors, Trk and P75NTR, there are many auxiliary proteins, which can also bind neurotrophins and regulate the signaling pathways. The versatility of interactions between them could explain multiple and completely opposite biological outcomes such as cell survival or apoptosis. Membrane protein SorCS2, a vacuolar protein sorting 10 protein-domain receptor, interacts with P75NTR and controls the activity of Trk receptors. The abnormal functioning of SorCS2 is associated with neurodegenerative diseases, such as Alzheimer's and Huntington's disease. But the mechanism of SorCS2 activation and basis of the interaction with P75NTR has remained elusive. Herein, we describe two efficient approaches for the intracellular domain of the SorCS2 production employing bacterial and cell-free expression systems, as well as purification and refolding protocols. Finally, we characterized the purified protein by DLS and NMR and demonstrated that the protein sample is suitable for structural studies.

Probing the effect of membrane contents on transmembrane protein-protein interaction using solution NMR and computer simulations.

The interaction between the secondary structure elements is the key process, determining the spatial structure and activity of a membrane protein. Transmembrane (TM) helix-helix interaction is known to be especially important for the function of so-called type I or bitopic membrane proteins. In the present work, we present the approach to study the helix-helix interaction in the TM domains of membrane proteins in various lipid environment using solution NMR spectroscopy and phospholipid bicelles. The technique is based on the ability of bicelles to form particles with the size, depending on the lipid/detergent ratio. To implement the approach, we report the experimental parameters of "ideal bicelle" models for four kinds of zwitterionic phospholipids, which can be also used in other structural studies. We show that size of bicelles and type of the rim-forming detergent do not affect substantially the spatial structure and stability of the model TM dimer. On the other hand, the effect of bilayer thickness on the free energy of the dimer is dramatic, while the structure of the protein is unchanged in various lipids with fatty chains having a length from 12 to 18 carbon atoms. The obtained data is analyzed using the computer simulations to find the physical origin of the observed effects.

Behavior of Most Widely Spread Lipids in Isotropic Bicelles.

Isotropic bicelles are a widely used membrane mimetic for structural studies of membrane proteins and their transmembrane domains. Simple and cheap in preparation, they contain a patch of lipid bilayer that reproduces the native environment of membrane proteins. Despite the obvious power of bicelles in reproducing the various kinds of environments, the vast majority of structural studies employ the single lipid/detergent system. On the other hand, even if the alternative bicelle composition is used, the properties of mixtures are not characterized, and the mere presence of lipid bilayer and discoidal shape of bicelle particles is not confirmed. Here we present an extensive investigation of various bicellar mixtures and describe the behavior of bicelles with lipids other than classical DMPC, namely sphingomyelins (SM), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), phosphatidylserines (PS), and cholesterol. These lipids are rarely used in modern structural biology, but can help a lot in understanding the influence of the membrane composition on the properties of both integral and peripheral membrane proteins. Additionally, the ability of diheptanoylphosphatidylcholine (DH7PC) to serve as a rim-forming agent was investigated. We followed the phase transitions as revealed by 31P NMR and size of particles measured by 1H NMR diffusion as the criteria of the proper morphology and structure of bicelles. As an outcome, we state that SM exclusively, and PG/PS in mixtures with zwitterionic lipids can form small isotropic bicelles, which reproduce the key features of lipid behavior in bilayers. Mixtures, containing exclusively the anionic lipids, fail to reveal the lipid phase transition and do not follow the size predicted for the ideal bicelle particles. PE and DH7PC are the unwanted components of bicellar mixtures, and cholesterol can be added to bicelles, however, with certain precautions. In combination with our several most recent works, this study provides a practical guide for the preparation of small isotropic bicelles.

NMR relaxation parameters of methyl groups as a tool to map the interfaces of helix-helix interactions in membrane proteins.

In the case of soluble proteins, chemical shift mapping is used to identify the intermolecular interfaces when the NOE-based calculations of spatial structure of the molecular assembly are impossible or impracticable. However, the reliability of the membrane protein interface mapping based on chemical shifts or other relevant parameters was never assessed. In the present work, we investigate the predictive power of various NMR parameters that can be used for mapping of helix-helix interfaces in dimeric TM domains. These parameters are studied on a dataset containing three structures of helical dimers obtained for two different proteins in various membrane mimetics. We conclude that the amide chemical shifts have very little predictive value, while the methyl chemical shifts could be used to predict interfaces, though with great care. We suggest an approach based on conversion of the carbon NMR relaxation parameters of methyl groups into parameters of motion, and one of such values, the characteristic time of methyl rotation, appears to be a reliable sensor of interhelix contacts in transmembrane domains. The carbon NMR relaxation parameters of methyl groups can be measured accurately and with high sensitivity and resolution, making the proposed parameter a useful tool for investigation of protein-protein interfaces even in large membrane proteins. An approach to build the models of transmembrane dimers based on perturbations of methyl parameters and TMDOCK software is suggested.

Characterization of Small Isotropic Bicelles with Various Compositions.

Structural studies of membrane proteins are of great importance and interest, with solution and solid state NMR spectroscopy being very promising tools for that task. However, such investigations are hindered by a number of obstacles, and in the first place by the fact that membrane proteins need an adequate environment that models the cell membrane. One of the most widely used and prospective membrane mimetics is isotropic bicelles. While large anisotropic bicelles are well-studied, the field of small bicelles contains a lot of "white spots". The present work reports the radii of particles and concentration of the detergents in the monomeric state in solutions of isotropic bicelles, formed by 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and sodium cholate, as a function of lipid/detergent ratio and temperature. These parameters were measured using (1)H NMR diffusion spectroscopy for the bicelles composed of lipids with saturated fatty chains of different length and lipids, containing unsaturated fatty acid residue. The influence of a model transmembrane protein (membrane domain of rat TrkA) on the properties of bicelles and the effect of the bicelle size and composition on the properties of the transmembrane protein were investigated with heteronuclear NMR and nuclear Overhauser effect spectroscopy. We show that isotropic bicelles that are applicable for solution NMR spectroscopy behave as predicted by the theoretical models and are likely to be bicelles rather than mixed micelles. Using the obtained data, we propose a simple approach to control the size of bicelles at low concentrations. On the basis of our results, we compared different rim-forming agents and selected CHAPS as a detergent of choice for structural studies in bicelles, if the deuteration of the detergent is not required.

