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Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. Here we report the structure and dynamics of TLR1 toll-interleukin like (TIR) cytoplasmic domain in crystal and in solution. We found that the TLR1-TIR domain is capable of specific binding of Zn with nanomolar affinity. Interactions with Zn are mediated by cysteine residues 667 and 686 and C667 is essential for the Zn binding. Potential structures of the TLR1-TIR/Zn complex were predicted in silico. Using the functional assays for the heterodimeric TLR1/2 receptor, we found that both Zn addition and Zn depletion affect the activity of TLR1, and C667A mutation disrupts the receptor activity. Analysis of C667 position in the TLR1 structure and possible effects of C667A mutation, suggests that zinc-binding ability of TLR1-TIR domain is critical for the receptor activation., Lushpa et al report the structure and dynamics of the TLR1 toll-interleukin like (TIR) cytoplasmic domain in both crystal and solution. They demonstrate that the TLR1 TIR domain is capable of specific binding of Zn with nanomolar affinity, which appears to be critical for receptor activation, and provide potential structures TLR1-TIR/Zn complex based on in silico data.

Chemokine receptors form a major sub-family of G protein-coupled receptors (GPCRs) and they are involved in a number of cellular and physiological processes related to our immune response and regulation. A better structural understanding of ligand-binding, activation, signaling and regulation of chemokine receptors is very important to design potentially therapeutic interventions for human disorders arising from aberrant chemokine signaling. One of the key limitations in probing the structural details of chemokine receptors is the availability of large amounts of purified, homogenous and fully functional chemokine ligands, and the commercially available products, are not affordable for in-depth structural studies. Moreover, production of uniformly isotope-labeled chemokines, for example, suitable for NMR-based structural investigation, also remains challenging. Here, we have designed a streamlined approach to express and purify the human chemokine CCL7 as well as its15N-,15N/13C-,2H/15N/13C-isotope-labeled derivatives, at milligram levels usingE. coliexpression system. Purified CCL7 not only maintains a well-folded three-dimensional structure as analyzed using circular dichroism and1H/15N NMR but it also induces coupling of heterotrimeric G-proteins and β-arrestins for selected chemokine receptors in cellular system. Our strategy presented here may be applicable to other chemokines and therefore, provide a potentially generic and cost-effective approach to produce chemokines in large amounts for functional and structural studies.

T7 expression system is an extremely popular approach for the recombinant protein production in Escherichia coli for structural and functional studies and therapeutic applications. There are many useful tools and successful techniques that allow expressing the desired protein in this system. However, high yield of soluble protein often requires a systematic optimization of a wide range of cell cultivation parameters. Here we analyze the effect of three key cultivation parameters - chemical inductor, temperature and time of post-induction culturing on the expression level of TLR1 intracellular TIR domain in a soluble form. In addition, the influence of Triton X-100 detergent on the protein solubility during the cell lysis was investigated. We show that a high expression level of the correctly folded soluble protein can be obtained under different combinations of cultivation parameters.

Toll-like receptors (TLRs) play a key role in the innate and adaptive immune systems. While a lot of structural data is available for the extracellular and cytoplasmic domains of TLRs, and a model of the dimeric full-length TLR3 receptor in the active state was build, the conformation of the transmembrane (TM) domain and juxtamembrane regions in TLR dimers is still unclear. In the present work, we study the transmembrane and juxtamembrane parts of human TLR4 receptor using solution NMR spectroscopy in a variety of membrane mimetics, including phospholipid bicelles. We show that the juxtamembrane hydrophobic region of TLR4 includes a part of long TM α-helix. We report the dimerization interface of the TM domain and claim that long TM domains with transmembrane charged aminoacids is a common feature of human toll-like receptors. This fact is analyzed from the viewpoint of protein activation mechanism, and a model of full-length TLR4 receptor in the dimeric state has been proposed.

Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. At least 8 different pathogenic mutations, implicated in cancers and growth disorders, have been identified in FGFR3 transmembrane segment. Here we use heteronuclear NMR spectroscopy to determine the dimeric structure of FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, while others are located within a putative alternative interface. This implies that while the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the plasma membrane is complex. We propose a FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data.

Specific helix–helix interactions between the single-span transmembrane domains of receptor tyrosine kinases are believed to be important for their lateral dimerization and signal transduction. Establishing structure–function relationships requires precise structural-dynamic information about this class of biologically significant bitopic membrane proteins. ErbB4 is a ubiquitously expressed member of the HER/ErbB family of growth factor receptor tyrosine kinases that is essential for the normal development of various adult and fetal human tissues and plays a role in the pathobiology of the organism. The dimerization of the ErbB4 transmembrane domain in membrane-mimicking lipid bicelles was investigated by solution NMR. In a bicellar DMPC/DHPC environment, the ErbB4 membrane-spanning α-helices (651–678)2 form a right-handed parallel dimer through the N-terminal double GG4-like motif A655GxxGG660 in a fashion that is believed to permit proper kinase domain activation. During helix association, the dimer subunits undergo a structural adjustment (slight bending) with the formation of a network of inter-monomeric polar contacts. The quantitative analysis of the observed monomer–dimer equilibrium provides insights into the kinetics and thermodynamics of the folding process of the helical transmembrane domain in the model environment that may be directly relevant to the process that occurs in biological membranes. The lipid bicelles occupied by a single ErbB4 transmembrane domain behave as a true (“ideal”) solvent for the peptide, while multiply occupied bicelles are more similar to the ordered lipid microdomains of cellular membranes and appear to provide substantial entropic enhancement of the weak helix–helix interactions, which may be critical for membrane protein activity.

Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel alpha-helical bundle, region (544-569)(2), through the N-terminal glycine zipper motif A(550)X(3)G(554)X(3)G(558). Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu(547), whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A(560)X(3)G(564). This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pK(a) value of Glu(547) and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an "extracellular pH sensor," and can have relevant physiological implications.

BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.