Pépite | Biologie structurale des interactions protéines-glucides par les adhésines fimbriaires d'Escherichia coli
194
0
0
Texte intégral
(2) HDR de Julie Bouckaert, Lille 1, 2012. Foreword It was one of the most amazing times in my life when admitted to work for CNRS in France. Even more so it was to join UGSF filled with scientists that are passionate about glycobiology. Already for two years, I find it an inspiring place with a warm atmosphere. This is largely due to its director, Dr. Jean-‐Claude Michalski, who gives academic freedom as a right and supports the scientists in their undertakings. I am also grateful to Dr. Guy Lippens, a challenging personality with whom it is a pleasure to discuss and be inspired, and Isabelle Landrieu and Xavier Hanoulle and many others of the group who are protein structure companions. But then I have not talked yet about Yann Guérardel and the biodiversity laboratories where I perform research today. At the GFG (Groupe Français des Glucides) meeting in Ambleteuse, in 2000, Yann explained me structures of LAM (lipoarabinomannan) from Mycobacterium sp.. Later on I visited at UGSF in Villeneuve d’Ascq, to find Emmanuel Maes and Gerard Strecker over paper NMR spectra, and I got some "sulfated N-‐actyllactosamines" and other "froggy" sugars for co-‐crystallization with bacterial adhesins. The laboratory of "Diversité structurale associée aux glycoconjugués microbienne" has a multidisciplinary approach to research in glycobiology. Yann is the brain behind this by bringing together many excellent scientists and colleagues, among whom Emeline Fabre, Emmanuel Maes, Diane Jouanneau, Bernadette Coddeville, Elizabeth Elass, Fréderic Krzewinski, Florence Delpace, Nao Yamakawa, Xavier Trivelli, Christophe Biot, Ossarath Kohl, Christophe Mariller, José Provato, Faustine Dubar, Jorick Vanbeselare. I would like to thank mostly everyone from UGSF and the jury for their interest and for being here today. It is a great pleasure for me to present the HDR to all of you. . . © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(3) HDR de Julie Bouckaert, Lille 1, 2012. INDEX. Foreword Parcours scientifique 1. Introduction 1.1 Infection by bacterial fimbrial adhesion 1.2 Biogenesis of fimbrial adhesins 1.3 Medical applications 1.3.1 Vaccines 1.3.2 Anti-adhesives 2. The FimH adhesin of type-1 fimbriae 2.1 The mannose-binding pocket of FimH 2.2 Recognition of oligosaccharides by FimH and fine specificity 2.3 The type-1 fimbrial adhesin FimH of Escherichia coli is an important virulence factor in urinary tract infections and inflammatory bowel diseases 2.4 Lead optimization based on thermodynamics and structures 2.5 Characterizing binding interactions by ITC 2.6 Fundamentals of interactions applied in drug design 3. Factors that enhance adhesion 3.1 Shear force enhanced adhesion 3.2 Multivalency in bacterial adhesion - glycan receptor interactions 3.3 Glycan multivalency is tightly regulated in cell biology and in the innate system 3.4 Analyzing multivalency in FimH-mannose interactions 4. Receptors for bacterial adhesins 4.1 Glycan receptors in the urinary tract 4.2 Bacterial adhesion, invasion and replication 4.3 Role of fimbrial (ultra)structure in receptor recognition 4.4 Dynamics in glycosylation of cells in relation to bacterial adhesion References Projects List of publications. © 2014 Tous droits réservés.. page 1 4 4 5 16 16 17 19 20 23 27. 28 29 30 33 33 35 38 39 40 40 41 43 47 48 60 63. doc.univ-lille1.fr.
(4) HDR de Julie Bouckaert, Lille 1, 2012. Added manuscripts, published or in submission 1) Paper to be submitted to ACS Chemical Biology, ‘O- and C-linked mannoside inhibitors to chemically disarm uropathogenic Escherichia coli by targeting their type-1 fimbriae mediated adhesion’ p. 68 2) For several high-affinity compounds of the synthetic library, the enthalpic contribution and the entropic compensation have been measured and X-ray crystal structures have been refined. Biochemistry 2012 ‘The tyrosine gate as a potential entropic lever in the receptor-binding site of the bacterial adhesin FimH’ p. 96 3) In Chem. Commun. 2011 'The functional valency of dodecamannosylated fullerenes with Escherichia coli FimH—towards novel bacterial antiadhesives' Here, in addition to molar ratio (n) determination of FimH-mannosylated fullurene interactions using ITC, a method to determine stoichiometries in a solution affinity SPR assay has been explored. p. 122 4) In Chemistry, a European Journal 2011 'Clustering of Escherichia coli type-1 fimbrial adhesins by using multimeric heptyl α-D-mannoside probes with a carbohydrate core' p. 139 5) Manuscript submitted to J. Med. Chem. 'β-cyclodextrins grafted with n-heptyl α-Dmannoside interfere with Escherichia coli adhesion by binding the FimH fimbrial adhesin at very low doses in aggregation-prone complexes' p. 149 6) Molecular Microbiology 2012 ‘Structural insight in histo—blood group binding by the F18 fimbrial adhesin FedF’ p. 171 7) Biochemical Society Transactions 2011 ‘Glycosylation changes as important factors for the susceptibility to urinary tract infection’ p. 185. © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(5) HDR de Julie Bouckaert, Lille 1, 2012. Parcours scientifique JULIE BOUCKAERT, Ph.D, CNRS Chargé de Recherche CR1, UGSF. Julie Bouckaert, born 2nd June 1967 in Roeselare, Belgium, Belgian nationality Female, married, 3 children Imoh (22-06-97) Niels (31-12-01) Emilie (23-07-03) Unité de Glycobiologie Structurale et Fonctionelle (UGSF), UMR 8576 du CNRS, Avenue Mendeleiev, Bâtiment C9, Université Lille 1, Sciences et Technologies, 59655 Villeneuve d’Ascq , France, Tel +33320336347 Email [email protected]. PROFESSIONAL EXPERIENCE AND EDUCATION. Oct 2010-present. Chargé de Recherche classe 1 du CNRS, team of Yann Guérardel “ Diversité structurale associée aux glycoconjugués ”, UMR 8576, UGSF, Université Lille 1. March ‘08-Sept ‘10 VIB staff scientist (VIB = Flemish Institute for Biotechnology) 2000-2002. Visiting postdoc at Hultgren laboratories, Department of Molecular Microbiology Washington School of St. Louis, USA. 1996-Feb. 2008. 3 consecutive Postdoc grants by FWO (=Fund for Scientific Research) postdoctoral Researcher and project leader at VUB (=Vrije Universiteit Brussel)-VIB, (Belgium), DU Dr. Lode Wyns, inclusive 1 FWO-mobility and 1 FWO long-term leave grants for USA: 2000-2002. 1991-1996. PhD “Mapping the structural features of metal- and carbohydrate binding of concanavalin A ” promotor Dr. Lode Wyns, VUB, Brussels, Belgium. FIELDS OF INTEREST: Structure-to-function relationships of microbial lectins Structural study of multivalent protein-carbohydrate interactions Analysis of differential glycosylation and bacterial infection pathology during metabolic imbalance Structure-based design of anti-bacterial and anti-fungal drugs. SCIENTIFIC ACTIVITY •. 49 publications published in peer-reviewed journals, 1 paper under review. •. H-index of 19 - total of non-self-citations 1170. •. 1 patent with WU St. Louis (2002), 1 with VIB (2004), 1 with CNRS (2012). Page 1 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(6) HDR de Julie Bouckaert, Lille 1, 2012. •. •. Coordinator of ANR Blanc “ STARLET: Enhancing the therapeutic value of synthetic carbohydrate polymers against chronic Escherichia coli infections in Crohn’s disease by the interplay with glycosylation dynamics on host cell surfaces ” 1 Oct. 2012 - 2015 Coordinator of Pre-PICs and bilateral seminars 29-30 Mai 2012 Ukrainian (National Academy of Science)-French CNRS on bacterial adhesion and relation with apoptosis of eukaryotic cells. •. Contributing to a crystallographic platform within the glycobiology unit UGSF (DU JC Michalski) and with the IRI (Institute for Interdisciplinary Research), Biologie structurale intégrée (DU Vincent Villeret). • • • • •. Bonus Qualité de Recherche 2011 for a crystallization robot for UGSF-IRI Crystalllography within ANR Blanc MIEN on a Candida albicans glycosyltransferase family, 2010-2014 FWO-MDEIE Flanders-Quebec January 2011- June 2013. Synchrotron trips (SOLEIL, ESRF, BESSY), coordinator Proxima1 and Swing in 2008-2010 2001-2012 Invited speaker for 11 international conferences. TUTORING • 2011-2012 : 2 BTS stagiares, 1 master2 student • 2010-2011 : 1 BTS stagiare • 2009 – 2012 : International VIB-PhD student “Hoodwinking uropathogenic bacteria with sugar to keep them away from diabetics”, VUB, Belgium • 2008-2012 : co-promotor of Phd thesis (FWO aspirant grant), “Biomacromolecular interactions of polyadhesins in fimbrial biogenesis at the cell envelope of Escherichia coli, in biofilms and with their eukaryot receptors” VUB, Belgium • 2004 – 2011: co-promotor of Phd thesis (university assistantship) “Biochemical and structural study of the interaction of carbohydrate-based inhibitors with the FimH adhesin”, VUB, Belgium • 2003 – 2009: co-pormotor of PhD thesis (university assistantship) “Structure-function of the proteins involved in biogenesis and receptor binding of F4 