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

Publication date: Available online 17 April 2013
Source:Journal of Molecular Biology

Author(s): Benjamin D. Pope , David M. Gilbert

The “Replicon Theory” of Jacob, Brenner, and Cuzin has reliably served as the paradigm for regulating the sites where individual replicons initiate replication. Concurrent with the replicon model was Taylor's demonstration that plant and animal chromosomes replicate segmentally in a defined temporal sequence, via cytologically defined units too large to be accounted for by a single replicon. Instead, there seemed to be a program to choreograph when chromosome units replicate during S phase, executed by inititation at clusters of individual replicons within each segment. Here, we summarize recent molecular evidence for the existence of such units, now known as “replication domains”, and discuss how the organization of large chromosomes into structural units has added additional layers of regulation to the original replicon model.
Graphical abstract

Publication date: Available online 17 April 2013
Source:Journal of Molecular Biology

Author(s): Camilla Trovesi , Nicola Manfrini , Marco Falcettoni , Maria Pia Longhese

The eukaryotic cell cycle comprises a series of events, whose ordering and correct progression depends on the oscillating activity of cyclin-dependent kinases (Cdks), which safeguard timely duplication and segregation of the genome. Cell division is intimately connected to an evolutionarily conserved DNA damage response (DDR), which involves DNA repair pathways that reverse DNA lesions, as well as checkpoint pathways that inhibit cell cycle progression while repair occurs. There is increasing evidence that Cdks are involved in the DDR, in particular in DNA repair by homologous recombination and in activation of the checkpoint response. However, Cdks have to be carefully regulated, because even an excess of their activity can affect genome stability. In this review, we consider the physiological role of Cdks in the DDR.
Graphical abstract

Publication date: Available online 16 April 2013
Source:Journal of Molecular Biology

Author(s): Bret D. Wallace , Laurie Betts , Garrick Talmage , Rebecca M. Pollet , Natalie S. Holman , Matthew R. Redinbo

The human nuclear xenobiotic receptor PXR recognizes a range of potentially harmful drugs and endobiotic chemicals but must complex with the nuclear receptor RXRα to control the expression of numerous drug metabolism genes. To date, the structural basis and functional consequences of this interaction have remained unclear. Here we present 2.8-Å-resolution crystal structures of the heterodimeric complex formed between the ligand-binding domains of human PXR and RXRα. These structures establish that PXR and RXRα form a heterotetramer unprecedented in the nuclear receptor family of ligand-regulated transcription factors. We further show that both PXR and RXRα bind to the transcriptional coregulator SRC-1 with higher affinity when they are part of the PXR/RXRα heterotetramer complex than they do when each ligand-binding domain is examined alone. Furthermore, we purify the full-length forms of each receptor from recombinant bacterial expression systems and characterize their interactions with a range of direct and everted repeat DNA elements. Taken together, these data advance our understanding of PXR, the master regulator of drug metabolism gene expression in humans, in its functional partnership with RXRα.
Graphical abstract

Publication date: Available online 16 April 2013
Source:Journal of Molecular Biology

Author(s): Markus G. Rudolph , Dagmar Klostermeier

Type II DNA topoisomerases alter the supercoiling state of DNA in an ATP-dependent fashion that requires large conformational changes. The directionality of DNA strand transfer is controlled by three transient protein interfaces, termed the N-gate, DNA-gate, and C-gate. Bacterial gyrase is a type II DNA topoisomerase of A2B2 composition. The N-gate is formed by the two GyrB subunits and the GyrA subunits form the DNA- and C-gates. In structures of type II topoisomerase fragments, the DNA- and C-gates delimit a cavity for DNA and can be open or closed. However, the conformational space accessible has not yet been mapped. Here, we describe the crystal structure of the Bacillus subtilis DNA gyrase A subunit lacking the C-terminal DNA-wrapping domains. Five dimeric states of the GyrA N-terminal domain are observed, with their DNA- and C-gates either closed or open to different extents. All of these conformations can in principle accommodate double-stranded DNA in the central cavity but only one conformation has its DNA-gate open wide enough for DNA to enter. The structure thus reflects the lower limit of DNA-gate opening that must occur during gyrase catalysis. The DNA-gate is formed by two flat surfaces, with few interactions. In contrast, the C-gate exhibits a highly undulated surface and forms a large number of interactions. None of the dimers in the crystal structures display an open C-gate that would allow DNA passage, in agreement with a transient opening of this gate during the catalytic cycle of DNA supercoiling.
Graphical abstract

Publication date: Available online 16 April 2013
Source:Journal of Molecular Biology

Author(s): Anita M. Preininger , Jens Meiler , Heidi Hamm

Structure and dynamics of G proteins and their cognate receptors, both alone and in complex, are becoming increasingly accessible to experimental techniques. Understanding the conformational changes and timelines that govern these changes can lead to new insights into the processes of ligand binding and associated G protein activation. Experimental systems may involve the use of, or otherwise stabilize, non-native environments. This can complicate our understanding of structural and dynamic features of processes such as the ionic lock, tryptophan toggle, and G protein flexibility. While elements in the receptor's transmembrane helices and the C-terminal α5 helix of Gα undergo well-defined structural changes, regions subject to conformational flexibility may be important in fine-tuning the interactions between activated receptors and G proteins. The pairing of computational and experimental approaches will continue to provide powerful tools to probe the conformation and dynamics of receptor-mediated G protein activation.
Graphical abstract

