Journal of Molecular Biology

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  • Editorial Board
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3









    Categories: Journal Articles
  • Contents List
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3









    Categories: Journal Articles
  • Making Sense of Vps4
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): James H. Hurley , Bei Yang







    Categories: Journal Articles
  • Jamming Up the “β-Staple”: Regulation of SIRT1 Activity by Its C-Terminal Regulatory Segment (CTR)
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Ruth Anne Pumroy , Gino Cingolani







    Categories: Journal Articles
  • The Oligomeric State of the Active Vps4 AAA ATPase
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Nicole Monroe , Han Han , Malgorzata D. Gonciarz , Debra M. Eckert , Mary Anne Karren , Frank G. Whitby , Wesley I. Sundquist , Christopher P. Hill

    The cellular ESCRT (endosomal sorting complexes required for transport) pathway drives membrane constriction toward the cytosol and effects membrane fission during cytokinesis, endosomal sorting, and the release of many enveloped viruses, including the human immunodeficiency virus. A component of this pathway, the AAA ATPase Vps4, provides energy for pathway progression. Although it is established that Vps4 functions as an oligomer, subunit stoichiometry and other fundamental features of the functional enzyme are unclear. Here, we report that although some mutant Vps4 proteins form dodecameric assemblies, active wild-type Saccharomyces cerevisiae and Sulfolobus solfataricus Vps4 enzymes can form hexamers in the presence of ATP and ADP, as assayed by size-exclusion chromatography and equilibrium analytical ultracentrifugation. The Vta1p activator binds hexameric yeast Vps4p without changing the oligomeric state of Vps4p, implying that the active Vta1p–Vps4p complex also contains a single hexameric ring. Additionally, we report crystal structures of two different archaeal Vps4 homologs, whose structures and lattice interactions suggest a conserved mode of oligomerization. Disruption of the proposed hexamerization interface by mutagenesis abolished the ATPase activity of archaeal Vps4 proteins and blocked Vps4p function in S. cerevisiae. These data challenge the prevailing model that active Vps4 is a double-ring dodecamer, and argue that, like other type I AAA ATPases, Vps4 functions as a single ring with six subunits.
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    Categories: Journal Articles
  • Structural and Functional Analysis of Human SIRT1
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Andrew M. Davenport , Ferdinand M. Huber , André Hoelz

    SIRT1 is a NAD+-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a β hairpin structure that complements the β sheet of the NAD+-binding domain, covering an essentially invariant hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD+-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain.
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    Categories: Journal Articles
  • Intramolecular Donor Strand Complementation in the E. coli Type 1 Pilus Subunit FimA Explains the Existence of FimA Monomers As Off-Pathway Products of Pilus Assembly That Inhibit Host Cell Apoptosis
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Michal J. Walczak , Chasper Puorger , Rudi Glockshuber , Gerhard Wider

    Type 1 pili are filamentous organelles mediating the attachment of uropathogenic Escherichia coli to epithelial cells of host organisms. The helical pilus rod consists of up to 3000 copies of the main structural subunit FimA that interact via donor strand complementation, where the incomplete Ig-like fold of FimA is completed by insertion of the N-terminal extension (donor strand) of the following FimA subunit. Recently, it was shown that FimA also exists in a monomeric, assembly-incompetent form and that FimA monomers act as inhibitors of apoptosis in infected host cells. Here we present the NMR structure of monomeric wild-type FimA with its natural N-terminal donor strand complementing the Ig fold. Compared to FimA subunits in the assembled pilus, intramolecular self-complementation in the monomer stabilizes the FimA fold with significantly less interactions, and the natural FimA donor strand is inserted in the opposite orientation. In addition, we show that a motif of two glycine residues in the FimA donor strand, separated by five residues, is the prerequisite of the alternative, parallel donor strand insertion mechanism in the FimA monomer and that this motif is preserved in FimA homologs of many enteroinvasive pathogens. We conclude that FimA is a unique case of a protein with alternative, functionally relevant folding possibilities, with the FimA polymer forming the highly stable pilus rod and the FimA monomer promoting pathogen propagation by apoptosis suppression of infected epithelial target cells.
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    Categories: Journal Articles
  • Protein-Mediated Chromosome Pairing of Repetitive Arrays
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Ekaterina V. Mirkin , Frederick S. Chang , Nancy Kleckner

