| 1. |
- Aronsson, Anna, et al.
(författare)
-
Structural insights of RmXyn10A – A prebiotic-producing GH10 xylanase with a non-conserved aglycone binding region
- 2018
-
Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639. ; 1866:2, s. 292-306
-
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.
|
|
| 2. |
- Bernado, Pau, et al.
(författare)
-
Structure and Dynamics of Ribosomal Protein L12: An Ensemble Model Based on SAXS and NMR Relaxation
- 2010
-
Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 98:10, s. 2374-2382
-
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.
|
|
| 3. |
- Duelli, Annette, 1980, et al.
(författare)
-
The C-Terminal Random Coil Region Tunes the Ca2+-Binding Affinity of S100A4 through Conformational Activation.
- 2014
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:5
-
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.
|
|
| 4. |
- Gupta, Arun A., et al.
(författare)
-
Formation of a Secretion-Competent Protein Complex by a Dynamic Wrap-around Binding Mechanism
- 2018
-
Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 430:18, Part B, s. 3157-3169
-
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.
|
|
| 5. |
|
|
| 6. |
- Josts, Inokentijs, et al.
(författare)
-
Conformational States of ABC Transporter MsbA in a Lipid Environment Investigated by Small-Angle Scattering Using Stealth Carrier Nanodiscs
- 2018
-
Ingår i: Structure. - : Elsevier. - 0969-2126 .- 1878-4186. ; 26:8, s. 1072-1079.e4
-
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.
|
|
| 7. |
- Nitsche, Julius, et al.
(författare)
-
Structural basis for activation of plasma-membrane Ca(2+)-ATPase by calmodulin.
- 2018
-
Ingår i: Communications Biology. - : Nature Publishing Group. - 2399-3642. ; 1
-
Tidskriftsartikel (refereegranskat)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.
|
|
| 8. |
- Söderberg, Christopher, et al.
(författare)
-
Oligomerization Propensity and Flexibility of Yeast Frataxin Studied by X-ray Crystallography and Small-Angle X-ray Scattering.
- 2011
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 414:5, s. 783-797
-
Tidskriftsartikel (refereegranskat)abstract
- Frataxin is a mitochondrial protein with a central role in iron homeostasis. Defects in frataxin function lead to Friedreich's ataxia, a progressive neurodegenerative disease with childhood onset. The function of frataxin has been shown to be closely associated with its ability to form oligomeric species; however, the factors controlling oligomerization and the types of oligomers present in solution are a matter of debate. Using small-angle X-ray scattering, we found that Co(2+), glycerol, and a single amino acid substitution at the N-terminus, Y73A, facilitate oligomerization of yeast frataxin, resulting in a dynamic equilibrium between monomers, dimers, trimers, hexamers, and higher-order oligomers. Using X-ray crystallography, we found that Co(2+) binds inside the channel at the 3-fold axis of the trimer, which suggests that the metal has an oligomer-stabilizing role. The results reveal the types of oligomers present in solution and support our earlier suggestions that the trimer is the main building block of yeast frataxin oligomers. They also indicate that different mechanisms may control oligomer stability and oligomerization in vivo.
|
|
| 9. |
- Söderberg, Christopher, et al.
(författare)
-
The molecular basis of iron-induced oligomerization of frataxin and the role of the ferroxidation reaction in oligomerization.
- 2013
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 288:12, s. 8156-8167
-
Tidskriftsartikel (refereegranskat)abstract
- The role of the mitochondrial protein frataxin in iron storage and detoxification, iron delivery to iron-sulfur cluster biosynthesis, heme biosynthesis and aconitase repair has been extensively studied during the last decade. However, still no general consensus exists on the details of the mechanism of frataxin function and oligomerization. Here, using small-angle X-ray scattering (SAXS) and X-ray crystallography, we describe the solution structure of the oligomers formed during the iron-dependent assembly of yeast (Yfh1) and E. coli (CyaY) frataxin. At an iron-to-protein ratio of 2, the initially monomeric Yfh1 is converted to a trimeric form in solution. The trimer in turn serves as the assembly unit for higher-order oligomers induced at higher iron-to-protein ratios. The X-ray crystallographic structure obtained from iron-soaked crystals demonstrates that iron binds at the trimer-trimer interaction sites, presumably contributing to oligomer stabilization. For the ferroxidation-deficient D79A;D82A variant of Yfh1, iron-dependent oligomerization may still take place, although more than 50% of the protein is found in the monomeric state at the highest iron-to-protein ratio used. This demonstrates that the ferroxidation reaction controls frataxin assembly and presumably the iron chaperone function of frataxin and its interactions with target proteins. For E. coli CyaY, the assembly unit of higher order oligomers is a tetramer, which could be an effect of the much shorter N-terminal region of this protein. The results show that understanding of the mechanistic features of frataxin function requires detailed knowledge of the interplay between the ferroxidation reaction, iron-induced oligomerization and the structure of oligomers formed during assembly.
|
|
| 10. |
|
|
