Publication date: Available online 29 April 2013
Source:Journal of Molecular Biology

Author(s): Justin M. Miller , Jiabei Lin , Tao Li , Aaron L. Lucius

There are five known ATP-dependent proteases in Escherichia coli, Lon, ClpAP, ClpXP, HslUV, and the membrane-associated FtsH, that catalyze the removal of both misfolded and properly folded proteins in cellular protein quality control pathways. Hexameric ClpA rings associate with one or both faces of the cylindrically-shaped tetradecameric ClpP protease. ClpA catalyzes unfolding and translocation of polypeptide substrates into the proteolytic core of ClpP for degradation through repeated cycles of ATP binding and hydrolysis at two nucleotide binding domains on each ClpA monomer. We previously reported a molecular mechanism for ClpA catalyzed polypeptide translocation in the absence of ClpP, including elementary rate constants, overall rate, and the kinetic step-size. However, the potential allosteric effect of ClpP on the mechanism of ClpA catalyzed translocation remains unclear. Using single-turnover fluorescence stopped flow methods, here we report that ClpA, when associated with ClpP, translocates polypeptide with an overall rate of ~35 amino acids per second and, on average, traverses ~5 amino acids between two rate limiting steps with reduced cooperativity between ATP binding sites in the hexameric ring. This is in direct contrast to our previously reported observation that, in the absence of ClpP, ClpA translocates polypeptide substrates with a maximum translocation rate of ~20 amino acids per second with cooperativity between ATPase sites. Our results demonstrate that ClpP allosterically impacts the polypeptide translocation activity of ClpA by reducing the cooperativity between ATP binding sites.
Graphical abstract

Publication date: Available online 29 April 2013
Source:Journal of Molecular Biology

Author(s): Jun Zhang , Sanya Hashmi , Fatima Cheema , Nafla Al-Nasser , Razan Bakheet , Ranjit S. Parhar , Futwan Al-Mohanna , Randy Gaugler , M. Mahmood Hussain , Sarwar Hashmi

Lipid metabolism is coordinately regulated through signaling networks that integrate biochemical pathways of fat assimilation, mobilization and utilization. Excessive diversion of fat for storage is a key risk factor for many fat related human diseases. Dietary lipids are absorbed from the intestines and transported to various organs and tissues to provide energy and maintain lipid homeostasis. In humans, disparity between TG synthesis and removal, via mitochondrial beta-oxidation and VLDL secretion, causes excessive TG accumulation in the liver. The mutation in Caenorhabditis elegans KLF-3 leads to high TG accumulation in the worm’s intestine. Our previous data suggested that klf-3 regulates lipid metabolism by promoting FA β-oxidation. Depletion of cholesterol in the diet has no effect on fat deposition in klf-3 (ok1975) mutants. Addition of vitamin D in the diet however, increases fat levels in klf-3 worms. This suggests that excess vitamin D may be lowering the rate of FA beta-oxidation, with the eventual increase in fat accumulation. We also demonstrate that mutation in klf-3 reduces expression of C. elegans dsc-4 and/or vit genes, the orthologues of mammalian MTP and apoB respectively. Both MTP and apoB are essential for mammalian lipoprotein assembly and transport and mutation in both dsc-4 (qm182) and vit-5 (ok3239), results in high fat accumulation in worm intestine. Genetic interactions between klf-3 and dsc-4, as well as vit-5 genes, suggest that klf-3 may have an important role in regulating lipid assembly and secretion.
Graphical abstract

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Author(s): Thomas M. Moon , Fernando Correa , Lisa N. Kinch , Alexander T. Piala , Kevin H. Gardner , Elizabeth J. Goldsmith

