Journal Articles

  • Molecular Interactions and Residues Involved in Force Generation in the T4 Viral DNA Packaging Motor
    [Dec 2014]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Amy D. Migliori , Douglas E. Smith , Gaurav Arya

    Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of the bacteriophage T4 motor protein gp17 suggests that this motor generates large forces by undergoing a conformational change from an extended to a compact state. This transition is proposed to be driven by electrostatic interactions between complementarily charged residues across the interface between the N- and C-terminal domains of gp17. Here we use atomistic molecular dynamics simulations to investigate in detail the molecular interactions and residues involved in such a compaction transition of gp17. We find that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identify five charged residues and six uncharged residues at the interface that play a dominant role in the compaction transition and also reveal salt bridging, van der Waals, and solvent hydrogen-bonding interactions mediated by these residues in stabilizing the compact form of gp17. The formation of a salt bridge between Glu309 and Arg494 is found to be particularly crucial, consistent with experiments showing complete abrogation in packaging upon Glu309Lys mutation. The computed contributions of several other residues are also found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations.
    Graphical abstract




    Categories: Journal Articles
  • Traceless Splicing Enabled by Substrate-Induced Activation of the Nostoc punctiforme Npu DnaE Intein after Mutation of a Catalytic Cysteine to Serine
    [Dec 2014]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Manoj Cheriyan , Siu-Hong Chan , Francine Perler

    Inteins self-catalytically cleave out of precursor proteins while ligating the surrounding extein fragments with a native peptide bond. Much attention has been lavished on these molecular marvels with the hope of understanding and harnessing their chemistry for novel biochemical transformations including coupling peptides from synthetic or biological origins and controlling protein function. Despite an abundance of powerful applications, the use of inteins is still hampered by limitations in our understanding of their specificity (defined as flanking sequences that permit splicing) and the challenge of inserting inteins into target proteins. We examined the frequently used Nostoc punctiforme Npu DnaE intein after the C-extein cysteine nucleophile (Cys+1) was mutated to serine or threonine. Previous studies demonstrated reduced rates and/or splicing yields with the Npu DnaE intein after mutation of Cys+1 to Ser+1. In this study, genetic selection identified extein sequences with Ser+1 that enabled the Npu DnaE intein to splice with only a 5-fold reduction in rate compared to the wild-type Cys+1 intein and without mutation of the intein itself to activate Ser+1 as a nucleophile. Three different proteins spliced efficiently after insertion of the intein flanked by the selected sequences. We then used this selected specificity to achieve traceless splicing in a targeted enzyme at a location predicted by primary sequence similarity to only the selected C-extein sequence. This study highlights the latent catalytic potential of the Npu DnaE intein to splice with an alternative nucleophile and enables broader intein utility by increasing insertion site choices.
    Graphical abstract




    Categories: Journal Articles
  • The High-Risk HPV16 E7 Oncoprotein Mediates Interaction between the Transcriptional Coactivator CBP and the Retinoblastoma Protein pRb
    [Dec 2014]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Ariane L. Jansma , Maria A. Martinez-Yamout , Rong Liao , Peiqing Sun , H. Jane Dyson , Peter E. Wright

    The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the cyclic-AMP response element binding binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high-risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low-risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and on the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high-risk HPV16 E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control.
    Graphical abstract




    Categories: Journal Articles
  • Rad23 Interaction with the Proteasome Is Regulated by Phosphorylation of Its Ubiquitin-Like (UbL) Domain
    [Dec 2014]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Ruei-Yue Liang , Li Chen , Bo-Ting Ko , Yu-Han Shen , Yen-Te Li , Bo-Rong Chen , Kuan-Ting Lin , Kiran Madura , Show-Mei Chuang

