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Fighting Flu With Ready-Made Antibodies

Author: Bruce Alberts

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Author(s): M. Amin-ul Mannan , William R. Shadrick , Gabriel Biener , Byung-Sik Shin , Ashish Anshu , Valerica Raicu , David N. Frick , Madhusudan Dey

The endoplasmic reticulum transmembrane receptor Ire1 senses over-accumulation of unfolded proteins in the endoplasmic reticulum and initiates the unfolded protein response (UPR). The cytoplasmic portion of Ire1 has a protein kinase domain (KD) and a kinase extension nuclease (KEN) domain that cleaves an mRNA for encoding the Hac1 transcription factor needed to express UPR genes. During this UPR signaling, Ire1 proteins self-assemble into an oligomer of dimers, which essentially requires autophosphorylation of a constituent activation loop in the KD. However, it is not clear how dimerization, autophosphorylation, and KEN domain function are precisely coordinated. In this study, we uncoupled the KD and KEN domain functions, by removing the activation loop along with an extended region that we called the auto-inhibitory region (AIR), or by swapping the activation loop with a homologous loop from phosphorylase kinase 1 (Ire1PHK). Both Ire1ΔAIR and Ire1PHK activated the UPR even when either protein contained a mutation (D797A) that abolished the ability of Ire1 KD to transfer phosphates to the AIR. Neither protein functioned when containing mutations in key ATP binding residues (E746A and N749A) or in residues that disrupted Ire1 dimer interface (W426A or R697D). We interpret these results as evidence supporting the notion that the primary function of the kinase domain is to autophosphorylate the AIR in order to relieve auto-inhibition and that ADP acts as a switch to activate the KEN domain-catalyzed HAC1 mRNA cleavage.
Graphical abstract

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Author(s): Alexei A. Adzhubei , Michael J.E. Sternberg , Alexander A. Makarov

The poly-l-proline type II (PPII) helix in recent years has emerged clearly as a structural class not only of fibrillar proteins (in collagen, PPII is a dominant conformation) but also of the folded and unfolded proteins. Although much less abundant in folded proteins than the α-helix and β-structure, the left-handed, extended PPII helix represents the only frequently occurring regular structure apart from these two structure classes. Natively unfolded proteins have a high content of the PPII helices identified by spectroscopic methods. Apart from the structural function, PPII is favorable for protein–protein and protein–nucleic acid interactions and plays a major role in signal transduction and protein complex assembly, as this structure is often found in binding sites, specifically binding sites of widely spread SH3 domains. PPII helices do not necessarily contain proline, but proline has high PPII propensity. Commonly occurring proline-rich regions, serving as recognition sites, are likely to have PPII structure. PPII helices are involved in transcription, cell motility, self-assembly, elasticity, and bacterial and viral pathogenesis, and has an important structural role in amyloidogenic proteins. However, PPII helices are not always assigned in experimentally solved structures, and they are rarely used in protein structure modeling. We aim to give an overview of this structural class and of the place it holds in our current understanding of protein structure and function. This review is subdivided into three main parts: the first part covers PPII helices in unfolded peptides and proteins, the second part includes studies of the PPII helices in folded proteins, and the third part discusses the functional role of the PPII.
Graphical abstract Highlights ► The PPII helix is an extended, flexible left-handed helix without regular hydrogen bonds. ► PPII commonly occurs in folded proteins; it is abundant in unfolded proteins. ► PPII helices do not necessarily contain proline but proline has high PPII propensity. ► PPII has an important structural role and forms protein binding motifs. ► The PPII helix is a structure class comparable with the α-helix and β-structure.

