Enzyme stability and function can be affected by various environmental factors, such as temperature, pH, and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species. An AP from the marine bacterium Vibrio splendidus (VAP) shows complex pH-dependent activation and stabilization in the 0–1.0 M range of halogen salts and has been hypothesized to specifically bind chloride anions. Here, using X-ray crystallography and anomalous scattering, we have located two chloride binding sites in the structure of VAP, one in the active site and another one at a peripheral site. Further characterization of the binding sites using site-directed mutagenesis and small-angle X-ray scattering showed that upon binding of chloride to the peripheral site, structural dynamics decreased locally, resulting in thermal stabilization of the VAP active conformation. Binding of the chloride ion in the active site did not displace the bound inorganic phosphate product, but it may promote product release by facilitating rotational stabilization of the substrate-binding Arg129. Overall, these results reveal the complex nature and dynamics of chloride binding to enzymes through long-range modulation of electronic potential in the vicinity of the active site, resulting in increased catalytic efficiency and stability.
2.52 Å X-ray diffraction data set of mouse CNPase phosphodiesterase domain, mutant V318I, tetragonal crystal form. Data were collected on the ID30A-3 beamline at the Europian Synchrotron Radiation Facility (ESRF). Also includes the XDS.INP and XSCALE.INP files used for data processing in XDS and XSCALE, processing results (XSCALE.LP) and the processed reflection file (CNP_11manual.HKL).
1.80 Å X-ray diffraction data set of mouse CNPase phosphodiesterase domain, mutant G324D. Data were collected on the ID30A-3 beamline at the Europian Synchrotron Radiation Facility (ESRF). Also includes the XDS.INP file used for data processing in XDS, processing results (XSCALE.LP) and the processed reflection file (CNP_2manual.HKL).
Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-tagged ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N- and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and C-terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc, and interrogation of Arc capsid domain specific functions.
MD simulation trajectory for G324D mutant of mouse CNPase phosphodiesterase domain. The mutation corresponds to the human CNPase myopia 2 mutation. The simulation was run for 1 µs using GROMACS.
2.20 Å resolution anomalous diffraction dataset for Vibrio alkaline phosphatase, crystallised in 1.0 M NaCl. Data were collected with an X-ray energy of 6 keV at the P14 beamline at the DESY-PETRA III synchrotron in Hamburg, Germany. This dataset was used to estimate the location of chloride ions bound to the enzyme. "NaClAnon.hkl" is the final non-merged anomalous reflection file from data processing in XDS and XSCALE.
Abstract Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is little understood. Arc has an NTD involved in membrane binding and a CTD which binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerization, including assembly of retrovirus-like capsid involved in intercellular signaling. We produced and characterised six ultra-high-affinity anti-Arc nanobodies (Nb). The CTD of both rat and human Arc could be crystallised in ternary complexes with two Nbs simultaneously bound (H11 and C11). H11 binding deep into the stargazing-binding pocket of Arc CTD suggested competitive binding with Arc ligand peptides, which was confirmed in vitro . This indicates that the H11 Nb could serve as a genetically-encoded tool for inhibition of endogenous Arc N-lobe interactions in study of neuronal function and plasticity. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. Dynamics were affected by mutations known to inhibit capsid formation, implying a role for Arc CTD dynamics in oligomerisation. Dimerisation of the NTD, together with structural dynamics of the CTD, suggest a mechanism, by which structural dynamics of the CTD may promote capsomer formation, and dimerisation of the NTD links capsomers, facilitating the formation of capsids. The described recombinant ultrahigh-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function in vitro and in vivo .
Abstract Peptides available in biological niches inhabited by the human pathogen Staphylococcus aureus serve as a rich source of amino acids required for growth and successful host colonisation. Uptake of peptides by S. aureus involves at least two transport systems: the POT family di/tri-peptide permease DtpT and the oligopeptide ABC transporter Opp3. Here we study the individual and combined functions of DtpT and Opp3 in enabling utilisation of 282 di-/tri-peptides via a high-throughput phenotypic screen. We reveal that many peptides can be utilised via either transporter, though DtpT appears to be the primary route of uptake for dipeptides. Intriguingly, we demonstrate a preference for Asp/Glu-containing peptides among putative DtpT substrates. To measure DtpT-mediated peptide transport directly, the protein was purified and reconstituted into proteoliposomes and active transport of diverse di- and tri-peptides was demonstrated, supporting the conclusions of the phenotypic screen. During this in vitro analysis, we also found that DtpT could transport the biologically prevalent tripeptide reduced glutathione (GSH). In the absence of the known glutathione transporter Gis, dtpT is essential for growth under conditions where GSH is supplied as the sole sulphur source, identifying DtpT as the predicted second GSH uptake system of S. aureus . Site-directed mutagenesis of the predicted ligand binding site of DtpT suggests GSH binds the protein vertically in a manner dependent on interactions with Gln310. Finally, we demonstrate that GSH transport is required by S. aureus for complete fitness during in vitro macrophage infection experiments, and our data suggest that GSH uptake plays a role in modulating host-pathogen interactions in these infections. Together, these data reveal important new functions for DtpT, both in the utilisation of diverse peptides for growth but also providing the first clues toward a distinct role of DtpT during intracellular infection.
Abstract Synaptic plasticity is vital for brain function and memory formation. One of the key proteins in long-term synaptic plasticity and memory is the activity-regulated cytoskeleton-associated protein (Arc). Mammalian Arc forms virus-like capsid-like structures in a process requiring the N-terminal domain and contains two C-terminal lobes that are structural homologues to retroviral capsids. Drosophila has two isoforms of Arc, dArc1 and dArc2, with low sequence similarity to mammalian Arc, but lacking the mammalian Arc N-terminal domain. Both dArc isoforms have a capsid homology domain consisting of N- and C-terminal lobes. We carried out structural characterization of the four individual dArc lobe domains. As opposed to the corresponding mammalian Arc lobe domains, which are monomeric, the dArc lobes were all oligomeric in solution, indicating a strong propensity for homophilic interactions. The N-lobe from dArc2 formed a domain-swapped dimer in the crystal structure, resulting in a novel dimer interaction that could be relevant for capsid assembly or other dArc functions. This domain-swapped structure resembles the dimeric protein C of flavivirus capsids, as well as the structure of histones dimers, domain-swapped transcription factors, and membrane-interacting BAK domains. The strong oligomerization properties of the isolated dArc lobe domains explain the ability of dArc to form capsids in the absence of any large N-terminal domain, in contrast to the mammalian protein.
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