The guidance receptor plexin D1 is a mechanosensor in endothelial cells.

Shear stress on arteries produced by blood flow is important for vascular development and homeostasis but can also initiate atherosclerosis1. Endothelial cells that line the vasculature use molecular mechanosensors to directly detect shear stress profiles that will ultimately lead to atheroprotective or atherogenic responses2. Plexins are key cell-surface receptors of the semaphorin family of cell-guidance signalling proteins and can regulate cellular patterning by modulating the cytoskeleton and focal adhesion structures3-5. However, a role for plexin proteins in mechanotransduction has not been examined. Here we show that plexin D1 (PLXND1) has a role in mechanosensation and mechanically induced disease pathogenesis. PLXND1 is required for the response of endothelial cells to shear stress in vitro and in vivo and regulates the site-specific distribution of atherosclerotic lesions. In endothelial cells, PLXND1 is a direct force sensor and forms a mechanocomplex with neuropilin-1 and VEGFR2 that is necessary and sufficient for conferring mechanosensitivity upstream of the junctional complex and integrins. PLXND1 achieves its binary functions as either a ligand or a force receptor by adopting two distinct molecular conformations. Our results establish a previously undescribed mechanosensor in endothelial cells that regulates cardiovascular pathophysiology, and provide a mechanism by which a single receptor can exhibit a binary biochemical nature.

Structural basis of semaphorin-plexin cis interaction.

Semaphorin ligands interact with plexin receptors to contribute to functions in the development of myriad tissues including neurite guidance and synaptic organisation within the nervous system. Cell-attached semaphorins interact in trans with plexins on opposing cells, but also in cis on the same cell. The interplay between trans and cis interactions is crucial for the regulated development of complex neural circuitry, but the underlying molecular mechanisms are uncharacterised. We have discovered a distinct mode of interaction through which the Drosophila semaphorin Sema1b and mouse Sema6A mediate binding in cis to their cognate plexin receptors. Our high-resolution structural, biophysical and in vitro analyses demonstrate that monomeric semaphorins can mediate a distinctive plexin binding mode. These findings suggest the interplay between monomeric vs dimeric states has a hereto unappreciated role in semaphorin biology, providing a mechanism by which Sema6s may balance cis and trans functionalities.

Anaerobic peroxisomes in Entamoeba histolytica metabolize myo-inositol.

Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90-100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0.044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.

Oligomeric Architecture of Mouse Activating Nkrp1 Receptors on Living Cells.

Mouse activating Nkrp1 proteins are commonly described as type II transmembrane receptors with disulfide-linked homodimeric structure. Their function and the manner in which Nkrp1 proteins of mouse strain (C57BL/6) oligomerize are still poorly understood. To assess the oligomerization state of Nkrp1 proteins, mouse activating EGFP-Nkrp1s were expressed in mammalian lymphoid cells and their oligomerization evaluated by Förster resonance energy transfer (FRET). Alternatively, Nkrp1s oligomers were detected by Western blotting to specify the ratio between monomeric and dimeric forms. We also performed structural characterization of recombinant ectodomains of activating Nkrp1 receptors. Nkrp1 isoforms c1, c2 and f were expressed prevalently as homodimers, whereas the Nkrp1a displays larger proportion of monomers on the cell surface. Cysteine-to-serine mutants revealed the importance of all stalk cysteines for protein dimerization in living cells with a major influence of cysteine at position 74 in two Nkrp1 protein isoforms. Our results represent a new insight into the oligomerization of Nkrp1 receptors on lymphoid cells, which will help to determine their function.

Impact of Chemical Cross-Linking on Protein Structure and Function.

Chemical cross-linking coupled with mass spectrometry is a popular technique for deriving structural information on proteins and protein complexes. Also, cross-linking has become a powerful tool for stabilizing macromolecular complexes for single-particle cryo-electron microscopy. However, an effect of cross-linking on protein structure and function should not be forgotten, and surprisingly, it has not been investigated in detail so far. Here, we used kinetic studies, mass spectrometry, and NMR spectroscopy to systematically investigate an impact of cross-linking on structure and function of human carbonic anhydrase and alcohol dehydrogenase 1 from Saccharomyces cerevisiae. We found that cross-linking induces rather local structural disturbances and the overall fold is preserved even at a higher cross-linker concentration. The results establish general experimental conditions for chemical cross-linking with minimal effect on protein structure and function.

Solution structure of the lymphocyte receptor Nkrp1a reveals a distinct conformation of the long loop region as compared to in the crystal structure.

Mouse Nkrp1a receptor is a C-type lectin-like receptor expressed on the surface of natural killer cells that play an important role against virally infected and tumor cells. The recently solved crystal structure of Nkrp1a raises questions about a long loop region which was uniquely extended from the central region in the crystal. To understand the functional significance of the loop, the solution structure of Nkrp1a using nuclear magnetic resonance (NMR) spectroscopy was determined. A notable difference between the crystal and NMR structure of Nkrp1a appears in the conformation of the long loop region. While the extended loop points away from the central core and mediates formation of a domain swapped dimer in the crystal, the solution structure is monomeric with the loop tightly anchored to the central region. The findings described the first solution structure in the Nkrp1 family and revealed intriguing similarities and differences to the crystal structure. Proteins 2016; 84:1304-1311. © 2016 Wiley Periodicals, Inc.

