Journal Articles

  • Nitro-Assisted Brønsted Acid Catalysis: Application to a Challenging Catalytic Azidation
    [Jul 2015]

    Journal of the American Chemical SocietyDOI: 10.1021/jacs.5b06055
    Categories: Journal Articles
  • Effect of Intrinsic and Extrinsic Factors on the Simulated D-band Length of Type I Collagen
    [Jul 2015]

    Abstract

    A signature feature of collagen is its axial periodicity visible in TEM as alternating dark and light bands. In mature, type I collagen, this repeating unit, D, is 67 nm long. This periodicity reflects an underlying packing of constituent triple-helix polypeptide monomers wherein the dark bands represent gaps between axially adjacent monomers. This organization is visible distinctly in the microfibrillar model of collagen obtained from fiber diffraction. However, to date, no atomistic simulations of this diffraction model under zero-stress conditions have reported a preservation of this structural feature. Such a demonstration is important as it provides the baseline to infer response functions of physiological stimuli. In contrast, simulations predict a considerable shrinkage of the D-band (11-19%). Here we evaluate systemically the effect of several factors on D-band shrinkage. Using force fields employed in previous studies we find that irrespective of the temperature/pressure coupling algorithms, assumed salt concentration or hydration level, and whether or not the monomers are cross-linked, the D-band shrinks considerably. This shrinkage is associated with the bending and widening of individual monomers, but employing a force field whose backbone dihedral energy landscape matches more closely with our computed CCSD(T) values produces a small D-band shrinkage of < 3%. Since this force field also performs better against other experimental data, it appears that the large shrinkage observed in earlier simulations is a force-field artifact. The residual shrinkage could be due to the absence of certain atomic-level details, such as glycosylation sites, for which we do not yet have suitable data. This article is protected by copyright. All rights reserved.

    Categories: Journal Articles
  • Probing protease sensitivity of recombinant human erythropoietin reveals α3-α4 inter-helical loop as a stability determinant
    [Jul 2015]

    ABSTRACT

    Although unglycosylated HuEpo is fully functional, it has very short serum half-life. However, the mechanism of in vivo clearance of human Epo (HuEpo) remains largely unknown. In this study, the relative importance of protease-sensitive sites of recombinant HuEpo (rHuEpo) has been investigated by analysis of structural data coupled with in vivo half-life measurements. Our results identify α3-α4 inter-helical loop region as a target site of lysosomal protease Cathepsin L. Consistent with previously-reported lysosomal degradation of HuEpo, these results for the first time identify cleavage sites of rHuEpo by specific lysosomal proteases. Furthermore, in agreement with the lowered exposure of the peptide backbone around the cleavage site, remarkably substitutions of residues with bulkier amino acids result in significantly improved in vivo stability. Together, these results have implications for the mechanism of in vivo clearance of the protein in humans. This article is protected by copyright. All rights reserved.

    Categories: Journal Articles
  • Selective refinement and selection of near-native models in protein structure prediction
    [Jul 2015]

    Abstract

    In recent years in silico protein structure prediction reached a level where fully automated servers can generate large pools of near-native structures. However, the identification and further refinement of the best structures from the pool of models remain problematic. To address these issues, we have developed (i) a target-specific selective refinement (SR) protocol; and (ii) molecular dynamics (MD) simulation based ranking (SMDR) method. In SR the all-atom refinement of structures is accomplished via the Rosetta Relax protocol, subject to specific constraints determined by the size and complexity of the target. The best-refined models are selected with SMDR by testing their relative stability against gradual heating through all-atom MD simulations. Through extensive testing we have found that Mufold-MD, our fully automated protein structure prediction server updated with the SR and SMDR modules consistently outperformed its previous versions. This article is protected by copyright. All rights reserved.