WNK1 [with no lysine (K)-1] is a 250-kDa serine/threonine protein kinase involved in the maintenance of cellular salt levels and is directly linked to a hereditary form of hypertension. Here, we report the solution NMR structure of the autoinhibitory domain of WNK1 (WNK1-AI), a small regulatory subunit that lies immediately C-terminal of the kinase domain. We show that this domain is a homolog of the RFXV-binding PASK/FRAY homology 2 (PF2) domain found in OSR (oxidative stress responsive) and SPAK (serine/threonine proline–alanine-rich) kinases, which are substrates of WNK1. The WNK1-AI has a circularly permuted topology relative to the OSR1–PF2 domain. Nevertheless, like PF2 domains, WNK1-AI binds peptides that contain an RFXV motif with micromolar affinities as assessed by changes in 1H,15N heteronuclear single quantum coherence spectra. Mutations to the WNK1-AI and binding peptides confirm a similar binding mode.
Graphical abstract Highlights ► The structure of the WNK1 autoinhibitory domain was solved by high-resolution solution NMR methods. ► The structure is similar to that of the PF2 domain of OSR, a WNK1 substrate. ► The autoinhibitory domain binds an RFXV peptide derived from the WNK1 kinase domain.

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Author(s): Xiaojie Yu , Snezana Vasiljevic , Daniel A. Mitchell , Max Crispin , Christopher N. Scanlan

Intravenous immunoglobulin (IVIg) therapy is used to treat a wide range of autoimmune conditions and consists of pooled immunoglobulin G (IgG) from healthy donors. The immunosuppressive effects of IVIg are, in part, attributed to terminal α2,6-linked sialic acid residues on the N-linked glycans of the IgG Fc (fragment crystallizable) domain. This α2,6-sialylated Fc (sFc) has been reported to bind to the carbohydrate recognition domain (CRD) of the cell-surface lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) and its murine orthologue SIGN-R1 (specific intracellular adhesion molecule-grabbing non-integrin R1) and, via this interaction, to signal the downstream expression of immunosuppressive cytokines and receptors. Consistent with this model, the antiinflammatory effect of IVIg treatment is abolished in a murine knock-out of SIGN-R1 and can be restored by a knock-in with human DC-SIGN. In contrast, however, existing glycan array and X-ray crystallographic studies indicate that the CRDs of both SIGN-R1 and DC-SIGN bind to a restricted set of primarily oligomannose-type glycans that does not include the glycans found on sFc. We attempted to reconcile these immunological and biophysical observations. We first generated hypersialylated, desialylated, deglycosylated and untreated serum IgG and found that the affinity for the complete extracellular region of the DC-SIGN tetramer was similar for all antibody glycoforms. Moreover, the binding could be attributed to cross-reactive, polyclonal Fab (fragment antigen-binding) specificities in serum as neither recombinant Fc nor sFc bound to DC-SIGN. In addition, serum IgG exhibited no competition against known ligands of the DC-SIGN CRD. These findings lead us to suggest that IVIg therapy does not involve binding of IgG Fc to DC-SIGN and that alternative cell-surface lectins are required for the antiinflammatory activity of sFc.
Graphical abstract Highlights ► Sialylated IgG Fc glycans are essential for IVIg therapy. ► The DC-SIGN lectin has been proposed to mediate binding to sialylated Fc. ► Binding is attributed to Fab specificities and is independent of glycosylation. ► We propose that alternative lectins may be the therapeutic target of IVIg.

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Author(s): Robert J. Bauer , Brian W. Graham , Michael A. Trakselis

