Crystallization of the F41 Fragment of Flagellin and Data Collection from Extremely Thin Crystals

doi:10.1006/jsbi.2000.4312

Journal of Structural Biology

Volume 132, Issue 2, November 2000, Pages 106-111

https://doi.org/10.1006/jsbi.2000.4312 Get rights and content

Abstract

Flagellin, which constructs supercoiled filaments of the bacterial flagellum, is very difficult to crystallize because of its strong tendency to polymerize. We therefore crystallized the F41 fragment of flagellin, which does not polymerize because terminal regions that play important roles in polymerization are cleaved off. F41 was crystallized by the hanging drop vapor diffusion method in a mixture of polyethylene glycol, glycerol, and isopropanol, with a reservoir solution covered with silicon oil. The two key factors for success in growing sufficiently large crystals were isopropanol and silicon oil, which worked well to reduce the otherwise very high nucleation rate that resulted in hundreds of tiny crystals. The crystals were grown to very thin plates with thickness less than 10 μm, which made the collection of diffraction data very difficult. Freezing and annealing of the crystals and irradiation at synchrotron beamlines had to be carried out by specific methods and under specific conditions for its structure analysis at 2.0-Å resolution.

References (24)

S Kanto et al.
Amino acids responsible for flagellar shape are distributed in terminal regions of flagellin
J. Mol. Biol.
(1991)
R.M Macnab et al.
Normal-to-curly flagellar transitions and their role in bacterial tumbling: Stabilization of an alternative quaternary structure by mechanical force
J. Mol. Biol.
(1977)
Y Mimori et al.
The structure of the R-type straight flagellar filament of Salmonella at 9 Å resolution by electron cryomicroscopy
J. Mol. Biol.
(1995)
Y Mimori-Kiyosue et al.
Locations of terminal segments of flagellin in the filament structure and their roles in polymerization and polymorphism
J. Mol. Biol.
(1997)
Y Mimori-Kiyosue et al.
Role of the outermost subdomain of Salmonella flagellin in the filament structure revealed by electron cryomicroscopy
J. Mol. Biol.
(1998)
D.G Morgan et al.
Structure of bacterial flagellar filaments at 11 Å resolution: Packing of the α-helices
J. Mol. Biol.
(1995)
F Vonderviszt et al.
Terminal regions of flagellin are disordered in solution
J. Mol. Biol.
(1989)
F Vonderviszt et al.
Structural organization of flagellin
J. Mol. Biol.
(1990)
F Vonderviszt et al.
Role of the disordered terminal regions of flagellin in filament formation and stability
J. Mol. Biol.
(1991)
I Yamashita et al.
Radial mass analysis of the flagellar filament of Salmonella: Implications for subunit folding
J. Mol. Biol.
(1995)

S Asakura

Polymerization of flagellin and polymorphism of flagella

Adv. Biophys.

(1970)

Cited by (29)

