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Of spiders, bugs, and men: Structural and functional studies of proteins involved in assembly
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
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 [en]
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: urn:nbn:se:uu:diva-366703ISBN: 978-91-513-0513-4 (print)OAI: oai:DiVA.org:uu-366703DiVA, id: diva2:1265339
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
List of papers
1. The Escherichia coli CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite
Open this publication in new window or tab >>The Escherichia coli CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite
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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.

Keywords
CysZ, Sulfate, Transport, Membrane protein, Inhibition
National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
urn:nbn:se:uu:diva-227987 (URN)10.1016/j.bbamem.2014.03.003 (DOI)000336695300014 ()
Available from: 2014-07-04 Created: 2014-07-02 Last updated: 2018-11-23Bibliographically approved
2. Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis
Open this publication in new window or tab >>Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus mirabilis
2018 (English)In: Acta crystallographica. Section D, Structural biology, ISSN 2059-7983, Vol. 74, no Pt 11, p. 1053-1062Article in journal (Refereed) Published
Abstract [en]

The important uropathogen Proteus mirabilis encodes a record number of chaperone/usher-pathway adhesive fimbriae. Such fimbriae, which are used for adhesion to cell surfaces/tissues and for biofilm formation, are typically important virulence factors in bacterial pathogenesis. Here, the structures of the receptor-binding domains of the tip-located two-domain adhesins UcaD (1.5 Å resolution) and AtfE (1.58 Å resolution) from two P. mirabilis fimbriae (UCA/NAF and ATF) are presented. The structures of UcaD and AtfE are both similar to the F17G type of tip-located fimbrial receptor-binding domains, and the structures are very similar despite having only limited sequence similarity. These structures represent an important step towards a molecular-level understanding of P. mirabilis fimbrial adhesins and their roles in the complex pathogenesis of urinary-tract infections.

Keywords
Proteus mirabilis, adhesins, fimbriae, urinary-tract infection
National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-366693 (URN)10.1107/S2059798318012391 (DOI)000449044300003 ()30387764 (PubMedID)
Funder
Swedish Research Council
Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2019-01-07Bibliographically approved
3. Structural basis for MrpH-dependent Proteus mirabilis biofilm formation
Open this publication in new window or tab >>Structural basis for MrpH-dependent Proteus mirabilis biofilm formation
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Proteus mirabilis is a Gram-negative uropathogen and the major causative agent in catheter-associated  (CAUTI) and complicated UTIs. Mannose resistant Proteus-like fimbriae (MR/P) are crucially important for P. mirabilis infectivity and are required for biofilm formation and auto-aggregation, as well as for bladder and kidney colonisation. Here, the X-ray structure of the MR/P tip-located MrpH adhesin is reported. The structure has an unusual fold not previously observed, and contains a transition metal centre with Cu2+ or Zn2+ ligated by three conserved histidine residues and a ligand. Using metal complementation biofilm assays and site directed mutagenesis of the three histidines we show that an intact metal binding site occupied by zinc or copper is essential for MR/P-mediated biofilm formation. The studies presented here provide important clues as to the mechanism of MR/P-mediated biofilm formation and will serve as a starting point for identifying the physiological MR/P receptor(s).

Keywords
fimbriae; adhesins; biofilm; urinary tract infection; Proteus mirabilis
National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-366696 (URN)
Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2018-11-23
4. Conversion of spidroin dope to spider silk involves an asymmetric dimer intermediate
Open this publication in new window or tab >>Conversion of spidroin dope to spider silk involves an asymmetric dimer intermediate
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(English)Manuscript (preprint) (Other academic)
National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-366699 (URN)
Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2018-11-23
5. Bri2 BRICHOS domain binds Bri23 and depends on conserved face A tyrosine residues for anti-amyloid activity
Open this publication in new window or tab >>Bri2 BRICHOS domain binds Bri23 and depends on conserved face A tyrosine residues for anti-amyloid activity
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(English)Manuscript (preprint) (Other academic)
Keywords
Bri2, BRICHOS, amyloid, Alzheimer's disease, molecular chaperone
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-366698 (URN)
Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2018-11-23

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