DNA priming and unwinding activities are coupled within bacterial primosome complexes to initiate synthesis on the lagging strand during DNA replication. Archaeal organisms contain conserved primase genes homologous to both the bacterial DnaG and archaeo-eukaryotic primase families. The inclusion of multiple DNA primases within a whole domain of organisms complicates the assignment of the metabolic roles of each. In support of a functional bacterial-like DnaG primase participating in archaeal DNA replication, we have detected an interaction of Sulfolobus solfataricus DnaG (SsoDnaG) with the replicative S. solfataricus minichromosome maintenance (SsoMCM) helicase on DNA. The interaction site has been mapped to the N-terminal tier of SsoMCM analogous to bacterial primosome complexes. Mutagenesis within the metal binding site of SsoDnaG verifies a functional homology with bacterial DnaG that perturbs priming activity and DNA binding. The complex of SsoDnaG with SsoMCM stimulates the ATPase activity of SsoMCM but leaves the priming activity of SsoDnaG unchanged. Competition for binding DNA between SsoDnaG and SsoMCM can reduce the unwinding ability. Fluorescent gel shift experiments were used to quantify the binding of the ternary SsoMCM–DNA–SsoDnaG complex. This direct interaction of a bacterial-like primase with a eukaryotic-like helicase suggests that formation of a unique but homologous archaeal primosome complex is possible but may require other components to stimulate activities. Identification of this archaeal primosome complex broadly impacts evolutionary relationships of DNA replication.
Graphical abstract Highlights ► Primase–helicase interactions occur in all domains to couple unwinding and priming. ► Bacterial-like DnaG primase interacts with eukaryotic-like MCM helicase in archaea. ► The archaeal DnaG primase has a conserved active site with bacterial DnaG. ► An archaeal DnaG–MCM complex is structurally analogous to the bacterial primosome. ► Archaeal DnaG as a homolog of the bacterial primase is validated.

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Author(s): Christopher M. Yates , Michael J.E. Sternberg

The widespread application of whole-genome sequencing is identifying numerous non-synonymous single nucleotide polymorphisms (nsSNPs), many of which are associated with disease. We analyzed nsSNPs from Humsavar and the 1000 Genomes Project to investigate why some proteins and domains are more tolerant of mutations than others. We identified 311 proteins and 112 Pfam families, corresponding to 2910 domains, as disease susceptible and 32 proteins and 67 Pfam families (10,783 domains) as disease resistant based on the relative numbers of disease-associated and neutral polymorphisms. Proteins with no significant difference from expected numbers of disease and polymorphism nsSNPs are classified as other. This classification takes into account the phenotypes of all known mutations in the protein or domain rather than simply classifying based on the presence or absence of disease nsSNPs. Of the two hypotheses suggested, our results support the model that disease-resistant domains and proteins are more able to tolerate mutations rather than having more lethal mutations that are not observed. Disease-resistant proteins and domains show significantly higher mutation rates and lower sequence conservation than disease-susceptible proteins and domains. Disease-susceptible proteins are more likely to be encoded by essential genes, are more central in protein–protein interaction networks and are less likely to contain loss-of-function mutations in healthy individuals. We use this classification for nsSNP phenotype prediction, predicting nsSNPs in disease-susceptible domains to be disease and those in disease-resistant domains to be polymorphism. In this way, we achieve higher accuracy than SIFT, a state-of-the-art algorithm.
Graphical abstract Highlights ► Many disease-associated nsSNPs and the proteins containing them have been studied. ► We use the numbers of disease and neutral nsSNPs to classify proteins and domains. ► Tolerance of proteins and domains for mutations is related to conservation. ► It is also related to interaction networks and function. ► This classification is useful for predicting phenotypes of nsSNPs.

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Author(s): Mathieu Gissot , Robert Walker , Stephane Delhaye , Tchilabalo Dilezitoko Alayi , Ludovic Huot , David Hot , Isabelle Callebaut , Christine Schaeffer-Reiss , Alain Van Dorsselaer , Stanislas Tomavo

