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Journal of molecular biology [journal]
- Control of RecBCD Enzyme Activity by DNA Binding- and Chi Hotspot-dependent Conformational Changes. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 26.
Faithful repair of DNA double-strand breaks by homologous recombination is crucial to maintain functional genomes. The major Escherichia coli pathway of DNA break repair requires RecBCD enzyme, a complex protein machine with multiple activities. Upon encountering a Chi recombination hotspot (5'GCTGGTGG3') during DNA unwinding, RecBCD's unwinding, nuclease, and RecA-loading activities change dramatically, but the physical basis for these changes is unknown. Here we identify, during RecBCD's DNA unwinding, two Chi-stimulated conformational changes involving RecC. One produced a marked, long-lasting, Chi-dependent increase in protease-sensitivity of a small patch, near the Chi recognition domain, on the solvent-exposed RecC surface. The other change was identified by crosslinking of an artificial amino acid inserted in this RecC patch to RecB. Small angle X-ray scattering analysis confirmed a major conformational change upon binding of DNA to the enzyme and is consistent with two changes. We propose that upon DNA binding the RecB nuclease domain swings from one side of RecC to the other; when RecBCD encounters Chi, the nuclease domain returns to its initial position determined by crystallography, where it nicks DNA exiting from RecC and loads RecA onto the newly generated 3'-ended single-stranded DNA during continued unwinding; a crevice between RecB and RecC increasingly narrows during these steps. This model provides a physical basis for the intramolecular "signal transduction" from Chi to RecC to RecD to RecB inferred previously from genetic and enzymatic analyses, and it accounts for the enzymatic changes that accompany Chi's stimulation of recombination.
- Regulation of HIV-1 latency by chromatin structure and nuclear architecture. [REVIEW]
- J Mol Biol 2014 Jul 26.
Current antiretroviral therapies fail to cure HIV-1 infection because HIV-1 persists as a transcriptionally inactive provirus in resting memory CD4(+) T cells. Multiple molecular events are known to regulate HIV-1 gene expression, yet the mechanisms governing the establishment and maintenance of latency remain incompletely understood. Here we summarize different molecular aspects of viral latency, from its establishment in resting CD4(+) T cells to the mechanisms involved in the reactivation of latent viral reservoirs. We focus on the relevance of chromatin structure and nuclear architecture in determining the transcriptional fate of integrated HIV-1 genomes, in light of recent findings indicating that proximity to specific subnuclear neighborhoods regulate HIV-1 gene expression.
- A fibrin-specific monoclonal antibody from a designed phage display library inhibits clot formation and localizes to tumors in vivo. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 26.
Fibrin formation from fibrinogen is a rare process in the healthy organism, but is a pathological feature of thrombotic events, cancer and a wide range of inflammatory conditions. We have designed and constructed an antibody phage display library (containing 13 billion clones), for the selective recognition of the N-terminal peptide of fibrin alpha chain. The key structural feature for selective fibrin binding was a K94E mutation in the VH domain. From this library, an antibody was isolated (termed AP2), which recognizes the five N-terminal aminoacids of fibrin with high-affinity (Kd=44 nM), but does not bind to fibrinogen. The AP2 antibody could be expressed in various formats (scFv, SIP and IgG) and inhibited fibrin clot formation in a concentration-dependent manner. Moreover, the AP2 antibody stained the fibrin-rich provisional stroma in solid tumors, but did not exhibit any detectable staining towards normal tissues. Using a radioiodinated antibody preparation and quantitative biodistribution studies in tumor-bearing mice, AP2 was shown to selectively localize to fibrin-rich F9 murine teratocarcinomas, but not to SKRC-52 human kidney cancer xenografts. Collectively, the experiments indicate that the AP2 antibody recognizes fibrin in vitro and in vivo. The antibody may facilitate the development of fibrin-specific therapeutic agents.