fimbriae”, VUB, Belgium • 1997 – 2009 (postdoctoral): co-promoting 18 master2 students PEER REVIEW Peer reviewer for structure-(micro)biological journals, about 16 articles per year Biochemistry, Molecular Microbiology, PLoS One, FEBS Letts, J. Mol. Biol., PNAS, PloS Pathogens, Central European Journal of Biology, Yonsei Medical Journal, J. Biol. Chem., PLoS One, Analyst, Molecular Biosystems, Acta Crystallographica volume D (Biological Crystallography), editorial board of Acta Crystallographica volume F (Structure and Crystallization reports). AWARDS 9th Jenner Glycobiology and Medicine Symposium Sept 13 – 15 2009 2nd poster price. Page 2 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(7) HDR de Julie Bouckaert, Lille 1, 2012. INVITED LECTURES (with funding). Conference. Place. Start End. Titel. Conferences on structurebased drug design. Basel, Switserland. February. Structure and thermodynamics of. 22, 2012. FimH – mannoside interactions. Participating Investigators Bethesda, Meeting < Consortium for MD, USA Functional Glycomics. July 27 to 29, 2011. Bacterial adhesins: F17G / GafD as a paradigm. Carbohydrates Gordon Research Conference. NewHampshire, USA. 19-24 June 2011. Structural and Thermodynamic Basis of Mannose Binding to the Type-1 Fimbrial Adhesin of Escherichia coli. Workshop on Glycan. Emory. Structural sampling of glycan interaction. Arrays, Consortium for Functional Glycomics. Atlanta. 19-21 Sept 2010. Glycomarkers for Disease. Mikolajki. Polish Natl. Academy of Sci.. Poland. 12-16 Sept 2010. Urinary tract infections in diabetics, the story of uropathogenic E. coli. Leveraging Glycan Arrays with Biol., Comput. and Struct. Data. Bethesda. 22-23. USA. Oct 2009. Glycan binding by bacterial fimbrial adhesins: serving multiple functions?. Royal Society of Chemistry. Dublin. 26 – 28 Apr 2009. Debugging urinary tract infections with mannose-based anti-adhesives. IFR 147 Journée de Biologie Structurale. Lille. 9 April 2009. Structural biology of lectin and adhesin interactions by X-ray crystallography. XVIII International. Firenze. A novel class of high-affinity inhibitors of. symposium on Glycoconjugates. Italy. 4-9 Sept 2005. XVII International Glycobiology Congres. Bangalore India. 12-16 Jan 2003. Yet another way to bind mannose: the bacterial adhesin FimH. Medimmune Inc.. Maryland. 8 Jul 2002 Structural characterization of E. coli J96 FimH. Georgia. Ireland. France. profiles pinpoints the natural receptors of bacterial fimbrial adhesins. type 1 fimbrial adherence of E. coli disclosed in the FimH crystal structure. Page 3 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(8) HDR de Julie Bouckaert, Lille 1, 2012. 1. Introduction 1.1 Infection by bacterial fimbrial adhesion Bacterial infections constitute a major global health problem, acutely accentuated by the rapid spread of antibiotic resistant bacterial strains. The World Health Report 2004 1 estimates that respiratory and diarrhoeal infections were responsible for 4.0 and 1.8 million deaths, respectively, in 2002, to be compared to the toll of diseases such as AIDS (2.8 million deaths), tuberculosis (1.6 million deaths), and malaria (1.3 million deaths). A significant proportion of respiratory and diarrhoeal infections are caused by Gram-negative pathogens such as Salmonella enterica (typhoid fever, enterocolitis), Haemophilus influenzae (pneumonia), Bordetella pertussis (whooping cough), and Escherichia coli (diarrhoea). The overwhelming majority of the world’s annual 4 million neonatal deaths occur in developing countries. Bacterial infection is the major cause of neonatal admissions to hospitals, and probably the biggest cause of morbidity in the community. The most common serious neonatal infections involve bacteraemia, meningitis, and respiratory tract infections, and case fatality rates may be as high as 45%. Key pathogens in these infections are E. coli, Klebsiella sp., Staphylococcus aureus and Streptococcus pyogenes 2.. Figure 1. Examples of bacterial ‘hair styles’. A) Thick and straight: type 1 fimbriated E. coli. Type 1 fimbriae are composite structures with a relatively rigid 8-nm wide rod tipped by a thin (2.5 nm) more flexible tip fibrillum. B) Thin and wavy: F17 fimbriated E. coli covered with 1-3 µm long, flexible, 2-3 nm wide fimbriae. C) Tangled (capsules & sheaths): Y. pestis F1 capsular antigen 3. Here no individual fibres are visible, but the capsule consists of a tangle of thin (~2 nm) flexible fibres. Most bacteria depend on the expression of specialized adhesive organelles on the bacterial cell surface to mediate attachment to target tissues. Gram-negative bacteria can grow hair-like adhesive organelles referred to as pili (from the Latin word for ‘hair’) or fimbriae (from the Latin word for ‘thread’) arranged in a multitude of ‘hairstyles’ ranging from soft, long, wavy. Page 4 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(9) HDR de Julie Bouckaert, Lille 1, 2012. hair, to afro style (Figure 1). In one common class of such fibrillar organelles, many copies of a receptor binding ‘single-domain adhesin’ (SDA) are incorporated in a thin (2-5 nm wide) and flexible fibre. Examples of such ‘polyadhesins’ include the Dr adhesins expressed by many UPEC strains, SEF14 fimbriae of Salmonella enteritidis, Myf fimbriae of Yersinia enterocolitica, and the pH6 and F1 antigens expressed by Yersinia pestis. A second class of fibrillar adhesive organelle commonly expressed by Gram-negative pathogens display a carbohydrate binding ‘two-domain adhesin’ (TDA) at the tip of complex pili or fimbriae. These composite structures frequently incorporate the TDA at the tip of a thin (~2 nm) and flexible ‘tip fibrillum’ linked to a relatively rigid 1-2 µm long and ~8 nm wide ‘stalk’, or ‘rod’, consisting of a helically wound fibre of fimbrial subunits (‘pilins’). Examples of TDA-displaying adhesive organelles include the much studied P pili and type 1 fimbriae, that provide UPEC with the ability to bind to Galα14Gal-containing and mannose-containing receptors respectively, and that are important for UPEC colonization of the urinary tract. The specialized SDA and TDA bacterial adhesive organelles provide promising targets for development of new classes of anti-bacterial drugs that may either directly block adhesion or interfere with the biogenesis of adhesive organelles, and for the development of novel vaccines to prevent bacterial infections.. 1.2 Biogenesis of fimbrial adhesins . Results from structure-based studies of components of the P pilus, type-1 fimbriae, and F1 antigen systems have led to a relatively refined understanding of how SDA and TDA fimbrial adhesins are constructed, and of how periplasmic chaperons direct and promote their assembly. Key to the success of these studies has been the realization that the fimbrial subunits, although very unstable on their own, can be isolated as stable complexes with their cognate periplasmic chaperon. These studies have shown that, in spite of a great deal of variation in appearance and binding specificity, a large group of bacterial pili/fimbriae share the same underlying fibre structure, consisting of a string of non-covalently linked immunoglobulin (Ig)-like modules. This linear fibre structure is assembled from monomeric, incomplete Ig subunits by a periplasmic chaperon together with an outer membrane usher 4-6. The fibre morphology is determined by additional interactions between pilin subunits in the fibre, allowing some fibres to coil into rigid helical structures such as the type-1 fimbrial rods, while others remain as relatively flexible extended linear fibres that sometimes collapse into amorphous ‘sheaths’ or ‘capsules’ 7 (Figure 1). Further quaternary interactions between pilin subunits, among which winding up into the. Page 5 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(10) HDR de Julie Bouckaert, Lille 1, 2012. right-handed helical rod for the type-1 pilus, typically inholds a regulation of function (such as affinity) of the bacterial cell surface organelles 8. Helper proteins for the biogenesis of fimbriae and pili from Gram-negative bacteria are present within the operon, at least comprising a periplasmic chaperon and an outer membrane usher protein. The common factor of all structural proteins building up pili and fimbriae is the lack of the 7 , C-terminal G β-strand in a variable immunoglobulin (Ig)-fold 5,9,10. The periplasmic chaperon is a steric chaperon that prevents misfolding of the pilin because of the lack of the C-terminal β-strand. It stabilizes the pilin by lending it the G β-strand of its Nterminal domain and subsequently presents the pilin assembly-competent to the usher. The usher has the molecular machinery in the periplasm for donor strand exchange of the chaperon β-strand with the N-terminal extension of the pilin of a next incoming chaperon-pilin complex at de usher (Figure 2). Upon donor strand exchange (DSE), the folding of the pilin is completed and the stability of the fimbriae