    Categories: Journal Articles
  • Editorial Board
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15









    Categories: Journal Articles
  • Contents List
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15









    Categories: Journal Articles
  • Circular RNA and Splicing: Skip Happens
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15

    Author(s): Jeffrey Wilusz







    Categories: Journal Articles
  • Exon Skipping Is Correlated with Exon Circularization
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15

    Author(s): Steven Kelly, Chris Greenman, Peter R. Cook, Argyris Papantonis

    Circular RNAs are found in a wide range of organisms and it has been proposed that they perform disparate functions. However, how RNA circularization is connected to alternative splicing remains largely unexplored. Here, we stimulated primary human endothelial cells with tumor necrosis factor α or tumor growth factor β, purified RNA, generated &gt;2.4 billion RNA-seq reads, and used a custom pipeline to characterize circular RNAs derived from coding exons. We find that circularization of exons is widespread and correlates with exon skipping, a feature that adds considerably to the regulatory complexity of the human transcriptome.
    Graphical abstract




    Categories: Journal Articles
  • Limited Proteolysis Reveals That Amyloids from the 3D Domain-Swapping Cystatin B Have a Non-Native β-Sheet Topology
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15

    Author(s): Peter J. Davis, David Holmes, Jonathan P. Waltho, Rosemary A. Staniforth

    3D domain-swapping proteins form multimers by unfolding and then sharing of secondary structure elements, often with native-like interactions. Runaway domain swapping is proposed as a mechanism for folded proteins to form amyloid fibres, with examples including serpins and cystatins. Cystatin C amyloids cause a hereditary form of cerebral amyloid angiopathy whilst cystatin B aggregates are found in cases of Unverricht-Lundborg Syndrome, a progressive form of myoclonic epilepsy. Under conditions that favour fibrillisation, cystatins populate stable 3D domain-swapped dimers both in vitro and in vivo that represent intermediates on route to the formation of fibrils. Previous work on cystatin B amyloid fibrils revealed that the α-helical region of the protein becomes disordered and identified the conservation of a continuous 20-residue elongated β-strand (residues 39–58), the latter being a salient feature of the dimeric 3D domain-swapped structure. Here we apply limited proteolysis to cystatin B amyloid fibrils and show that not only the α-helical N-terminal of the protein (residues 1–35) but also the C-terminal of the protein (residues 80–98) can be removed without disturbing the underlying fibril structure. This observation is incompatible with previous models of cystatin amyloid fibrils where the β-sheet is assumed to retain its native antiparallel arrangement. We conclude that our data favour a more generic, at least partially parallel, arrangement for cystatin β-sheet structure in mature amyloids and propose a model that remains consistent with available data for amyloids from either cystatin B or cystatin C.
    Graphical abstract




    Categories: Journal Articles
  • E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15

    Author(s): Emily F. Ruff, Amanda C. Drennan, Michael W. Capp, Mikaela A. Poulos, Irina Artsimovitch, M. Thomas Record

    In transcription initiation by Escherichia coli RNA polymerase (RNAP), initial binding to promoter DNA triggers large conformational changes, bending downstream duplex DNA into the RNAP cleft and opening 13bp to form a short-lived open intermediate (I2). Subsequent conformational changes increase lifetimes of λPR and T7A1 open complexes (OCs) by &gt;105-fold and &gt;102-fold, respectively. OC lifetime is a target for regulation. To characterize late conformational changes, we determine effects on OC dissociation kinetics of deletions in RNAP mobile elements σ70 region 1.1 (σ1.1), β′ jaw and β′ sequence insertion 3 (SI3). In very stable OC formed by the wild type WT RNAP with λPR (RPO) and by Δσ1.1 RNAP with λPR or T7A1, we conclude that downstream duplex DNA is bound to the jaw in an assembly with SI3, and bases −4 to +2 of the nontemplate strand discriminator region are stably bound in a positively charged track in the cleft. We deduce that polyanionic σ1.1 destabilizes OC by competing for binding sites in the cleft and on the jaw with the polyanionic discriminator strand and downstream duplex, respectively. Examples of σ1.1-destabilized OC are the final T7A1 OC and the λPR I3 intermediate OC. Deleting σ1.1 and either β′ jaw or SI3 equalizes OC lifetimes for λPR and T7A1. DNA closing rates are similar for both promoters and all RNAP variants. We conclude that late conformational changes that stabilize OC, like early ones that bend the duplex into the cleft, are primary targets of regulation, while the intrinsic DNA opening/closing step is not.
    Graphical abstract