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Author(s): Barry C. Johnson , Mathieu Métifiot , Andrea Ferris , Yves Pommier , Stephen H. Hughes

Although there are structures of the different domains of human immunodeficiency virus type 1 (HIV-1) integrase (IN), there is no structure of the entire protein. The recently determined crystal structures of the prototype foamy virus (PFV) IN tetramer, in complexes with viral DNA, led to the generation of models of full-length HIV-1 IN. These models were generated, in part, by superimposing the structures of the domains of HIV-1 IN onto the structure of full-length PFV IN. We developed a model for HIV-1 IN—based solely on its sequence alignment with PFV IN—that differs in several ways from the previous models. Specifically, in our model, the junction between the catalytic core domain and C-terminal domain adopts a helix–loop–helix motif that is similar to the corresponding segment of PFV IN and differs from the crystal structures of these two HIV-1 IN domains. The alignment of residues in the C-terminal domain also differs from the previous models. Our model can be used to explain the phenotype of previously published HIV-1 IN mutants. We made additional mutants, and the behavior of these new mutants provides additional support for the model.
Graphical abstract

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Author(s): Mercedes Spínola-Amilibia , José Rivera , Miguel Ortiz-Lombardía , Antonio Romero , José L. Neira , Jerónimo Bravo

The breast cancer metastasis suppressor 1 (BRMS1) gene suppresses metastasis without affecting the primary tumor growth. Cellular localization of BRMS1 appears to be important for exerting its effects on metastasis inhibition. We recently described a nucleo-cytoplasmic shuttling for BRMS1 and identified a nuclear export signal within the N-terminal coiled coil. The structure of these regions shows an antiparallel coiled coil capable of oligomerizing, which compromises the accessibility to the nuclear export signal consensus residues. We have studied the structural and biophysical features of this region to further understand the contribution of the N-terminal coiled coil to the biological function of BRMS1. We have observed that residues 85 to 98 might be important in defining the oligomerization state of the BRMS1 N-terminal coiled coil. The fragments are mainly disordered in solution, with evidence of residual structure. In addition, we report the presence of a conformational dynamic equilibrium (oligomeric folded species ↔ oligomeric unfolded) in solution in the BRMS1 N-terminal coiled coil that might facilitate the nuclear export of BRMS1 to the cytoplasm.
Graphical abstract Highlights ► BRMS151–84 and BRMS151–98 are coiled coils with different oligomerization states. ► Both isolated coiled coils behave as highly dynamic protein fragments in solution. ► Intrinsic disorder in solution can be important to accomplish function of BRMS1.

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Author(s): Christopher H.S. Aylett , Thierry Izoré , Linda A. Amos , Jan Löwe

Pseudomonas ΦKZ-like bacteriophages encode a group of related tubulin/FtsZ-like proteins believed to be essential for the correct centring of replicated bacteriophage virions within the bacterial host. In this study, we present crystal structures of the tubulin/FtsZ-like protein TubZ from Pseudomonas bacteriophage ΦKZ in both the monomeric and protofilament states, revealing that ΦKZ TubZ undergoes structural changes required to polymerise, forming a canonical tubulin/FtsZ-like protofilament. Combining our structures with previous work, we propose a polymerisation–depolymerisation cycle for the Pseudomonas bacteriophage subgroup of tubulin/FtsZ-like proteins. Electron cryo-microscopy of ΦKZ TubZ filaments polymerised in vitro implies a long-pitch helical arrangement for the constituent protofilaments. Intriguingly, this feature is shared by the other known subgroup of bacteriophage tubulin/FtsZ-like proteins from Clostridium species, which are thought to be involved in partitioning the genomes of bacteriophages adopting a pseudo-lysogenic life cycle.
Graphical abstract Highlights ► Bacteriophage ΦKZ encodes a tubulin/FtsZ homologue, TubZ, similar to 201Φ2-1 PhuZ. ► We have resolved crystal structures of ΦKZ TubZ as both a monomer and a protofilament. ► ΦKZ TubZ undergoes structural changes only at its surface during polymerisation. ► Electron microscopy shows that ΦKZ TubZ forms intertwined helical filaments in vitro. ► This feature is common to other TubZs in Bacillus plasmids and Clostridium prophage.

