The Structure and Binding Behavior of the Bacterial Cell Surface Layer Protein SbsC

Pavkov, Tea; Egelseer, Eva M.; Tesarz, Manfred; Svergun, Dmitri I.; Sleytr, Uwe B.; Keller, Walter

doi:10.1016/j.str.2008.05.012

Cited by 72 publications

(90 citation statements)

References 70 publications

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“…The radius of gyration is in good agreement with the hydrodynamic radius (2.8 nm) determined by DLS measurements. From I 0 we calculated the apparent molecular weight of TraMÁ in solution, using BSA (bovine serum albumin) as a molecular-weight standard (Pavkov et al, 2008). The value of 20.1 kDa is in good agreement with the theoretical molecular weight of TraMÁ (18.6 kDa) and with the observation from gel filtration (24.4 kDa).…”

Section: Biophysical Characterizationsupporting

confidence: 67%

Crystallization and preliminary structure determination of the transfer protein TraM from the Gram-positive conjugative plasmid pIP501

et al. 2013

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The major means of horizontal gene spread (e.g. of antibiotic resistance) is conjugative plasmid transfer. It presents a serious threat especially for hospitalized and immuno-suppressed patients, as it can lead to the accelerated spread of bacteria with multiple antibiotic resistances. Detailed information about the process is available only for bacteria of Gram-negative (GÀ) origin and little is known about the corresponding mechanisms in Gram-positive (G+) bacteria. Here we present the purification, biophysical characterization, crystallization and preliminary structure determination of the TraM C-terminal domain (TraMÁ, comprising residues 190-322 of the full-length protein), a putative transfer protein from the G+ conjugative model plasmid pIP501. The crystals diffracted to 2.5 Å resolution and belonged to space group P1, with unitcell parameters a = 39.21, b = 54.98, c = 93.47 Å , = 89.91, = 86.44, = 78.63 and six molecules per asymmetric unit. The preliminary structure was solved by selenomethionine single-wavelength anomalous diffraction.

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Section: Biophysical Characterizationsupporting

confidence: 67%

Crystallization and preliminary structure determination of the transfer protein TraM from the Gram-positive conjugative plasmid pIP501

et al. 2013

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“…As a basis, a detailed understanding of the mechanisms underlying S-layer protein display on the bacterial cell is required. Above that, the involvement of S-layers in cell adhesion and surface recognition and their function as virulence factors (2)(3)(4)(5) have prompted in-depth research on that subject.…”

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confidence: 99%

Are the Surface Layer Homology Domains Essential for Cell Surface Display and Glycosylation of the S-Layer Protein from Paenibacillus alvei CCM 2051T?

Janesch

Messner

Schäffer

2012

Journal of Bacteriology

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Paenibacillus alvei CCM 2051T cells are decorated with a two-dimensional (2D) crystalline array comprised of the glycosylated S-layer protein SpaA. At its N terminus, SpaA possesses three consecutive surface layer (S-layer) homology (SLH) domains containing the amino acid motif TRAE, known to play a key role in cell wall binding, as well as the TVEE and TRAQ variations thereof. SpaA is predicted to be anchored to the cell wall by interaction of the SLH domains with a peptidoglycan (PG)-associated, nonclassical, pyruvylated secondary cell wall polymer (SCWP). In this study, we have analyzed the role of the three predicted binding motifs within the SLH domains by mutating them into TAAA motifs, either individually, pairwise, or all of them. Effects were visualized in vivo by homologous expression of chimeras made of the mutated S-layer proteins and enhanced green fluorescent protein and in an in vitro binding assay using His-tagged SpaA variants and native PG-containing cell wall sacculi that either contained SCWP or were deprived of it. Experimental data indicated that (i) the TRAE, TVEE, and TRAQ motifs are critical for the binding function of SLH domains, (ii) two functional motifs are sufficient for cell wall binding, regardless of the domain location, (iii) SLH domains have a dual-recognition function for the SCWP and the PG, and (iv) cell wall anchoring is not necessary for SpaA glycosylation. Additionally, we showed that the SLH domains of SpaA are sufficient for in vivo cell surface display of foreign proteins at the cell surface of P. alvei. Bacterial cell surface layers (S-layers) are a distinct type of cell surface decoration (1) enabled by monomolecular self-assembly of individual (glyco)proteins on the supporting cell envelope layer into a two-dimensional (2D) crystalline array exhibiting periodicity on the nanometer scale. These structures hold a great promise for in vivo cell surface display of biofunctional epitopes by protein and/or glycosylation engineering, with possible relevance for basic as well as applied research, encompassing biotechnology and therapy. As a basis, a detailed understanding of the mechanisms underlying S-layer protein display on the bacterial cell is required. Above that, the involvement of S-layers in cell adhesion and surface recognition and their function as virulence factors (2-5) have prompted in-depth research on that subject.Bacterial cell surface display is an evolutionary optimized strategy to express molecules of interest on the exterior of cells by using natural microbial functional components. Many of these cell surface anchors are proteins exhibiting functions in pathogenesis or cell wall maintenance; they are either membrane associated or integrated or cell wall associated, with either covalent or noncovalent linkage (6, 7). Also other components, such as choline residues of (lipo)teichoic acids, can be involved in the binding mechanism (8). For S-layers of Gram-positive bacteria, still another binding mechanism exists: S-layers are noncovalently attached to th...

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“…It has been assumed that the difficulty in obtaining three-dimensional crystals is the consequence of the propensity for two-dimensional assembly, which prevents the proteins from being sufficiently well behaved for crystallization. Despite that, there are a few examples of limited success for portions of S layers (38,39). Moreover, many studies have been conducted on isolated in vitro S-layer sheets using negative-stain electron microscopy.…”

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Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

et al. 2010

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The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.Surface layers (S layers) are the outermost cell wall component in many archaea and bacteria (6, 44). Most S layers are composed of a single protein or glycoprotein species that selforganizes into two-dimensional (2D) lattices of various sizes, usually with square or hexagonal symmetry (7,14,43). This geometrical arrangement is almost the only commonality among species, since sequence homology between S-layer proteins is low and functionality differs in many cases. In many archaea, the S layer is the only cell wall component, so it may have a role in shape determination. However, in bacteria such as Caulobacter crescentus, the role is more likely related to protection against a variety of predatorial assaults (8).One interest in understanding S layers comes from their potential applications in nanotechnology (46) and therapeutic applications, such as anti-HIV microbicide development (37) and cancer therapy (9). The concept is to display heterologous proteins from within the S-layer structure in order to create dense arrays of foreign insertions. Resolving the S-layer organization and structure at high resolution in cells as close to a native state as possible is crucial to understand or predict where proteins are displayed in the array, particularly when more than one foreign peptide is being displayed simultaneously.A significant limitation has been the dif...

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The Structure and Binding Behavior of the Bacterial Cell Surface Layer Protein SbsC

Cited by 72 publications

References 70 publications

Crystallization and preliminary structure determination of the transfer protein TraM from the Gram-positive conjugative plasmid pIP501

Crystallization and preliminary structure determination of the transfer protein TraM from the Gram-positive conjugative plasmid pIP501

Are the Surface Layer Homology Domains Essential for Cell Surface Display and Glycosylation of the S-Layer Protein from Paenibacillus alvei CCM 2051T?

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

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