The structure of the second CysD domain of MUC2 and role in mucin organization by transglutaminase-based cross-linking.

The MUC2 mucin protects the colonic epithelium by a two-layered mucus with an inner attached bacteria-free layer and an outer layer harboring commensal bacteria. CysD domains are 100 amino-acid-long sequences containing 10 cysteines that separate highly O-glycosylated proline, threonine, serine (PTS) regions in mucins. The structure of the second CysD, CysD2, of MUC2 is now solved by nuclear magnetic resonance. CysD2 shows a stable stalk region predicted to be partly covered by adjacent O-glycans attached to neighboring PTS sequences, whereas the CysD2 tip with three flexible loops is suggested to be well exposed. It shows transient dimer interactions at acidic pH, weakened at physiological pH. This transient interaction can be stabilized in vitro and in vivo by transglutaminase 3-catalyzed isopeptide bonds, preferring a specific glutamine residue on one flexible loop. This covalent dimer is modeled suggesting that CysD domains act as connecting hubs for covalent stabilization of mucins to form a protective mucus.

Effective synthesis, development and application of a highly fluorescent cyanine dye for antibody conjugation and microscopy imaging.

An asymmetric cyanine-type fluorescent dye was designed and synthesized via a versatile, multi-step process, aiming to conjugate with an Her2+ receptor specific antibody by an azide-alkyne click reaction. The aromaticity and the excitation and relaxation energetics of the fluorophore were characterized by computational methods. The synthesized dye exhibited excellent fluorescence properties for confocal microscopy, offering efficient applicability in in vitro imaging due to its merits such as a high molar absorption coefficient (36 816 M-1 cm-1), excellent brightness, optimal wavelength (627 nm), larger Stokes shift (26 nm) and appropriate photostability compared to cyanines. The conjugated cyanine-trastuzumab was constructed via an effective, metal-free, strain-promoted azide-alkyne click reaction leading to a regulated number of dyes being conjugated. This novel cyanine-labelled antibody was successfully applied for in vitro confocal imaging and flow cytometry of Her2+ tumor cells.

Cortex-wide response mode of VIP-expressing inhibitory neurons by reward and punishment.

Neocortex is classically divided into distinct areas, each specializing in different function, but all could benefit from reinforcement feedback to inform and update local processing. Yet it remains elusive how global signals like reward and punishment are represented in local cortical computations. Previously, we identified a cortical neuron type, vasoactive intestinal polypeptide (VIP)-expressing interneurons, in auditory cortex that is recruited by behavioral reinforcers and mediates disinhibitory control by inhibiting other inhibitory neurons. As the same disinhibitory cortical circuit is present virtually throughout cortex, we wondered whether VIP neurons are likewise recruited by reinforcers throughout cortex. We monitored VIP neural activity in dozens of cortical regions using three-dimensional random access two-photon microscopy and fiber photometry while mice learned an auditory discrimination task. We found that reward and punishment during initial learning produce rapid, cortex-wide activation of most VIP interneurons. This global recruitment mode showed variations in temporal dynamics in individual neurons and across areas. Neither the weak sensory tuning of VIP interneurons in visual cortex nor their arousal state modulation was fully predictive of reinforcer responses. We suggest that the global response mode of cortical VIP interneurons supports a cell-type-specific circuit mechanism by which organism-level information about reinforcers regulates local circuit processing and plasticity.

Gram-positive bacteria are held at a distance in the colon mucus by the lectin-like protein ZG16.