Flagellin glycoproteomics of the periodontitis associated pathogen selenomonas sputigena reveals previously not described O-glycans and rhamnose fragment rearrangement occurring on the glycopeptides
2018, Molecular and Cellular Proteomics
Flagellated, Gram-negative, anaerobic, crescent-shaped Selenomonas species are colonizers of the digestive system, where they act at the interface between health and disease. Selenomonas sputigena is also considered a potential human periodontal pathogen, but information on its virulence factors and underlying pathogenicity mechanisms is scarce. Here we provide the first report of a Selenomonas glycoprotein, showing that S. sputigena produces a diversely and heavily O-glycosylated flagellin C9LY14 as a major cellular protein, which carries various hitherto undescribed rhamnose- and N-acetylglucosamine linked O-glycans in the range from mono- to hexasaccharides. A comprehensive glycomic and glycoproteomic assessment revealed extensive glycan macro- and microheterogeneity identified from 22 unique glycopeptide species. From the multiple sites of glycosylation, five were unambiguously identified on the 437-amino acid C9LY14 protein (Thr149, Ser182, Thr199, Thr259, and Ser334), the only flagellin protein identified. The O-glycans additionally showed modifications by methylation and putative acetylation. Some O-glycans carried hitherto undescribed residues/modifications as determined by their respective m/z values, reflecting the high diversity of native S. sputigena flagellin. We also found that monosaccharide rearrangement occurred during collision-induced dissociation (CID) of protonated glycopeptide ions. This effect resulted in pseudo Y1-glycopeptide fragment ions that indicated the presence of additional glycosylation sites on a single glycopeptide. CID oxonium ions and electron transfer dissociation, however, confirmed that just a single site was glycosylated, showing that glycan-to-peptide rearrangement can occur on glycopeptides and that this effect is influenced by the molecular nature of the glycan moiety. This effect was most pronounced with disaccharides. This study is the first report on O-linked flagellin glycosylation in a Selenomonas species, revealing that C9LY14 is one of the most heavily glycosylated flagellins described to date. This study contributes to our understanding of the largely under-investigated surface properties of oral bacteria. The data have been deposited to the ProteomeXchange with identifier PXD005859.
Immune response of Atlantic salmon to recombinant flagellin
2011, Vaccine
Many viral vaccines used in aquaculture are unable to stimulate an appropriate level of immunity to withstand infection. By targeting specific components of the immune system it may be possible to trigger stronger, more effective responses to antigens. Flagellin has the ability to stimulate both the soluble and membrane-bound forms of toll-like receptor 5 (TLR5) in salmon leading to a proinflammatory response and activation of both the innate and adaptive immune system. In this study flagellin (FlaD from Vibrio anguillarum) was recombinantly produced in two forms, full-length (FDL) and a truncated form (FDS) with portions of the N- and C-termini removed to prevent polymerization. FDS was used to produce an antibody that was able to bind both forms of flagellin in immunoblot analysis. In cell culture using COS-7 cells, FDL was shown to stimulate the NF-κB pathway more effectively than FDS. Both forms of flagellin were used as an adjuvant with the antigen LPH (Hemocyanin from Limulus polyphemus hemolymph) in an immunization dose–response study. FDS and FDL stimulated the innate immune system of salmon inducing proinflammatory effects on days 2, 4 and 7 and the gene expression of important cytokines such as TNFα, IL-6, IL-8, and IL-1β were significantly up-regulated (p < 0.05) in the spleen. TLR5S was more highly up-regulated than TLR5M indicating that the soluble form of TLR5 may play an important role in the innate immune response in salmon. ELISA analysis showed that the use of flagellin as an adjuvant with LPH was not able to significantly induce flagellin or LPH antibodies. This study shows that flagellin has the potential to be a highly effective adjuvant for salmon immunization, but further research is needed.
Recombinant flagellins with partial deletions of the hypervariable domain lose antigenicity but not mucosal adjuvancy
2010, Biochemical and Biophysical Research Communications
Flagellin contains conserved N/C domains for TLR5 binding to activate innate immunity and a middle hypervariable domain harboring the major antigenic epitopes. However, conflict results existed in the previous studies as to whether the hypervariable domain was involved in the cytokine production and adjuvancy of flagellin. Here we constructed three flagellin variants (designated as FliCΔ190–278, FliCΔ220–320, and FliCΔ180–400) with deletions in the hypervariable domain. Our data demonstrated that all deletion variants lost substantial antigenicity but not mucosal adjuvancy. Surprisingly, the variant with deletion of amino acids 220–320 (FliCΔ220–320) induced higher production of IL-8, MCP-1, and TNF-α, and showed higher mucosal adjuvancy than full-length FliC flagellin. Our data supported the notion that the hypervariable domain was involved in the cytokine production by flagellin and more importantly demonstrated that the hypervariable domain was important for the mucosal adjuvancy of flagellin.
Glycosylation of Bacterial and Archaeal Flagellins
2010, Microbial Glycobiology
This chapter focuses on the structural diversity of flagellar glycans, methods for characterization of flagellin glycoproteins, and novel glycan biosynthetic pathways. The relevance of the glycosylation process to assembly as well as other novel biological roles is also discussed. The fllagellin protein of archaeabacteria Halobacterium halobium was shown to contain N-glycosidically linked oligosaccharides of sulfated (1→4)-linked glucuronic acid. More recently, the complete structural characterization of each of the four flagellin proteins FlaA, FlaB1, FlaB2, and FlaB3 from M. voltae revealed a novel trisaccharide of mass 779 attached in N-linkage to a total of 14 sites. Structural characterization of Gram-negative bacteria Listeria monocytogenes revealed that the FlaA structural protein monomer is covalently modified with O-linked β-GlcNAc at 3–6 sites per subunit. Motility has been recognized as critical to the pathogenic process for the human pathogen Pseudomas aeruginosa as well as the plant pathogen Pseudomonas syringae, and both of these organisms produce glycosylated flagella. The use of mass spectrometry for characterization of flagellin glycan structures has been substantial. Typically, structural analysis begins with accurate mass measurements of the purified intact glycoprotein to determine if the measured mass of the flagellin is consistent with the predicted protein mass and to deduce the mass of the carbohydrate residues. This is then followed by determination of the nature of the carbohydrate modifications and the sites at which the modifications occur.
Glycosylation of bacterial and archaeal flagellins
2009, Microbial Glycobiology: Structures, Relevance and Applications
This chapter focuses on the structural diversity of flagellar glycans, methods for characterization of flagellin glycoproteins, and novel glycan biosynthetic pathways. The relevance of the glycosylation process to assembly as well as other novel biological roles is also discussed. The fllagellin protein of archaeabacteria Halobacterium halobium was shown to contain N-glycosidically linked oligosaccharides of sulfated (1→4)-linked glucuronic acid. More recently, the complete structural characterization of each of the four flagellin proteins FlaA, FlaB1, FlaB2, and FlaB3 from M. voltae revealed a novel trisaccharide of mass 779 attached in N-linkage to a total of 14 sites. Structural characterization of Gram-negative bacteria Listeria monocytogenes revealed that the FlaA structural protein monomer is covalently modified with O-linked β-GlcNAc at 3–6 sites per subunit. Motility has been recognized as critical to the pathogenic process for the human pathogen Pseudomas aeruginosa as well as the plant pathogen Pseudomonas syringae, and both of these organisms produce glycosylated flagella. The use of mass spectrometry for characterization of flagellin glycan structures has been substantial. Typically, structural analysis begins with accurate mass measurements of the purified intact glycoprotein to determine if the measured mass of the flagellin is consistent with the predicted protein mass and to deduce the mass of the carbohydrate residues. This is then followed by determination of the nature of the carbohydrate modifications and the sites at which the modifications occur.
The FliS chaperone selectively binds the disordered flagellin C-terminal D0 domain central to polymerisation
2003, FEMS Microbiology Letters
Assembly of each Salmonella typhimurium flagellum filament requires export and polymerisation of ca. 30 000 flagellin (FliC) subunits. This is facilitated by the cytosolic chaperone FliS, which binds to the 494 residue FliC and inhibits its polymerisation. Yeast two-hybrid assays, co-purification and affinity blotting showed that FliS binds specifically to the C-terminal 40 amino acid component of the disordered D0 domain central to polymerisation. Without FliS binding, the C-terminus is degraded. Our data provide further support for the view that FliS is a domain-specific bodyguard preventing premature monomer interaction.