    Chromosomally integrated arrays of lacO and tetO operator sites visualized by LacI and TetR repressor proteins fused with GFP (green fluorescent protein) (or other fluorescent proteins) are widely used to monitor the behavior of chromosomal loci in various systems. However, insertion of such arrays and expression of the corresponding proteins is known to perturb genomic architecture. In several cases, juxtaposition of such arrays located on different chromosomes has been inferred to reflect pairing of the corresponding loci. Here, we report that a version of TetR-GFP mutated to disrupt GFP dimerization (TetR-A206KGFP or “TetR-kGFP”) abolishes pairing of tetO arrays in vivo and brings spatial proximity of chromosomal loci marked with those arrays back to the wild-type level. These data argue that pairing of arrays is caused by GFP dimerization and thus presents an example of protein-assisted interaction in chromosomes. Arrays marked with another protein, TetR-tdTomato, which has a propensity to form intramolecular dimers instead of intermolecular dimers, also display reduced level of pairing, supporting this idea. TetR-kGFP provides an improved system for studying chromosomal loci with a low pairing background.
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    Categories: Journal Articles
  • Insights into Eukaryotic Primer Synthesis from Structures of the p48 Subunit of Human DNA Primase
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Sivaraja Vaithiyalingam , Diana R. Arnett , Amit Aggarwal , Brandt F. Eichman , Ellen Fanning , Walter J. Chazin

    DNA replication in all organisms requires polymerases to synthesize copies of the genome. DNA polymerases are unable to function on a bare template and require a primer. Primases are crucial RNA polymerases that perform the initial de novo synthesis, generating the first 8–10 nucleotides of the primer. Although structures of archaeal and bacterial primases have provided insights into general priming mechanisms, these proteins are not well conserved with heterodimeric (p48/p58) primases in eukaryotes. Here, we present X-ray crystal structures of the catalytic engine of a eukaryotic primase, which is contained in the p48 subunit. The structures of p48 reveal that eukaryotic primases maintain the conserved catalytic prim fold domain, but with a unique subdomain not found in the archaeal and bacterial primases. Calorimetry experiments reveal that Mn2+ but not Mg2+ significantly enhances the binding of nucleotide to primase, which correlates with higher catalytic efficiency in vitro. The structure of p48 with bound UTP and Mn2+ provides insights into the mechanism of nucleotide synthesis by primase. Substitution of conserved residues involved in either metal or nucleotide binding alter nucleotide binding affinities, and yeast strains containing the corresponding Pri1p substitutions are not viable. Our results reveal that two residues (S160 and H166) in direct contact with the nucleotide were previously unrecognized as critical to the human primase active site. Comparing p48 structures to those of similar polymerases in different states of action suggests changes that would be required to attain a catalytically competent conformation capable of initiating dinucleotide synthesis.
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    Categories: Journal Articles
  • Interaction of HoloCcmE with CcmF in Heme Trafficking and Cytochrome c Biosynthesis
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Brian San Francisco , Robert G. Kranz

    The periplasmic heme chaperone holoCcmE is essential for heme trafficking in the cytochrome c biosynthetic pathway in many bacteria, archaea, and plant mitochondria. This pathway, called system I, involves two steps: (i) formation and release of holoCcmE (by the ABC-transporter complex CcmABCD) and (ii) delivery of the heme in holoCcmE to the putative cytochrome c heme lyase complex, CcmFH. CcmFH is believed to facilitate the final covalent attachment of heme (from holoCcmE) to the apocytochrome c. Although most models for system I propose that holoCcmE delivers heme directly to CcmF, no interaction between holoCcmE and CcmF has been demonstrated. Here, a complex between holoCcmE and CcmF is “trapped”, purified, and characterized. HoloCcmE must be released from the ABC-transporter complex CcmABCD to interact with CcmF, and the holo-form of CcmE interacts with CcmF at levels at least 20-fold higher than apoCcmE. Two conserved histidines (here termed P-His1 and P-His2) in separate periplasmic loops in CcmF are required for interaction with holoCcmE, and evidence that P-His1 and P-His2 function as heme-binding ligands is presented. These results show that heme in holoCcmE is essential for complex formation with CcmF and that the heme of holoCcmE is coordinated by P-His1 and P-His2 within the WWD domain of CcmF. These features are strikingly similar to formation of the CcmC:heme:CcmE ternary complex [Richard-Fogal C, Kranz RG. The CcmC:heme:CcmE complex in heme trafficking and cytochrome c biosynthesis. J Mol Biol 2010;401:350–62] and suggest common mechanistic and structural aspects.
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    Categories: Journal Articles
  • Molecular Dynamics Simulations and Structure-Guided Mutagenesis Provide Insight into the Architecture of the Catalytic Core of the Ectoine Hydroxylase
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Nils Widderich , Marco Pittelkow , Astrid Höppner , Daniel Mulnaes , Wolfgang Buckel , Holger Gohlke , Sander H.J. Smits , Erhard Bremer