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Author(s): Cristiane R. Guzzo , German Dunger , Roberto Kopke Salinas , Chuck S. Farah

Signal transduction pathways mediated by cyclic-bis(3′→5′)-dimeric GMP (c-di-GMP) control many important and complex behaviors in bacteria. C-di-GMP is synthesized through the action of GGDEF domains that possess diguanylate cyclase activity and is degraded by EAL or HD-GYP domains with phosphodiesterase activity. There is mounting evidence that some important c-di-GMP-mediated pathways require protein–protein interactions between members of the GGDEF, EAL, HD-GYP and PilZ protein domain families. For example, interactions have been observed between PilZ and the EAL domain from FimX of Xanthomonas citri (Xac). FimX and PilZ are involved in the regulation of type IV pilus biogenesis via interactions of the latter with the hexameric PilB ATPase associated with the bacterial inner membrane. Here, we present the crystal structure of the ternary complex made up of PilZ, the FimX EAL domain (FimXEAL) and c-di-GMP. PilZ interacts principally with the lobe region and the N-terminal linker helix of the FimXEAL. These interactions involve a hydrophobic surface made up of amino acids conserved in a non-canonical family of PilZ domains that lack intrinsic c-di-GMP binding ability and strand complementation that joins β-sheets from both proteins. Interestingly, the c-di-GMP binds to isolated FimXEAL and to the PilZ–FimXEAL complex in a novel conformation encountered in c-di-GMP–protein complexes in which one of the two glycosidic bonds is in a rare syn conformation while the other adopts the more common anti conformation. The structure points to a means by which c-di-GMP and PilZ binding could be coupled to FimX and PilB conformational states.
Graphical abstract Highlights ► C-di-GMP signaling controls complex behaviors in bacteria. ► The c-di-GMP receptor FimX and PilZ are regulators of type IV pilus biogenesis. ► We report the crystal structure of the PilZ–FimX EAL domain–c-di-GMP complex. ► The c-di-GMP ligand adopts a rare syn/anti conformation in the complex ► C-di-GMP and PilZ binding could be coupled to FimX and PilB conformational states.

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Author(s): Saba Abdul-Hussein , Juni Andréll , Christopher G. Tate

Structure determination of mammalian integral membrane proteins is challenging due to their instability upon detergent solubilisation and purification. Recent successes in the structure determination of G-protein-coupled receptors (GPCRs) resulted from the development of GPCR-specific protein engineering strategies. One of these, conformational thermostabilisation, could in theory facilitate structure determination of other membrane proteins by improving their tolerance to detergents and locking them in a specific conformation. We have therefore used this approach on the cocaine-sensitive rat serotonin transporter (SERT). Out of a panel of 554 point mutants throughout SERT, 10 were found to improve its thermostability. The most stabilising mutations were combined to make the thermostabilised mutants SAH6 (L99A+G278A+A505L) and SAH7 (L405A+P499A+A505L) that were more stable than SERT by 18°C and 16°C, respectively. Inhibitor binding assays showed that both of the thermostabilised SERT mutants bound [125I]RTI55 (β-CIT) with affinity similar to that of the wild-type transporter, although cocaine bound with increased affinity (17- to 56-fold) whilst ibogaine, imipramine and paroxetine all bound with lower affinity (up to 90-fold). Neither SAH6 nor SAH7 was capable of transporting [3H]serotonin into HEK293 cell lines stably expressing the mutants, although serotonin bound to them with an apparent K i of 155μM or 82μM, respectively. These data combined suggest that SAH6 and SAH7 are thermostabilised in a specific cocaine-bound conformation, making them promising candidates for crystallisation. Conformational thermostabilisation is thus equally applicable to membrane proteins that are transporters in addition to those that are GPCRs.
Graphical abstract Highlights ► The serotonin transporter was thermostabilised to facilitate structural studies. ► Mutant SERT-SAH6 contained three point mutations that improved thermostability up to 18°C. ► SERT-SAH6 preferentially bound cocaine compared to other inhibitors. ► SERT-SAH6 bound serotonin with high affinity but could not transport it. ► SERT-SAH6 is locked in a cocaine-bound conformation ideal for crystallisation.