    Rad23 was identified as a DNA repair protein, although a role in protein degradation has been described. The protein degradation function of Rad23 contributes to cell cycle progression, stress response, endoplasmic reticulum proteolysis, and DNA repair. Rad23 binds the proteasome through a UbL (ubiquitin-like) domain and contains UBA (ubiquitin-associated) motifs that bind multiubiquitin chains. These domains allow Rad23 to function as a substrate shuttle-factor. This property is shared by structurally similar proteins (Dsk2 and Ddi1) and is conserved among the human and mouse counterparts of Rad23. Despite much effort, the regulation of Rad23 interactions with ubiquitinated substrates and the proteasome is unknown. We report here that Rad23 is extensively phosphorylated in vivo and in vitro. Serine residues in UbL are phosphorylated and influence Rad23 interaction with proteasomes. Replacement of these serine residues with acidic residues, to mimic phosphorylation, reduced proteasome binding. We reported that when UbL is overexpressed, it can compete with Rad23 for proteasome interaction and can inhibit substrate turnover. This effect is not observed with UbL containing acidic substitutions, consistent with results that phosphorylation inhibits interaction with the proteasome. Loss of both Rad23 and Rpn10 caused pleiotropic defects that were suppressed by overexpressing either Rad23 or Rpn10. Rad23 bearing a UbL domain with acidic substitutions failed to suppress rad23Δ rpn10Δ, confirming the importance of regulated Rad23/proteasome binding. Strikingly, threonine 75 in human HR23B also regulates interaction with the proteasome, suggesting that phosphorylation is a conserved mechanism for controlling Rad23/proteasome interaction.
    Graphical abstract




    Categories: Journal Articles
  • Regional Discrimination and Propagation of Local Rearrangements along the Ribosomal Exit Tunnel
    [Dec 2014]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Jianli Lu , Carol Deutsch

    All proteins, from bacteria to man, are made in the ribosome and are elongated, one residue at a time, at the peptidyl transferase center. This growing peptide chain wends its way through the ribosomal tunnel to the exit port, ~100Å from the peptidyl transferase center. We have identified locations in the tunnel that sense and respond to single side chains of the nascent peptide to induce local conformational changes. Moreover, side-chain sterics and rearrangements deep in the tunnel influence the disposition of residues 45Å away at the exit port and are consistent with side-chain-induced axial retraction of the peptide backbone. These coupled responses are neither haphazard nor uniform along the tunnel. Rather, they are confined to discriminating zones in the tunnel and are sequence specific. Such discerning communication may contribute to folding events and mechanisms governing sequence-specific signaling between different regions of the tunnel during translation.
    Graphical abstract




    Categories: Journal Articles
  • Lysophospholipid-Containing Membranes Modulate the Fibril Formation of the Repeat Domain of a Human Functional Amyloid, Pmel17
    [Dec 2014]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Zhiping Jiang , Jennifer C. Lee

    Pmel17 is an important protein for pigmentation in human skin and eyes. Proteolytic fragments from Pmel17 form fibrils upon which melanin is deposited in melanosomes. The repeat domain (RPT) derived from Pmel17 only forms fibrils under acidic melanosomal conditions. Here, we examined the effects of lipids on RPT aggregation to explore whether intramelanosomal vesicles can facilitate fibrillogenesis. Using transmission electron microscopy, circular dichroism, and fluorescence spectroscopy, we monitored fibril formation at the ultrastructural, secondary conformational, and local levels, respectively. Phospholipid vesicles and lysophospholipid (lysolipid) micelles were employed as membrane mimics. The surfactant-like lysolipids are particularly pertinent due to their high content in melanosomal membranes. Interestingly, RPT aggregation kinetics were influenced only by lysolipid-containing phospholipid vesicles. While both vesicles containing either anionic lysophosphatidylglycerol (LPG) or zwitterionic lysophosphatidylcholine (LPC) stimulate aggregation, LPG exerted a greater effect on reducing the apparent nucleation time. A detailed comparison showed distinct behaviors of LPG versus LPC monomers and micelles plausibly originating from their headgroup hydrogen bonding capabilities. Acceleration and retardation of aggregation were observed for LPG monomers and micelles, respectively. Because a specific interaction between LPG and RPT was identified by intrinsic W423 fluorescence and induced α-helical structure, it is inferred that binding of LPG near the C-terminal amyloid core initiates intermolecular association, whereas stabilization of α-helical conformation inhibits β-sheet formation. Contrastingly, LPC promotes RPT aggregation at both submicellar and micellar concentrations via non-specific binding with undetectable secondary structural change. Our findings suggest that protein–lysolipid interactions within melanosomes may regulate amyloid formation in vivo.
    Graphical abstract