Molecular mechanisms controlling gene expression in apicomplexan parasites remain poorly understood. Here, we report the characterization of two Toxoplasma gondii homologs of the ancient archeal Alba proteins named TgAlba1 and TgAlba2. The targeted disruption of TgAlba1 and TgAlba2 genes in both virulent type I and avirulent type II strains of T. gondii reveals that TgAlba proteins may have an important role in regulating stress response. We found that although the steady-state level of the Tgalba2 transcript is increased in the ΔTgalba1 null mutant parasites, the cognate TgAlba2 protein is undetectable, suggesting that TgAlba1 is required for translation of TgAlba2. Using a tandem affinity purification tag strategy combined with proteomic analyses, we provide evidence that many factors known to be involved in the translation machinery are co-purified with TgAlba1 and TgAlba2. We further performed RNA pull-down and microarray analyses to show that TgAlba1 and TgAlba2 bind to more than 30 RNAs including their own transcripts. Moreover, we demonstrate that the tight translational regulation of the TgAlba2 endogenous transcript relies on the presence of both its 3′ untranslated region and that of the TgAlba1 protein. Thus, our findings on TgAlba1 and TgAlba2 are consistent with a role in gene-specific translation.
Graphical abstract Highlights ► T. gondii Alba proteins are involved in gene-specific translation. ► TgAlba proteins co-localize with RNA granules and are involved in stress responses. ► TgAlba proteins bind RNA and co-purify with proteins involved in translation regulation. ► Translation of Tgalba2 depends on the presence of its 3′ untranslated region and the TgAlba1 protein.

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Author(s): Thomas Hofmeyer , Stefan Schmelz , Matteo T. Degiacomi , Matteo Dal Peraro , Matin Daneschdar , Andrea Scrima , Joop van den Heuvel , Dirk W. Heinz , Harald Kolmar

The complement system as a major part of innate immunity is the first line of defense against invading microorganisms. Orchestrated by more than 60 proteins, its major task is to discriminate between host cells and pathogens and to initiate immune response. Additional recognition of necrotic or apoptotic cells demands a fine-tune regulation of this powerful system. C4b-binding protein (C4BP) is the major inhibitor of the classical complement and lectin pathway. The crystal structure of the human C4BP oligomerization domain in its 7α isoform and molecular simulations provide first structural insights of C4BP oligomerization. The heptameric core structure is stabilized by intermolecular disulfide bonds. In addition, thermal shift assays indicate that layers of electrostatic interactions mainly contribute to the extraordinary thermodynamic stability of the complex. These findings make C4BP a promising scaffold for multivalent ligand display with applications in immunology and biological chemistry.
Graphical abstract Highlights ► The core crystal structure of a major modulator of complement system is presented. ► The human C4BP core complex reveals a heptameric ring structure. ► Seven disulfide bonds and three layers of electrostatic interactions provide high stability. ► Molecular modeling provides insights into the structure of heterooligomeric isoforms.

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Author(s): Daniel H. Lin , Stephan Zimmermann , Tobias Stuwe , Evelyn Stuwe , André Hoelz

The nuclear pore complex is the sole mediator of bidirectional transport between the nucleus and cytoplasm. Nup358 is a metazoan-specific nucleoporin that localizes to the cytoplasmic filaments and provides several binding sites for the mobile nucleocytoplasmic transport machinery. Here we present the crystal structure of the C-terminal domain (CTD) of Nup358 at 1.75Å resolution. The structure reveals that the CTD adopts a cyclophilin-like fold with a non-canonical active-site configuration. We determined biochemically that the CTD possesses weak peptidyl-prolyl isomerase activity and show that the active-site cavity mediates a weak association with the human immunodeficiency virus-1 capsid protein, supporting its role in viral infection. Overall, the surface is evolutionarily conserved, suggesting that the CTD serves as a protein–protein interaction platform. However, we demonstrate that the CTD is dispensable for nuclear envelope localization of Nup358, suggesting that the CTD does not interact with other nucleoporins.
Graphical abstract Highlights ► Crystal structure of the CTD of Nup358. ► Nup358 CTD has peptidyl-prolyl isomerase activity. ► Nup358 CTD has weak binding affinity for human immunodeficiency virus-1 capsid protein. ► Nup358 CTD is dispensable for nuclear envelope localization.

