Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Naoko Imamoto

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Junya Kobayashi , Yoshiyuki Matsuura

Kap121p (also known as Pse1p) is an essential karyopherin that mediates nuclear import of a plethora of cargoes including cell cycle regulators, transcription factors, and ribosomal proteins in Saccharomyces cerevisiae. It has been proposed that the spindle assembly checkpoint signaling triggers molecular rearrangements of nuclear pore complexes and thereby arrests Kap121p-mediated nuclear import at metaphase, while leaving import mediated by other karyopherins unaffected. The Kap121p-specific import inhibition is required for normal progression through mitosis. To understand the structural basis for Kap121p-mediated nuclear import and its unique regulatory mechanism during mitosis, we determined crystal structures of Kap121p in isolation and also in complex with either its import cargoes or nucleoporin Nup53p or RanGTP. Kap121p has a superhelical structure composed of 24 HEAT repeats. The structures of Kap121p–cargo complexes define a non-conventional nuclear localization signal (NLS) that has a consensus sequence of KV/IxKx1-2K/H/R. The structure of Kap121p–Nup53p complex shows that cargo and Nup53p compete for the same high-affinity binding site, explaining how Nup53p binding forces cargo release when the Kap121p-binding site of Nup53p is exposed during mitosis. Comparison of the NLS and RanGTP complexes reveals that RanGTP binding not only occludes the cargo-binding site but also forces Kap121p into a conformation that is incompatible with NLS recognition.
Graphical abstract

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Qing Chang , Ryo Nitta , Shigeyuki Inoue , Nobutaka Hirokawa

Kinesin superfamily proteins (KIFs) are microtubule-based molecular motors driven by the energy derived from the hydrolysis of ATP. Previous studies have revealed that the ATP binding step is crucial both for the power stroke to produce motility and for the inter-domain regulation of ATPase activity to guarantee the processive movement of dimeric KIFs. Here, we report the first crystal structure of KIF4 complexed with the non-hydrolyzable ATP analog, AMPPNP (adenylyl imidodiphosphate), at 1.7Å resolution. By combining our structure with previously solved KIF1A structures complexed with two ATP analogs, molecular snapshots during ATP binding reveal that the closure of the nucleotide-binding pocket during ATP binding is achieved by closure of the backdoor. Closure of the backdoor stabilizes two mobile regions, switch I and switch II, to generate the phosphate tube from which hydrolyzed phosphate is released. Through the stabilization of switch II, the local conformational change at the catalytic center is further relayed to the neck-linker element that fully docks to the catalytic core to produce the power stroke. Because the neck linker is a sole element that connects the partner heads in dimeric KIFs, this tight structural coordination between the catalytic center and neck linker enables inter-domain communication between the partner heads. This study also revealed the putative microtubule-binding site of KIF4, thus providing structural insights that describe the specific binding of KIF4 to the microtubule.
Graphical abstract Highlights ► First crystal structure of KIF4 was solved at 1.7Å resolution. ► New KIF4 structure adopts the pre-hydrolysis state with ATP. ► Closure of the backdoor triggers the hydrolysis of ATP. ► Backdoor closure also regulates the conformations of switch II and the neck linker. ► The KIF4-specific microtubule binding sequences are concentrated on switch II.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Charanya Kumar , Gregory M. Williams , Brett Havens , Michelle K. Dinicola , Jennifer A. Surtees