    Categories: Journal Articles
  • Mia40 Combines Thiol Oxidase and Disulfide Isomerase Activity to Efficiently Catalyze Oxidative Folding in Mitochondria
    [Dec 2014]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Johanna R. Koch , Franz X. Schmid

    Mia40 (a mitochondrial import and assembly protein) catalyzes disulfide bond formation in proteins in the mitochondrial intermembrane space. By using Cox17 (a mitochondrial copper-binding protein) as a natural substrate, we discovered that, in the presence of Mia40, the formation of native disulfides is strongly favored. The catalytic mechanism of Mia40 involves a functional interplay between the chaperone site and the catalytic disulfide. Mia40 forms a specific native disulfide in Cox17 much more rapidly than other disulfides, in particular, non-native ones, which originates from the recently described high affinity for hydrophobic regions near target cysteines and the long lifetime of the mixed disulfide. In addition to its thiol oxidase function, Mia40 is active also as a disulfide reductase and isomerase. We found that species with inadvertently formed incorrect disulfides are rebound by Mia40 and reshuffled, revealing a proofreading mechanism that is steered by the conformational folding of the substrate protein.
    Graphical abstract




    Categories: Journal Articles
  • Evidence for New Homotypic and Heterotypic Interactions between Transmembrane Helices of Proteins Involved in Receptor Tyrosine Kinase and Neuropilin Signaling
    [Dec 2014]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Paul Sawma , Lise Roth , Cécile Blanchard , Dominique Bagnard , Gérard Crémel , Emmanuelle Bouveret , Jean-Pierre Duneau , James N. Sturgis , Pierre Hubert

    Signaling in eukaryotic cells frequently relies on dynamic interactions of single-pass membrane receptors involving their transmembrane (TM) domains. To search for new such interactions, we have developed a bacterial two-hybrid system to screen for both homotypic and heterotypic interactions between TM helices. We have explored the dimerization of TM domains from 16 proteins involved in both receptor tyrosine kinase and neuropilin signaling. This study has revealed several new interactions. We found that the TM domain of Mucin-4, a putative intramembrane ligand for erbB2, dimerizes not only with erbB2 but also with all four members of the erbB family. In the Neuropilin/Plexin family of receptors, we showed that the TM domains of Neuropilins 1 and 2 dimerize with themselves and also with Plexin-A1, Plexin-B1, and L1CAM, but we were unable to observe interactions with several other TM domains notably those of members of the VEGF receptor family. The potentially important Neuropilin 1/Plexin-A1 interaction was confirmed using a surface plasmon resonance assay. This work shows that TM domain interactions can be highly specific. Exploring further the propensities of TM helix–helix association in cell membrane should have important practical implications related to our understanding of the structure–function of bitopic proteins' assembly and subsequent function, especially in the regulation of signal transduction.
    Graphical abstract




    Categories: Journal Articles
  • Cu,Zn-Superoxide Dismutase without Zn Is Folded but Catalytically Inactive
    [Dec 2014]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Sean Nedd , Rachel L. Redler , Elizabeth A. Proctor , Nikolay V. Dokholyan , Anastassia N. Alexandrova