set. th. b . c a . Page 6 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(11) HDR de Julie Bouckaert, Lille 1, 2012. Figure 2. Type-1 pili biogenesis. (a) The production of type 1 fimbriae requires at least eight genes localized within the fim gene cluster and is subject to phase variation. fimA, fimI, fimF, fimG, and fimH code for the five structural protein components of type 1 fimbriae, with a FimA polymer forming the helical rod and a single FimH TDA strategically located at the distal tip of each fimbriae. FimH is attached to the rod via single copies of FimG and FimF, forming the short and stubby tip fibrillum. The location of FimI in the fimbrius is unknown. Assembly of type 1 fimbriae is mediated by the FimC chaperon together with the OM usher FimD. fimB and fimE encode regulatory proteins that control the expression of type 1 fimbriae. (b) Schematic model of type 1 pilus assembly by the chaperon–usher pathway. The periplasmic chaperon FimC forms stoichiometric complexes with the newly translocated pilus subunits (FimA, FimG, FimF, FimH). In these complexes, FimC donates its G1 donor strand to the individual subunits, thereby completing the immunoglobulin-like fold of the subunits. FimC–subunit complexes diffuse to the assembly platform (usher) FimD, which specifically recognizes FimC–subunit complexes via its periplasmic, N-terminal segment of residues 1–139. Subsequently, FimC is released to the periplasm, and the subunit is delivered to the translocation pore of FimD, where it is supposed to interact with the previously incorporated subunit via donor strand exchange (DSE, inset (c)). The pilus rod, composed of FimA subunits, assembles into its helical quaternary structure on the cell surface. From 11. IM, inner membrane; OM, outer membrane. The subunits of fimbriae are constructed essentially as Ig-like β-sandwiches, but with a circular permutation that positions the sequence corresponding to the seventh, C-terminal, Ig βstrand (strand G of a canonical Ig domain) at the N-terminus of the polypeptide sequence. In a typical Ig fold, the ‘top’ edge of the sandwich, defined by the A and F strands, is capped by the C-terminal G strand, which is hydrogen bonded to the F strand and provides hydrophobic residues to the core of the fold. The hydrophobic effect drives folding of globular proteins by favoring the packing of hydrophobic side chains together in a hydrophobic core, shielded from the surrounding water. In pilins however, owing to the absence of a seventh, C-terminal (G) strand, the polypeptide chain simply cannot fold in such a way as to create a shielded hydrophobic core, explaining the instability of free pilin subunits. The N-terminal extension of pilins is flexible in solution and does not contribute to the subunit’s globular fold. It carries a β-strand motif of alternating hydrophobic and hydrophilic residues. The inserted N-terminal segment adopts a β-strand conformation running antiparallel to the F strand, with hydrophobic side chains bound in three (TDA systems) or five (SDA systems) acceptor cleft sub-pockets, hence completing the Ig-fold of the subunit. This mode of binding, termed ‘donor strand complementation’ (DSC), is likely to be present in all surface polymers assembled through the chaperon/usher pathway. The resulting linear fibre is composed of. Page 7 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(12) HDR de Julie Bouckaert, Lille 1, 2012. globular modules each having an intact Ig-topology generated by DSC. In this fibre, each Ig module is made from two polypeptide chains, with the G-strand being provided in trans (because the N-terminal segment of one subunit is donated to fulfil the role of the Ig-fold G strand in a second subunit, the N-terminal sequence is also referred to as the Gd (d for donor) sequence) (Figure 3). TDAs lack the N-terminal Gd polymerization sequence and instead have an entire receptor-binding domain coupled to the N-terminus of the pilin domain. As a consequence, TDAs can only be incorporated in a single copy at the tip of fimbriae.. Figure 3. Model of the F4 fimbrius based on the crystal structure of the F4 SDA, FaeG 12 In spite of their non-covalent nature, and in contrast to free pilins or pilins bound to their periplasmic chaperon, studies have shown that the fibre Ig modules are extremely stable 13,14. The folding free energy estimated from reversible unfolding of an engineered monomeric F1 fibre module (Caf1-SC) in GdmCl at 37°C is in the range 70-80 kJ mol-1 15! This should be compared to the typical range for proteins, about 20-60 kJ mol-1. Indeed, the function of adhesive surface fibres requires them to be mechanically resilient, and in the absence of a counteracting evolutionary pressure (e.g. there is no need for protein turnover outside of the cell) no functional constraints on maximum stability apply. Biogenesis of stable polymeric surface fibres such as those of pili, fimbriae, or capsules, poses many challenges to the Gram-negative bacterial cell. It must be able to protect the unstable and highly aggregative fibre subunits from aggregation and proteolytic degradation during their transport from the site of production in the cytoplasm, across the inner membrane (IM) and the periplasm, to the site of assembly at the outer membrane (OM) of the cell. Having reached the OM, subunit assembly must be controlled to form the desired polymeric structure, which must then be secreted to the cell surface. In the cytoplasm, pilin subunits are expressed as pre-proteins with an N-terminal export signal that targets them for export via the general secretion (Sec) pathway. In the periplasm, a periplasmic chaperon, together with an OM usher, handle the subsequent events that lead to assembly of pilin monomers into surface located fibrillar structures (Figure 2b).. Page 8 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(13) HDR de Julie Bouckaert, Lille 1, 2012. Figure 4. Chaperon structure. Ribbon diagram of FimC chaperon from the structure of the FimC: FimH complex 5. β-strands in the N-terminal domain are labelled. The hydrophobic residues in the Gd donor strand are shown as stick models and labelled. Also shown are the two invariant residues in the subunit binding cleft between the two Ig-fold domains that are crucial for subunit binding and chaperon function. The periplasmic chaperons are steric chaperons that bind to fibre subunits as they emerge in the periplasm, ensure their correct folding, and deliver the folded subunits to the usher where they are assembled into fibrillar polymeric structures. For TDA-carrying structures, binding of a chaperon: TDA complex to an empty usher initiates assembly 16 which then proceeds by sequential addition of pilin subunits to the base of the growing fibre and simultaneous secretion of the fibre through the usher pore 17. In the absence of chaperon, subunit folding is slow and leads to a marginally stable and aggregation prone structure, whereas in the presence of chaperon, stable and soluble chaperon: subunit complexes are rapidly formed 18. Subunits bind in the cleft between the two chaperon domains with the subunit C-terminal carboxyl group anchored by two positively charged residues (Arg8 and Lys112 in FimC) at the bottom of the subunit-binding cleft (Figure 4). These two residues are strictly conserved in all the periplasmic chaperons and are crucial for chaperon function 19. Periplasmic chaperons deliver folded pilin subunits to the OM usher. At the usher, chaperon: subunit interactions must be replaced by subunit: subunit interactions. Hence, the A1 and G1 strands of the chaperon capping the subunit at the base of the growing structure, on the periplasmic side of the usher, must dissociate to allow the N-terminal sequence of the next subunit to be bound in the polymerization cleft (Figure 2). DSE can occur even in the absence of. Page 9 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(14) HDR de Julie Bouckaert, Lille 1, 2012. the usher as evidenced e.g. by the accumulation of low molecular weight Caf1 polymers in the periplasm of Caf1A usher negative bacteria expressing Caf1M chaperon and Caf1 subunit, or in vitro following incubation of Caf1M:Caf1 complex 20. However, compared to usher-mediated assembly, this process is slow and inefficient, and DSE is catalyzed by the usher (Figure 6).. a. b. c. d. Figure 5. (a) Topology diagram of the SDA FaeGntd (ntd=N-terminus deleted) in yellow, that is expressed in complex with the FaeE chaperon. Only the G1 donor strand of FaeE chaperon is shown, in green. (b) Topology diagram of FaeGntd/dsc (N-terminal extension was deleted and complementation at the C-terminus using a DNKQ sequence spacer (according to 21) or a longer and flexible 10 glycine-rich linker attached for covalent expression to the C-terminus of FaeG to. Page 10 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(15) HDR de Julie Bouckaert, Lille 1, 2012. enable reaching the P* complementation site, see later in Figure 