    Categories: Journal Articles
  • The Role of Packaging Sites in Efficient and Specific Virus Assembly
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15

    Author(s): Jason D. Perlmutter, Michael F. Hagan

    During the life cycle of many single-stranded RNA viruses, including many human pathogens, a protein shell called the capsid spontaneously assembles around the viral genome. Understanding the mechanisms by which capsid proteins selectively assemble around the viral RNA amidst diverse host RNAs is a key question in virology. In one proposed mechanism, short sequences (packaging sites) within the genomic RNA promote rapid and efficient assembly through specific interactions with the capsid proteins. In this work, we develop a coarse-grained particle-based computational model for capsid proteins and RNA that represents protein–RNA interactions arising both from nonspecific electrostatics and from specific packaging site interactions. Using Brownian dynamics simulations, we explore how the efficiency and specificity of assembly depend on solution conditions (which control protein–protein and nonspecific protein–RNA interactions) and the strength and number of packaging sites. We identify distinct regions in parameter space in which packaging sites lead to highly specific assembly via different mechanisms and others in which packaging sites lead to kinetic traps. We relate these computational predictions to in vitro assays for specificity in which cognate viral RNAs compete against non-cognate RNAs for assembly by capsid proteins.
    Graphical abstract




    Categories: Journal Articles
  • Locating Herpesvirus Bcl-2 Homologs in the Specificity Landscape of Anti-Apoptotic Bcl-2 Proteins
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15

    Author(s): Glenna Wink Foight, Amy E. Keating

    Viral homologs of the anti-apoptotic Bcl-2 proteins are highly diverged from their mammalian counterparts, yet they perform overlapping functions by binding and inhibiting BH3 (Bcl-2 homology 3)-motif-containing proteins. We investigated the BH3 binding properties of the herpesvirus Bcl-2 homologs KSBcl-2, BHRF1, and M11, as they relate to those of the human Bcl-2 homologs Mcl-1, Bfl-1, Bcl-w, Bcl-xL, and Bcl-2. Analysis of the sequence and structure of the BH3 binding grooves showed that, despite low sequence identity, M11 has structural similarities to Bcl-xL, Bcl-2, and Bcl-w. BHRF1 and KSBcl-2 are more structurally similar to Mcl-1 than to the other human proteins. Binding to human BH3-like peptides showed that KSBcl-2 has similar specificity to Mcl-1, and BHRF1 has a restricted binding profile; M11 binding preferences are distinct from those of Bcl-xL, Bcl-2, and Bcl-w. Because KSBcl-2 and BHRF1 are from human herpesviruses associated with malignancies, we screened computationally designed BH3 peptide libraries using bacterial surface display to identify selective binders of KSBcl-2 or BHRF1. The resulting peptides bound to KSBcl-2 and BHRF1 in preference to Bfl-1, Bcl-w, Bcl-xL, and Bcl-2 but showed only modest specificity over Mcl-1. Rational mutagenesis increased specificity against Mcl-1, resulting in a peptide with a dissociation constant of 2.9nM for binding to KSBcl-2 and &gt;1000-fold specificity over other Bcl-2 proteins, as well as a peptide with &gt;70-fold specificity for BHRF1. In addition to providing new insights into viral Bcl-2 binding specificity, this study will inform future work analyzing the interaction properties of homologous binding domains and designing specific protein interaction partners.
    Graphical abstract




    Categories: Journal Articles
  • Structure and Mechanism of Dimer–Monomer Transition of a Plant Poly(A)-Binding Protein upon RNA Interaction: Insights into Its Poly(A) Tail Assembly
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15

    Author(s): Mariane Noronha Domingues, Mauricio Luis Sforça, Adriana Santos Soprano, Jack Lee, Tatiana de Arruda Campos Brasil de Souza, Alexandre Cassago, Rodrigo Villares Portugal, Ana Carolina de Mattos Zeri, Mario Tyago Murakami, Ari Sadanandom, Paulo Sergio Lopes de Oliveira, Celso Eduardo Benedetti