    Amyotrophic lateral sclerosis has been linked to the gain of aberrant function of superoxide dismutase, Cu,Zn-SOD1 upon protein misfolding. The mechanism of SOD1 misfolding is thought to involve mutations leading to the loss of Zn, followed by protein unfolding and aggregation. We show that the removal of Zn from SOD1 may not lead to an immediate unfolding but immediately deactivates the enzyme through a combination of subtle structural and electronic effects. Using quantum mechanics/discrete molecular dynamics, we showed that both Zn-less wild-type (WT)-SOD1 and its D124N mutant that does not bind Zn have at least metastable folded states. In those states, the reduction potential of Cu increases, leading to the presence of detectable amounts of Cu(I) instead of Cu(II) in the active site, as confirmed experimentally. The Cu(I) protein cannot participate in the catalytic Cu(I)–Cu(II) cycle. However, even without the full reduction to Cu(I), the Cu site in the Zn-less variants of SOD1 is shown to be catalytically incompetent: unable to bind superoxide in a way comparable to the WT-SOD1. The changes are more radical and different in the D124N Zn-less mutant than in the Zn-less WT-SOD1, suggesting D124N being perhaps not the most adequate model for Zn-less SOD1. Overall, Zn in SOD1 appears to be influencing the Cu site directly by adjusting its reduction potential and geometry. Thus, the role of Zn in SOD1 is not just structural, as was previously thought; it is a vital part of the catalytic machinery.
    Graphical abstract




    Categories: Journal Articles
  • A “Fuzzy”-Logic Language for Encoding Multiple Physical Traits in Biomolecules
    [Dec 2014]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Shira Warszawski , Ravit Netzer , Dan S. Tawfik , Sarel J. Fleishman

    To carry out their activities, biological macromolecules balance different physical traits, such as stability, interaction affinity, and selectivity. How such often opposing traits are encoded in a macromolecular system is critical to our understanding of evolutionary processes and ability to design new molecules with desired functions. We present a framework for constraining design simulations to balance different physical characteristics. Each trait is represented by the equilibrium fractional occupancy of the desired state relative to its alternatives, ranging from none to full occupancy, and the different traits are combined using Boolean operators to effect a “fuzzy”-logic language for encoding any combination of traits. In another paper, we presented a new combinatorial backbone design algorithm AbDesign where the fuzzy-logic framework was used to optimize protein backbones and sequences for both stability and binding affinity in antibody-design simulation. We now extend this framework and find that fuzzy-logic design simulations reproduce sequence and structure design principles seen in nature to underlie exquisite specificity on the one hand and multispecificity on the other hand. The fuzzy-logic language is broadly applicable and could help define the space of tolerated and beneficial mutations in natural biomolecular systems and design artificial molecules that encode complex characteristics.
    Graphical abstract




    Categories: Journal Articles
  • Quality Control in Eukaryotic Membrane Protein Overproduction
    [Dec 2014]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Jennifer A. Thomas , Christopher G. Tate

    The overexpression of authentically folded eukaryotic membrane proteins in milligramme quantities is a fundamental prerequisite for structural studies. One of the most commonly used expression systems for the production of mammalian membrane proteins is the baculovirus expression system in insect cells. However, a detailed analysis by radioligand binding and comparative Western blotting of G protein-coupled receptors and a transporter produced in insect cells showed that a considerable proportion of the expressed protein was misfolded and incapable of ligand binding. In contrast, production of the same membrane proteins in stable inducible mammalian cell lines suggested that the majority was folded correctly. It was noted that detergent solubilisation of the misfolded membrane proteins using either digitonin or dodecylmaltoside was considerably less efficient than using sodium dodecyl sulfate or foscholine-12, whilst these detergents were equally efficient at solubilising correctly folded membrane proteins. This provides a simple and rapid test to suggest whether heterologously expressed mammalian membrane proteins are indeed correctly folded, without requiring radioligand binding assays. This will greatly facilitate the high-throughput production of fully functional membrane proteins for structural studies.
    Graphical abstract




    Categories: Journal Articles
  • Corrigendum to “Statistical Mechanics of Monod–Wyman–Changeux” [J Mol Biol 425 (9) (May 13 2013) 1433-1460]
    [Dec 2014]

    Publication date: 12 December 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 24

    Author(s): Sarah Marzen , Hernan G. Garcia , Rob Phillips







    Categories: Journal Articles
  • Experimental validation of plant peroxisomal targeting prediction algorithms by systematic comparison of in vivo import efficiency and in vitro PTS1 binding affinity
    [Dec 2014]

    Publication date: Available online 10 December 2014
    Source:Journal of Molecular Biology

    Author(s): Nicola S. Skoulding , Gopal Chowdhary , Mara J. Deus , Alison Baker , Sigrun Reumann , Stuart L. Warriner