7). The N-terminal extension of FaeG is shown in magenta, and the DNKQ or Gly10 linker is shown in red. (c) Cartoon representation of the FaeE–FaeGntd complex. The donor strand complementation of FaeGntd (yellow) by the chaperon FaeE (light green) is shown. The donor strand G1 of the FaeE chaperon is shown in bright green. (d) Cartoon representation of the FaeGntd/dsc. The N-terminal extension (Nte) is pictured in magenta. Notice the large conformational changes near the α2helix-D” strand – E1 strand where the D”-E loop, indicated in carbohydrate binding by sitedirected mutageneses 12, gains structure upon donor strand exchange.. Page 11 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(16) HDR de Julie Bouckaert, Lille 1, 2012. Figure 6. (a) Integrated, hypothetical model of pilus subunit incorporation at the OM usher. The model shows the twin-pore usher FimD2:C:F:G:H complex as derived by cryo-EM (left), for FimD usher pores 1 (blue) and 2 (dark blue), the FimC chaperon in yellow), FimF in red), FimG (in orange), and the FimH adhesion (green). In addition, the model shows the tentative position (shaded in light gray) of an incoming FimC:A complex bound to usher 2nd N-terminal domain (FimDN2:C:A, colored dark blue:yellow:cyan and labeled N2, FimC, and FimA, respectively). N1 indicates the N-terminal domain of usher 1. (b) The schematic diagram shows the PapC twinned pores and N-terminal domains (in blue and dark blue for usher 1 and 2, respectively), the plug domain (P) in usher 2 and usher 1 for the two alternative models of gating, in magenta). For clarity, the C-terminal domains of ushers 1 and 2 are not shown. step 1 The FimD2:C:F:G:H complex recruits an incoming FimC:FimA complex through binding to the N2. steps 2 and 3 The complex is brought within donor-strand exchange of FimF, resulting in the release of the FimF-bound chaperon (C1) and the dissociation of N1. step 4 N1 is now free to recruit another FimC:A complex and step 5, bring the complex within proximity of the N2-bound FimC:A complex. step 6: Donor-strand exchange releases N2 for recruitment of the next chaperonsubunit complex. Iteration of alternating binding to released usher N-terminal domains, followed by DSE with the penultimate chaperon-subunit complex, leads to stepwise growth of the pilus fiber. No energy input from external sources is required to convert periplasmic chaperon: subunit pre-assembly complexes into free chaperons and secreted fibres 22, Instead, assembly is driven by subunit folding energy conserved by the chaperon. Comparison of the structure of the FaeG bound to the FaeE chaperon and its structure in the F4 fibre module, revealed a large conformational difference (Figure DSE.png). In the FaeG:FaeE super-barrel, the chaperon G1 donor strand occupies the polymerization cleft, with large hydrophobic residues from the G1 donor strand inserted between the A and F β-strands, preventing them from contacting each other (Figure 5c). Molecular dynamics simulations predict that this open, partially folded moltenglobule like conformation is not stable and would not be maintained in solution. In contrast to the chaperon donor residues, the often smaller donor residues in the subunit N-terminal Gd donor segment involve a register shift from P1-P4 sub-site binding in the chaperon complex to P2-P5 sub-site binding in the fibre module, allowing narrowing of the hydrophobic groove and condensation of the Ig-fold (P1-P5 designated in the nomenclature of Sauer et al. 23, Figure 7). Several observations suggest that periplasmic chaperons target and bind subunits in an unfolded or at least partially unfolded state. The high efficiency of chaperon/usher-mediated assembly in vivo 11,22 suggests that this process cannot rely on the slow self-folding of subunits.. Page 12 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(17) HDR de Julie Bouckaert, Lille 1, 2012. The periplasmic chaperon binds to unfolded subunits and chaperon binding was shown to increase the rate of folding by a factor of 100 18. To bind unfolded subunits, the periplasmic chaperon, presumably, has to recognize some common feature of the (partially) unfolded conformations. The extensive interactions between the hydrophobic cores of the N-terminal domain of the chaperon and the subunit observed in the structures of chaperon: subunit complexes suggest that the chaperon might recognize and attract hydrophobic core residues that are exposed in unfolded subunits. The surface exposed hydrophobic patch created by the bulky hydrophobic side chains in the G1 donor strand of free chaperons might attract (partially) unfolded subunits and provide a template onto which the subunit core can condense, facilitating folding. By providing a folding platform consisting of a pair of template β-strands (A1 and G1), and large hydrophobic donor residues, periplasmic chaperons promote subunit folding and partition intrinsically aggregative protein subunits away from non-productive aggregation pathways. Subunit folding onto this platform results in chaperon donor residues being incorporated into the core of the subunit and formation of a fused super-barrel. Subunit folding coupled to chaperon binding thus does not reach the native state, and the subunit is trapped in an open, activated, high-energy conformation. The resulting meta-stable complex provides a convenient substrate for fibre assembly. Upon dissociation of the activated subunit from the chaperon, folding can be completed to form a condensed hydrophobic core. By arresting subunit folding and trapping subunits in a molten globule-like high-energy conformation, the chaperons preserve the necessary folding energy that drives fimbrial assembly.. Page 13 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(18) HDR de Julie Bouckaert, Lille 1, 2012. Figure 7. Finalising the Ig-fold of FaeG and formation of the tryptophan 1-specific P* pocket upon exchange of the donor strand of the chaperon with the N-terminal extension of FaeG. (a) DSC of FaeGntd by the chaperon FaeE. The P1–P3 pockets on the surface of FaeGntd,ad (yellow surface representation) are indicated, and the P1–P3 residues (Leu103, Val101, Ile99) of the G1 strand of FaeE (green sticks, from PDB entry 3GFU) are labeled. The N-terminal extension of plant-expressed FaeG (derived from PDB entry 2J6G) is labeled in orange stick representation on the surface of FaeGntd, showing the different registers of both donor strands and the inaccessibility of the P* pocket before DSE. (b) Superposition of the N-terminal extension of the plant-expressed FaeG (orange sticks, labelled) and the structure of FaeGntd/dsc (yellow surface representation with blue sticks for its residues 1–10 (WMTGHis6) representation and red stick for the DNKQ linker, and the dsc is shown in magenta stick representation. The P1–P5 pockets are indicated. (c) Close-up view of the insertion of the Trp1 residue of the Nterminal extension of FaeGntd/dsc (magenta, PDB entry 3GEA) in the P* pocket showing that the different conformation of FaeG in complex with its chaperon (green, PDB entry 3GEW) and the FaeG Glu25 side chain precludes formation of the P* pocket. The usher must interact with chaperon:pilin subunit complexes in the periplasm to facilitate dissociation of the chaperon from the pilin subunits and polymerization of subunits with translocation across the outer membrane (OM) to the bacterial cell surface. Fimbrial ushers are large (80-90 kDa) porin-like integral OM proteins 24. Both the PapC (P pilus) and the FimD (type 1 fimbrial) ushers form 7x10 nm2 homo-dimers with a ~2 nm pore in the middle area of each monomer. Such a pore is wide enough to allow translocation of folded structural subunits or their polymers through the OM. Release of the chaperon G1 donor strand and insertion of the subunit Gd donor strand in a happens concerted in a zip-out-zip-in mechanism, inititated at the P5 acceptor cleft sub-pocket 25. Based on the recent crystal structure of FimD-FimC-FimH, a model for the transient quaternary complex of the usher with two chaperon:subunit complexes during assembly of type 1 fimbriae can be assumed (Figure 8) 6. The sequestration of chaperon-pilin subunit complexes is helped by the N-terminal periplasmic domain of the usher (Figure 6). release of the chaperon is helped by the two C-terminal periplasmic domains of the usher (CTD1 and CTD2, Figure 8). A ‘plug’ with a β-sandwich fold and located in the central β-barrel region of the resting usher, is dislocated towards the periplasmic side of the OM during pilus assembly (Figure 8) 6.. Page 14 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(19) HDR de Julie Bouckaert, Lille 1, 2012. Figure 8. Chaperon–subunit incorporation cycle at the FimD usher: (a) Side view of the FimD (blue)–FimC(yellow surface)–FimH (green surface) complex with a new incoming FimC chaperon (light yellow)–FimG (orange) complex modelled at the NTD binding site (the model is from PDB 3BWU; that is, based on the crystal