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Author(s): Mark Ultsch , Jack Bevers , Gerald Nakamura , Richard Vandlen , Robert F. Kelley , Lawren C. Wu , Charles Eigenbrot

The cytokine interleukin 13 (IL-13) is a major effector molecule for T-helper type 2 inflammation and is pathogenic in allergic diseases such as asthma. The effects of IL-13 are mediated via a pathway that is initiated by binding to a heterodimeric receptor consisting of IL-13Rα1 and IL-4Rα. Antibodies raised against IL-13 can block its inflammatory effects by interfering with binding to either of the two receptor polypeptides. Lebrikizumab is a monoclonal anti-IL-13 antibody that has shown clinical benefit in a phase II study for the treatment of moderate-to-severe uncontrolled asthma. Here we report the molecular structure of IL-13 in complex with the Fab from lebrikizumab by X-ray crystallography at 1.9Å resolution. We show that lebrikizumab inhibits IL-13 signaling by binding to IL-13 with very high affinity and blocking IL-13 binding to IL-4Rα. In addition, we use site-directed mutations to identify the most important antibody contributors to binding. Our studies define key features of lebrikizumab binding and its mechanism of action that may contribute to its clinical effects.
Graphical abstract Highlights ► The cytokine IL-13 is a major immune effector associated with asthma. ► Anti-IL-13 lebrikizumab has a dissociation constant less than 10pM. ► Lebrikizumab prevents binding of IL-4Rα, a receptor required for signaling.

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Author(s): Andreas Funkner , Christoph Parthier , Mike Schutkowski , Johnny Zerweck , Hauke Lilie , Natalya Gyrych , Gunter Fischer , Milton T. Stubbs , David M. Ferrari

The protein disulfide isomerase (PDI) family member ERp46/endoPDI/thioredoxin domain-containing protein 5 is preferentially expressed in a limited number of tissues, where it may function as a survival factor for nitrosative stress in vivo. It is involved in insulin production as well as in adiponectin signaling and interacts specifically with the redox-regulatory endoplasmic reticulum proteins endoplasmic oxidoreductin 1α (Ero1α) and peroxiredoxin-4. Here, we show that ERp46, although lacking a PDI-like redox-inactive b′-thioredoxin domain with its hydrophobic substrate binding site, is able to bind to a large pool of peptides containing aromatic and basic residues via all three of its catalytic domains (a0, a and a′), though the a0 domain may contain the primary binding site. ERp46, which shows relatively higher activity as a disulfide-reductase than as an oxidase/isomerase in vitro compared to PDI and ERp57, possesses chaperone activity in vivo, a property also shared by the C-terminal a′ domain. A crystal structure of the a′ domain is also presented, offering a view of possible substrate binding sites within catalytic domains of PDI proteins.
Graphical abstract Highlights ► Do redox-active PDI-like domains have redox-independent peptide binding activity? ► We present a novel approach to study the weak PDI protein: peptide interactions. ► ERp46 lacks a PDI-like redox-inactive domain but binds a large pool of peptides. ► The major binding site is in the a0 domain, but all three ERp46 domains bind peptides. ► Redox-active domains of a PDI protein show redox-independent peptide binding.

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Author(s): Benjamin P. Roscoe , Kelly M. Thayer , Konstantin B. Zeldovich , David Fushman , Daniel N.A. Bolon

The amino acid sequence of a protein governs its function. We used bulk competition and focused deep sequencing to investigate the effects of all ubiquitin point mutants on yeast growth rate. Many aspects of ubiquitin function have been carefully studied, which enabled interpretation of our growth analyses in light of a rich structural, biophysical and biochemical knowledge base. In one highly sensitive cluster on the surface of ubiquitin, almost every amino acid substitution caused growth defects. In contrast, the opposite face tolerated virtually all possible substitutions. Surface locations between these two faces exhibited intermediate mutational tolerance. The sensitive face corresponds to the known interface for many binding partners. Across all surface positions, we observe a strong correlation between burial at structurally characterized interfaces and the number of amino acid substitutions compatible with robust growth. This result indicates that binding is a dominant determinant of ubiquitin function. In the solvent-inaccessible core of ubiquitin, all positions tolerated a limited number of substitutions, with hydrophobic amino acids especially interchangeable. Some mutations null for yeast growth were previously shown to populate folded conformations indicating that, for these mutants, subtle changes to conformation caused functional defects. The most sensitive region to mutation within the core was located near the C-terminus that is a focal binding site for many critical binding partners. These results indicate that core mutations may frequently cause functional defects through subtle disturbances to structure or dynamics.
Graphical abstract Highlights ► Mutations provide a powerful probe of protein mechanism. ► Bulk competition and deep sequencing was used to monitor ubiquitin mutants. ► Sensitivity to mutation correlated with binding interfaces at surface positions. ► In the core, positions near critical binding sites were the most sensitive to mutation. ► Binding interactions impose dominant binding constraints throughout ubiquitin.