The distal colon functions as a bioreactor and harbors an enormous amount of bacteria in a mutualistic relationship with the host. The microbiota have to be kept at a safe distance to prevent inflammation, something that is achieved by a dense inner mucus layer that lines the epithelial cells. The large polymeric nets made up by the heavily O-glycosylated MUC2 mucin forms this physical barrier. Proteomic analyses of mucus have identified the lectin-like protein ZG16 (zymogen granulae protein 16) as an abundant mucus component. To elucidate the function of ZG16, we generated recombinant ZG16 and studied Zg16-/- mice. ZG16 bound to and aggregated Gram-positive bacteria via binding to the bacterial cell wall peptidoglycan. Zg16-/- mice have a distal colon mucus layer with normal thickness, but with bacteria closer to the epithelium. Using distal colon explants mounted in a horizontal perfusion chamber we demonstrated that treatment of bacteria with recombinant ZG16 hindered bacterial penetration into the mucus. The inner colon mucus of Zg16-/- animals had a higher load of Gram-positive bacteria and showed bacteria with higher motility in the mucus close to the host epithelium compared with cohoused littermate Zg16+/+ The more penetrable Zg16-/- mucus allowed Gram-positive bacteria to translocate to systemic tissues. Viable bacteria were found in spleen and were associated with increased abdominal fat pad mass in Zg16-/- animals. The function of ZG16 reveals a mechanism for keeping bacteria further away from the host colon epithelium.

Structural Basis of Ribosomal S6 Kinase 1 (RSK1) Inhibition by S100B Protein: MODULATION OF THE EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) SIGNALING CASCADE IN A CALCIUM-DEPENDENT WAY.

Mitogen-activated protein kinases (MAPK) promote MAPK-activated protein kinase activation. In the MAPK pathway responsible for cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by Ca(2+)-binding S100 proteins in malignant melanomas. Here, we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The Ca(2+)-dependent binding of S100B to the calcium/calmodulin-dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent of the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca(2+)-binding proteins could influence the activity of the CaMK domain-containing protein kinases. Our crystallographic, small angle x-ray scattering, and NMR analysis revealed that S100B forms a "fuzzy" complex with RSK1 peptide ligands. Based on fast-kinetics experiments, we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas.

Survivin co-ordinates formation of follicular T-cells acting in synergy with Bcl-6.

Follicular T helper (Tfh) cells are recognized by the expression of CXCR5 and the transcriptional regulator Bcl-6. Tfh cells control B cell maturation and antibody production, and if deregulated, may lead to autoimmunity. Here, we study the role of the proto-oncogene survivin in the formation of Tfh cells. We show that blood Tfh cells of patients with the autoimmune condition rheumatoid arthritis, have intracellular expression of survivin. Survivin was co-localized with Bcl-6 in the nuclei of CXCR5+CD4 lymphocytes and was immunoprecipitated with the Bcl-6 responsive element of the target genes. Inhibition of survivin in arthritic mice led to the reduction of CXCR5+ Tfh cells and to low production of autoantibodies. Exposure to survivin activated STAT3 and induced enrichment of PD-1+Bcl-6+ subset within Tfh cells. Collectively, our study demonstrates that survivin belongs to the Tfh cell phenotype and ensures their optimal function by regulating transcriptional activity of Bcl-6.

Terahertz radiation induces non-thermal structural changes associated with Fröhlich condensation in a protein crystal.

Whether long-range quantum coherent states could exist in biological systems, and beyond low-temperature regimes where quantum physics is known to be applicable, has been the subject to debate for decades. It was proposed by Fröhlich that vibrational modes within protein molecules can order and condense into a lowest-frequency vibrational mode in a process similar to Bose-Einstein condensation, and thus that macroscopic coherence could potentially be observed in biological systems. Despite the prediction of these so-called Fröhlich condensates almost five decades ago, experimental evidence thereof has been lacking. Here, we present the first experimental observation of Fröhlich condensation in a protein structure. To that end, and to overcome the challenges associated with probing low-frequency molecular vibrations in proteins (which has hampered understanding of their role in proteins' function), we combined terahertz techniques with a highly sensitive X-ray crystallographic method to visualize low-frequency vibrational modes in the protein structure of hen-egg white lysozyme. We found that 0.4 THz electromagnetic radiation induces non-thermal changes in electron density. In particular, we observed a local increase of electron density in a long α-helix motif consistent with a subtle longitudinal compression of the helix. These observed electron density changes occur at a low absorption rate indicating that thermalization of terahertz photons happens on a micro- to milli-second time scale, which is much slower than the expected nanosecond time scale due to damping of delocalized low frequency vibrations. Our analyses show that the micro- to milli-second lifetime of the vibration can only be explained by Fröhlich condensation, a phenomenon predicted almost half a century ago, yet never experimentally confirmed.