In Saccharomyces cerevisiae, repair of insertion/deletion loops is carried out by Msh2–Msh3-mediated mismatch repair (MMR). Msh2–Msh3 is also required for 3′ non-homologous tail removal (3′ NHTR) in double-strand break repair. In both pathways, Msh2–Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, the kinetics of the two processes appear different; MMR is likely rapid in order to coordinate with the replication fork, whereas 3′ NHTR has been shown to be a slower process. To understand the molecular requirements in both repair pathways, we performed an in vivo analysis of well-conserved residues in Msh3 that are hypothesized to be required for MMR and/or 3′ NHTR. These residues are predicted to be involved in either communication between the DNA-binding and ATPase domains within the complex or nucleotide binding and/or exchange within Msh2–Msh3. We identified a set of aromatic residues within the FLY motif of the predicted Msh3 nucleotide binding pocket that are essential for Msh2–Msh3-mediated MMR but are largely dispensable for 3′ NHTR. In contrast, mutations in other regions gave similar phenotypes in both assays. Based on these results, we suggest that the two pathways have distinct requirements with respect to the position of the bound ATP within Msh3. We propose that the differences are related, at least in part, to the kinetics of each pathway. Proper binding and positioning of ATP is required to induce rapid conformational changes at the replication fork, but is less important when more time is available for repair, as in 3′ NHTR.
Graphical abstract Highlights ► Msh2–Msh3 participates in the removal of 3′ non-homologous tails in recombination. ► Msh2–Msh3 initiates MMR of DNA slippage events in replication. ► Msh2–Msh3 undergoes conformational changes in ATP binding pocket upon DNA binding. ► Mutations in the Msh3 ATP binding pocket disrupt MMR but not recombination. ► Kinetic differences in the pathways may dictate distinct ATP positioning constraints.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Homer Pantua , Jingyu Diao , Mark Ultsch , Meredith Hazen , Mary Mathieu , Krista McCutcheon , Kentaro Takeda , Shailesh Date , Tommy K. Cheung , Qui Phung , Phil Hass , David Arnott , Jo-Anne Hongo , David J. Matthews , Alex Brown , Arvind H. Patel , Robert F. Kelley , Charles Eigenbrot , Sharookh B. Kapadia

Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody–peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the β-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies.
Graphical abstract Highlights ► The role of glycosylation in HCV resistance to neutralizing antibodies is unclear. ► Two affinity-matured broadly neutralizing antibodies against HCV were generated. ► A mutation at a conserved glycosylation Asn (N417) confers antibody resistance. ► N417S and N417T mutations result in a glycosylation shift to N415. ► Fab–peptide structures confirm that N415 is buried in the antibody–peptide interface.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Elizabeth A. Yates , Sherry L. Owens , Michael F. Lynch , Elena M. Cucco , C. Samuel Umbaugh , Justin Legleiter

A hallmark of Alzheimer's disease, a late-onset neurodegenerative disease, is the deposition of neuritic amyloid plaques composed of aggregated forms of the β-amyloid peptide (Aβ). Aβ forms a variety of nanoscale, toxic aggregate species ranging from small oligomers to fibrils. Aβ and many of its aggregate forms strongly interact with lipid membranes, which may represent an important step in several toxic mechanisms. Understanding the role that specific regions of Aβ play in regulating its aggregation and interaction with lipid membranes may provide insights into the fundamental interaction between Aβ and cellular surfaces. We investigated the interaction and aggregation of several Aβ fragments (Aβ1–11, Aβ1–28, Aβ10–26, Aβ12–24, Aβ16–22, Aβ22–35, and Aβ1–40) in the presence of supported model total brain lipid extract (TBLE) bilayers. These fragments represent a variety of chemically unique domains within Aβ, that is, the extracellular domain, the central hydrophobic core, and the transmembrane domain. Using scanning probe techniques, we elucidated aggregate morphologies for these different Aβ fragments in free solution and in the presence of TBLE bilayers. These fragments formed a variety of oligomeric and fibrillar aggregates under free solution conditions. Exposure to TBLE bilayers resulted in distinct aggregate morphologies compared to free solution and changes in bilayer stability dependent on the Aβ sequence. Aβ10–26, Aβ16–22, Aβ22–35, and Aβ1–40 aggregated into a variety of distinct fibrillar aggregates and disrupted the bilayer structure, resulting in altered mechanical properties of the bilayer. Aβ1–11, Aβ1–28, and Aβ12–24 had minimal interaction with lipid membranes, forming only sparse oligomers.
Graphical abstract Highlights ► The toxicity of Aβ may be mediated via lipid membranes. ► Aβ fragments formed distinct aggregates in free solution and on lipid surfaces. ► Mechanical properties of supported lipid bilayers were modulated by Aβ fragments. ► The interaction of Aβ with lipids is facilitated by domains within the peptide.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Don Vu , De-Bin Huang , Annapurna Vemu , Gourisankar Ghosh

