The Structure of RdDddP from Roseobacter denitrificans Reveals That DMSP Lyases in the DddP-Family Are Metalloenzymes

Marine microbes degrade dimethylsulfoniopropionate (DMSP), which is produced in large quantities by marine algae and plants, with DMSP lyases into acrylate and the gas dimethyl sulfide (DMS). Approximately 10% of the DMS vents from the sea into the atmosphere and this emission returns sulfur, which arrives in the sea through rivers and runoff, back to terrestrial systems via clouds and rain. Despite their key role in this sulfur cycle DMSP lyases are poorly understood at the molecular level. Here we report the first X-ray crystal structure of the putative DMSP lyase RdDddP from Roseobacter denitrificans, which belongs to the abundant DddP family. This structure, determined to 2.15 Å resolution, shows that RdDddP is a homodimeric metalloprotein with a binuclear center of two metal ions located 2.7 Å apart in the active site of the enzyme. Consistent with the crystallographic data, inductively coupled plasma mass spectrometry (ICP-MS) and total reflection X-ray fluorescence (TRXF) revealed the bound metal species to be primarily iron. A 3D structure guided analysis of environmental DddP lyase sequences elucidated the critical residues for metal binding are invariant, suggesting all proteins in the DddP family are metalloenzymes

Identification of NS2 determinants stimulating intrinsic HCV NS2 protease activity

Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2pro) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2pro activity by the NS3 protease domain. NS2pro activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2pro activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2pro activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2pro could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2pro activity mimic structural changes occurring during NS3-mediated NS2pro activation. Introducing these activating NS2pro mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2pro activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2pro activation by NS3

CHL1 Is a Selective Organizer of the Presynaptic Machinery Chaperoning the SNARE Complex

Proteins constituting the presynaptic machinery of vesicle release undergo substantial conformational changes during the process of exocytosis. While changes in the conformation make proteins vulnerable to aggregation and degradation, little is known about synaptic chaperones which counteract these processes. We show that the cell adhesion molecule CHL1 directly interacts with and regulates the activity of the synaptic chaperones Hsc70, CSP and αSGT. CHL1, Hsc70, CSP and αSGT form predominantly CHL1/Hsc70/αSGT and CHL1/CSP complexes in synapses. Among the various complexes formed by CHL1, Hsc70, CSP and αSGT, SNAP25 and VAMP2 induce chaperone activity only in CHL1/Hsc70/αSGT and CHL1/CSP complexes, respectively, indicating a remarkable selectivity of a presynaptic chaperone activity for proteins of the exocytotic machinery. In mice with genetic ablation of CHL1, chaperone activity in synapses is reduced and the machinery for synaptic vesicle exocytosis and, in particular, the SNARE complex is unable to sustain prolonged synaptic activity. Thus, we reveal a novel role for a cell adhesion molecule in selective activation of the presynaptic chaperone machinery

RETRACTED: Induction of a Homeostatic Circuit in Lung Tissue by Microbial Compounds

This article has been retracted at the request of the inhouse Editor, Peter Lee, and the authors. The text below has been agreed between Peter Lee and the corresponding author. Please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).Reason: The authors discovered that a duplication of several gel images occurred during the preparation of the above manuscript. Specifically, the gel images in Figure 2A were duplicated within Figure 2A, and in Figures 5F and 6B. These figures are important in showing the amount of Smad 2/3 as an indicator of alveolar macrophage-epithelial cell interactions, which explains the mechanism of the proposed homeostatic circuit in the lungs, and thus we are retracting the manuscript. The authors stand by the validity of the other figures, and sincerely apologize for the inconvenience caused by this retraction

A simple vapor-diffusion method enables protein crystallization inside the HARE serial crystallography chip

