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Of Spiders, Bugs, and Men

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Of Spiders, Bugs, and Men Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1748 Of spiders, bugs, and men Structural and functional studies of proteins involved in assembly WANGSHU JIANG ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0513-4 UPPSALA urn:nbn:se:uu:diva-366703 2018 Dissertation presented at Uppsala University to be publicly examined in Room B21, BMC, Husargatan 3, Uppsala, Friday, 18 January 2019 at 09:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Steve J. Matthews (Imperial College London). Abstract Jiang, W. 2018. Of spiders, bugs, and men. Structural and functional studies of proteins involved in assembly. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1748. 87 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0513-4. 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. 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 Wangshu Jiang, Department of Cell and Molecular Biology, Structural Biology, Box 596, Uppsala University, SE-751 24 Uppsala, Sweden. © Wangshu Jiang 2018 ISSN 1651-6214 ISBN 978-91-513-0513-4 urn:nbn:se:uu:diva-366703 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-366703) To Mikael List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Zhang, L., Jiang, W., Nan, J., Alqvist, J, Huang, Y. (2014) The Escherichia coli CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite. Biochimica et Biophysica Acta, 1838 (7): 1809-1816 II Jiang, W., Ubhayasekera, W., Pearson, M.M., Knight, S.D. (2018) Structures of two fimbrial adhesins, AtfE and UcaD, from the uropathogen Proteus Mirabilis. Acta Crystallographica Sec- tion D, 74 (11): 1053-1062 III Jiang, W., Ubhayasekera, W., Breed, M.C., Serr, N., Pearson, M.M., Knight, S.D. (2018) Structural basis for MrpH-dependent Proteus mirabilis biofilm formation. Manuscript IV Jiang, W., Askarieh, G., Shkumatov, A., Hedhammar, M., Knight, S.D. (2018) Conversion of spidroin dope to silk involves an asymmetric dimer intermediate. Manuscript V Jiang, W., Chen, G., Achterhold, A., Johansson, J., Knight, S.D. (2018) Bri2 BRICHOS domain binds Bri23 and depends on con- served face A tyrosine residues for anti-amyloid activity. Manu- script Reprints were made with permission from the respective publishers. Contents Introduction ............................................................................................... 11 Case 1: Escherichia coli Inner Membrane Transporter CysZ (Paper I) ... 13 Purification and Functional Characterization of CysZ .................... 14 Oligomerization State of CysZ ........................................................ 15 Case 2: Fimbriae Adhesins: UcaD, AtfE and MrpH from Uropathogen Proteus mirabilis (Paper II and III) .......................................................... 21 Chaperone-Usher Pathway in Gram-negative bacteria ........................ 22 Fimbriae adhesins in P. mirabilis ......................................................... 25 X-ray Structure Determination of UcaD, AtfE and MrpH ................... 27 Structure-aided functional studies on MrpH ........................................ 31 Preliminary investigations on possible receptors for MrpH, AtfE and UcaD ..................................................................................................... 33 Case 3: Structural Transitions of Spider Silk N-Terminal Domain (Paper IV) ............................................................................................................. 35 Spider silk proteins and dope-to-silk transition .................................... 35 Lock-and-Trigger mechanism: how NT and CT respond to pH drop .. 37 X-ray structure determination of NT alternative dimer ....................... 39 SAXS studies of NT under near physiological conditions ................... 43 Case 4: Human Bri2 BRICHOS domain as a molecular chaperone (Paper V) .............................................................................................................. 48 Amyloid diseases and amyloidogenic peptides .................................... 50 Properties and structures of amyloid fibrils ......................................... 52 The amyloid β hypothesis .................................................................... 55 Chaperones and their role in amyloid diseases .................................... 55 BRICHOS domains and BRICHOS-containing proteins ..................... 57 Bri2 processing and its involvement in dementia ................................ 60 Functions of Bri2 BRICHOS ............................................................... 62 Structural and functional studies of Bri2 BRICHOS ........................... 63 Construct screening for Bri2 BRICHOS recombinant expression .. 63 Crystallization trials with promising Bri2 BRICHOS constructs .... 67 Structural characterization of Bri2 BRICHOS with SAXS ................. 68 Functional studies of interactions between Bri2 BRICHOS and its client peptides ....................................................................................... 71 Populärvetenskaplig sammanfattning ....................................................... 74 Acknowledgements ................................................................................... 77 References ................................................................................................. 80 Abbreviations TM transmembrane IMAC immobilized metal ion affinity chromatography SEC size exclusion chromatography ITC isothermal titration calorimetry Tm melting temperature UTIs urinary tract infections CAUTIs catheter-associated urinary tract infection CUP chaperone-usher pathway TDA two-domain adhesin FGCs fimbrial gene clusters NTD N-terminal lectin domain (of two-domain adhesin) UPEC uropathogenic Escherichia coli SAD single-wavelength anomalous dispersion NT N-terminal domain of spider silk protein CT C-terminal domain of spider silk protein SAXS small angle x-ray scattering Rg radius of gyration EOM ensemble optimization method AD Alzheimer’s disease Aβ amyloid β peptide APP amyloid precursor protein NFT neurofibrillary tangles IDPs intrinsically disordered proteins PDB Protein Data Bank NMR nuclear magnetic resonance spectroscopy Cryo-EM cryo-electron microscopy ThT thioflavin T BBB blood brain barrier SP-C lung surfactant protein C IAPP islet amyloid polypeptide HSP heat shock protein NUCB1 nucleobindin-1 aa amino acids GKN1 gastrokine 1 ChM-1 chondromodulin-1 TNMD tenomodulin FBD familial British dementia FDD familial Danish dementia ER endoplasmic reticulum IDE insulin degrading enzyme Trx thioredoxin TRB thrombin TEV tobacco etch virus MST micro-scale thermophoresis Kd dissociation constant BACE1 β-secretase 1 DLS dynamic light scattering PDI polydispersity index wt wild type Introduction As the driving force for most biological
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