| 1. |
- A. Strumpfer, Johan, et al.
(författare)
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Stretching of Twitchin Kinase
- 2012
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Ingår i: Biophysical Journal. - St. Louis, MO, United States : Cell Press. - 0006-3495 .- 1542-0086. ; 102:3 Supplement 1, s. 361a-362a
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Tidskriftsartikel (refereegranskat)abstract
- The giant proteins from the titin family, that form cytoskeletal filaments, have emerged as key mechanotransducers in the sarcomere. These proteins contain a conserved kinase region, which is auto-inhibited by a C-terminal tail domain. The inhibitory tail domain occludes the active sites of the kinases, thus preventing ATP from binding. It was proposed that through application of a force, such as that arising during muscle contraction, the inhibitory tail becomes detached, lifting inhibition. The force-sensing ability of titin kinase was demonstrated in AFM experiments and simulations [Puchner, et al., 2008, PNAS:105, 13385], which showed indeed that mechanical forces can remove the autoinhibitory tail of titin kinase. We report here steered molecular dynamics simulations (SMD) of the very recently resolved crystal structure of twitchin kinase, containing the kinase region and flanking fibronectin and immuniglobulin domains, that show a variant mechanism. Despite the significant structural and sequence similarity to titin kinase, the autoinhibitory tail of twitchin kinase remains in place upon stretching, while the N-terminal lobe of the kinase unfolds. The SMD simulations also show that the detachment and stretching of the linker between fibronectin and kinase regions, and the partial extension of the autoinhibitory tail, are the primary force-response. We postulate that this stretched state, where all structural elements are still intact, may represent the physiologically active state.
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| 2. |
- Aronsson, Anna, et al.
(författare)
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Structural insights of RmXyn10A – A prebiotic-producing GH10 xylanase with a non-conserved aglycone binding region
- 2018
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639. ; 1866:2, s. 292-306
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Tidskriftsartikel (refereegranskat)abstract
- Hydrolysis of arabinoxylan (AX) by glycoside hydrolase family 10 (GH10) xylanases produces xylo- and arabinoxylo-oligosaccharides ((A)XOS) which have shown prebiotic effects. The thermostable GH10 xylanase RmXyn10A has shown great potential to produce (A)XOS. In this study, the structure of RmXyn10A was investigated, the catalytic module by homology modelling and site-directed mutagenesis and the arrangement of its five domains by small-angle X-ray scattering (SAXS). Substrate specificity was explored in silico by manual docking and molecular dynamic simulations. It has been shown in the literature that the glycone subsites of GH10 xylanases are well conserved and our results suggest that RmXyn10A is no exception. The aglycone subsites are less investigated, and the modelled structure of RmXyn10A suggests that loop β6α6 in the aglycone part of the active site contains a non-conserved α-helix, which blocks the otherwise conserved space of subsite +2. This structural feature has only been observed for one other GH10 xylanase. In RmXyn10A, docking revealed two alternative binding regions, one on either side of the α-helix. However, only one was able to accommodate arabinose-substitutions and the mutation study suggests that the same region is responsible for binding XOS. Several non-conserved structural features are most likely to be responsible for providing affinity for arabinose-substitutions in subsites +1 and +2. The SAXS rigid model of the modular arrangement of RmXyn10A displays the catalytic module close to the cell-anchoring domain while the carbohydrate binding modules are further away, likely explaining the observed lack of contribution of the CBMs to activity.
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| 3. |
- Bernado, Pau, et al.
(författare)
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Structure and Dynamics of Ribosomal Protein L12: An Ensemble Model Based on SAXS and NMR Relaxation
- 2010
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 98:10, s. 2374-2382
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomal protein L12 is a two-domain protein that forms dimers mediated by its N-terminal domains. A 20-residue linker separates the N- and C-terminal domains. This linker results in a three-lobe topology with significant flexibility, known to be critical for efficient translation. Here we present an ensemble model of spatial distributions and correlation times for the domain reorientations of L12 that reconciles experimental data from small-angle x-ray scattering and nuclear magnetic resonance. We generated an ensemble of L12 conformations in which the structure of each domain is fixed but the domain orientations are variable. The ensemble reproduces the small-angle x-ray scattering data and the optimized correlation times of its reorientational eigenmodes fit the N-15 relaxation data. The ensemble model reveals intrinsic conformational properties of L12 that help explain its function on the ribosome. The two C-terminal domains sample a large volume and extend further away from the ribosome anchor than expected for a random-chain linker, indicating that the flexible linker has residual order. Furthermore, the distances between each C-terminal domain and the anchor are anticorrelated, indicating that one of them is more retracted on average. We speculate that these properties promote the function of L12 to recruit translation factors and control their activity on the ribosome.
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| 4. |
- Duelli, Annette, 1980, et al.
(författare)
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The C-Terminal Random Coil Region Tunes the Ca2+-Binding Affinity of S100A4 through Conformational Activation.