Publication date: 12 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 7

Publication date: 12 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 7

Publication date: 12 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 7

Author(s): P.H. Stokes , C.W. Liew , A.H. Kwan , P. Foo , H.E. Barker , A. Djamirze , V. O'Reilly , J.E. Visvader , J.P. Mackay , J.M. Matthews

LIM-only protein 4 (LMO4) is strongly linked to the progression of breast cancer. Although the mechanisms underlying this phenomenon are not well understood, a role is emerging for LMO4 in regulation of the cell cycle. We determined the solution structure of LMO4 in complex with CtIP (C-terminal binding protein interacting protein)/RBBP8, a tumour suppressor protein that is involved in cell cycle progression, DNA repair and transcriptional regulation. Our data reveal that CtIP and the essential LMO cofactor LDB1 (LIM-domain binding protein 1) bind to the same face on LMO4 and cannot simultaneously bind to LMO4. We hypothesise that overexpression of LMO4 may disrupt some of the normal tumour suppressor activities of CtIP, thereby contributing to breast cancer progression.
Graphical abstract Highlights ► LMO4 is overexpressed in breast cancer and can bind cell cycle control protein CtIP. ► LMO4 cannot bind CtIP and key partner LDB1 simultaneously. ► CtIP interacts with LMO4 using a short domain that forms an extended conformation on binding. ► The structure of a CtIP:LMO4 complex reveals similarity in LMO4–LDB1/CtIP binding. ► Excess LMO4 in cancer may sequester CtIP and may influence cell cycle control.

Publication date: 12 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 7

Author(s): Imad Shams , Assaf Malik , Irena Manov , Alma Joel , Mark Band , Aaron Avivi

The tumor suppressor gene p53 induces growth arrest and/or apoptosis in response to DNA damage/hypoxia. Inactivation of p53 confers a selective advantage to tumor cells under a hypoxic microenvironment during tumor progression. The subterranean blind mole rat, Spalax, spends its life underground at low-oxygen tensions, hence developing a wide range of respiratory/molecular adaptations to hypoxic stress, including critical changes in p53 structure and signaling pathway. The highly conserved p53 Arg(R)-172 is substituted by lysine (K) in Spalax, identical with a tumor-associated mutation. Functionality assays revealed that Spalax p53 is unable to activate apoptotic target genes but is still capable of activating cell cycle arrest genes. Furthermore, we have shown that the transcription patterns of representative p53-induced genes (Apaf1 and Mdm2) in Spalax are influenced by hypoxia. Cell cycle arrest allows the cells to repair DNA damage via different DNA repair genes. We tested the transcription pattern of three p53-related DNA repair genes (p53R2, Mlh1, and Msh2) under normoxia and short-acute hypoxia in Spalax, C57BL/6 wild-type mice, and two strains of mutant C57BL/6 mice, each carrying a different mutation at the R172 position. Our results show that while wild-type/mutant mice exhibit strong hypoxia-induced reductions of repair gene transcript levels, no such inhibition is found in Spalax under hypoxia. Moreover, unlike mouse p53R2, Spalax p53R2 transcript levels are strongly elevated under hypoxia. These results suggest that critical repair functions, which are known to be inhibited under hypoxia in mice, remain active in Spalax, as part of its unique hypoxia tolerance mechanisms.
Graphical abstract Highlights ► The subterranean Spalax is hypoxia tolerant and is resistant to cancer and vascular diseases. ► Spalax p53R172K substitution abrogates apoptosis and enhances cell cycle arrest activity. ► Expression of Spalax p53-induced apoptotic and homeostasis genes is hypoxia linked. ► Three p53-induced DNA repair genes were studied in Spalax and p53-mutated mice. ► DNA repair genes' expression is related to hypoxia and to p53 amino acid 172.

Publication date: 12 April 2013
Source:Journal of Molecular Biology, Volume 425, Issue 7

Author(s): Daegeun Kim , Jihye Park , Soo Jin Kim , Young-Min Soh , Ho Min Kim , Byung-Ha Oh , Ji-Joon Song

An outer membrane protein BP26/OMP28 of Brucella, BP26, is identified as a major immunodominant antigen and widely used as a diagnostic marker and for vaccination against Brucellosis. BP26 belongs to the family of proteins that contains a SIMPL (signaling molecule that associates with the mouse pelle-like kinase) domain, whose structure and function have been unknown. Here, we present the crystal structure of BP26 revealing that 16 BP26 molecules form a novel channel-like assembly as also shown by electron microscopy analysis. Eight BP26 molecules forming a ring structure contain a hole at the center of the octamer, and another octamer interacts with each other to form a channel having a large internal cavity. BP26 is found to be structurally similar to a bacteriophage protein involved in infection, implicating that BP26 might function during Brucella infection. In addition, the BP26 structure suggests that the protein functions as a multimeric channel-like form and provides a canonical model for the SIMPL domains.
Graphical abstract Highlights ► Crystal structure of an immunogenic protein BP26 from Brucella abortus. ► BP26 forms a homo-hexadecamer with an internal channel. ► The structure of BP26 represents the first structure of the SIMPL domains.