    Most peroxisomal matrix proteins possess a C-terminal targeting signal type 1 (PTS1). Accurate prediction of functional PTS1 sequences and their relative strength by computational methods is essential for determination of peroxisomal proteomes in silico, but has proved challenging, due to high sequence variability of non-canonical targeting signals, particularly in higher plants, and low availability of experimentally validated non-canonical examples. In this study in silico predictions were compared with in vivo targeting analyses and in vitro thermodynamic binding of mutated variants within the context of one model targeting sequence. There was broad agreement between the methods for entire PTS1 domains and position-specific single amino acid (aa) residues, including residues upstream of the PTS1 tripeptide. The hierarchy Leu>Met>Ile>Val at the C-terminal position was determined for all methods but both experimental approaches suggest Tyr is under weighted in the prediction algorithm due to the absence of this residue in the positive training dataset. A combination of methods better defines the score range that discriminates a functional PTS1. In vitro binding to the PEX5 receptor could discriminate amongst strong targeting signals whilst in vivo targeting assays were more sensitive, allowing detection of weak functional import signals that were below the limit of detection in the binding assay. Together the data provide a comprehensive assessment of the factors driving PTS1 efficacy and provide a framework for the more quantitative assessment of the protein import pathway in higher plants.
    Graphical abstract




    Categories: Journal Articles
  • Human Apurinic/Apyrimidinic Endonuclease 1 (APE1) has 3’ RNA Phosphatase and 3’ Exoribonuclease Activities
    [Dec 2014]

    Publication date: Available online 10 December 2014
    Source:Journal of Molecular Biology

    Author(s): Manbir Chohan , Sebastian Mackedenski , Wai-Ming Li , Chow H. Lee

    Apurinic/apyrimidinic endonuclease 1 (APE1) is the predominant mammalian enzyme in DNA base excision repair pathway that cleaves the DNA backbone immediately 5’ to abasic sites. In addition to its abasic endonuclease activity, APE1 has 3’ phosphatase and 3’-5’ exonuclease activities against DNA. We recently identified APE1 as an endoribonuclease that preferentially cleaves at UA, UG, and CA sites in single-stranded regions of RNAs, and can regulate c-myc mRNA level and half-life in cells. APE1 can also endonucleolytically cleave abasic single-stranded RNA. Here, we show for the first time that the human APE1 has 3’ RNA phosphatase and 3’ exoribonuclease activity. Using three distinct RNA substrates, we show that APE1, but not RNase A, can remove the phosphoryl group from the 3’ end of RNA decay products. Studies using various site-directed APE1 mutant proteins (H309N, H309S, D283N, N68A, D210N, Y171F, D308A, F266A, and D70A) suggest that the 3’ RNA phosphatase activity shares the same active centre as its other known nuclease activities. A number of APE1 variants previously identified in the human population, including the most common D148E variant, have greater than 80% reduction in the 3’ RNA phosphatase activity. APE1 can remove a ribonucleotide from the 3’ overhang of RNA decay product, but its 3’-5’ exoribonuclease activity against unstructured poly(A), poly(C), and poly(U) RNAs is relatively weak. This study further underscores the significance of understanding the role of APE1 in RNA metabolism in vivo.
    Graphical abstract




    Categories: Journal Articles
  • Computational de novo design of a self-assembling peptide with predefined structure
    [Dec 2014]

    Publication date: Available online 10 December 2014
    Source:Journal of Molecular Biology

    Author(s): Sabine Kaltofen , Chenge Li , Po-Ssu Huang , Louise C. Serpell , Andreas Barth , Ingemar André

