Background: Biochemical characterization of voltage-dependent anion channel 2 (VDAC2) is limited due to an inability to obtain functional protein. Results:The crystal structure of VDAC2 suggests a dimer interface that is confirmed by double electron-electron resonance and cross-linking. Conclusion: zfVDAC2 has a fractional dimeric population. Significance: VDAC isoforms are structurally similar, but this study has identified a number of hot spots that require further exploration.

Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na+ gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na+ ions. One Na+ binds to the conserved Na2 site, while the second Na+ binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na+ sites regulate N-acetylneuraminic acid transport.

The voltage-dependent anion channel (VDAC) mediates metabolite and ion flow across the outer mitochondrial membrane of all eukaryotic cells. The open channel passes millions of ATP molecules per second, while the closed state exhibits no detectable ATP flux. High-resolution structures of VDAC1 revealed a 19-stranded β-barrel with an α-helix partially occupying the central pore. To understand ATP permeation through VDAC, we solved the crystal structure of mouse VDAC1 (mVDAC1) in the presence of ATP, revealing a low-affinity binding site. Guided by these coordinates, we initiated hundreds of molecular dynamics (MD) simulations to construct a Markov State Model (MSM) of ATP permeation. These simulations indicate that ATP flows through VDAC using multiple pathways, consistent with our structural data and experimentally determined physiological rates.

Intrinsically unstructured proteins (IUPs), also known as natively unfolded proteins, lack well-defined secondary and tertiary structure under physiological conditions. In recent years, growing experimental and theoretical evidence has accumulated, indicating that many entire proteins and protein sequences are unstructured under physiological conditions, and that they play significant roles in diverse cellular processes. Bioinformatic algorithms have been developed to identify such sequences in proteins for which structural data are lacking, but still generate substantial numbers of false positives and negatives. We describe here a simple and reliable in vitro assay for identifying IUP sequences based on their susceptibility to 20S proteasomal degradation. We show that 20S proteasomes digest IUP sequences, under conditions in which native, and even molten globule states, are resistant. Furthermore, we show that protein-protein interactions can protect IUPs against 20S proteasomal action. Taken together, our results thus suggest that the 20S proteasome degradation assay provides a powerful system for operational definition of IUPs.

The biennial CASP experiment is a crucial way to evaluate, in an unbiased way, the progress in predicting novel 3D protein structures. In this article, we assess the quality of prediction of template free models, that is, ab initio prediction of 3D structures of proteins based solely on the amino acid sequences, that is, proteins that did not have significant sequence identity to any protein in the Protein Data Bank. There were 13 targets in this category and 102 groups submitted predictions. Analysis was based on the GDT_TS analysis, which has been used in previous CASP experiments, together with a newly developed method, the OK_Rank, as well as by visual inspection. There is no doubt that in recent years many obstacles have been removed on the long and elusive way to deciphering the protein‐folding problem. Out of the 13 targets, six were predicted well by a number of groups. On the other hand, it must be stressed that for four targets, none of the models were judged to be satisfactory. Thus, for template free model prediction, as evaluated in this CASP, successes have been achieved for most targets; however, a great deal of research is still required, both in improving the existing methods and in development of new approaches. Proteins 2009. © 2009 Wiley‐Liss, Inc.

Background:The flagellar motor generates bidirectional rotation using the proteins FliG, FliM, and CheY and membrane-bound stator complexes. Results: The structures of portions of FliG and FliM in complex allow for insights into switching and structure. Conclusion: FliG MC and FliM M in a 1:1 complex may form the C-ring of the motor. Significance: Understanding motor structure is key to understanding its mechanism.

More than 60 members of the Rab GTPase family exist in the human genome. However, our current understanding is only limited to the role of small Rab GTPases in membrane trafficking. Here we show that CRACR2A encodes a lymphocyte-specific “large Rab GTPase” containing multiple functional domains including EF-hand motifs, proline-rich and Rab GTPase domains with an unconventional prenylation site. We demonstrate its direct role in activation of the Ca2+ and the Jnk signaling pathways upon T cell receptor (TCR) stimulation using gene silencing and transgenic animal models. Mechanistically, vesicles containing this Rab GTPase translocate from the Golgi into the immunological synapse (IS) to activate these signaling pathways. The interaction between proline-rich domain of this Rab GTPase and a guanidine nucleotide exchange factor/scaffold protein Vav1 is essential for accumulation of these vesicles at the IS. Furthermore, we demonstrate that GTP binding and prenylation are closely linked to membrane association, stability, and thereby activation of downstream signaling by this large GTPase. Our findings reveal a novel function of a large Rab GTPase in TCR signaling pathways, which is potentially shared by other GTPases with similar domain architecture.

Objective To describe the use of a new lymph-node nodes identified by conventional and the LNRS methods was recorded and classified according the revealing solution (LNRS) for detecting lymph node involvement in total cystectomy specimens from TNM system. Result Twenty-two lymph nodes were detected by the patients with locally confined invasive transitional cell carcinoma (TCC) of the bladder, and to compare the conventional method, of which four were positive for tumour metastasis. Using the LNRS, an additional 21 results obtained with those using the conventional method (palpation and sectioning perivesical fat) that nodes were identified among which 12 were positive. The mean size of the lymph nodes detected by the may fail to detect very small lymph nodes. Materials and methods Of 12 cystectomy specimens conventional and LNRS methods was 7.96 mm and 3.81 mm, respectively. The stage of three patients was obtained from patients with TCC, six in which 0-3 metastatic nodes were identified by the conventional increased (Nx to N2, N0 to N2 and N1 to N2) and therefore two of these patients received adjuvant method were further investigated using LNRS. The revealing solution comprised 95% ethanol, diethyl chemotherapy. Conclusions LNRS significantly enhanced the yield of ether, glacial acetic acid and buÂered formalin (6552055:10 v/v) prepared under a fume-hood. After normal and metastatic nodes of cystectomy specimens and may identify smaller nodes. The LNRS method evaluation using the conventional method, the specimens were immersed for 6-12 h in the solution, allows a more accurate staging with better assessment of the prognosis and need for adjuvant therapy. washed under running tap water and the adipose tissue sectioned at intervals of 2-3 mm. Lymph nodes