Transcription factors of the nuclear factor kappaB (NF-κB) family arise through the combinatorial association of five distinct Rel subunits into functional dimers. However, not every dimer combination is observed in cells. The RelB subunit, for example, does not appear as a homodimer and forms heterodimers exclusively in combination with p50 or p52 subunits. We previously reported that the RelB homodimer could be forced to assemble through domain swapping in vitro. In order to understand the mechanism of selective dimerization among Rel subunits, we have determined the x-ray crystal structures of five RelB dimers. We find that RelB forms canonical side-by-side heterodimers with p50 and p52. We observe that, although mutation of four surface hydrophobic residues that are unique to RelB does not affect its propensity to form homodimers via domain swapping, alteration of two interfacial residues converts RelB to a side-by-side homodimer. Surprisingly, these mutant RelB homodimers remain distinct from canonical side-by-side NF-κB dimers in that the two monomers move away from one another along the 2-fold axis to avoid non-complementary interactions at the interface. The presence of distinct residues buried within the hydrophobic core of the RelB dimerization domain appears to influence the conformations of the surface residues that mediate the dimer interface. This conclusion is consistent with prior observations that alterations of domain core residues change dimerization propensity in the NF-κB family transcription factors. We suggest that RelB has evolved into a specialized NF-κB subunit with unique amino acids optimized for selective formation of heterodimers with p50 and p52.
Graphical abstract Highlights ► RelB forms two types of dimers—homodimers (domain swap) and heterodimers (canonical). ► Sequence variation of the domain core in Rel proteins reported to change dimerization propensity. ► Both core and interfacial residues coordinately dictate dimerization in the NF-κB family. ► Evolution of RelB as a specialized subunit may permit it to interact with non-NF-κB proteins.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Tatiana Nikitina , Difei Wang , Misha Gomberg , Sergei A. Grigoryev , Victor B. Zhurkin

Micrococcal nuclease (MNase) is extensively used in genome-wide mapping of nucleosomes but its preference for AT-rich DNA leads to errors in establishing precise positions of nucleosomes. Here, we show that the MNase digestion of nucleosomes assembled on a strong nucleosome positioning sequence, Widom's clone 601, releases nucleosome cores whose sizes are strongly affected by the linker DNA sequence. Our experiments produced nucleosomal DNA sizes varying between 147 and 155bp, with positions of the MNase cuts reflecting positions of the A⋅T pairs rather than the nucleosome core/linker junctions determined by X-ray crystallography. Extent of chromatosomal DNA protection by linker histone H1 also depends on the linker DNA sequence. Remarkably, we found that a combined treatment with MNase and exonuclease III (exoIII) overcomes MNase sequence preference producing nucleosomal DNA trimmed symmetrically and precisely at the core/linker junctions regardless of the underlying DNA sequence. We propose that combined MNase/exoIII digestion can be applied to in situ chromatin for unbiased genome-wide mapping of nucleosome positions that is not influenced by DNA sequences at the core/linker junctions. The same approach can be also used for the precise mapping of the extent of linker DNA protection by H1 and other protein factors associated with nucleosome linkers.
Graphical abstract Highlights ► MNase mapping of nucleosomes is biased by its preference for AT-rich DNA. ► For nucleosome 601, the “apparent” size of MNase-produced core DNA varies between 147 and 155bp, depending on the linker DNA sequence. ► Combined MNase and exoIII treatment overcomes this bias and cuts nucleosomal DNA at the core/linker junctions. ► This approach can be used for the high-resolution mapping of nucleosomes.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Kanagasabai Vadivel , Sayeh Agah , Amanda S. Messer , Duilio Cascio , Madhu S. Bajaj , Sriram Krishnaswamy , Charles T. Esmon , Kaillathe Padmanabhan , S. Paul Bajaj

