| 1. |
- Crennell, SJ, et al.
(author)
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Dimerisation and an increase in active site aromatic groups as adaptations to high temperatures: X-ray solution scattering and substrate-bound crystal structures of Rhodothermus marinus endoglucanase Cel12A
- 2006
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In: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 356:1, s. 57-71
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Journal article (peer-reviewed)abstract
- Cellulose, a polysaccharide consisting of beta-1,4-linked glucose, is the major component of plant cell walls and consequently one of the most abundant biopolymers on earth. Carbohydrate polymers such as cellulose are molecules with vast diversity in structure and function, and a multiplicity of hydrolases operating in concert are required for depolymerisation. The bacterium Rhodothermus marinus, isolated from shallow water marine hot springs, produces a number of carbohydrate-degrading enzymes including a family 12 cellulase Cel12A. The structure of R. marinus Cel12A in the ligand-free form (at 1.54 angstrom) and structures of RmCel12A after crystals were soaked in cellopentaose for two different lengths of time, have been determined. The shorter soaked complex revealed the conformation of unhydrolysed cellotetraose, while cellopentaose had been degraded more completely during the longer soak. Comparison of these structures with those of mesophilic family 12 cellulases in complex with inhibitors and substrate revealed that RmCel12A has a more extensive aromatic network in the active site cleft which ejects products after hydrolysis. The substrate structure confirms that during hydrolysis by family 12 cellulases glucose does not pass through a 2,5 B conformation. Small-angle X-ray scattering analysis of RmCel12A showed that the enzyme forms a loosely associated antiparallel dimer in solution, which may target the enzyme to the antiparallel polymer strands in cellulose. (c) 2005 Elsevier Ltd. All rights reserved.
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| 2. |
- Holdbrook, D. A., et al.
(author)
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A Spring-Loaded Mechanism Governs the Clamp-like Dynamics of the Skp Chaperone
- 2017
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In: Structure. - : Elsevier BV. - 0969-2126. ; 25:7
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Journal article (peer-reviewed)abstract
- The trimeric periplasmic holdase chaperone Skp binds and stabilizes unfolded outer membrane proteins (OMPs) as part of bacterial OMP biogenesis. Skp binds client proteins in its central cavity, thereby reducing its backbone dynamics, but the molecular mechanisms that govern Skp dynamics and adaptation to differently sized clients remains unknown. Here, we employ a combination of microsecond time-scale molecular dynamics simulation, small-angle X-ray scattering, and nuclear magnetic resonance spectroscopy to reveal that Skp is remarkably flexible, and features a molecular spring-loaded mechanism in its "tentacle'' arms that enables switching between two distinct conformations on sub-millisecond timescales. The conformational switch is executed around a conserved pivot element within the coiled-coil structures of the tentacles, allowing expansion of the cavity and thus accommodation of differently sized clients. The spring-loaded mechanism shows how a chaperone can efficiently modulate its structure and function in an ATP-independent manner.
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| 3. |
- Lazar, Tamas, et al.
(author)
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PED in 2021 : A major update of the protein ensemble database for intrinsically disordered proteins
- 2021
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 49:D1, s. 404-411
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Journal article (peer-reviewed)abstract
- The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs.
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| 4. |
- Macpherson, Alex, et al.
(author)
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The allosteric modulation of complement C5 by knob domain peptides
- 2021
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In: eLIFE. - : ELife Sciences Publications Ltd. - 2050-084X. ; 10
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Journal article (peer-reviewed)abstract
- Bovines have evolved a subset of antibodies with ultra-long heavy chain complementarity determining regions that harbour cysteine-rich knob domains. To produce high-affinity peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 angstrom from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors, and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.
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| 5. |
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| 6. |
- Valencia-Sanchez, M. I., et al.
(author)
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Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases
- 2016
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 291:28, s. 14430-14446
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Journal article (peer-reviewed)abstract
- Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric ((2)) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form ((22)) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the -subunit of the (22) GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the -subunit in the complex with an analog of glycyl adenylate at 2.8 angstrom resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of (22) GlyRS, convergent with (2) GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor.
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| 7. |
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| 8. |
- Josts, Inokentijs, et al.
(author)
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Conformational States of ABC Transporter MsbA in a Lipid Environment Investigated by Small-Angle Scattering Using Stealth Carrier Nanodiscs
- 2018
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In: Structure. - : Elsevier. - 0969-2126 .- 1878-4186. ; 26:8, s. 1072-1079.e4
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Journal article (peer-reviewed)abstract
- Structural studies of integral membrane proteins (IMPs) are challenging, as many of them are inactive or insoluble in the absence of a lipid environment. Here, we describe an approach making use of fractionally deuterium labeled "stealth carrier'' nanodiscs that are effectively invisible to low-resolution neutron diffraction and enable structural studies of IMPs in a lipidic native-like solution environment. We illustrate the potential of the method in a joint small-angle neutron scattering (SANS) and X-ray scattering (SAXS) study of the ATP-binding cassette (ABC) transporter protein MsbA solubilized in the stealth nanodiscs. The data allow for a direct observation of the signal from the solubilized protein without contribution from the surrounding lipid nanodisc. Not only the overall shape but also differences between conformational states of MsbA can be reliably detected from the scattering data, demonstrating the sensitivity of the approach and its general applicability to structural studies of IMPs.
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| 9. |
- Liu, Goksin, et al.
(author)
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Conformational multiplicity of bacterial ferric binding protein revealed by small angle x-ray scattering and molecular dynamics calculations
- 2023
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In: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 158:8
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Journal article (peer-reviewed)abstract
- This study combines molecular dynamics (MD) simulations with small angle x-ray scattering (SAXS) measurements to investigate the range of conformations that can be adopted by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations in solution. To explore how the conformational distribution of proteins may be modified in the environmental niches of biological media, we focus on the periplasmic ferric binding protein A (FbpA) from Haemophilus influenzae involved in the mechanism by which bacteria capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment. While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms, detection of conformational changes due to the point mutation D52A and changes in ionic strength (IS) from SAXS scattering profiles have been challenging. Here, to reach conclusions, statistical analyses with SAXS profiles and results from different techniques were combined in a complementary fashion. The SAXS data complemented by size exclusion chromatography point to multiple and/or alternative conformations at physiological IS, whereas they are well-explained by single crystallographic structures in low IS buffers. By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates. We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type.
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| 10. |
- Nitsche, Julius, et al.
(author)
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Structural basis for activation of plasma-membrane Ca(2+)-ATPase by calmodulin.
- 2018
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In: Communications Biology. - : Nature Publishing Group. - 2399-3642. ; 1
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Journal article (peer-reviewed)abstract
- Plasma-membrane Ca(2+)-ATPases expel Ca(2+) from the cytoplasm and are key regulators of Ca(2+) homeostasis in eukaryotes. They are autoinhibited under low Ca(2+) concentrations. Calmodulin (CaM)-binding to a unique regulatory domain releases the autoinhibition and activates the pump. However, the structural basis for this activation, including the overall structure of this calcium pump and its complex with calmodulin, is unknown. We previously determined the high-resolution structure of calmodulin in complex with the regulatory domain of the plasma-membrane Ca(2+)-ATPase ACA8 and revealed a bimodular mechanism of calcium control in eukaryotes. Here we show that activation of ACA8 by CaM involves large conformational changes. Combining advanced modeling of neutron scattering data acquired from stealth nanodiscs and native mass spectrometry with detailed dissection of binding constants, we present a structural model for the full-length ACA8 Ca(2+) pump in its calmodulin-activated state illustrating a displacement of the regulatory domain from the core enzyme.
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