- 2014
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:5
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Tidskriftsartikel (refereegranskat)abstract
- S100A4 interacts with many binding partners upon Ca2+ activation and is strongly associated with increased metastasis formation. In order to understand the role of the C-terminal random coil for the protein function we examined how small angle X-ray scattering of the wild-type S100A4 and its C-terminal deletion mutant (residues 1-88, Δ13) changes upon Ca2+ binding. We found that the scattering intensity of wild-type S100A4 changes substantially in the 0.15-0.25 Å-1 q-range whereas a similar change is not visible in the C-terminus deleted mutant. Ensemble optimization SAXS modeling indicates that the entire C-terminus is extended when Ca2+ is bound. Pulsed field gradient NMR measurements provide further support as the hydrodynamic radius in the wild-type protein increases upon Ca2+ binding while the radius of Δ13 mutant does not change. Molecular dynamics simulations provide a rational explanation of the structural transition: the positively charged C-terminal residues associate with the negatively charged residues of the Ca2+-free EF-hands and these interactions loosen up considerably upon Ca2+-binding. As a consequence the Δ13 mutant has increased Ca2+ affinity and is constantly loaded at Ca2+ concentration ranges typically present in cells. The activation of the entire C-terminal random coil may play a role in mediating interaction with selected partner proteins of S100A4.
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| 5. |
- Grela, Przemysłw, et al.
(författare)
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Structural characterization of the ribosomal P1A-P2B protein dimer by small-angle X-ray scattering and NMR spectroscopy
- 2007
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:7, s. 1988-1998
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Tidskriftsartikel (refereegranskat)abstract
- The five ribosomal P-proteins, denoted P0-(P1-P2)(2), constitute the stalk structure of the large subunit of eukaryotic ribosomes. In the yeast Saccharomyces cerevisiae, the group of P1 and P2 proteins is differentiated into subgroups that form two separate P1A-P2B and P1B-P2A heterodimers on the stalk. So far, structural studies on the P-proteins have not yielded any satisfactory information using either X-ray crystallography or NMR spectroscopy, and the structures of the ribosomal stalk and its individual constituents remain obscure. Here we outline a first, coarse-grained view of the P1A-P2B solution structure obtained by a combination of small-angle X-ray scattering and heteronuclear NMR spectroscopy. The complex has an elongated shape with a length of 10 nm and a cross section of similar to 2.5 nm. N-15 NMR relaxation measurements establish that roughly 30% of the residues are present in highly flexible segments, which belong primarily to the linker region and the C-terminal part of the polypeptide chain. Secondary structure predictions and NMR chemical shift analysis, together with previous results from CD spectroscopy, indicate that the structured regions involve alpha-helices. NMR relaxation data further suggest that several helices are arranged in a nearly parallel or antiparallel topology. These results provide the first structural comparison between eukaryotic P1 and P2 proteins and the prokaryotic L12 counterpart, revealing considerable differences in their overall shapes, despite similar functional roles and similar oligomeric arrangements. These results present for the first time a view of the structure of the eukaryotic stalk constituents, which is the only domain of the eukaryotic ribosome that has escaped successful structural characterization.
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| 6. |
- Gupta, Arun A., et al.
(författare)
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Formation of a Secretion-Competent Protein Complex by a Dynamic Wrap-around Binding Mechanism
- 2018
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 430:18, Part B, s. 3157-3169
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial virulence is typically initiated by translocation of effector or toxic proteins across host cell membranes. A class of gram-negative pathogenic bacteria including Yersinia pseudotuberculosis and Yersinia pestis accomplishes this objective with a protein assembly called the type III secretion system. Yersinia effector proteins (Yop) are presented to the translocation apparatus through formation of specific complexes with their cognate chaperones (Syc). In the complexes where the structure is available, the Yops are extended and wrap around their cognate chaperone. This structural architecture enables secretion of the Yop from the bacterium in early stages of translocation. It has been shown previously that the chaperone-binding domain of YopE is disordered in its isolation but becomes substantially more ordered in its wrap-around complex with its chaperone SycE. Here, by means of NMR spectroscopy, small-angle X-ray scattering and molecular modeling, we demonstrate that while the free chaperone-binding domain of YopH (YopHCBD) adopts a fully ordered and globular fold, it populates an elongated, wrap-around conformation when it engages in a specific complex with its chaperone SycH2. Hence, in contrast to YopE that is unstructured in its free state, YopH transits from a globular free state to an elongated chaperone-bound state. We demonstrate that a sparsely populated YopHCBD state has an elevated affinity for SycH2 and represents an intermediate in the formation of the protein complex. Our results suggest that Yersinia has evolved a binding mechanism where SycH2 passively stimulates an elongated YopH conformation that is presented to the type III secretion system in a secretion-competent conformation.
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| 7. |
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| 8. |
- Josts, Inokentijs, et al.
(författare)
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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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Ingår i: Structure. - : Elsevier. - 0969-2126 .- 1878-4186. ; 26:8, s. 1072-1079.e4
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Tidskriftsartikel (refereegranskat)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. |
- Lazar, Tamas, et al.
(författare)
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PED in 2021 : A major update of the protein ensemble database for intrinsically disordered proteins
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 49:D1, s. 404-411
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Tidskriftsartikel (refereegranskat)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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| 10. |
- Liu, Goksin, et al.
(författare)
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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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Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 158:8
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Tidskriftsartikel (refereegranskat)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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