Crystal structures of factor (F) VIIa/soluble tissue factor (TF), obtained under high Mg2+ (50mM Mg2+/5mM Ca2+), have three of seven Ca2+ sites in the γ-carboxyglutamic acid (Gla) domain replaced by Mg2+ at positions 1, 4, and 7. We now report structures under low Mg2+ (2.5mM Mg2+/5mM Ca2+) as well as under high Ca2+ (5mM Mg2+/45mM Ca2+). Under low Mg2+, four Ca2+ and three Mg2+ occupy the same positions as in high-Mg2+ structures. Conversely, under low Mg2+, reexamination of the structure of Gla domain of activated Protein C (APC) complexed with soluble endothelial Protein C receptor (sEPCR) has position 4 occupied by Ca2+ and positions 1 and 7 by Mg2+. Nonetheless, in direct binding experiments, Mg2+ replaced three Ca2+ sites in the unliganded Protein C or APC. Further, the high-Ca2+ condition was necessary to replace Mg4 in the FVIIa/soluble TF structure. In biological studies, Mg2+ enhanced phospholipid binding to FVIIa and APC at physiological Ca2+. Additionally, Mg2+ potentiated phospholipid-dependent activations of FIX and FX by FVIIa/TF and inactivation of activated factor V by APC. Since APC and FVIIa bind to sEPCR involving similar interactions, we conclude that under the low-Mg2+ condition, sEPCR binding to APC-Gla (or FVIIa-Gla) replaces Mg4 by Ca4 with an attendant conformational change in the Gla domain ω-loop. Moreover, since phospholipid and sEPCR bind to FVIIa or APC via the ω-loop, we predict that phospholipid binding also induces the functional Ca4 conformation in this loop. Cumulatively, the data illustrate that Mg2+ and Ca2+ act in concert to promote coagulation and anticoagulation.
Graphical abstract Highlights ► Plasma Ca2+ is insufficient to fold Gla domains in vitamin-K-dependent proteins. ► Ca2+ and Mg2+ occupy specific sites in the Gla domains of factor VIIa and Protein C. ► High Ca2+ or ligand binding to the ω-loop of Gla replaces Mg2+ at site 4 by Ca2+. ► Ca2+ occupancy at site 4 results in the active conformation of the ω-loop. ► Physiological Ca2+/Mg2+ act in concert to promote coagulation and anticoagulation.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Nathalie Goudreau , Oliver Hucke , Anne-Marie Faucher , Chantal Grand-Maître , Olivier Lepage , Pierre R. Bonneau , Stephen W. Mason , Steve Titolo

The nucleocapsid (NC) protein is an essential factor with multiple functions within the human immunodeficiency virus type 1 (HIV-1) replication cycle. In this study, we describe the discovery of a novel series of inhibitors that targets HIV-1 NC protein by blocking its interaction with nucleic acids. This series was identified using a previously described capsid (CA) assembly assay, employing a recombinant HIV-1 CA-NC protein and immobilized TG-rich deoxyoligonucleotides. Using visible absorption spectroscopy, we were able to demonstrate that this new inhibitor series binds specifically and reversibly to the NC with a peculiar 2:1 stoichiometry. A fluorescence-polarization-based binding assay was also developed in order to monitor the inhibitory activities of this series of inhibitors. To better characterize the structural aspect of inhibitor binding onto NC, we performed NMR studies using unlabeled and 13C,15N-double-labeled NC(1–55) protein constructs. This allowed the determination of the solution structure of a ternary complex characterized by two inhibitor molecules binding to the two zinc knuckles of the NC protein. To the best of our knowledge, this represents the first report of a high-resolution structure of a small-molecule inhibitor bound to NC, demonstrating sub-micromolar potency and moderate antiviral potency with one analogue of the series. This structure was compared with available NC/oligonucleotide complex structures and further underlined the high flexibility of the NC protein, allowing it to adopt many conformations in order to bind its different oligonucleotide/nucleomimetic targets. In addition, analysis of the interaction details between the inhibitor molecules and NC demonstrated how this novel inhibitor series is mimicking the guanosine nucleobases found in many reported complex structures.
Graphical abstract Highlights ► Discovery of a novel series of inhibitors that targets HIV-1 NC protein. ► Specific binding of inhibitor series to NC characterized by a 2:1 stoichiometry. ► Solution structure determination of the inhibitor/NC ternary complex by NMR. ► This work could also serve as a starting point for structure-based design efforts.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Olivier Preux , Dominique Durand , Alexis Huet , James F. Conway , Aurélie Bertin , Claire Boulogne , Jeannine Drouin-Wahbi , Didier Trévarin , Javier Pérez , Patrice Vachette , Pascale Boulanger