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Author(s): Ekaterina I. Biterova , Maria Svärd , Dominik D.D. Possner , Jodie E. Guy

Erv41p is a conserved integral membrane protein that is known to play a role in transport between the endoplasmic reticulum and Golgi apparatus, part of the early secretory pathway of eukaryotes. However, the exact function of the protein is not known, and it shares very low sequence identity with proteins of known structure or function. Here we present the structure of the full lumenal domain of Erv41p from Saccharomyces cerevisiae, determined by X-ray crystallography to a resolution of 2.0Å. The structure reveals the protein to be composed predominantly of two large β-sheets that form a twisted β-sandwich. Comparison to structures in the Protein Data Bank shows that the Erv41p lumenal domain displays only limited similarity to β-sandwich domains of other proteins. Analysis of the surface properties of the protein identifies an extensive patch of negative electrostatic potential on the exposed surface of one of the β-sheets, which likely forms a binding site for a ligand or interaction partner. A predominantly hydrophobic region close to the membrane interface is identified as a likely site for protein–protein interaction. This structure, the first of Erv41p or any of its homologues, provides a new starting point for studies of the roles of Erv41p and its interaction partners in the eukaryotic secretory pathway.
Graphical abstract Highlights ► Erv41p is a membrane protein that plays a role in the early secretory pathway. ► The crystal structure of the Erv41p lumenal domain has been determined at 2.0Å. ► Erv41p is composed of two β-sheets in a twisted β-sandwich fold. ► The structure will facilitate new studies to elucidate the function of Erv41p.

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Author(s): Dorothy Koveal , Michael W. Clarkson , Thomas K. Wood , Rebecca Page , Wolfgang Peti

Tyrosine phosphatase related to biofilm formation A (TpbA) is a periplasmic dual-specificity phosphatase (DUSP) that controls biofilm formation in the pathogenic bacterium Pseudomonas aeruginosa. While DUSPs are known to regulate important cellular functions in both prokaryotes and eukaryotes, very few structures of bacterial DUSPs are available. Here, we present the solution structure of TpbA in the ligand-free open conformation, along with an analysis of the structural and dynamic changes that accompany ligand/phosphate binding. While TpbA adopts a typical DUSP fold, it also possesses distinct structural features that distinguish it from eukaryotic DUSPs. These include additional secondary structural elements, β0 and α6, and unique conformations of the variable insert, the α4–α5 loop and helix α5 that impart TpbA with a flat active-site surface. In the absence of ligand, the protein tyrosine phosphatase loop is disordered and the general acid loop adopts an open conformation, placing the catalytic aspartate, Asp105, more than 11Å away from the active site. Furthermore, the loops surrounding the active site experience motions on multiple timescales, consistent with a combination of conformational heterogeneity and fast (picosecond to nanosecond) timescale dynamics, which are significantly reduced upon ligand binding. Taken together, these data structurally distinguish TpbA and possibly other bacterial DUSPs from eukaryotic DUSPs and provide a rich picture of active-site dynamics in the ligand-free state that are lost upon ligand binding.
Graphical abstract Highlights ► The NMR structure of TpbA is the first structure of a bacterial periplasmic DUSP. ► TpbA has unique structural features that distinguish it from eukaryotic DUSPs. ► Ligand-free TpbA shows active-site motions at multiple timescales. ► Ligand binding significantly reduces active-site dynamics. ► TpbA structure and dynamics provide new insights into enzyme regulation of DUSPs.

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Author(s): Andrea Grafmüller , Eva G. Noya , Gregory A. Voth

Microtubule (MT) stability is related to the hydrolysis of the guanosine triphosphate nucleotide (NT) bound to β-tubulin. However, the molecular mechanism by which the NT state influences the stability of the contacts in the MT lattice remains elusive. Here, we present large-scale atomistic simulations of different tubulin aggregates, including individual dimers, short protofilaments, a small lattice patch, and a piece of the MT lattice with two infinite protofilaments in both NT states. Together with a coarse-grained (CG) analysis of the fluctuations, these simulations highlight several regions of the protein where local changes are induced by the NT state or by the lateral and longitudinal contacts in the aggregates. Additionally, the CG analysis provides an indication of how the structural changes affect the bonds between the proteins. The results suggest a consistent picture of a possible molecular mechanism by which the NT state induces changes in the H1–S2 loop and more stable longitudinal bonds, both of which locate the H1–S2 and M-loop in more favorable positions to form lateral contacts.
Graphical abstract