    Poly(A)-binding proteins (PABPs) play crucial roles in mRNA biogenesis, stability, transport and translational control in most eukaryotic cells. Although animal PABPs are well-studied proteins, the biological role, three-dimensional structure and RNA-binding mode of plant PABPs remain largely uncharacterized. Here, we report the structural features and RNA-binding mode of a Citrus sinensis PABP (CsPABPN1). CsPABPN1 has a domain architecture of nuclear PABPs (PABPNs) with a single RNA recognition motif (RRM) flanked by an acidic N-terminus and a GRPF-rich C-terminus. The RRM domain of CsPABPN1 displays virtually the same three-dimensional structure and poly(A)-binding mode of animal PABPNs. However, while the CsPABPN1 RRM domain specifically binds poly(A), the full-length protein also binds poly(U). CsPABPN1 localizes to the nucleus of plant cells and undergoes a dimer–monomer transition upon poly(A) interaction. We show that poly(A) binding by CsPABPN1 begins with the recognition of the RNA-binding sites RNP1 and RNP2, followed by interactions with residues of the β2 strands, which stabilize the dimer, thus leading to dimer dissociation. Like human PABPN1, CsPABPN1 also seems to form filaments in the presence of poly(A). Based on these data, we propose a structural model in which contiguous CsPABPN1 RRM monomers wrap around the RNA molecule creating a superhelical structure that could not only shield the poly(A) tail but also serve as a scaffold for the assembly of additional mRNA processing factors.
    Graphical abstract




    Categories: Journal Articles
  • Anti-PolyQ Antibodies Recognize a Short PolyQ Stretch in Both Normal and Mutant Huntingtin Exon 1
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15

    Author(s): Gwen E. Owens, Danielle M. New, Anthony P. West, Pamela J. Bjorkman

    Huntington's disease is caused by expansion of a polyglutamine (polyQ) repeat in the huntingtin protein. A structural basis for the apparent transition between normal and disease-causing expanded polyQ repeats of huntingtin is unknown. The “linear lattice” model proposed random-coil structures for both normal and expanded polyQ in the preaggregation state. Consistent with this model, the affinity and stoichiometry of the anti-polyQ antibody MW1 increased with the number of glutamines. An opposing “structural toxic threshold” model proposed a conformational change above the pathogenic polyQ threshold resulting in a specific toxic conformation for expanded polyQ. Support for this model was provided by the anti-polyQ antibody 3B5H10, which was reported to specifically recognize a distinct pathologic conformation of soluble expanded polyQ. To distinguish between these models, we directly compared binding of MW1 and 3B5H10 to normal and expanded polyQ repeats within huntingtin exon 1 fusion proteins. We found similar binding characteristics for both antibodies. First, both antibodies bound to normal, as well as expanded, polyQ in huntingtin exon 1 fusion proteins. Second, an expanded polyQ tract contained multiple epitopes for fragments antigen-binding (Fabs) of both antibodies, demonstrating that 3B5H10 does not recognize a single epitope specific to expanded polyQ. Finally, small-angle X-ray scattering and dynamic light scattering revealed similar binding modes for MW1 and 3B5H10 Fab–huntingtin exon 1 complexes. Together, these results support the linear lattice model for polyQ binding proteins, suggesting that the hypothesized pathologic conformation of soluble expanded polyQ is not a valid target for drug design.
    Graphical abstract




    Categories: Journal Articles
  • Crystal Structure of Human DNA Methyltransferase 1
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15

    Author(s): Zhi-Min Zhang, Shuo Liu, Krystal Lin, Youfu Luo, John Jefferson Perry, Yinsheng Wang, Jikui Song