DYNLL2 dynein light chain binds to an extended linear motif of myosin 5a tail that has structural plasticity.

LC8 dynein light chains (DYNLL) are conserved homodimeric eukaryotic hub proteins that participate in diverse cellular processes. Among the binding partners of DYNLL2, myosin 5a (myo5a) is a motor protein involved in cargo transport. Here we provide a profound characterization of the DYNLL2 binding motif of myo5a in free and DYNLL2-bound form by using nuclear magnetic resonance spectroscopy, X-ray crystallography, and molecular dynamics simulations. In the free form, the DYNLL2 binding region, located in an intrinsically disordered domain of the myo5a tail, has a nascent helical character. The motif becomes structured and folds into a ß-strand upon binding to DYNLL2. Despite differences of the myo5a sequence from the consensus binding motif, one peptide is accommodated in each of the parallel DYNLL2 binding grooves, as for all other known partners. Interestingly, while the core motif shows a similar interaction pattern in the binding groove as seen in other complexes, the flanking residues make several additional contacts, thereby lengthening the binding motif. The N-terminal extension folds back and partially blocks the free edge of the ß-sheet formed by the binding motif itself. The C-terminal extension contacts the dimer interface and interacts with symmetry-related residues of the second myo5a peptide. The involvement of flanking residues of the core binding site of myo5a could modify the quaternary structure of the full-length myo5a and affect its biological functions. Our results deepen the knowledge of the diverse partner recognition of DYNLL proteins and provide an example of a Janus-faced linear motif.

The C-terminal random coil region tunes the Ca²âº-binding affinity of S100A4 through conformational activation.

S100A4 interacts with many binding partners upon Ca2+ activation and is strongly associated with increased metastasis formation. In order to understand the role of the C-terminal random coil for the protein function we examined how small angle X-ray scattering of the wild-type S100A4 and its C-terminal deletion mutant (residues 1-88, Δ13) changes upon Ca2+ binding. We found that the scattering intensity of wild-type S100A4 changes substantially in the 0.15-0.25 Å-1 q-range whereas a similar change is not visible in the C-terminus deleted mutant. Ensemble optimization SAXS modeling indicates that the entire C-terminus is extended when Ca2+ is bound. Pulsed field gradient NMR measurements provide further support as the hydrodynamic radius in the wild-type protein increases upon Ca2+ binding while the radius of Δ13 mutant does not change. Molecular dynamics simulations provide a rational explanation of the structural transition: the positively charged C-terminal residues associate with the negatively charged residues of the Ca2+-free EF-hands and these interactions loosen up considerably upon Ca2+-binding. As a consequence the Δ13 mutant has increased Ca2+ affinity and is constantly loaded at Ca2+ concentration ranges typically present in cells. The activation of the entire C-terminal random coil may play a role in mediating interaction with selected partner proteins of S100A4.

Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser.

The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.

Crystal structure of the S100A4-nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism.

S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. It binds to the nonmuscle myosin IIA (NMIIA) tail near the assembly competence domain (ACD) promoting filament disassembly, which could be associated with increasing metastatic potential of tumor cells. Here, we investigate the mechanism of S100A4-NMIIA interaction based on binding studies and the crystal structure of S100A4 in complex with a 45-residue-long myosin heavy chain fragment. Interestingly, we also find that S100A4 binds as strongly to a homologous heavy chain fragment of nonmuscle myosin IIC as to NMIIA. The structure of the S100A4-NMIIA complex reveals a unique mode of interaction in the S100 family: A single, predominantly α-helical myosin chain is wrapped around the Ca(2+)-bound S100A4 dimer occupying both hydrophobic binding pockets. Thermal denaturation experiments of coiled-coil forming NMIIA fragments indicate that the coiled-coil partially unwinds upon S100A4 binding. Based on these results, we propose a model for NMIIA filament disassembly: Part of the random coil tailpiece and the C-terminal residues of the coiled-coil are wrapped around an S100A4 dimer disrupting the ACD and resulting in filament dissociation. The description of the complex will facilitate the design of specific drugs that interfere with the S100A4-NMIIA interaction.

