Crystallization and preliminary crystallographic analysis of an Ig-domain-encompassing fragment of the giant adhesion protein SiiE from<i>Salmonella enterica</i>

Sturm, Karina; Griessl, Martin H.; Wagner, Carolin; Deiwick, Jörg; Hensel, Michael; Muller, Yves A.

doi:10.1107/s1744309111032039

Cited by 4 publications

(6 citation statements)

References 11 publications

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“…Models for SiiE secretion, surface retention and SiiE-mediated adhesion. ( A ) SiiE is secreted in an unfolded conformation starting with the C-terminus that possesses the secretion signal [63]. Extracellular Ca 2+ ions, which are present in the LPS layer, bind to conserved D residues and pull SiiE out of the secretion system and facilitate protein folding after secretion (unpublished observations, MH and BB).…”

Section: Discussionmentioning

confidence: 99%

The Giant Adhesin SiiE of Salmonella enterica

Barlag

Hensel

2015

Molecules

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Salmonella enterica is a Gram-negative, food-borne pathogen, which colonizes the intestinal tract and invades enterocytes. Invasion of polarized cells depends on the SPI1-encoded type III secretion system (T3SS) and the SPI4-encoded type I secretion system (T1SS). The substrate of this T1SS is the non-fimbrial giant adhesin SiiE. With a size of 595 kDa, SiiE is the largest protein of the Salmonella proteome and consists of 53 repetitive bacterial immunoglobulin (BIg) domains, each containing several conserved residues. As known for other T1SS substrates, such as E. coli HlyA, Ca 2+ ions bound by conserved D residues within the BIg domains stabilize the protein and facilitate secretion. The adhesin SiiE mediates the first contact to the host cell and thereby positions the SPI1-T3SS to initiate the translocation of a cocktail of effector proteins. This leads to actin remodeling, membrane ruffle formation and bacterial internalization. SiiE binds to host cell apical membranes in a lectin-like manner. GlcNAc and α2-3 linked sialic acid-containing structures are ligands of SiiE. Since SiiE shows repetitive domain architecture, we propose a zipper-like binding mediated by each individual BIg domain. In this review, we discuss the characteristics of the SPI4-T1SS and the giant adhesin SiiE.

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Section: Discussionmentioning

confidence: 99%

The Giant Adhesin SiiE of Salmonella enterica

Barlag

Hensel

2015

Molecules

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“…We also produced a deletion version of SiiE termed mini-SiiE that lacks the central residues 404 to 5053. Mini-SiiE was recombinantly produced in Escherichia coli and purified to homogeneity as described in detail elsewhere (Sturm et al, 2011). The crystal structure determination was achieved with a smaller fragment of SiiE that covers BIg domains 50-52 of SiiE.…”

Section: Production Of Siie Variantsmentioning

confidence: 99%

“…The crystal structure determination was achieved with a smaller fragment of SiiE that covers BIg domains 50-52 of SiiE. SiiE BIg50:52 was obtained via limited proteolysis of mini-SiiE with proteinase K and its identity confirmed by mass spectrometry (Sturm et al, 2011). Single-molecule force spectroscopy experiments were performed in filtered PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4) with a MFP three-dimensional atomic force microscope (Asylum Research, Santa Barbara, CA).…”

Section: Production Of Siie Variantsmentioning

confidence: 99%

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Structural Insight into the Giant Ca2+-Binding Adhesin SiiE: Implications for the Adhesion of Salmonella enterica to Polarized Epithelial Cells

Griessl¹,

Schmid²,

Kassler³

et al. 2013

Structure

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SiiE from Salmonella enterica is a giant 5,559-residue-long nonfimbrial adhesin that is secreted by a type 1 secretion system (T1SS) and initiates bacterial adhesion to polarized host cells. Structural insight has been gained into the 53 bacterial Ig-like (BIg) domains of SiiE, which account for 94% of the entire SiiE sequence. The crystal structure of a fragment comprising BIg domains 50 to 52 of SiiE reveals the BIg domain architecture and highlights two types of SiiE-specific Ca²⁺-binding sites. Sequence homology considerations suggest that full-length SiiE interacts with more than 100 Ca²⁺ ions. Molecular dynamics simulations and single-molecule imaging indicate that Ca²⁺ binding confers SiiE with a rigid 200 nm rod-like habitus that is required to reach out beyond the Salmonella lipopolysaccharide layer and to promote adhesion to host cells. The crystal structure suggests plausible routes for the establishment of the initial contact between Salmonella and host cells.

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“…Crystallization trials with an engineered deletion variant where the N-terminal half of BIg2 was fused to the C-terminal half of BIg49 resulting in a variant containing aa 5-403 and aa 5053-5559 (mini-SiiE) were unsuccessful until now. Limited proteolysis of mini-SiiE identified a stable fragment, SiiE-BIg50-52, which readily crystallized [28] and finally led to its high resolution 3D structure [14]. With the aim to gain structural insight into the N-terminal region, two variants were selected for protein production.…”

Section: Siie N-terminal Regionmentioning

confidence: 99%

Recombinant protein production and purification of SiiD, SiiE and SiiF - components of the SPI4-encoded type I secretion system fromSalmonellaTyphimurium

Klingl

Kordes

Schmid

et al. 2020

Preprint

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AbstractIn humans, Salmonella enterica infections are responsible for a pleiotropy of medical conditions. These include intestinal inflammation and typhoid fever. The initial contact between Salmonella and polarized epithelial cells is established by the SPI4-encoded type I secretion system (T1SS), which secrets SiiE, a giant non-fimbrial adhesin. We have recombinantly produced various domains of this T1SS from Salmonella enterica serovar Typhimurium in Escherichia coli for further experimental characterization. We purified three variants of SiiD, the periplasmic adapter protein spanning the space between the inner and outer membrane, two variants of the SiiE N-terminal region and the N-terminal domain of the SiiF ATP-binding cassette (ABC) transporter. In all three proteins, at least one variant yielded high amounts of pure soluble protein. Proper folding and cooperative unfolding were investigated by circular dichroism (CD) spectroscopy. Secondary structure content estimations from CD spectra were in good agreement with the values derived from SiiD and SiiF homology models or, in case of the SiiE N-terminal region, a secondary structure prediction. For one SiiD variant, protein crystals could be obtained that diffracted X-rays to approximately 4 Å resolution.

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Crystallization and preliminary crystallographic analysis of an Ig-domain-encompassing fragment of the giant adhesion protein SiiE fromSalmonella enterica

Cited by 4 publications

References 11 publications

The Giant Adhesin SiiE of Salmonella enterica

The Giant Adhesin SiiE of Salmonella enterica

Structural Insight into the Giant Ca2+-Binding Adhesin SiiE: Implications for the Adhesion of Salmonella enterica to Polarized Epithelial Cells

Recombinant protein production and purification of SiiD, SiiE and SiiF - components of the SPI4-encoded type I secretion system fromSalmonellaTyphimurium

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