    DNMT1 (DNA methyltransferase 1) is responsible for propagating the DNA methylation patterns during DNA replication. DNMT1 contains, in addition to a C-terminal methyltransferase domain, a large N-terminal regulatory region that is composed of an RFTS (replication foci targeting sequence) domain, a CXXC zinc finger domain and a pair of BAH (bromo adjacent homology) domains. The regulatory domains of DNMT1 mediate a network of protein–protein and protein–DNA interactions to control the recruitment and enzymatic activity of DNMT1. Here we report the crystal structure of human DNMT1 with all the structural domains (hDNMT1, residues 351–1600) in complex with S-adenosyl-l-homocysteine at 2.62Å resolution. The RFTS domain directly associates with the methyltransferase domain, thereby inhibiting the substrate binding of hDNMT1. Through structural analysis, mutational, biochemical and enzymatic studies, we further identify that a linker sequence between the CXXC and BAH1 domains, aside from its role in the CXXC domain-mediated DNMT1 autoinhibition, serves as an important regulatory element in the RFTS domain-mediated autoinhibition. In comparison with the previously determined structure of mouse DNMT1, this study also reveals a number of distinct structural features that may underlie subtle functional diversity observed for the two orthologues. In addition, this structure provides a framework for understanding the functional consequence of disease-related hDNMT1 mutations.
    Graphical abstract




    Categories: Journal Articles
  • Engineering Synthetic Antibody Inhibitors Specific for LD2 or LD4 Motifs of Paxillin
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15

    Author(s): Malgorzata Nocula-Lugowska, Mateusz Lugowski, Ravi Salgia, Anthony A. Kossiakoff

    Focal adhesion protein paxillin links integrin and growth factor signaling to actin cytoskeleton. Most of paxillin signaling activity is regulated via leucine-rich LD motifs (LD1–LD5) located at the N-terminus. Here, we demonstrate a method to engineer highly selective synthetic antibodies (sABs) against LD2 and LD4 that are binding sites for focal adhesion kinase (FAK) and other proteins. Phage display selections against peptides were used to generate sABs recognizing each LD motif. In the obtained X-ray crystal structures of the LD-sAB complexes, the LD motifs are helical and bind sABs through a hydrophobic side, similarly as in the structures with natural paxillin partners. The sABs are capable of pulling down endogenous paxillin in complex with FAK and can visualize paxillin in focal adhesions in cells. They were also used as selective inhibitors to effectively compete with focal adhesion targeting domain of FAK for the binding to LD2 and LD4. The sABs are tools for investigation of paxillin LD binding “platforms” and are capable of inhibiting paxillin interactions, thereby useful as potential therapeutics in the future.
    Graphical abstract




    Categories: Journal Articles
  • Structural and Functional Analysis of the Signal-Transducing Linker in the pH-Responsive One-Component System CadC of Escherichia coli
    [Jul 2015]

    Publication date: 31 July 2015
    Source:Journal of Molecular Biology, Volume 427, Issue 15

    Author(s): Sophie Buchner, Andreas Schlundt, Jürgen Lassak, Michael Sattler, Kirsten Jung

    The pH-responsive one-component signaling system CadC in Escherichia coli belongs to the family of ToxR-like proteins, whose members share a conserved modular structure, with an N-terminal cytoplasmic winged helix–turn–helix DNA-binding domain being followed by a single transmembrane helix and a C-terminal periplasmic pH-sensing domain. In E. coli CadC, a cytoplasmic linker comprising approximately 50 amino acids is essential for transmission of the signal from the sensor to the DNA-binding domain. However, the mechanism of transduction is poorly understood. Using NMR spectroscopy, we demonstrate here that the linker region is intrinsically disordered in solution. Furthermore, mutational analyses showed that it tolerates a range of amino acid substitutions (altering polarity, rigidity and α-helix-forming propensity), is robust to extension but is sensitive to truncation. Indeed, truncations either reversed the expression profile of the target operon cadBA or decoupled expression from external pH altogether. CadC dimerizes via its periplasmic domain, but light-scattering analysis provided no evidence for dimerization of the isolated DNA-binding domain, with or without the linker region. However, bacterial two-hybrid analysis revealed that CadC forms stable dimers in a stimulus- and linker-dependent manner, interacting only at pH&lt;6.8. Strikingly, a variant with inversed cadBA expression profile, which lacks most of the linker, dimerizes preferentially at higher pH. Thus, we propose that the disordered CadC linker is required for transducing the pH-dependent response of the periplasmic sensor into a structural rearrangement that facilitates dimerization of the cytoplasmic CadC DNA-binding domain.
    Graphical abstract




    Categories: Journal Articles
  • Syndicate content