Publication date: 26 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 8

Author(s): Trevor A. Addington , Robert W. Mertz , Justin B. Siegel , James M. Thompson , Andrew J. Fisher , Vladimir Filkov , Nicholas M. Fleischman , Alisa A. Suen , Chensong Zhang , Michael D. Toney

Identification of residues responsible for functional specificity in enzymes is a challenging and important problem in protein chemistry. Active-site residues are generally easy to identify, but residues outside the active site are also important to catalysis and their identities and roles are more difficult to determine. We report a method based on analysis of multiple sequence alignments, embodied in our program Janus, for predicting mutations required to interconvert structurally related but functionally distinct enzymes. Conversion of aspartate aminotransferase into tyrosine aminotransferase is demonstrated and compared to previous efforts. Incorporation of 35 predicted mutations resulted in an enzyme with the desired substrate specificity but low catalytic activity. A single round of DNA back-shuffling with wild-type aspartate aminotransferase on this variant generated mutants with tyrosine aminotransferase activities better than those previously realized from rational design or directed evolution. Methods such as this, coupled with computational modeling, may prove invaluable in furthering our understanding of enzyme catalysis and engineering.
Graphical abstract Highlights ► Introduces new computational method for identifying functionally relevant residues. ► Janus greatly reduces sequence space needed for conversion of enzyme function. ► Structures of highly active mutants provide examples of redesigned enzymes. ► Computational modeling shown to have potential for screening deleterious mutations.

Publication date: Available online 26 April 2013
Source:Journal of Molecular Biology

Author(s): Wen-Tao Hou , Wen-Zhe Li , Yuxing Chen , Yong-Liang Jiang , Cong-Zhao Zhou

The homeostasis of intracellular diadenosine 5',5'''-P 1,P 4-tetraphosphate (Ap4A) in the yeast Saccharomyces cerevisiae is maintained by two 60% sequence-identical paralogs of Ap4A phosphorylases (Apa1 and Apa2). Enzymatic assays show that, compared to Apa1, Apa2 has a relatively higher phosphorylase activity towards Ap3A, Ap4A and Ap5A, and Ap4A is the favorable substrate for both enzymes. To decipher the catalytic insights, we determined the crystal structures of Apa2 in the apo-, AMP- and Ap4A-complexed forms at 2.30, 2.80 and 2.70 Å resolution, respectively. Apa2 is an α/β protein with a core domain of a twisted eight-stranded antiparallel β-sheet flanked by several α-helices, similar to the galactose-1-phosphate uridylyltransferase (GalT) members of the histidine triad (HIT) superfamily. However, a unique auxiliary domain enables an individual Apa2 monomer to possess an intact substrate-binding cleft, which is distinct from previously reported dimeric GalT proteins. This cleft is perfectly complementary to the favorable substrate Ap4A, the AMP and ATP moieties of which are perpendicular to each other, leaving the α-phosphate group exposed at the sharp turn against the catalytic residue His161. Structural comparisons combined with site-directed mutagenesis and activity assays enable us to define the key residues for catalysis. Furthermore, multiple-sequence alignment reveals that Apa2 and homologs represent canonical Ap4A phosphorylases which could be grouped as a unique branch in the GalT family.
Graphical abstract

Publication date: Available online 26 April 2013
Source:Journal of Molecular Biology

