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The Escherichia coli CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
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2014 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 7, p. 1809-1816Article in journal (Refereed) Published
Abstract [en]

The Escherichia coli inner membrane protein CysZ mediates the sulfate uptake subsequently utilized for the synthesis of sulfur-containing compounds in cells. Here we report the purification and functional characterization of CysZ. Using Isothermal Titration Calorimetry, we have observed interactions between CysZ and its putative substrate sulfate. Additional sulfur-containing compounds from the cysteine synthesis pathway have also been analyzed for their abilities to interact with CysZ. Our results suggest that CysZ is dedicated to a specific pathway that assimilates sulfate for the synthesis of cysteine. Sulfate uptake via CysZ into E. coil whole cells and proteoliposome offers direct evidence of CysZ being able to mediate sulfate uptake. In addition, the cysteine synthesis pathway intermediate sulfite can interact directly with CysZ with higher affinity than sulfate. The sulfate transport activity is inhibited in the presence of sulfite, suggesting the existence of a feedback inhibition mechanism in which sulfite regulates sulfate uptake by CysZ. Sulfate uptake assays performed at different extracellular pH and in the presence of a proton uncoupler indicate that this uptake is driven by the proton gradient. (C) 2014 Elsevier B.V. All rights reserved.

Place, publisher, year, edition, pages
2014. Vol. 1838, no 7, p. 1809-1816
Keywords [en]
CysZ, Sulfate, Transport, Membrane protein, Inhibition
National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
URN: urn:nbn:se:uu:diva-227987DOI: 10.1016/j.bbamem.2014.03.003ISI: 000336695300014OAI: oai:DiVA.org:uu-227987DiVA, id: diva2:732475
Available from: 2014-07-04 Created: 2014-07-02 Last updated: 2018-11-23Bibliographically approved
In thesis
1. Of spiders, bugs, and men: Structural and functional studies of proteins involved in assembly
Open this publication in new window or tab >>Of spiders, bugs, and men: Structural and functional studies of proteins involved in assembly
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein assembly enables complex machineries while being economical with genetic information. However, protein assembly also constitutes a potential threat to the host, and needs to be carefully regulated.

Sulfate is a common source of sulfur for cysteine synthesis in bacteria. A putative sulfate permease CysZ from Escherichia coli appears much larger than its apparent molecular mass when analyzed by chromatography and native gel. Clearly CysZ undergoes homo-oligomerization. Using isothermal titration calorimetry, we confirmed that CysZ binds to its putative substrate sulfate, and also sulfite with higher affinity. CysZ-mediated sulfate transport—in both E. coli whole cells and proteoliposomes—was inhibited in the presence of sulfite, indicating a feedback inhibition mechanism.

Proteus mirabilis is a Gram-negative bacterium causing urinary tract infections. Its simultaneous expression of multiple fimbriae enables colonization and biofilm formation. Fimbriae are surface appendages assembled from protein subunits, with distal adhesins specifically recognizing host-cell receptors. We present the first three structures of P. mirabilis fimbrial adhesins. While UcaD and AtfE adopt the canonical immunoglobulin-like fold, MrpH has a previously unknown fold. The coordination of Zn or Cu ion by three conserved histidine residues in MrpH is required for MrpH-dependent biofilm formation.

Spider silk is an assembly of large proteins called spidroins. The N-terminal domain (NT) of spidroins senses the pH decrease along the silk spinning gland, and transits from monomer to dimer. A locked NT dimer interlinks spidroin molecules into polymers. We identified a new asymmetric dimer form of NT by x-ray crystallography. With additional evidence from small angle x-ray scattering (SAXS), we propose the asymmetric dimer as a common intermediate of NT in silk formation.

Alzheimer’s disease is a life-threatening dementia, where aggregation-prone Aβ peptides self-assemble into amyloid fibrils. Bri2 BRICHOS is a molecular chaperone that efficiently delays Aβ fibrillation, and protects the region of its pro-protein with high β-propensity from aggregation. Combining SAXS and microscale thermophoresis data, we confirmed binding between Bri2 BRICHOS and its native client peptide. Using site-directed mutagenesis, we showed that three conserved tyrosine residues in Bri2 BRICHOS are important for its anti-Aβ fibrillation activity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 87
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1748
Keywords
Protein assembly, sulfate transporter, crystallography, Proteus mirabilis, fimbriae, adhesin, urinary tract infection, biofilm, spider silk, asymmetric dimer, Bri2, BRICHOS, molecular chaperone, Alzheimer's disease, amyloid
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biology with specialization in Structural Biology
Identifiers
urn:nbn:se:uu:diva-366703 (URN)978-91-513-0513-4 (ISBN)
Public defence
2019-01-18, Room B21, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2018-12-14 Created: 2018-11-23 Last updated: 2018-12-14

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Zhang, LiJiang, WangshuAlmqvist, Jonas

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