structure of FimD NTD alone bound to FimC– FimF). (b) Clipped view of the (FimC–FimG)–(FimC–FimH) contact zone (boxed area in (a)), showing positioning of the FimG N-terminal extension (FimG Nte; in red) above the P5 pocket in the FimC–FimH complex (FimC–FimH in yellow–green, the P5 pocket shown in aqua green). The crystal structure of a FimC: FimHpilin complex (FimHpilin is the C-terminal pilin domain of FimH) bound to the N-terminal domain of the type 1 pilus usher (FimDN) 11, as well as that of a similar complex from the F1 antigen system 26, showed earlier that the ushers have a chaperon binding surface formed by the folded core of the usher domain and by an extended Nterminal ‘tail’ of the usher. The usher recognizes a patch of conserved hydrophobic residues on the ‘back’ of the chaperon N-terminal domain. A general allosteric mechanism for selective targeting of chaperon:subunit to the usher that permits the release and recycling of the free chaperons has just recently been unvealed 27. Free chaperons present a pair of conserved proline residues at the beginning of the pilin-binding loop. These prolines block binding to the Nterminal part of the usher by occlusion of the usher-binding surface. Binding of a pilin subunit to the chaperon rotates the proline lock away from the usher-binding surface, allowing the. Page 15 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(20) HDR de Julie Bouckaert, Lille 1, 2012. chaperon-subunit complex to bind to the usher. Besides chaperon-usher interactions, in the FimC: FimHpilin: FimDN complex there are also significant interactions between the usher Nterminal tail and the chaperon-bound pilin subunit. Very few such interactions are present in the F1 antigen complex, which might reflect the need to distinguish several different subunit types and to assemble these in a particular sequence in the more complex type-1 fimbrial (TDA) system, but not in the F1 antigen (SDA) system. 1.3 Medical Applications 1.3.1 Vaccines Whole cell antigen presentation may shield potentially efficient broad-range antigens from the immune system. This is the case with TDA fimbriae, where a conserved and intrinsically highly antigenic receptor-binding adhesin is incorporated as a minor component of a large complex protein structure on the bacterial cell surface, leading to efficient production of antibodies directed against the much less conserved bulk components of the organelle but not against the adhesin. Because of their critical role in pathogenesis and because they are naturally expressed on the surface of bacteria, bacterial adhesins have long been considered as attractive components of vaccines, so far with only limited success. Many adhesin vaccine formulations have been based on intact adhesive organelles (e.g. fimbriae), which are antigenically highly variable and hence induce protection limited to bacteria expressing the same fimbrial variant. For example, antibodies directed against purified whole type 1 fimbriae or P pili protect against cystitis and pyelonephritis respectively, in both murine and primate models for these diseases 28,29. However, protection is limited to either E. coli strains homologous to that from which the fimbriae used for immunization were derived, or to a small subset of serologically cross-reactive heterologous strains. Therefore, any vaccine composed predominantly of the major structural proteins of fimbriae (e.g. FimA or PapA) will be of limited value because antibodies developed against these highly variable proteins are specific for the strains from which the protein used for immunization was derived. The realization that TDAs could be obtained as stable and soluble complexes with their cognate chaperon inspired new hope for the development of adhesin-based vaccines. Promising preclinical trials with UTI vaccines based on the FimC: FimH chaperon: adhesin complex 30 and on the PapD: PapG chaperon: adhesin complex 31 have been reported. Both vaccine candidates were shown to protect against UPEC mucosal infection in murine and primate models. In vitro binding data suggested that the ability of anti-FimH antibodies to block type 1 fimbrial adhesion. Page 16 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(21) HDR de Julie Bouckaert, Lille 1, 2012. contributed significantly to FimC: FimH-induced protection. Development of the FimC: FimH vaccine candidate was dropped during phase II clinical trials because of limited protection 30. The discovery of FimH binding to glycoprotein 2 (GP2) on M cells in Peyer's patches opens opportunities for mucosal vaccination 32. GP2 is a previously unrecognized transcytotic receptor on M cells for type-1 piliated bacteria (E. coli and Salmonella thyphimurium) and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2dependent transcytotic pathway could provide a new target for the development of M-celltargeted mucosal vaccines. Formulation of the vaccine becomes very important and multivalent antigen presentation generally results in a significantly improved response 33 over monomeric presentation of antigens such as those used for the FimC:FimH and PapD:PapG vaccines. Multivalent antigen presentation may be achieved by coupling subunit antigens to a suitable carrier particle. Since the carbohydrate binding site is located in the top half of the receptorbinding domains, distal from the C-terminal linker region that connects it to the pilin domain in the full length TDAs, modified proteins consisting of the isolated receptor-binding domain coupled to a suitable C-terminal tag could be used to incorporate functional adhesins into multivalent antigen-carrying particles for use as vaccines. 1.3.2 Anti-adhesives One aspect of the pathogen-host interaction that shows great promise as a target for development of novel means of interfering with bacterial infections is the early establishment of physical contact between pathogen and host. Adhesion of bacteria to target tissues is frequently a necessary first step in pathogenesis 34,35. For example, uropathogenic E. coli (UPEC) depend on specific binding to mannose-containing receptors on the luminal surface of the bladder epithelium for the establishment of cystitis (bladder infection) 36,37, whereas binding to Galα14Gal-containing receptors in the upper urinary tract is a prerequisite for the establishment of pyelonephritis (kidney infection) 38. An alternative to adhesin-based vaccines as a means to block bacterial adhesion is the use of small compounds that interact tightly with target adhesins 28,39. Aromatically substituted mannosides have long been known to be particularly potent inhibitors (nanomolar binding constants) of FimH-mediated bacterial adhesion 40. Fruit juice, in particular cranberry juice, has traditionally been considered to be useful in the treatment of UTIs. Fructose has been shown to be the active compound in fruit juices that inhibits adhesion of type 1 fimbriated E. coli 41. Fructose binding to FimH is only ~15 times weaker than mannose binding and thus fructose binds as tight to FimH as the physiological P pilus globotetraoside receptor to PapG-II 42.. Page 17 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(22) HDR de Julie Bouckaert, Lille 1, 2012. In two independent crystal structures for the FimH receptor-binding domain, we found a butyl mannoside, derived from the yeast extract used to grow bacteria for protein expression, bound in the mannose-binding site. The serendipitous discovery of this ‘sticky’ ligand led to the identification of alkyl mannosides as a new class of high-affinity FimH ligands. The mannose group of the butyl mannoside binds identically to D-mannose (Fig. 9C). The alkyl chain extends out of the pocket towards Tyr48 and Tyr137, making van der Waals contacts to both tyrosine rings. We discovered that butyl α-D-mannoside binds to FimH significantly better (Kd ~150 nM) than mannose (Kd ~2.3 µM). To investigate the effect of sequential addition of methyl groups to the O1 oxygen of D-mannose, a series of alkyl mannosides were synthesized and the dissociation constants determined using two different binding assays. We discovered a linear correlation between the binding free energy, as calculated from the measured dissociation constants, and the number of methyl groups (between 1 and 7) in the alkyl mannoside, with each additional methyl group contributing on average -0.6 kcal mol-1 of binding energy. The best binding alkyl mannoside, heptyl mannoside, binds a few hundred times stronger than mannose, equivalent in affinity to the most tightly-binding aromatically substituted mannosides for FimH and of mannose dendrimers for type 1 fimbriated E. coli 40,43. Alkyl mannosides had not previously been recognized as strong binders to FimH. Since they are easily synthesized and highly soluble in water, they became potential blocking agents for FimH-mediated adhesion. Type 1 fimbriated UPEC have probably been the most extensively studied target of glycodendrimer chemistry 43-46. Nevertheless, glycodendrimer chemistry designed to inhibit type 1 mediated bacterial adherence has proven to be excruciatingly difficult. A successful example of a large glycodendrimer FimH inhibitor is the DP16 dendrimer designed by Nagahori et al.43. This inhibitor inhibits type 1 fimbrial adhesion considerably better (sub-nanomolar IC50 values) than monovalent mannose but still not significantly better than the best-known small-molecule inhibitors. The difficulties in finding strong multivalent adhesion inhibitors could be solely due to incorrect linkage of the mannose residue in the dendrimer, preventing mannose from binding in the mannose binding pocket of FimH 47, but most often reflects the difficulties in trying to use molecular ligands to simulate the complexity of multivalent cellular host-pathogen interactions (this will be further discussed in chapter 2).. Page 18 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(23) HDR de Julie Bouckaert, Lille 1, 2012. 