Capsids of double-stranded DNA (dsDNA) bacteriophages initially assemble into compact procapsids, which undergo expansion upon the genome packaging. This shell remodeling results from a structural rearrangement of head protein subunits. It is a critical step in the capsid maturation pathway that yields final particles capable to withstand the huge internal pressure generated by the packed DNA. Here, we report on the expansion process of the large capsid (T =13) of bacteriophage T5. We purified the intermediate prohead II form, which is competent for packaging the 121-kbp dsDNA genome, and we investigated its morphology and dimensions using cryo-electron microscopy and small-angle X-ray scattering. Decreasing the pH or the ionic strength triggers expansion of prohead II, converting them into thinner and more faceted capsids isomorphous to the mature virion particles. At low pH, prohead II expansion is a highly cooperative process lacking any detectable intermediate. This two-state reorganization of the capsid lattice per se leads to a remarkable stabilization of the particle. The melting temperature of expanded T5 capsid is virtually identical with that of more complex shells that are reinforced by inter-subunit cross-linking (HK97) or by additional cementing proteins (T4). The T5 capsid with its “simple” two-state conversion thus appears to be a very attractive model for investigating the mechanism of the large-scale allosteric transition that takes place upon the genome packaging of dsDNA bacteriophages.
Graphical abstract Highlights ►The prohead II precursor form of phage T5 capsid expands upon DNA packaging. ► Decreasing the pH or the ionic strength triggers T5 prohead II expansion. ► The acid-induced expansion of prohead II is a two-state transition. ► The concerted rearrangement of the head proteins yields a highly stable mature capsid.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Daniel J. Merkel , Sarah B. Wells , Bryce C. Hilburn , Fatima Elazzouzi , Gabriela C. Pérez-Alvarado , Brian M. Lee

Cytoplasmic polyadenylation element binding protein (CPEB) provides temporal and spatial control of protein synthesis required for early development and neuronal synaptic plasticity. CPEB regulates protein expression by inhibiting polyadenylation of selected mRNA transcripts, which prevents binding of the ribosome for protein synthesis. Two RNA recognition motif domains and a C-terminal binuclear zinc-binding domain are required for mRNA binding, but the zinc-binding domain is not required for sequence-specific recognition of the targeted mRNA transcript. The structure and function of the zinc-binding domain of CPEB are unknown. The C-terminal region of CPEB may participate in assembly of the ribonucleoprotein complex that includes the scaffold protein, Symplekin, and the cleavage and polyadenylation specificity factor. Sumoylation of Symplekin is required for polyadenylation, and both cleavage and polyadenylation specificity factor and poly(A) polymerase are sumoylated. The foreshortened poly(A) tail is maintained by poly(A) ribonuclease, which associates with CPEB. While zinc-binding domains are renowned for nucleic acid recognition, binuclear zinc-binding structural motifs, such as LIM (Lin-11, Isl-1, Mec-3), RING (really interesting new gene), PHD (plant homeodomain) and ZZ (ZZ-type zinc finger) domains, participate in protein–protein interactions. Here, we report the solution structure of the C-terminal zinc-binding domain of CPEB1 (CPEB1-ZZ), which has a cross-braced zinc binding topology. The structural similarity to other ZZ domains suggests that the CPEB1-ZZ domain recruits sumoylated proteins during assembly of the ribonucleoprotein complex prior to mRNA export from the nucleus.
Graphical abstract Highlights ► CPEB regulates translation of mRNA in early development and synaptic plasticity. ► The C-terminal zinc-binding domain of CPEB1 adopts a ZZ structural motif. ► The ZZ domain of CPEB1 reveals a potential SUMO-binding surface. ► The ZZ domain may mediate nuclear assembly of the ribonucleoprotein complex.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): AnthonyA. Armstrong , James E.K. Hildreth , L.Mario Amzel

The mechanism by which antibodies elicited against protein-derived peptides achieve cross-reactivity with their cognate proteins remains unknown. To address this question, we have carried out the complete thermodynamic characterization of the association of a monoclonal antibody (260.33.12) raised against a peptide (SNpep) derived from staphylococcal nuclease (SNase) with both eliciting peptide and cognate protein. Although both ligands bind with similar affinity (K d =0.42μM and 0.30μM for protein and peptide, respectively), protein and peptide binding have highly different thermodynamic signatures: peptide binding is characterized by a large enthalpic contribution (ΔH =−7.7kcal/mol) whereas protein binding is dominated by a large entropic contribution (− TΔS =−7.2kcal/mol). The structure of the SNpep:Fab complex, determined by X-ray diffraction, reveals that the bound conformation of the peptide differs from the conformation of the corresponding loop region in crystal structures of free SNase. The energy difference, estimated by molecular dynamics simulations between native SNase and a model in which the Ω-loop is built in the conformation of the Fab-bound peptide, shows that the energetic cost of adopting this conformation is compatible with the enthalpic cost of binding the protein vis-à-vis the peptide. These results are compatible with a mechanism by which the anti-peptide antibody recognizes the cognate protein: high affinity is maintained upon binding a non-native conformation by offsetting enthalpic penalties with reduced entropic losses. These findings provide potentially useful guidelines for the identification of linear epitopes within protein sequences that are well suited for the development of synthetic peptide vaccines.
Graphical abstract Highlights ► A system was devised to study the mechanisms by which anti-peptide antibodies react with native proteins. ► Antibody 260.33.12 was raised against a peptide derived from a loop in SNase. ► Structure determination shows that 260.33.12 binds peptide in a SNase non-native conformation. ► 260.33.12 binds peptide and protein with similar affinities but different enthalpies. ► When binding SNase, relative entropic gains offset enthalpic penalties.