Fixed-target serial crystallography has become an important method for the study of protein structure and dynamics at synchrotrons and X-ray free-electron lasers. However, sample homogeneity, consumption and the physical stress on samples remain major challenges for these high-throughput experiments, which depend on high-quality protein microcrystals. The batch crystallization procedures that are typically applied require time- and sample-intensive screening and optimization. Here, a simple protein crystallization method inside the features of the HARE serial crystallography chips is reported that circumvents batch crystallization and allows the direct transfer of canonical vapor-diffusion conditions to in-chip crystallization. Based on conventional hanging-drop vapor-diffusion experiments, the crystallization solution is distributed into the wells of the HARE chip and equilibrated against a reservoir with mother liquor. Using this simple method, high-quality microcrystals were generated with sufficient density for the structure determination of four different proteins. A new protein variant was crystallized using the protein concentrations encountered during canonical crystallization experiments, enabling structure determination from ∼55 µg of protein. Additionally, structure determination from intracellular crystals grown in insect cells cultured directly in the features of the HARE chips is demonstrated. In cellulo crystallization represents a comparatively un­explored space in crystallization, especially for proteins that are resistant to crystallization using conventional techniques, and eliminates any need for laborious protein purification. This in-chip technique avoids harvesting the sensitive crystals or any further physical handling of the crystal-containing cells. These proof-of-principle experiments indicate the potential of this method to become a simple alternative to batch crystallization approaches and also as a convenient extension to canonical crystallization screens

Real-time investigation of dynamic protein crystallization in living cells

X-ray crystallography requires sufficiently large crystals to obtain structural insights at atomic resolution, routinely obtained in vitro by time-consuming screening. Recently, successful data collection was reported from protein microcrystals grown within living cells using highly brilliant free-electron laser and third-generation synchrotron radiation. Here, we analyzed in vivo crystal growth of firefly luciferase and Green Fluorescent Protein-tagged reovirus μNS by live-cell imaging, showing that dimensions of living cells did not limit crystal size. The crystallization process is highly dynamic and occurs in different cellular compartments. In vivo protein crystallization offers exciting new possibilities for proteins that do not form crystals in vitroL.R., M.K., D.R., and C.B. thank the German Federal Ministry for Education and Research (BMBF) for funding (Grant Nos. 01KX0806 and 01KX0807). L.R., M.D., and C.B. acknowledge support from the BMBF in the context of the Röntgen-Angström-Cluster (Grant No. 05K12GU3). J.M.-C. and A.B.-N. acknowledge support from the Spanish Ministerio Economía y Competitividad (MINECO, Grant No. BFU2013-43513-R). I.V.M., R.D., and L.R. are grateful for support from the DFG Cluster of Excellence “Inflammation at Interfaces” (EXC 306)S

Characterization of prion protein function by focal neurite stimulation

The cellular prion protein (PrPC), encoded by the PRNP gene, is a ubiquitous glycoprotein, which is highly expressed in the brain. This protein, mainly known for its role in neurodegenerative diseases, is involved in several physiological processes including neurite outgrowth. By using a novel focal stimulation technique, we explored the potential function of PrPC, in its soluble form, as a signaling molecule. Thus, soluble recombinant prion proteins (recPrP) encapsulated in micro-vesicles were released by photolysis near the hippocampal growth cones. Local stimulation of wild-type growth cones with full-length recPrP induced neurite outgrowth and rapid growth cone turning towards the source. This effect was shown to be concentration dependent. Notably, PrPC-knockout growth cones were insensitive to recPrP stimulation, but this property was rescued in PrPknockout growth cones expressing GFP-PrP. Taken together, our findings indicate that recPrP functions as a signaling molecule, and that its homophilic interaction with membrane-anchored PrPC might promote neurite outgrowth and facilitate growth cone guidance. \ua9 2016. Published by The Company of Biologists Ltd

Electronic damage in S atoms in a native protein crystal induced by an intense X-ray free-electron laser pulse

Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed

Microtubules control cellular shape and coherence in amoeboid migrating cells

Cells navigating through complex tissues face a fundamental challenge: while multiple protrusions explore different paths, the cell needs to avoid entanglement. How a cell surveys and then corrects its own shape is poorly understood. Here, we demonstrate that spatially distinct microtubule dynamics regulate amoeboid cell migration by locally promoting the retraction of protrusions. In migrating dendritic cells, local microtubule depolymerization within protrusions remote from the microtubule organizing center triggers actomyosin contractility controlled by RhoA and its exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: (1) impaired cell edge coordination during path finding and (2) defective adhesion resolution. Compromised shape control is particularly hindering in geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. We thus demonstrate that microtubules can act as a proprioceptive device: they sense cell shape and control actomyosin retraction to sustain cellular coherence