    Many bacteria amass compatible solutes to fend-off the detrimental effects of high osmolarity on cellular physiology and water content. These solutes also function as stabilizers of macromolecules, a property for which they are referred to as chemical chaperones. The tetrahydropyrimidine ectoine is such a compatible solute and is widely synthesized by members of the Bacteria. Many ectoine producers also synthesize the stress protectant 5-hydroxyectoine from the precursor ectoine, a process that is catalyzed by the ectoine hydroxylase (EctD). The EctD enzyme is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. A crystal structure of the EctD protein from the moderate halophile Virgibacillus salexigens has previously been reported and revealed the coordination of the iron catalyst, but it lacked the substrate ectoine and the co-substrate 2-oxoglutarate. Here we used this crystal structure as a template to assess the likely positioning of the ectoine and 2-oxoglutarate ligands within the active site by structural comparison, molecular dynamics simulations, and site-directed mutagenesis. Collectively, these approaches suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine. Our study thus provides a view into the catalytic core of the ectoine hydroxylase and suggests an intricate network of interactions between the three ligands and evolutionarily highly conserved residues in members of the EctD protein family.
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    Categories: Journal Articles
  • Light-Induced Differences in Conformational Dynamics of the Circadian Clock Regulator VIVID
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Chung-Tien Lee , Erik Malzahn , Michael Brunner , Matthias P. Mayer

    The LOV (light–oxygen–voltage) domain protein VIVID (VVD) is a negative regulator of the circadian transcription factor White Collar Complex and controls light response and photoadaptation in Neurospora. Blue light converts VIVID from the dark state into the light state (VVDL) with concomitant homodimerization. Upon return to low-light conditions, VVD very slowly reverts back into the monomeric dark state (VVDD). To better understand the nature of the conformational changes that are the basis for the light–dark switch in VVD, we used hydrogen exchange mass spectrometry to probe solvent accessibility of backbone amide protons. Our data demonstrate that all structural elements of VVDD except for the N-cap region exchange according to the rare EX1 mechanism indicating a reversible unfolding with rather slow refolding rate. Interestingly, the unfolding halftimes of different elements were not identical but varied from 400 to 900s. VVDL also exchanges according to the EX1 mechanism, albeit with a halftime of 6h. Surprisingly, the dimerization interface showed very little protection suggesting a rapid dimer–monomer interconversion.
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    Categories: Journal Articles
  • DRoP: A Water Analysis Program Identifies Ras-GTP-Specific Pathway of Communication between Membrane-Interacting Regions and the Active Site
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Bradley M. Kearney , Christian W. Johnson , Daniel M. Roberts , Paul Swartz , Carla Mattos

    Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras.
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    Categories: Journal Articles
  • Structural Determinants of Unique Properties of Human IgG4-Fc
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Anna M. Davies , Theo Rispens , Pleuni Ooijevaar-de Heer , Hannah J. Gould , Roy Jefferis , Rob C. Aalberse , Brian J. Sutton