In science news around the world, two billionaires have bought the former building of drug company Merck Serono in Geneva, the $12 million Laboratory of Neuro Imaging is moving to the University of Southern California Keck Medical Center, and Saudi Deputy Health Minister Ziad Memish complained that intellectual property considerations are slowing down the development of diagnostic tests for Middle East respiratory syndrome coronavirus.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Miloslava Maninová , Zuzana Klímová , J. Thomas Parsons , Michael J. Weber , Marcin P. Iwanicki , Tomáš Vomastek

The establishment of cell polarity is an essential step in the process of cell migration. This process requires precise spatiotemporal coordination of signaling pathways that in most cells create the typical asymmetrical profile of a polarized cell with nucleus located at the cell rear and the microtubule organizing center (MTOC) positioned between the nucleus and the leading edge. During cell polarization, nucleus rearward positioning promotes correct microtubule organizing center localization and thus the establishment of front–rear polarity and directional migration. We found that cell polarization and directional migration require also the reorientation of the nucleus. Nuclear reorientation is manifested as temporally restricted nuclear rotation that aligns the nuclear axis with the axis of cell migration. We also found that nuclear reorientation requires physical connection between the nucleus and cytoskeleton mediated by the LINC (linker of nucleoskeleton and cytoskeleton) complex. Nuclear reorientation is controlled by coordinated activity of lysophosphatidic acid (LPA)-mediated activation of GTPase Rho and the activation of integrin, FAK (focal adhesion kinase), Src, and p190RhoGAP signaling pathway. Integrin signaling is spatially induced at the leading edge as FAK and p190RhoGAP are predominantly activated or localized at this location. We suggest that integrin activation within lamellipodia defines cell front, and subsequent FAK, Src, and p190RhoGAP signaling represents the polarity signal that induces reorientation of the nucleus and thus promotes the establishment of front–rear polarity.
Graphical abstract Highlights ► During nuclear reorientation, nuclear axis aligns with the direction of movement. ► Nuclear reorientation requires LPA/Rho and integrin/FAK/p190RhoGAP signaling. ► Integrin/FAK/p190RhoGAP antagonizes LPA/Rho signaling at the leading edge. ► Nuclear reorientation is important for the spatial organization of migrating cell.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Elvira Vitu , Sapna Sharma , Samuel D. Stampfer , Ekaterina E. Heldwein

Viral fusogens mediate the merger of the viral envelope and cellular membrane during viral entry. These proteins share little sequence similarity but all are thought to act by refolding through a series of conformational intermediates from the metastable prefusion form to the stable postfusion form. Crystal structures of both prefusion and postfusion forms have illuminated the conformational pathways of several viral fusogens. By contrast, only the structure of the postfusion form is available for glycoprotein B (gB), the conserved fusogen of herpesviruses. To gain insight into the nature of the fusogenic conformational changes in gB, we used several approaches aimed at engineering the prefusion form of the herpes simplex virus type 1 gB ectodomain, including modifications intended to stabilize the prefusion form and novel mutations aimed at destabilizing the postfusion form. We found that the postfusion conformation of gB is remarkably stable and resistant to perturbations. Several mutations successfully destabilized the gB trimer, identifying regions that are critical for the stability of the postfusion form. Yet, none of the constructs adopted the prefusion conformation. We propose that the soluble ectodomain of gB folds into the postfusion form without first adopting the prefusion intermediate. These results suggest that other regions of gB, including the transmembrane region and the cytoplasmic domain, may be necessary to establish and maintain the metastable prefusion conformation.
Graphical abstract Highlights ► The ectodomain of herpesvirus fusogen gB spontaneously folds into the postfusion form. ► Postfusion conformation of gB is remarkably stable and resistant to perturbations. ► Domain dV is critical for the stability of the postfusion form. ► The isolated gB ectodomain folds into the postfusion form without prefusion intermediate. ► The transmembrane region and the cytodomain may be necessary to maintain the metastable prefusion form.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Fabian Schreiber , Erik L.L. Sonnhammer