2. The FimH adhesin of type 1 fimbriae Adhesion of UPEC as well as other strains of E. coli to physiological receptors or other surfaces displaying mannose-containing receptors depends on the FimH TDA located at the tip of type 1 fimbriae. The first crystal structure of FimH was determined in complex with its periplasmic chaperone, FimC and clearly demonstrated that FimH is divided into two domains; a receptor-binding, or lectin, domain (LD), and a pilin domain 5 (Figures 2, 6, 8). The pilin domain connects FimH to FimG, which is incorporated in the tip fibrillum of type 1 fimbriae after FimH. The two domains of FimH are coupled via a very flexible linker made of two glycine residues, Gly159 and Gly160, that precede the third conserved cysteine Cys161 making a disulphide bond (to Cys187) within the pilin domain.. Figure 9. The receptor-binding domains of three TDA lectin domains, of FimH, PapG-II and F17a-G, each viewed in the same orientation obtained by superimpositioning of the structural core of their Ig-fold (designated by labels), and in complex with α-D-mannose, globotetraoside and N-acetyl β-D-glucosamine, respectively. The FimH receptor-binding domain was originally described as an 11-stranded β-barrel, with a fold unrelated to any other protein fold known at that time. With the second and third three-dimensional structures of TDA receptor-binding domains, from PapG-II 38 and F17G 9, becoming revealed, the overall similar fold among these structures became apparent (Figure 9).. Page 19 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(24) HDR de Julie Bouckaert, Lille 1, 2012. From then on, the receptor-binding domains have been characterized as Ig-folds, with large structural variation in the loops joining the Ig-core made up of strands B, C, E, and F 9. The pilin domain of FimH has the same fold as pilin subunits with an incomplete Ig sandwich lacking the 7th, G, strand. Hence, FimH comprises one complete and one incomplete Ig fold. In the receptorbinding domain, the G strand leads directly into the double-glycine linker (Gly159 and Gly160) that joins the receptor-binding domain to the FimH pilin domain, and is the structural equivalent of the donor strand in the pilin fibre modules. Dr fimbriae SDAs incorporate a specialised ‘invasin’ at the very tip of the fibrillar structure 48,49. To trigger invasion, an invasin must bind to and interact with target receptors, and so is also an adhesin. Invasins have the same basic fold as pilins but lack an N-terminal polymerization sequence and hence, like the TDAs, can only be located at the tip of the fibre. FimH has the dual role of binding and triggering invasion and might be thought of as a special case of invasin molecule where the/invasin domain is covalently coupled to a pilin domain, rather than linked via DSC as in SDA polyadhesins.. 2.1 The mannose-binding pocket of FimH In the FimC:FimH crystals, the receptor-binding domain of FimH was found to have bound to the open ring sugar glucamide of cyclohexylbutanoyl-N-hydroxyethyl glucamide (C-HEGA) 5. C-HEGA had been added at 300 mM to the crystallization medium in order to stabilize crystal formation, but was not known to bind FimH. Its binding led to the identification of the mannosebinding pocket, because the glucamide part of C-HEGA was bent in a way to closely approximate the cyclic pyranose ring. Later, the structure of the FimC: FimH complex bound to 50 D-mannose confirmed that C-HEGA had bound in a way that mimicked binding of mannose . Mutation of the amino acids interacting with C-HEGA to alanine or closely resembling amino acids almost uniformly led to radical abolishment of type-1 mediated bacterial haemagglutination, bladder cell binding and bladder tissue colonization 50. The mutagenesis study gave the insight that the contribution of each single amino acid in the binding pocket is almost uniformly crucial for mannose binding, implying that the recognition by FimH is highly fine-tuned and specific. Furthermore, it showed that modification of the FimH binding pocket directly affected type-1 mediated bacterial adherence to its physiological target, the urothelial mannosylated receptors.. Page 20 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(25) HDR de Julie Bouckaert, Lille 1, 2012. Figure 10. (a) The three-dimensional structure of the FimH receptor-binding domain is an elongated 11-stranded β-barrel with an Ig-fold. Numbering is according to conventions established for antibody domains and for PapD and loop identifications according to previous descriptions (reference strand numbers A1 = 1, A' = 2, A2 = 3, B1 = 4, B2 = 4', C = 5, D1 = 6, D' = 7, D2 = 8, E = 9, F = 10, G = 11). The bound saccharide is butyl α-D-mannose. (b) The. Page 21 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(26) HDR de Julie Bouckaert, Lille 1, 2012. receptor-binding domain of FimH displaying the electrostatic potential surface, with positively charged residues shown in blue, negatively charged residues in red and neutral and hydrophobic residues in white. The residues of the hydrophobic ridge around the mannose-binding pocket are labelled. (c) Stereo image of the mannose binding pocket, viewed 90º away from the orientation in Figure 10a and seen from the inside of FimH. The 2Fo-Fc electron density for butyl α-Dmannoside is shown. FimH can make 14 possible hydrogen bonds (purple broken lines) with the non-reducing mannose. The only oxygen of mannose that is not involved in direct interaction with FimH is the axially oriented anomer O1, sticking outwards of the pocket and in this structure linked to butyl. This agrees with the receptor binding site of FimH being able to bind only to terminally exposed mannose residues on high-mannose glycans. The mannose-binding pocket is a small, deep and negatively charged pocket at the tip of the FimH adhesin (Figure 10). Bound mannose makes direct hydrogen bonds to the side chains of residues Asp54, Gln133, Asn135, and Asp140, to the positively charged amino terminus, and to the main chain of Asp47. There are also indirect water-mediated hydrogen bonds from O2 of mannose to the side chain of Gln133 and to the main chain oxygen of Phe1 and Gly14. The water molecule mediating these contacts fills up the space between O2 of mannose and the Phe144 side chain that together with the side chain of Ile13 defines the bottom of the binding site. A collar of hydrophobic residues extends from the mannose-binding pocket towards the tip of the FimH molecule. The high ridge of the collar is bordered by two tyrosine residues, Tyr48 and Tyr137, referred to as the tyrosine gate 51. These positioning of their aromatic side chains explain the relatively strong binding of aromatically substituted mannose residues, such as pnitrophenyl mannopyranoside and methyl umbelliferyl mannopyranoside. Moreover, the hydrophobic collar around the binding site provides a wide hydrophobic slide to direct electrostatic attraction of the polar mannose residues into the small and charged mannosebinding pocket. Analyses of FimH receptor specificity have initially been performed by using whole fimbriated bacteria. It has been possible to compare these results with those of the isolated and soluble receptor-binding domain of FimH (residues 1-158) and even purified type 1 fimbriae. Solution affinity equilibrium constants for FimH binding to a series of carbohydrates were obtained in two ways, using a competitive surface plasmon resonance (SPR) assay and in a tritiated mannose displacement assay 51. In these binding studies, FimH was confirmed not only to be highly specific, but also to have an unusual high affinity (Kd = 2.3 µM) for mannose. High affinity had already been suggested from the crystal structure of the FimH-mannose complex, because of the very tight network of hydrogen bonds involved in mannose binding, with in total 14 potential hydrogen bonds involving all of the mannose’s oxygen atoms except for the α-. Page 22 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(27) HDR de Julie Bouckaert, Lille 1, 2012. anomeric O1 atom50. Of all the tested mono- and disaccharides other than mannose, (methyl 2deoxy-α-D-mannopyranoside, glucose, galactose, fructose, sucrose, and turanose), only fructose has an affinity approaching that of FimH for mannose, binding with only 15-fold lower affinity. The relatively tight binding of fructose is presumably due to ring opening and conversion to the pyranose form of fructose (Frup). Docking predicts that Frup is differently oriented in the binding site compared to mannose (Figure 11), allowing it to bind with only one hydrogen bond less than mannose. This hydrogen bond is replaced by a hydroxyl-methyl group interaction.. Figure 11. Stereo figure of fructose superimposed on mannose in the FimH binding pocket. Fructose in green (for clarity of the picture, each atom was shifted 0.3 Å from the ideal superposition), mannose in grey. The two different Tyr48 side chain conformations are shown. Note the lack of O1 of mannose (anomeric oxygen) and the presence of (an extra) hydroxyl on C2 (the equivalent of C5 of mannose) that is in close contact (2.7 Å) with Ile52 (orange dashed bond).. 