    Human IgG4, normally the least abundant of the four subclasses of IgG in serum, displays a number of unique biological properties. It can undergo heavy-chain exchange, also known as Fab-arm exchange, leading to the formation of monovalent but bispecific antibodies, and it interacts poorly with FcγRII and FcγRIII, and complement. These properties render IgG4 relatively “non-inflammatory” and have made it a suitable format for therapeutic monoclonal antibody production. However, IgG4 is also known to undergo Fc-mediated aggregation and has been implicated in auto-immune disease pathology. We report here the high-resolution crystal structures, at 1.9 and 2.35Å, respectively, of human recombinant and serum-derived IgG4-Fc. These structures reveal conformational variability at the CH3–CH3 interface that may promote Fab-arm exchange, and a unique conformation for the FG loop in the CH2 domain that would explain the poor FcγRII, FcγRIII and C1q binding properties of IgG4 compared with IgG1 and -3. In contrast to other IgG subclasses, this unique conformation folds the FG loop away from the CH2 domain, precluding any interaction with the lower hinge region, which may further facilitate Fab-arm exchange by destabilisation of the hinge. The crystals of IgG4-Fc also display Fc–Fc packing contacts with very extensive interaction surfaces, involving both a consensus binding site in IgG-Fc at the CH2–CH3 interface and known hydrophobic aggregation motifs. These Fc–Fc interactions are compatible with intact IgG4 molecules and may provide a model for the formation of aggregates of IgG4 that can cause disease pathology in the absence of antigen.
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    Categories: Journal Articles
  • Structural Complementation of the Catalytic Domain of Pseudomonas Exotoxin A
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Erin L. Boland , Crystal M. Van Dyken , Rachel M. Duckett , Andrew J. McCluskey , Gregory M.K. Poon

    The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2. PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and protein translation assays demonstrate that the resulting fusions—supplied exogenously as genetic elements or purified protein fragments—had no significant catalytic activity or effect on protein synthesis individually but, in combination, catalyzed the ADP-ribosylation of eukaryotic elongation factor 2 and inhibited protein synthesis. Although complementing PE3 fragments are catalytically less efficient than intact PE3 in cell-free systems, co-expression in live cells transfected with transgenes encoding the toxin fusions inhibits protein synthesis and causes cell death comparably as intact PE3. Complementation of split PE3 offers a direct extension of the immunotoxin approach to generate bispecific agents that may be useful to target complex phenotypes.
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    Categories: Journal Articles
  • Destabilization of the Homotetrameric Assembly of 3-Deoxy-d-Arabino-Heptulosonate-7-Phosphate Synthase from the Hyperthermophile Pyrococcus furiosus Enhances Enzymatic Activity
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Ali Reza Nazmi , Linley R. Schofield , Renwick C.J. Dobson , Geoffrey B. Jameson , Emily J. Parker

    Many proteins adopt homomeric quaternary structures to support their biological function, including the first enzyme of the shikimate pathway that is ultimately responsible for the biosynthesis of the aromatic amino acids in plants and microorganisms. This enzyme, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS), adopts a variety of different quaternary structures depending on the organism in which it is found. The DAH7PS from the hyperthermophilic archaebacterium Pyrococcus furiosus was previously shown to be tetrameric in its crystalline form, and this quaternary association is confirmed in an improved structure in a different crystal system. This tetramer is also present in solution as revealed by small-angle X-ray scattering and analytical ultracentrifugation. This homotetrameric form has two distinct interfaces, both of which bury over 10% each of the surface area of a single monomer. Substitution of Ile for Asp in the hydrophobic region of one interface gives a protein with a remarkable 4-fold higher maximum catalytic rate than the wild-type enzyme. Analytical ultracentrifugation at pH7.5 reveals that the tetrameric form is destabilized; although the protein crystallizes as a tetramer, equilibrium exists between tetrameric and dimeric forms with a dissociation constant of 22μM. Thus, under the conditions of kinetic assay, the enzyme is primarily dimeric, revealing that the dimeric form is a fully functional catalyst. However, in comparison to the wild-type protein, the thermal stability of the dimeric protein is significantly compromised. Thus, an unusual compromise of enzymatic activity versus stability is observed for this DAH7PS from an organism that favors a hyperthermophilic environment.
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    Categories: Journal Articles
  • The Structure of TAX1BP1 UBZ1+2 Provides Insight into Target Specificity and Adaptability
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): M. Angeles Ceregido , Mercedes Spínola Amilibia , Lieven Buts , José Rivera-Torres , Abel Garcia-Pino , Jerónimo Bravo , Nico A.J. van Nuland