Author(s): Charalampos (Babis) Kalodimos

Publication date: Available online 25 April 2013
Source:Journal of Molecular Biology

Author(s): Agnieszka Szyk , Grzegorz Piszczek , Antonina Roll-Mecak

Tubulin partition between soluble and polymeric forms is tightly regulated in cells. Stathmin and tubulin tyrosine ligase (TTL) each form stable complexes with tubulin and inhibit tubulin polymerization. Here we explore the mutual relationship between these proteins in vitro and demonstrate that full-length stathmin and TTL compete for binding to tubulin and fail to make a stable tubulin:stathmin:TTL triple complex in solution. Moreover, stathmin depresses TTL tubulin tyrosination activity in vitro. These results suggest that TTL and stathmin have either a partially overlapping footprint on the tubulin dimer or that stathmin induces a tubulin conformation incompatible with stable TTL binding.
Graphical abstract

Publication date: Available online 23 April 2013
Source:Journal of Molecular Biology

Author(s): Nirupama Gupta , Louise W. DeMong , Sowmya Banda , Paul R. Copeland

Selenoproteins are present in all three domains of life and are responsible for a major part of a cell’s antioxidant defense against reactive oxygen species. Synthesis of selenoproteins requires the decoding of a UGA codon as selenocysteine (Sec) instead of translation termination. Sec is incorporated into the growing polypeptide chain during translation elongation and is known to require a set of highly specific factors: The Sec insertion sequence (SECIS) element in the 3’ untranslated region (3’ UTR) , Sec-tRNASec, the Sec-specific elongation factor eEFSec, and SECIS binding protein 2 (SBP2). Since reconstitution has not been reported, whether these factors are sufficient is unknown. Here we report a novel in vitro translation system in which Sec incorporation has been reconstituted from purified components introduced into a Sec naive system. In addition, we developed a novel method to purify Sec-tRNASec and active eEFSec/GTP/tRNA ternary complex. We found that the known basal factors are sufficient for Sec incorporation in vitro. Using this highly manipulable system, we have also found that ribosomes from non-Sec utilizing organisms cannot support Sec incorporation and that some SECIS elements are intrinsically less efficient than others. Having identified the essential set of factors, this work removes a significant barrier to our understanding of the mechanism of Sec incorporation.
Graphical abstract

Publication date: Available online 22 April 2013
Source:Journal of Molecular Biology

Author(s): Jorge A. Lamboy , Hajin Kim , Holly Dembinski , Taekjip Ha , Elizabeth A. Komives

Previous single-molecule fluorescence resonance energy transfer (smFRET) studies in which the second and sixth ankyrin repeats (ARs) of IκBα were labeled with FRET pairs showed slow fluctuations as if the IκBα AR domain was unfolding in its native state. To systematically probe where these slow dynamic fluctuations occur, we now present data from smFRET studies wherein FRET labels were placed at ARs 1 and 4 (mutant named AR 1–4), at ARs 2 and 5 (AR 2–5), and at ARs 3 and 6 (AR 3–6). The results presented here reveal that AR 6 most readily detaches/unfolds from the AR domain, undergoing substantial fluctuations at room temperature. AR 6 has fewer stabilizing consensus residues than the other IκBα ARs, probably contributing to the ease with which AR 6 “loses grip”. AR 5 shows almost no fluctuations at room temperature, but a significant fraction of molecules shows fluctuations at 37°C. Introduction of stabilizing mutations that are known to fold AR 6 dampen the fluctuations of AR 5, indicating that the AR 5 fluctuations are likely due to weakened inter-repeat stabilization from AR 6. AR 1 also fluctuates somewhat at room temperature, suggesting that fluctuations are a general behavior of ARs at ends of AR domains. Remarkably, AR1 still fluctuates in the bound state, but mainly between 0.6 and 0.9 FRET efficiency, whereas in the free IκBα, the fluctuations extend to <0.5 FRET efficiency. Overall, our results provide a more complete picture of the energy landscape of the native state dynamics of an AR domain.
Graphical abstract