2.2 Recognition of oligosaccharides by FimH and fine specificity The physiological receptors for FimH are N-glycosylated proteins carrying high-mannose structures. The largest high-mannose glycan, oligomannose-9 (Figure 12), is fully substituted with terminal α1-2 linked mannosides on its D1, D2, and D3 arms. It has long been known that only mannose at the end of such an arm can bind in the mannose binding pocket of FimH. In oligomannose-9, all three terminal mannose residues at its non-reducing ends are potential candidates for binding in the monosaccharide-binding pocket of FimH. The affinity indeed increases with a factor of three for oligomannose-9 over Manα1-2Man, which at the same time indicates that the binding of oligomannose-9 is not polyvalent. In other words, not all three. Page 23 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(28) HDR de Julie Bouckaert, Lille 1, 2012. terminal residues are bound simultaneously by one FimH molecule, but the probability to encounter Manα1-2Man has been increased three-fold. Prolongation of Manα1-2Man at its reducing end, as in Manα1-2Manα1-2Man, increases affinity only moderately and is glycosidic linkage-independent. These findings suggest that a complementary fit of the larger trisaccharides, providing more hydrogen bonds, more van der Waals interactions and burial of hydrophobic surface, is important for generating tighter binding.. Figure 12. Oligomannosidic glycans are recognized by the FimH adhesion. Epitope mapping on the N-linked high-mannose glycan with FimH on Biacore3000 revealed the highest affinity (Kd = 20 nM) for the Manα1-3Manα1-4GlcNAc epitope (boxed) on oligomannose-3 and -5, due to the presence of an unsubstituted D1 arm. Structures of the different oligomannosides are illustrated schematically to the right (squares = GlcNAc; circles = Man) together with their affinities for the FimH lectin domain (isolated from E. coli strain J96). Bottom right: Purified FimH lectin domains all have comparable affinity profiles. The group of Sharon first observed that other, higher-affinity epitopes, are hidden in oligomannose-9 and are only accessible in high-mannose substructures much smaller than oligomannose-9 52,53. Probing the fine specificity of FimH for high-mannose epitopes using a series of oligomannosides, corresponding to substructures of high-mannose N-linked glycans on. Page 24 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(29) HDR de Julie Bouckaert, Lille 1, 2012. proteins, revealed that for those oligomannosides (oligomannose-3 and -5) where the D1 arm is not capped at the non-reducing end by an α1-2 linked mannose residue (Fig. 11), the affinity is very high (Kd = 20 nM) 54. This affinity parallels the affinity of FimH for aryl (p-nitrophenyl αD-mannose Kd = 44 nM and methyl umbelliferyl α-D-mannose Kd = 20 nM), and alkyl mannosides (pentyl α-D-mannose, Kd = 25 nM, hexyl α-D-mannose, Kd = 10 nM, and heptyl αD-mannose,. Kd = 5 nM) 51.. The highest-affinity epitope for FimH, Manα1-3Manα1-4GlcNAc, is not free for binding to FimH in any of the recently elucidated glycan structures on mouse UPIa 37. The binding affinities of FimC:FimH chaperone-adhesin complexes with engineered point mutations in the mannose-binding pocket and the adhesion of recombinant type 1 fimbriated E. coli strains to human bladder tissue sections always correlated with in vitro binding to mono-mannose binding for each of the mutations, not with their (tighter) tri-mannose (Manα1-3(Manα1-6)Man) binding 50. . This may be understood from the fact that mannose residues that can function as FimH monomannose receptors are always present at the non-reducing termini (D1, D2 or D3 arm) of any high-mannose glycan substructure, whereas tri-mannose is only accessible at the non-reducing end of oligomannosides 7D1, 6, 5, and 3 (Figure 12). Only very low amounts of oligomannoside6 are found on mouse UPIa, and oligomannose 7, 8, and 9 occur in significantly higher amounts 37 . Hence tri-mannose is likely to be available for FimH binding to a much smaller extent than mono-mannose. Regardless of its origin, whether isolated from uropathogenic (J96, CI#4) or intestinal (F18, EH485, EH12) E. coli clinical isolates, the FimH lectin domain display its highest affinity towards oligomannosides-3 and -5 (Figure 12) 55. The recent crystal structure of FimH in complex with oligomannoside-3 disclosed the molecular basis for this observation. The moiety, Manα(1,3)Manβ(1,4)GlcNAcβ(1,4)GlcNAcβ, of oligomannoside-3 forms a close and extended interaction with amino acid residues, among which Tyr48 and Tyr137, that are indeed conserved among E. coli strains 56,57. An exception is the Asn135Lys mutation found in the EHEC strain O157:H7, that is functionally silent in mannose binding 50.. Page 25 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(30) HDR de Julie Bouckaert, Lille 1, 2012. Figure 13. Panel of FimH lectin domain interactions along the oligomannose-3 chain. A, Electron density map, contoured at 1 e/A° 3, for oligomannose-3 in the FimH receptor-binding site. The surface of the binding site is subdivided into its hydrophobic support platform (grey, residues Phe142, Phe1 and Ile13), its polar pocket (red, residues Asn46, Asp47, Asp54, Gln133, Asn135, Asn138 and Asp140), the tyrosine gate (blue, residues Tyr137, Ile52 and Tyr48) and residue Thr51 (green). B, Aromatic-to-saccharide stacking (green dashed lines) of the Tyr48 side chain onto the B-face of Man3. C, van der Waals support of the b1,4 linkage (yellow dashed lines) and an hydrophobic contact of C5 of GlcNAc2 by Tyr137 (green dashed lines). D, Thr51 tops of the site by hydrophilic (red), hydrophobic and van der Waals (yellow) interactions with the chitobiose. The anomeric O1 of GlcNAc1 would be exchanged for by the nitrogen of the amide of aspargine on a receptor glycoprotein for FimH.. Page 26 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(31) HDR de Julie Bouckaert, Lille 1, 2012. . 2.3 The type-1 fimbrial adhesin FimH of Escherichia coli is an important virulence factor in urinary tract infections and inflammatory bowel diseases FimH is the type-1 fimbrial adhesin and invasin of Escherichia coli, the most prevalent causative agent of urinary tract infection (UTI) or cystitis. Mannosides and also antibodies directed against the FimH lectin domain prevent the first step in the pathogenic cascade by interfering with FimH-mediated bacterial adhesion.28 Pioneering work by Sharon showed that aromatic α-D-mannosides were approximatively 500 to 1000 times more effective than was methyl α-D-mannoside in inhibiting adherence of mannose-specific E. coli to guinea pig red blood cells or yeast agglutination. Additionally, we have previously shown that adherence and invasion of host cells by E. coli. can be prevented by alkyl α-D-mannosides, which are potent antagonists of interactions mediated by FimH. 58 This is likely due to hydrophobic interactions with one isoleucine (52) and two tyrosines (48 and 137) forming the entrance of the FimH binding site. 50,51. FimH inhibitory compounds to eradicate resilient infections, that involve bacteria both growing in dynamic intracellular bacterial communities or quiescent in underlaying bladder cell layers, are being explored in order to develop non-antibiotic anti-adhesives against urinary tract infections 43,47,58-60. The increasing prevalence of E. coli isolates that are resistant to antimicrobial agents has stimulated interest in novel non-antibiotic methods for the prevention of UTIs. Women are significantly more likely to experience UTIs than men. During any given year, 11 percent of women report a UTI and nearly half of these women (44%) experience recurrent UTIs. Catheter-associated UTI is the most common nosocomial infection. Among the most important risk factors for UTIs is a history of previous UTIs, thus recurrent UTIs 61,62. The financial implications of all these UTIs are enormous, predominantly as a result of the high incidence.. The aromatic groups of ligands can pack between the two tyrosine side chains on the broad hydrophobic platform between the tyrosine rings. In a study around the serendipitous finding of a butyl mannoside in the mannose-binding pocket of FimH 51, it was shown that a linear heptyl chains on mannose increases the affinity for FimH beyond this for the aromatically substituted mannose. In addition to a strongly hydrophobic nature, alkyl chains retain significant conformational freedom while interacting to the broad hydrophobic platform of the tyrosine gate, as illustrated by the two alternative bound conformations observed in the two independent crystal structures obtained for the FimH: butyl mannoside complex 51. Alkyl chains substituted on α-D-. Page 27 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(32) HDR de Julie Bouckaert, Lille 1, 2012. mannosides may thus limit their loss in entropy upon binding by not choosing a defined conformation, which is indeed also apparent from their entropy contributions for binding FimH as measured using isothermal calorimetry 63.. 