    Protein and peptide self-assembly is a powerful design principle for engineering of new biomolecules. More sophisticated biomaterials could be built if both the structure of the overall assembly as well as that of the self-assembling building block could be controlled. To approach this problem we developed a computational design protocol to enable de novo design of self-assembling peptides with predefined structure. The protocol was used to design a peptide building block with a βαβ fold that self-assembles into fibrilar structures. The peptide associates into a double β-sheet structure with tightly packed α-helices decorating the exterior of the fibrils. Using circular dichroism, Fourier transform infrared spectroscopy, electron microscopy and X-ray fiber diffraction we demonstrate that the peptide adopts the designed conformation. The results demonstrate that computational protein design can be used to engineer protein and peptide assemblies with predefined three-dimensional structures, which can serve as scaffolds for the development of functional biomaterials. Rationally designed proteins and peptides could also be used to investigate the subtle energetic and entropic tradeoffs in natural self-assembly processes and the relation between assembly structure and assembly mechanism. We demonstrate that the de novo designed peptide self-assembles with a mechanism that is more complicated than expected, in a process where small changes in solution conditions can lead to significant differences in assembly properties and conformation. These results highlight that formation of structured protein/peptide assemblies is often dependent on the formation of weak but highly precise intermolecular interactions.
    Graphical abstract




    Categories: Journal Articles
  • The Crystal Structure of the Human Titin:Obscurin Complex Reveals a Conserved Yet Specific Muscle M-band Zipper Module
    [Dec 2014]

    Publication date: Available online 6 December 2014
    Source:Journal of Molecular Biology

    Author(s): Stefano Pernigo , Atsushi Fukuzawa , Alessandro Pandini , Mark Holt , Jens Kleinjung , Mathias Gautel , Roberto A. Steiner

    M10 is the most C-terminal immunoglobulin (Ig) domain of the giant protein titin and a frequent target of disease-linked mutations. Currently, it is the only known muscle Ig-domain able to interact with two alternative ligands – obscurin and obscurin-like-1 (Obsl1) – in different sarcomeric subregions. Obscurin and Obsl1 use their homologous N-terminal Ig domain (O1 in obscurin and OL1 in Obsl1) to bind M10 in a mutually exclusive manner. We present here the X-ray structure of the human titin:obscurin M10:O1 complex extending our previous work on the M10:OL1 interaction. Similar to M10:OL1, the M10:O1 complex displays a chevron-shaped antiparallel Ig-Ig architecture held together by a conserved molecular interface, which we validated by isothermal titration calorimetry and sorting experiments in neonatal rat cardiomyocytes (NRCs). O1 although structurally related to OL1 and M10, both members of the I-set Ig family, presents an intriguing switch of its βA’ strand. This leads to structural differences between the complexes, particularly, for the ‘open-side’ of the chevron-shaped assembly. A bioinformatics analysis reveals that the βA’-switch observed for O1 is rare and that it is involved in mediating protein-protein interactions. Molecular Dynamics simulations also suggest that this topological alteration substantially increases local flexibility compared to the conventional I-set Ig domains. The O1/OL1 Ig domains are candidate discriminatory structural modules potentially directing the binding of specific additional partners at the M-band. Cellular sorting experiments in NRCs are consistent with the view that the titin:obscurin/Obsl1 complexes might be a platform for higher order interactions.
    Graphical abstract




    Categories: Journal Articles
  • Structure of an APC3-APC16 complex: Insights into assembly of the Anaphase Promoting Complex/Cyclosome
    [Dec 2014]

    Publication date: Available online 6 December 2014
    Source:Journal of Molecular Biology

    Author(s): Masaya Yamaguchi , Shanshan Yu , Renping Qiao , Florian Weissmann , Darcie J. Miller , Ryan VanderLinden , Nicholas G. Brown , Jeremiah J. Frye , Jan-Michael Peters , Brenda A. Schulman

    The Anaphase Promoting Complex/Cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the “Platform” centers around a cullin-RING-like E3 ligase catalytic core; the “Arc Lamp” is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-twofold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3’s TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, via their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7, and contains a >200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3’s loop and IR-tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer, and together with biochemistry and prior data explain how APC16 recruits APC7 to APC3, show how APC3’s C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR-tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C.
    Graphical abstract




    Categories: Journal Articles
  • Mapping the gating and permeation pathways in the voltage-gated proton channel Hv1
    [Dec 2014]

    Publication date: Available online 4 December 2014
    Source:Journal of Molecular Biology

    Author(s): Adam Chamberlin , Feng Qiu , Yibo Wang , Sergei Y. Noskov , H. Peter Larsson