Membrane protein crystallization from lipidic phases.

Membrane protein structural biology is enjoying a steady acceleration in the rate of success. Nevertheless, numerous membrane protein targets are resistant to the traditional approach of directly crystallizing detergent solubilized and purified protein and the 'niche market' of lipidic phase crystallization is emerging as a powerful complement. These approaches, including lipidic cubic phase, lipidic sponge phase, and bicelle crystallization methods, all immerse purified membrane protein within a lipid rich matrix before crystallization. This environment is hypothesized to contribute to the protein's long-term structural stability and thereby favor crystallization. Spectacular recent successes include the high-resolution structures of the beta(2)-adrenergic G-protein-coupled receptor, the A(2A) adenosine G-protein-coupled receptor, and the mitochondrial voltage dependent anion channel. In combination with technical innovations aiming to popularize these methods, lipidic phase crystallization approaches can be expected to deliver an increasing scientific impact as the field develops.

Raman-assisted crystallography reveals end-on peroxide intermediates in a nonheme iron enzyme.

Iron-peroxide intermediates are central in the reaction cycle of many iron-containing biomolecules. We trapped iron(III)-(hydro)peroxo species in crystals of superoxide reductase (SOR), a nonheme mononuclear iron enzyme that scavenges superoxide radicals. X-ray diffraction data at 1.95 angstrom resolution and Raman spectra recorded in crystallo revealed iron-(hydro)peroxo intermediates with the (hydro)peroxo group bound end-on. The dynamic SOR active site promotes the formation of transient hydrogen bond networks, which presumably assist the cleavage of the iron-oxygen bond in order to release the reaction product, hydrogen peroxide.

Structural and evolutionary consequences of unpaired cysteines in trypsinogen.

Vertebrate trypsins usually contain six disulfide bonds but human trypsin 1 (PRSS1) contains only five and human trypsin 2 (PRSS2) contains only four. To elucidate possible evolutionary pathways leading to the loss of disulfide bonds, we have constructed mutants lacking one or two cysteines of four disulfide bonds (C22-C157, C127-C232, C136-C201, and C191-C220) in rat anionic trypsinogen and followed their expression in the periplasm of Escherichia coli. When both cysteines of any of the above-mentioned disulfide bonds were replaced by alanines we found, as expected, proteolytically active enzymes. In the case of C127-C232 (missing from both human trypsins) and C191-C220 both single mutants gave active enzymes although their yield was significantly reduced. In contrast, only one of the single mutants of disulfide bonds C22-C157 and C136-C201 (missing from human trypsin 2) was expressed in E. coli. In the case of these disulfide bonds, we obtained no expression when the solvent accessible molecular surface of the free cysteine residue was the smaller one, indicating that a buried unpaired cysteine was more deleterious than one on the surface of the molecule.

Crystal structure reveals basis for the inhibitor resistance of human brain trypsin.

Severe neurodegradative brain diseases, like Alzheimer, are tightly linked with proteolytic activity in the human brain. Proteinases expressed in the brain, such as human trypsin IV, are likely to be involved in the pathomechanism of these diseases. The observation of amyloid formed in the brain of transgenic mice expressing human trypsin IV supports this hypothesis. Human trypsin IV is also resistant towards all studied naturally occurring polypeptide inhibitors. It has been postulated that the substitution of Gly193 to arginine is responsible for this inhibitor resistance. Here we report the X-ray structure of human trypsin IV in complex with the inhibitor benzamidine at 1.7 A resolution. The overall fold of human trypsin IV is similar to human trypsin I, with a root-mean square deviation of only 0.5 A for all C(alpha) positions. The crystal structure reveals the orientation of the side-chain of Arg193, which occupies an extended conformation and fills the S2' subsite. An analysis of surface electrostatic potentials shows an unusually strong clustering of positive charges around the primary specificity pocket, to which the side-chain of Arg193 also contributes. These unique features of the crystal structure provide a structural basis for the enhanced inhibitor resistance, and enhanced substrate restriction, of human trypsin IV.