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Abstract

References (24)

Amino acids responsible for flagellar shape are distributed in terminal regions of flagellin

J. Mol. Biol.

Normal-to-curly flagellar transitions and their role in bacterial tumbling: Stabilization of an alternative quaternary structure by mechanical force

J. Mol. Biol.

The structure of the R-type straight flagellar filament of Salmonella at 9 Å resolution by electron cryomicroscopy

J. Mol. Biol.

Locations of terminal segments of flagellin in the filament structure and their roles in polymerization and polymorphism

J. Mol. Biol.

Role of the outermost subdomain of Salmonella flagellin in the filament structure revealed by electron cryomicroscopy

J. Mol. Biol.

Structure of bacterial flagellar filaments at 11 Å resolution: Packing of the α-helices

J. Mol. Biol.

Terminal regions of flagellin are disordered in solution

J. Mol. Biol.

Structural organization of flagellin

J. Mol. Biol.

Role of the disordered terminal regions of flagellin in filament formation and stability

J. Mol. Biol.

Radial mass analysis of the flagellar filament of Salmonella: Implications for subunit folding

J. Mol. Biol.

Polymerization of flagellin and polymorphism of flagella

Adv. Biophys.

Cited by (29)

Flagellin glycoproteomics of the periodontitis associated pathogen selenomonas sputigena reveals previously not described O-glycans and rhamnose fragment rearrangement occurring on the glycopeptides

Immune response of Atlantic salmon to recombinant flagellin

Recombinant flagellins with partial deletions of the hypervariable domain lose antigenicity but not mucosal adjuvancy

Glycosylation of Bacterial and Archaeal Flagellins

Glycosylation of bacterial and archaeal flagellins

The FliS chaperone selectively binds the disordered flagellin C-terminal D0 domain central to polymerisation

Regular ArticleCrystallization of the F41 Fragment of Flagellin and Data Collection from Extremely Thin Crystals

Abstract

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

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J. Mol. Biol.

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J. Mol. Biol.

J. Mol. Biol.

Polymerization of flagellin and polymorphism of flagella

Adv. Biophys.

Regular Article
Crystallization of the F41 Fragment of Flagellin and Data Collection from Extremely Thin Crystals