2.4 Lead optimization based on thermodynamics and structures Alkyl mannosides are antagonists for the FimH adhesin, located at the tip adhesin of type-1 fimbriae, by inhibiting the binding and invasion of type-1 fimbriated E. coli in bladder and intestinal cells. The binding of butyl α-D-mannose has been observed in two different crystal structures, where the butyl on O1 of mannose takes on 2 different conformations, along with the aromatic side chain of Tyr48. In all mannose-bound FimH structures, mannose is invariantly buried in a deep, charged pocket. On the contrary, the alkyl tail shuffles over a large hydrophobic platform extending from the mannose-binding pocket (Figure 14, right). This broad platform is composed of 2 tyrosines on either side and an isoleucine backing up this so-called tyrosine gate 51 . Tyr48, makes strong aromatic stacking interactions with the butyl tail sticking out of the mannose-binding pocket, in both crystal structures (Figure 14).. Figure 14. Tyr48 stacking with the butyl of butyl α-D-mannose in conformational concordancy Isothermal titration calorimetry (ITC) data indicate an increased contribution in enthalpy when the alkyl is substituted for by an aryl. A larger interaction interface between Tyr48 and a phenyl group on O1 of mannose has been predicted in models 51 and confirmed in several crystal structures 63. However, the decrease in entropy for the phenyl is also larger than for the alkyl and it is still the alkyl mannosides that have the best affinity for FimH. It thus appears that the moves of the heptyl over the hydrophobic interaction surface, in harmony with Tyr48 in an aromatic stacking interaction, are very important for the retention of entropy of the alkyl tail upon binding of heptyl α-D-mannose and that this explains the high affinity of this lead compound. This is revealing and moreover very much of relevance in development of carbohydrate-based drugs in. Page 28 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(33) HDR de Julie Bouckaert, Lille 1, 2012. the field of protein-carbohydrate interactions and, eg. to treat urinary and intestinal infections caused by type-1 fimbriated E. coli. In the crystal structures, we only obtain a static view of two butyl α-D-mannose and Tyr48 conformations. The glycan binding site is often used in crystal lattice interactions in several space groups, restricting Tyr48 movement and thus at least stabilizing and favoring certain alkyl conformations. Besides the snapshots obtained from crystal structures, there is a need for methods (NMR) to study the dynamics of the ligand and the receptor in the binding event.. 2.5 Characterizing binding interactions by ITC Since all binding events are accompanied by the evolution of heat (a change in enthalpy, ∆H), a full thermodynamic characterization of the binding reaction provides fundamental information about the molecular interactions driving the binding process. If the reaction being studied is, for example, the binding of a drug candidate to a mutant protein, this thermodynamic information could suggest alterations to the chemical structure of the drug which would improve molecular interactions at the protein-drug interface, increased specificity for the mutant protein, heightened binding and enhanced efficacy of the drug. At constant temperature and pressure, ITC measures the enthalpy of a binding reaction. A solution of one component (for example, a ligand such as a drug candidate) is titrated into a dilute solution of the second component (for example a protein). At each injection of ligand into sample, the equilibrium of free and bound ligand is established, and heat is released (exothermic) or absorbed (endothermic). At the end of the titration, all the binding sites in the sample are occupied and the equilibrium association constant (Ka) can be calculated 64. Since temperature (T) is held constant throughout, the free energy ∆G of the binding reaction can be determined by: ∆G = -RT ln Ka Integration of heat changes measures ∆H, and the change in entropy ∆S can be determined by: ∆S = (∆H - ∆G). T Knowing the concentration of the two components, the stoichiometry n of the binding reaction can be determined in addition to the association constant, and the enthalpy and entropy of the binding reaction. Quantification of these thermodynamic parameters reveals the physical processes involved in the binding reaction. A spontaneous binding process must have a negative ∆G, and ∆G will become increasingly negative as binding becomes tighter. The enthalpic contribution to binding is primarily due to an increased number of hydrogen bonds with optimal. Page 29 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
(34) HDR de Julie Bouckaert, Lille 1, 2012. (linear) donor-acceptor geometry and distance at the ligand-target interface, and to more favorable van der Waals interactions between the two interacting molecules; the hydrophilicity of the system (polar gate effect 65) will determine how important electrostatic, polar and dipolar interactions will be in driving the reaction. The entropic contribution has two primary components: conformational changes such as folding or unfolding of the macromolecules, and the release of bound solvent as hydrophobic groups interact. The large number of ordered water molecules released into the bulk solvent when the hydrophobic surfaces of the ligand and target interact provides the main driving force in hydrophobic interactions. This driving force is sufficient to compensate for the unfavorable conformational entropy of the macromolecule and ligand caused by the decreased conformational and rotational freedom following binding. In addition to the entropic effect, burial of surface area also affects the heat capacity of the sample, since water molecules ordered at hydrophobic surfaces have a different heat capacity from that of the water that has been released into the bulk solvent following binding.. 2.6 Fundamentals of interactions applied in drug design Biological interactions are bound to take place in aqueous liquid. When solutes are unbound, water is solvating their surface. The binding sites of lectins for their carbohydrates are evolutionary selected to react with their ligands in water and thermodynamics drives this interaction. The thermodynamics of interactions are equilibrium events between different molecular states and are determined by the number and type of bonds that are made by the component molecules. The stabilization of the system is likely to result to an important degree from the establishment of an energetically more favorable assortment of hydrogen bonds including the reorganizations in the aqueous phase when the complementary surfaces become associated. The potential for attractive and repulsive electrostatic interactions, hydrogen bond formation, polarization and induction and London dispersion forces all contribute to transition from one defined state to another. Monte Carlo simulations on the GSIV lectin of the changes in hydration which accompany complex formation indicate that special hydration effects are felt about the periphery of the combining site for about 6 A before these are attenuated to properties of bulk water. Changing a key polar hydroxyl group to a halogen, an amino group or a methoxy group essentially inactivates compounds, resembling virtually inviolate stereoelectronic complementarity requirements. From: Lemieux R.U., the chemical mapping of proteincarbohydrate complexes, in Complex carbohydrates in drug research, Alfred Benzon symposium, 36 p 188-197 (1994). Equally, changing mannose for 2-deoxymannose decreased affinity for FimH dramatically 51.. Page 30 of 190 © 2014 Tous droits réservés.. doc.univ-lille1.fr.
Documents relatifs