    TAX1BP1 is a novel ubiquitin-binding adaptor protein involved in the negative regulation of the NF-kappaB transcription factor, which is a key player in inflammatory responses, immunity and tumorigenesis. TAX1BP1 recruits A20 to the ubiquitinated signaling proteins TRAF6 and RIP1, leading to their A20-mediated deubiquitination and the disruption of IL-1-induced and TNF-induced NF-kappaB signaling, respectively. The two zinc fingers localized at its C-terminus function as novel ubiquitin-binding domains (UBZ, ubiquitin-binding zinc finger). Here we present for the first time both the solution and crystal structures of two classical UBZ domains in tandem within the human TAX1BP1. The relative orientation of the two domains is slightly different in the X-ray structure with respect to the NMR structure, indicating some degree of conformational flexibility, which is rationalized by NMR relaxation data. The observed degree of flexibility and stability between the two UBZ domains might have consequences on the recognition mechanism of interacting partners.
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    Categories: Journal Articles
  • From DARPins to LoopDARPins: Novel LoopDARPin Design Allows the Selection of Low Picomolar Binders in a Single Round of Ribosome Display
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Johannes Schilling , Jendrik Schöppe , Andreas Plückthun

    Antibodies are the most versatile binding proteins in nature with six loops creating a flexible continuous interaction surface. However, in some molecular formats, antibodies are aggregation prone. Designed ankyrin repeat proteins (DARPins) were successfully created as alternative design solutions. Nevertheless, their concave shape, rigidity and incompletely randomized binding surface may limit the epitopes that can be targeted by this extremely stable scaffold. Combining conformational diversity and a continuous convex paratope found in many antibodies with the beneficial biophysical properties of DARPins, we created LoopDARPins, a next generation of DARPins with extended epitope binding properties. We employed X-ray structure determination of a LoopDARPin for design validation. Biophysical characterizations show that the introduction of an elongated loop through consensus design does not decrease the stability of the scaffold, consistent with molecular dynamics simulations. Ribosome-display selections against extracellular signal-regulated kinase 2 (ERK2) and four members of the BCL-2 family (BCL-2, BCL-XL, BCL-W and MCL-1) of anti-apoptotic regulators yielded LoopDARPins with affinities in the mid-picomolar to low nanomolar range against all targets. The BCL-2 family binders block the interaction with their natural interaction partner and will be valuable reagents to test the apoptotic response in functional assays. With the LoopDARPin scaffold, binders for BCL-2 with an affinity of 30pM were isolated with only a single round of ribosome display, an enrichment that has not been described for any scaffold. Identical stringent one-round selections with conventional DARPins without loop yielded no binders. The LoopDARPin scaffold may become a highly valuable tool for biotechnological high-throughput applications.
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    Categories: Journal Articles
  • Exploring the Minimally Frustrated Energy Landscape of Unfolded ACBP
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Valéry Ozenne , Jeffrey K. Noel , Pétur O. Heidarsson , Søren Brander , Flemming M. Poulsen , Malene Ringkjøbing Jensen , Birthe B. Kragelund , Martin Blackledge , Jens Danielsson

    The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape.
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    Categories: Journal Articles
  • Flexible Gates Generate Occluded Intermediates in the Transport Cycle of LacY
    [Jan 2014]

    Publication date: 6 February 2014
    Source:Journal of Molecular Biology, Volume 426, Issue 3

    Author(s): Lukas S. Stelzl , Philip W. Fowler , Mark S.P. Sansom , Oliver Beckstein

    The major facilitator superfamily (MFS) transporter lactose permease (LacY) alternates between cytoplasmic and periplasmic open conformations to co-transport a sugar molecule together with a proton across the plasma membrane. Indirect experimental evidence suggested the existence of an occluded transition intermediate of LacY, which would prevent leaking of the proton gradient. As no experimental structure is known, the conformational transition is not fully understood in atomic detail. We simulated transition events from a cytoplasmic open conformation to a periplasmic open conformation with the dynamic importance sampling molecular dynamics method and observed occluded intermediates. Analysis of water permeation pathways and the electrostatic free-energy landscape of a solvated proton indicated that the occluded state contains a solvated central cavity inaccessible from either side of the membrane. We propose a pair of geometric order parameters that capture the state of the pathway through the MFS transporters as shown by a survey of available crystal structures and models. We present a model for the occluded state of apo-LacY, which is similar to the occluded crystal structures of the MFS transporters EmrD, PepTSo, NarU, PiPT and XylE. Our simulations are consistent with experimental double electron spin–spin distance measurements that have been interpreted to show occluded conformations. During the simulations, a salt bridge that has been postulated to be involved in driving the conformational transition formed. Our results argue against a simple rigid-body domain motion as implied by a strict “rocker-switch mechanism” and instead hint at an intricate coupling between two flexible gates.
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    Categories: Journal Articles