Lipid-protein nanodiscs offer new perspectives for structural and functional studies of water-soluble membrane-active peptides.

Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer stabilized in solution by the apolipoprotein or a special membrane scaffold protein (MSP). In this work, the applicability of LPN-based membrane mimetics in the investigation of water-soluble membrane-active peptides was studied. It was shown that a pore-forming antimicrobial peptide arenicin-2 from marine lugworm (charge of +6) disintegrates LPNs containing both zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids. In contrast, the spider toxin VSTx1 (charge of +3), a modifier of Kv channel gating, effectively binds to the LPNs containing anionic lipids (POPC/DOPG, 3 : 1) and does not cause their disruption. VSTx1 has a lower affinity to LPNs containing zwitterionic lipids (POPC), and it weakly interacts with the protein component of nanodiscs, MSP (charge of -6). The neurotoxin II (NTII, charge of +4) from cobra venom, an inhibitor of the nicotinic acetylcholine receptor, shows a comparatively low affinity to LPNs containing anionic lipids (POPC/DOPG, 3 : 1 or POPC/DOPS, 4 : 1), and it does not bind to LPNs/POPC. The obtained data show that NTII interacts with the LPN/POPC/DOPS surface in several orientations, and that the exchange process among complexes with different topologies proceeds fast on the NMR timescale. Only one of the possible NTII orientations allows for the previously proposed specific interaction between the toxin and the polar head group of phosphatidylserine from the receptor environment (Lesovoy et al., Biophys. J. 2009. V. 97. № 7. P. 2089-2097). These results indicate that LPNs can be used in structural and functional studies of water-soluble membrane-active peptides (probably except pore-forming ones) and in studies of the molecular mechanisms of peptide-membrane interaction.

Structural investigations of recombinant urokinase growth factor-like domain.

Urokinase-type plasminogen activator (uPA) is a serine protease that converts the plasminogen zymogen into the enzymatically active plasmin. uPA is synthesized and secreted as the single-chain molecule (scuPA) composed of an N-terminal domain (GFD) and kringle (KD) and C-terminal proteolytic (PD) domains. Earlier, the structure of ATF (which consists of GFD and KD) was solved by NMR (A. P. Hansen et al. (1994) Biochemistry, 33, 4847-4864) and by X-ray crystallography alone and in a complex with the soluble form of the urokinase receptor (uPAR, CD87) lacking GPI (C. Barinka et al. (2006) J. Mol. Biol., 363, 482-495). According to these data, GFD contains two ß-sheet regions oriented perpendicularly to each other. The area in the GFD responsible for binding to uPAR is localized in the flexible Ω-loop, which consists of seven amino acid residues connecting two strings of antiparallel ß-sheet. It was shown by site-directed mutagenesis that shortening of the Ω-loop length by one amino acid residue leads to the inability of GFD to bind to uPAR (V. Magdolen et al. (1996) Eur. J. Biochem., 237, 743-751). Here we show that, in contrast to the above-mentioned studies, we found no sign of the ß-sheet regions in GFD in our uPA preparations either free or in a complex with uPAR. The GFD seems to be a rather flexible and unstructured domain, demonstrating in spite of its apparent flexibility highly specific interaction with uPAR both in vitro and in cell culture experiments. Circular dichroism, tryptophan fluorescence during thermal denaturation of the protein, and heteronuclear NMR spectroscopy of ¹5N/¹³C-labeled ATF both free and in complex with urokinase receptor were used to judge the secondary structure of GFD of uPA.

Lipid-protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: comparison with detergent micelles, bicelles and liposomes.

Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.

Spatial structure and dimer--monomer equilibrium of the ErbB3 transmembrane domain in DPC micelles.

In present work the interaction of two TM α-helices of the ErbB3 receptor tyrosine kinase from the ErbB or HER family (residues 639-670) was studied by means of NMR spectroscopy in a membrane-mimicking environment provided by the DPC micelles. The ErbB3 TM segment appeared to form a parallel symmetric dimer in a left-handed orientation. The interaction between TM spans is accomplished via the non-standard motif and is supported by apolar contacts of bulky side chains and by stacking of aromatic rings together with π-cation interactions of Phe and Arg side chains. The investigation of the dimer--monomer equilibrium revealed thermodynamic properties of the assembly and the presence of two distinct regimes of the dimerization at low and at high peptide/detergent ratio. It was found that the detergent in case of ErbB3 behaves not as an ideal solvent, thus affecting the dimer--monomer equilibrium. Such behavior may account for the problems occurring with the refolding and stability of multispan helical membrane proteins in detergent solutions. The example of ErbB3 allows us to conclude that the thermodynamic parameters of dimerization, measured in micelles for two different helical pairs, cannot be compared without the investigation of their dependence on detergent concentration.

[Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members].

A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.

Dimeric structure of the transmembrane domain of glycophorin a in lipidic and detergent environments.

Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions between transmembrane α-helices by the example of dimerizing the transmembrane domain of the bitopic protein glycophorin A. However, a number of peculiarities place lipid bicelles closer to natural lipid bilayers in terms of their physical properties.