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Author(s): Xiaolei Ma , Pierre A. Barthelemy , Lionel Rouge , Christian Wiesmann , Sachdev S. Sidhu

We compared the capacity of an autonomous heavy chain variable (VH) domain (VH-B1a) to support diversity within its antigen-binding site relative to the conventional antigen-binding fragment (Fab) from which it was derived. We find that VH-B1a can tolerate significant diversity within all three complementarity-determining regions (CDRs) and also within framework 3, and thus, VH-B1a and the Fab are similar in terms of the regions of the antigen-binding site that can tolerate diversity without compromising stability. We constructed libraries of synthetic VH domains and isolated binders with moderate affinity for vascular endothelial growth factor (VEGF) from a library in which only CDR3 was randomized. One binder was subjected to affinity maturation to derive an autonomous VH domain (VH-V1a) that recognized both human and mouse VEGF with high affinity (K D =16nM or 10nM, respectively). Structural analysis revealed that VH-V1a binds to an epitope that is distinct from the epitopes of a natural VEGF receptor and six different anti-VEGF Fabs. Moreover, VH-V1a recognizes VEGF by using an unusual paratope consisting predominantly of CDR3 but with significant contributions from framework residues within the former light chain interface. These results suggest that VH-B1a and other autonomous VH domains may be useful scaffolds to support both conventional libraries with antigen-binding sites built from the three CDR loops and, also, nonconventional libraries with antigen-binding sites built from CDR3 and the former light chain interface.
Graphical abstract Highlights ► The engineered human VH domain VH-B1a folds and functions autonomously. ► The antigen-binding site of VH-B1a can tolerate significant sequence diversity. ► A VH domain library yielded a high-affinity-binder VH-V1a targeting VEGF. ► The anti-VEGF VH-V1a domain targets an unusual epitope. ► The VH-V1a paratope includes substantial contributions from the framework.

Publication date: 26 June 2013
Source:Journal of Molecular Biology, Volume 425, Issue 12

Author(s): Derya Meral , Brigita Urbanc

In Alzheimer's disease (AD), amyloid β-protein (Aβ) self-assembles into toxic oligomers. Of the two predominant Aβ alloforms, Aβ1–40 and Aβ1–42, the latter is particularly strongly linked to AD. N-terminally truncated and pyroglutamated Aβ peptides were recently shown to seed Aβ aggregation and contribute significantly to Aβ-mediated toxicity, yet their folding and assembly were not explored computationally. Discrete molecular dynamics approach previously captured in vitro-derived distinct Aβ1–40 and Aβ1–42 oligomer size distributions and predicted that the more toxic Aβ1–42 oligomers had more flexible and solvent-exposed N-termini than Aβ1–40 oligomers. Here, we examined oligomer formation of Aβ3–40, Aβ3–42, Aβ11–40, and Aβ11–42 by the discrete molecular dynamics approach. The four N-terminally truncated peptides showed increased oligomerization propensity relative to the full-length peptides, consistent with in vitro findings. Conformations formed by Aβ3–40/42 had significantly more flexible and solvent-exposed N-termini than Aβ1–40/42 conformations. In contrast, in Aβ11–40/42 conformations, the N-termini formed more contacts and were less accessible to the solvent. The compactness of the Aβ11–40/42 conformations was in part facilitated by Val12. Two single amino acid substitutions that reduced and abolished hydrophobicity at position 12, respectively, resulted in a proportionally increased structural variability. Our results suggest that Aβ11–40 and Aβ11–42 oligomers might be less toxic than Aβ1–40 and Aβ1–42 oligomers and offer a plausible explanation for the experimentally observed increased toxicity of Aβ3–40 and Aβ3–42 and their pyroglutamated forms.
Graphical abstract Highlights ► N-terminally truncated Aβ relevant to AD studied in silico. ► Increased assembly propensity of N-terminally truncated Aβ consistent with in vitro data. ► Aβ without the first two residues formed oligomers with strongly disordered N-termini. ► Aβ without the first 10 residues formed compact oligomer conformations. ► Oligomers formed by these different Aβ isoforms predicted to have distinct toxicities.

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

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