Nkrp1 family, from lectins to protein interacting molecules.

The C-type lectin-like receptors include the Nkrp1 protein family that regulates the activity of natural killer (NK) cells. Rat Nkrp1a was reported to bind monosaccharide moieties in a Ca2+-dependent manner in preference order of GalNac > GlcNAc >> Fuc >> Gal > Man. These findings established for rat Nkrp1a have been extrapolated to all additional Nkrp1 receptors and have been supported by numerous studies over the past two decades. However, since 1996 there has been controversy and another article showed lack of interactions with saccharides in 1999. Nevertheless, several high affinity saccharide ligands were synthesized in order to utilize their potential in antitumor therapy. Subsequently, protein ligands were introduced as specific binders for Nkrp1 proteins and three dimensional models of receptor/protein ligand interaction were derived from crystallographic data. Finally, for at least some members of the NK cell C-type lectin-like proteins, the "sweet story" was impaired by two reports in recent years. It has been shown that the rat Nkrp1a and CD69 do not bind saccharide ligands such as GlcNAc, GalNAc, chitotetraose and saccharide derivatives (GlcNAc-PAMAM) do not directly and specifically influence cytotoxic activity of NK cells as it was previously described.

Re-evaluation of binding properties of recombinant lymphocyte receptors NKR-P1A and CD69 to chemically synthesized glycans and peptides.

The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A) was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-D-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.

Structural model of lymphocyte receptor NKR-P1C revealed by mass spectrometry and molecular modeling.

NKR-P1C is an activating immune receptor expressed on the surface of mouse natural killer cells. It has been widely used as a marker for NK cell identification in different mice strains. Recently we solved a crystal structure of the C-type lectin-like domain of a homologous protein, NKR-P1A, using X-ray crystallography and also described the strategy for rapid characterization of the protein conformation in solution. This procedure utilized chemical cross-linking, hydrogen/deuterium exchange, and molecular modeling. It was found that the solution structure differs from the crystal structure in the conformation of the loop region. The loop, detached from the protein compact core in the crystal structure, is closely attached to the core of the protein in solution. Here we present and interpret the solution structure of the C-type lectin-like domain of NKR-P1C using chemical cross-linking and molecular modeling. The validation of the model and conformation of the loop region in NKR-P1C were addressed using ion-mobility mass spectrometry.

Chemical cross-linking and H/D exchange for fast refinement of protein crystal structure.

A combination of chemical cross-linking and hydrogen-deuterium exchange coupled to high resolution mass spectrometry was used to describe structural differences of NKR-P1A receptor. The loop region extended from the compact core in the crystal structure was found to be closely attached to the protein core in solution. Our approach has potential to refine protein structures in solution within a few days and has very low sample consumption.

Primary and secondary functions of HLA-E are determined by stability and conformation of the peptide-bound complexes.

MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8+ T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination.

Mouse and human antibodies bind HLA-E-leader peptide complexes and enhance NK cell cytotoxicity.

The non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we report the isolation of 3H4, a murine HLA-E-VL9-specific IgM antibody that enhances killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line. Structural analysis reveal that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9. Upon in vitro maturation, an affinity-optimized IgG form of 3H4 showes enhanced NK killing of HLA-E-VL9-expressing cells. HLA-E-VL9-specific IgM antibodies similar in function to 3H4 are also isolated from naïve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the naïve B cell antibody pool have the capacity to enhance NK cell cytotoxicity.

Molecular architecture of mouse activating NKR-P1 receptors.

Receptors belonging to NKR-P1 family and their specific Clr ligands form an alternative missing self recognition system critical in immunity against tumors and viruses, elimination of tumor cells subjected to genotoxic stress, activation of T cell dependent immune response, and hypertension. The three-dimensional structure of the extracellular domain of the mouse natural killer (NK) cell receptor mNKR-P1Aex has been determined by X-ray diffraction. The core of the C-type lectin domain (CTLD) is homologous to the other CTLD receptors whereas one quarter of the domain forms an extended loop interacting tightly with a neighboring loop in the crystal. This domain swapping mechanism results in a compact interaction interface. A second dimerization interface resembles the known arrangement of other CTLD NK receptors. A functional dimeric form of the receptor is suggested, with the loop, evolutionarily conserved within this family, proposed to participate in interactions with ligands.

High-level expression of soluble form of mouse natural killer cell receptor NKR-P1C(B6) in Escherichia coli.