    Voltage-gated proton channels (Hv1) are ubiquitous throughout nature and are implicated in numerous physiological processes. The gene encoding for Hv1 however was only identified in 2006. The lack of sufficient structural information of this channel has hampered the understanding of the molecular mechanism of channel activation and proton permeation. This study uses both simulation and experimental approaches to further develop existing models of the Hv1 channel. Our study provides insights into features of channel gating and proton permeation pathway. We compare open- and closed-state structures developed previously with a recent crystal structure that traps the channel in a presumably closed state. Insights into gating pathways were provided using a combination of all-atom MD simulations with a swarm-of-trajectories with the string method for extensive transition path sampling and evolution. A detailed residue-residue interaction profile and a hydration profile were studied to map the gating pathway in this channel. In particular it allows us to identify potential intermediate states and compare them to the experimentally observed crystal structure of Takeshita et al [1]. The mechanisms governing ion transport in the WT and mutant Hv1 channels were studied by a combination of electrophysiological recordings and free energy simulations. With these results we were able to further refine ideas about the location and function of the selectivity filter. The refined structural models will be essential for future investigations of this channel and the development of new drugs targeting cellular proton transport.
    Graphical abstract Highlights Our study reports on basic biophysical principles governing selective ion permeation in voltage-gated proton channels, which are membrane proteins with important roles in immune response and fertility. To further confirm and develop our model we compared it to recently reported crystal structures. To gain further insight into the mechanisms of ion selectivity in these channels were performed in-vivo and in-silico mutations on the channels and investigated their functioning. We found that targeted modifications around the constriction zone formed in an open state of the channel dramatically affects ion selectivity of the channel enabling transport of Na+. We also further investigate the gating behavior of the wild-type structures. Our in-silico predictions were confirmed experimentally both with regard to the mutant and the wild-type structures, further establishing the validity of the channel model for future applications in drug development targeting proton channels.




    Categories: Journal Articles
  • An Improved Single-chain Fab Platform for Efficient Display and Recombinant Expression
    [Dec 2014]

    Publication date: Available online 3 December 2014
    Source:Journal of Molecular Biology

    Author(s): James T. Koerber , Michael J. Hornsby , James A. Wells

    Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain Fab (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain Fv with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1-3mg per liter. Furthermore, rerouting of the scFab to the co-translational signal recognition particle (SRP) pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (81°C Tm), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.
    Graphical abstract




    Categories: Journal Articles
  • Specificity Determinants in Small Multidrug Transporters
    [Dec 2014]

    Publication date: Available online 3 December 2014
    Source:Journal of Molecular Biology

    Author(s): Shlomo Brill , Ofir Sade-Falk , Yael Elbaz-Alon , Shimon Schuldiner

    Multiple-antibiotic resistance has become a major global public health concern, and to overcome this problem, it is necessary to understand the resistance mechanisms that allow survival of the microorganisms at the molecular level. One mechanism responsible for such resistance involves active removal of the antibiotic from the pathogen cell by MDTs (multidrug transporters). A prominent MDT feature is their high polyspecificity allowing for a single transporter to confer resistance against a range of drugs. Here we present the molecular mechanism underlying substrate recognition in EmrE, a small MDT from Escherichia coli. EmrE is known to have a substrate preference for aromatic, cationic compounds, such as methyl viologen (MV2+). In this work, we use a combined bioinformatic and biochemical approach to identify one of the major molecular determinants involved in MV2+ transport and resistance. Replacement of an Ala residue with Ser in weakly resistant SMRs from Bacillus pertussis and Mycobacterium tuberculosis enables them to provide robust resistance to MV2+ and to transport MV2+ and has negligible effects on the interaction with other substrates. This shows that the residue identified herein is uniquely positioned in the binding site so as to be exclusively involved in the mediating of MV2+ transport and resistance, both in EmrE and in other homologues. This work provides clues toward uncovering how specificity is achieved within the binding pocket of a polyspecific transporter that may open new possibilities as to how these transporters can be manipulated to bind a designed set of drugs.
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