An accurate inference of orthologs is essential in many research fields such as comparative genomics, molecular evolution, and genome annotation. Existing methods for genome-scale orthology inference are mostly based on all-versus-all similarity searches that scale quadratically with the number of species. This limits their application to the increasing number of available large-scale datasets. Here, we present Hieranoid, a new orthology inference method using a hierarchical approach. Hieranoid performs pairwise orthology analysis using InParanoid at each node in a guide tree as it progresses from its leaves to the root. This concept reduces the total runtime complexity from a quadratic to a linear function of the number of species. The tree hierarchy provides a natural structure in multi-species ortholog groups, and the aggregation of multiple sequences allows for multiple alignment similarity searching techniques, which can yield more accurate ortholog groups. Using the recently published orthobench benchmark, Hieranoid showed the overall best performance. Our progressive approach presents a new way to infer orthologs that combines efficient graph-based methodology with aspects of compute-intensive tree-based methods. The linear scaling with the number of species is a major advantage for large-scale applications and makes Hieranoid well suited to cope with vast amounts of sequenced genomes in the future. Hieranoid is an open source and can be downloaded at Hieranoid.sbc.su.se.
Graphical abstract Highlights ► We applied the idea of progressive alignment algorithm to orthology prediction. ► It is based on progressively applying pairwise InParanoid along a guide tree. ► Our method scales linearly, as opposed to the quadratic scaling of most others. ► Benchmark results indicate that our new method is more accurate.

Publication date: 12 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 11

Author(s): Daniel Lightwood , Victoria O'Dowd , Bruce Carrington , Vaclav Veverka , Mark D. Carr , Markus Tservistas , Alistair J. Henry , Bryan Smith , Kerry Tyson , Sabrina Lamour , Marguerite Bracher , Kaushik Sarkar , Alison Turner , Alastair D. Lawson , Tim Bourne , Neil Gozzard , Roger Palframan

Researchers claim that a beautifully preserved, 170-million-year-old skeleton from China's fossil-rich Liaoning province, dubbed Aurornis xui, is the earliest known-for-sure bird. And the new Will Smith flick After Earth is an ecological fable with a stark moral about humans' impact on the planet—a movie that scientifically quickly goes off the rails but provides an opportunity for scientists to grab an interested audience's attention to counter misinformation.

Publication date: Available online 1 June 2013
Source:Journal of Molecular Biology

Author(s): Senthil K. Perumal , Scott W. Nelson , Stephen J. Benkovic

Bacteriophage T4 UvsW helicase contains both unwinding and annealing activities and displays some functional similarities to bacterial RecG and RecQ helicases. UvsW is involved in several DNA repair pathways, playing important roles in recombination-dependent DNA repair and the reorganization of stalled replication forks. The T4 single-stranded DNA binding protein, gp32, is a central player in nearly all DNA replication and repair processes and is thought to facilitate their coordination by recruiting and regulating the various proteins involved. Here, we show that the activities of the UvsW protein are modulated by gp32. UvsW catalyzed unwinding of recombination intermediates such as D-loops and static X-DNA (Holliday junction mimic) to ssDNA products is enhanced by the gp32 protein. The enhancement requires the presence of the protein interaction domain of gp32 (the acidic carboxy terminus), suggesting that a specific interaction between UvsW and gp32 is required. In the absence of this interaction, the ssDNA annealing and ATP-dependent translocation activities of UvsW are severely inhibited when gp32 coats the ssDNA lattice. However, when UvsW and gp32 do interact, UvsW is able to efficiently displace the gp32 protein from the ssDNA. This ability of UvsW to remove gp32 from ssDNA may explain its ability to enhance the strand invasion activity of the T4 recombinase (UvsX) and suggests a possible new role for UvsW in gp32-mediated DNA transactions.
Graphical abstract