Mouse NKR-P1C(B6) receptor corresponding to NK1.1 alloantigen is one of the most widespread surface markers of mouse NK and NKT cells in C57BL/6 mice detected by monoclonal antibody PK136. Although functional studies revealed the ability of this receptor to activate both natural killing and production of cytokines upon antibody crosslinking, the ligand for NKR-P1C(B6) remains unknown. In order to initiate ligand identification, structural studies, and epitope mapping experiments, we developed a simple and efficient expression and purification protocol allowing to produce large amounts of pure soluble monomeric mouse NKR-P1C(B6). Our protein encompassed approximately half of the stalk region and the entire C-terminal globular ligand binding domain. The identity of protein that was devoid of N-terminal initiation methionine and had all three expected disulfides closed was confirmed using high resolution ion cyclotron resonance mass spectrometry. Protein produced into inclusion bodies in Escherichia coli was efficiently refolded into a unique three dimensional structure as confirmed by NMR using (1)H-(15)N-HSQC spectra of uniformly labeled protein. The exceptional purity of the protein should allow its crystallization and detailed structural investigations, and is a prerequisite for its use as a probe in ligand identification and antibody epitope mapping experiments.

Structure of the H107R variant of the extracellular domain of mouse NKR-P1A at 2.3 Å resolution.

The structure of the H107R variant of the extracellular domain of the mouse natural killer cell receptor NKR-P1A has been determined by X-ray diffraction at 2.3 Å resolution from a merohedrally twinned crystal. Unlike the structure of the wild-type receptor in space group I4(1)22 with a single chain per asymmetric unit, the crystals of the variant belonged to space group I4(1) with a dimer in the asymmetric unit. Different degrees of merohedral twinning were detected in five data sets collected from different crystals. The mutation does not have a significant impact on the overall structure, but led to the binding of an additional phosphate ion at the interface of the molecules.

Cooperation between subunits is essential for high-affinity binding of N-acetyl-D-hexosamines to dimeric soluble and dimeric cellular forms of human CD69.

CD69 is an earliest lymphocyte activation antigen and a universal leukocyte triggering molecule expressed at sites of active immune response. The binding of GlcNAc to the dimeric human CD69 was followed by equilibrium dialysis, fluorescence titration, and NMR. Clear cooperation was observed in the high-affinity binding (K(d) = 4.0 x 10(-7) M) of the carbohydrate to two subunits of the dimeric CD69 (Hill coefficient 1.94). A control monosaccharide ManNAc was not bound by human CD69, and both monosaccharides had no effects on the structure of the receptor. However, a monomeric CD69 obtained by mutating Q93 and R134 at the dimer interface exhibited a much lower affinity for GlcNAc (K(d) = 1.3 x 10(-5) M) and no cooperativity (Hill coefficient 1.07). Perturbation of the dimer interface resulted in a severe impairment of the signaling ability of cellular CD69 when cross-linked with an antibody or with a bivalent high-affinity N-acetylhexosamine dimer-based ligand. The availability of stable preparations of soluble CD69 receptor with well-documented ligand binding properties will be beneficial for immunological experiments evaluating the role of this antigen in the complex environment of the immune system. Moreover, such preparations in combination with efficient ligand mimetics able to both activate CD69(+) lymphocytes and to block undesired hyperactivation caused by other cellular ligands will also become indispensable tools in explaining the exact role of the CD69 antigen in the interaction between the tumor cell and the effector natural killer lymphocyte.

Cell guidance ligands, receptors and complexes - orchestrating signalling in time and space.

Members of four cell guidance molecule families (the netrins, slits, ephrins and semaphorins) interact with their cognate cell surface receptors to guide cells during development and maintain tissue homeostasis. Integrated structure and cell-based analyses are providing insight into the mechanisms by which these signalling systems can deliver myriad outcomes that require exquisite accuracy in timing and location. Here we review recent advances in our understanding of the roles of oligomeric states, auto-inhibition, signalling assembly size and composition in cell guidance cue function.

Drosophila OTK Is a Glycosaminoglycan-Binding Protein with High Conformational Flexibility.

The transmembrane protein OTK plays an essential role in plexin and Wnt signaling during Drosophila development. We have determined a crystal structure of the last three domains of the OTK ectodomain and found that OTK shows high conformational flexibility resulting from mobility at the interdomain interfaces. We failed to detect direct binding between Drosophila Plexin A (PlexA) and OTK, which was suggested previously. We found that, instead of PlexA, OTK directly binds semaphorin 1a. Our binding analyses further revealed that glycosaminoglycans, heparin and heparan sulfate, are ligands for OTK and thus may play a role in the Sema1a-PlexA axon guidance system.

Diversity of oligomerization in Drosophila semaphorins suggests a mechanism of functional fine-tuning.

Semaphorin ligands and their plexin receptors are one of the major cell guidance factors that trigger localised changes in the cytoskeleton. Binding of semaphorin homodimer to plexin brings two plexins in close proximity which is a prerequisite for plexin signalling. This model appears to be too simplistic to explain the complexity and functional versatility of these molecules. Here, we determine crystal structures for all members of Drosophila class 1 and 2 semaphorins. Unlike previously reported semaphorin structures, Sema1a, Sema2a and Sema2b show stabilisation of sema domain dimer formation via a disulfide bond. Unexpectedly, our structural and biophysical data show Sema1b is a monomer suggesting that semaphorin function may not be restricted to dimers. We demonstrate that semaphorins can form heterodimers with members of the same semaphorin class. This heterodimerization provides a potential mechanism for cross-talk between different plexins and co-receptors to allow fine-tuning of cell signalling.