- Cyclophilin-facilitated membrane translocation as pharmacological target to prevent intoxication of mammalian cells by binary clostridial actin ADP-ribosylated toxins. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 21.
Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile CDT belong to the family of binary actin ADP-ribosylating toxins and are composed of a binding/translocation component and a separate enzyme component. The enzyme components ADP-ribosylate G-actin in the cytosol of target cells resulting in depolymerization of F-actin, cell rounding and cell death. The binding/translocation components bind to their cell receptors and form complexes with the respective enzyme components. After receptor-mediated endocytosis, the binding/translocation components form pores in membranes of acidified endosomes and the enzyme components translocate through these pores into the cytosol. This step is facilitated by the host cell chaperone heat shock protein 90 and peptidyl-prolyl cis/trans isomerases (PPIases) including cyclophilin A. Here, we demonstrate that a large isoform of cyclophilin A, the multi-domain enzyme cyclophilin-40, binds to the enzyme components C2I, Ia and CDTa in vitro. Isothermal titration calorimetry revealed a direct binding to C2I with a calculated affinity of 101nM and to Ia with an affinity of 1.01μM. Closer investigation for the prototypic C2I revealed that binding to cyclophilin-40 did not depend on its ADP-ribosyltransferase activity but was stronger for unfolded C2I. The interaction of C2I with cyclophilin-40 was also demonstrated in lysates from C2-treated cells by pull-down. Treatment of cells with a non-immunosuppressive cyclosporine A-derivative, which still binds to and inhibits the PPIase activity of cyclophilins, protected cells from intoxication with C2, iota and CDT toxins, offering an attractive approach for development of novel therapeutic strategies against binary actin ADP-ribosylating toxins.
- Structure of the entire stalk region of the dynein motor domain. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 21.
Dyneins are large microtubule-based motor complexes that power a range of cellular processes including the transport of organelles, and the beating of cilia and flagella. The motor domain is located within the dynein heavy chain and comprises an N-terminal mechanical linker element, a central ring of six AAA+modules of which four bind or hydrolyze ATP, and a long stalk extending from the AAA+ring with a microtubule-binding domain (MTBD) at its tip. A crucial mechanism underlying the motile activity of cytoskeletal motor proteins is precise coupling between the ATPase and track-binding activities. In dynein, a stalk region consisting of a long (~15nm) antiparallel coiled coil separates these two activities, which must facilitate communication between them. This communication is mediated by a small degree of helix sliding in the coiled coil. However, no high-resolution structure is available of the entire stalk region including the MTBD. Here, we have reported the structure of the entire stalk region of mouse cytoplasmic dynein in a weak microtubule-binding state, which was determined using X-ray crystallography, and have compared it with the dynein motor domain from Dictyostelium discoideum in a strong microtubule-binding state as well as with a mouse MTBD with its distal portion of the coiled coil fused to seryl-tRNA synthetase from Thermus thermophilus. Our results strongly support the helix-sliding model based on the complete structure of the dynein stalk with a different form of coiled-coil packing. We also propose a plausible mechanism of helix sliding together with further analysis using molecular dynamic simulations. Our results present the importance of conserved proline residues for an elastic motion of stalk coiled coil, and imply the manner of change between high-affinity and low-affinity state of MTBD.
- Diffusion of human Replication Protein A along single stranded DNA. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 21.
Replication Protein A (RPA) is a eukaryotic single stranded (ss) DNA binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA-DNA interactions, we combined ensemble and single molecule fluorescence approaches to examine human RPA diffusion along ssDNA and find that an hRPA hetero-trimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D1~5000 nucleotide(2)s(-1) at 37˚C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA.
- Unconventional Secretion of Fibroblast Growth Factor 2 - a Novel Type of Protein Translocation Across Membranes? [REVIEW]
- J Mol Biol 2014 Jul 19.
N-terminal signal peptides are a hallmark of the vast majority of soluble secretory proteins that are transported along the endoplasmic reticulum (ER)/Golgi-dependent pathway. They are recognized by signal recognition particle (SRP), a process that initiates membrane translocation into the lumen of the ER followed by vesicular transport to the cell surface and release into the extracellular space. Beyond this well-established mechanism of protein secretion from eukaryotic cells, a number of extracellular proteins with critical physiological functions in immune surveillance and tissue organization are known to be secreted in an SRP-independent manner. Such processes have collectively been termed 'unconventional protein secretion' and, while known for more than two decades, their underlying mechanisms are only beginning to emerge. Different types of unconventional secretory mechanisms have been described with the best-characterized example being based on direct translocation of cytoplasmic proteins across plasma membranes. The aim of this review is to critically assess our current knowledge of this type of unconventional secretion focusing on fibroblast growth factor 2 (FGF2) as the most established example.
- Dynactin 3D structure: Implications for assembly and dynein binding. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 18.
The multisubunit protein complex, dynactin, is an essential component of the cytoplasmic dynein motor. High resolution structural work on dynactin and the dynein/dynactin supercomplex has been limited to small subunits and recombinant fragments that do not report fully on either≈1 MDa assembly. In the present study, we used negative stain electron microscopy and image analysis based on random conical tilt reconstruction to obtain a three-dimensional structure of native vertebrate dynactin. The 35nm long dynactin molecule has a V-shaped shoulder at one end and a flattened tip at the other, both offset relative to the long axis of the actin-related protein (Arp) backbone. The shoulder projects dramatically away from the Arp filament core in a way that cannot be appreciated in 2D images, which has implications for the mechanism of dynein binding. The 3D structure allows the helical parameters of the entire Arp filament core, which includes the actin capping protein, CP, to be determined for the first time. This structure exhibits near identity to F-actin and can be well fitted into the dynactin envelope. Molecular fitting of modeled CP-Arp polymers into the envelope shows that the filament contains between 7 and 9 Arp protomers and is capped at both ends. In the 7-Arp model, which agrees best with measured Arp stoichiometry and other structural information, actin capping protein (CP) is not present at the distal tip of the structure, unlike what is seen in the other models. The 3D structure suggests a mechanism for dynactin assembly and length specification.
- Adaptive remodelling by FliN in the bacterial rotary motor. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 18.
Sensory adaptation in the E. coli chemosensory pathway has been the subject of interest for decades, with investigation focusing on the receptors that process extracellular inputs. Recent studies demonstrate that the flagellar motors responsible for cell locomotion also play a role, adding or subtracting FliM subunits to maximise sensitivity to pathway signals. It is difficult to reconcile this FliM remodelling with the observation that partner FliN subunits are relatively static fixtures in the motor. By fusing a fluorescent protein internally to FliN, we show that there is in fact significant FliN remodelling. The kinetics and stoichiometry of FliN in steady-state and in adapting motors are investigated and found to match the behaviour of FliM in all respects except for timescale, where FliN rates are about four times slower. We notice that motor adaptation is slower in the presence of the fluorescent protein, indicating a possible source for the difference. The behaviour of FliM and FliN is consistent with a kinetic and stoichiometric model that contradicts the traditional view of a packed, rigid motor architecture.
- Structural and biochemical characterization of the natural product kendomycin and the 20S proteasome. [JOURNAL ARTICLE]
- J Mol Biol 2014 Jul 16.
Natural products are a valuable source for novel lead structures in drug discovery, but for the majority of isolated bioactive compounds the cellular targets are unknown. The structurally unique ansa-polyketide kendomycin was reported to exert its potent cytotoxic effects via impairment of the ubiquitin proteasome system, but the exact mode of action remained unclear. Here, we present a systematic biochemical characterization of kendomycin-proteasome interactions in vitro as well as in vivo, including complex structures of wildtype and mutant yeast 20S proteasome with kendomycin. Our results provide evidence for a polypharmacological mode of action for kendomycin's cytotoxic effect on cancer cells.