Interaction of the Crystalline Bacterial Cell Surface Layer Protein SbsB and the Secondary Cell Wall Polymer of Geobacillus stearothermophilus PV72 Assessed by Real-Time Surface Plasmon Resonance Biosensor Technology

Abstract

The interaction between S-layer protein SbsB and the secondary cell wall polymer (SCWP) of Geobacillus stearothermophilus PV72/p2 was investigated by real-time surface plasmon resonance biosensor technology. The SCWP is an acidic polysaccharide that contains N-acetylglucosamine, N-acetylmannosamine, and pyruvic acid. For interaction studies, recombinant SbsB (rSbsB) and two truncated forms consisting of either the S-layerlike homology (SLH) domain (3SLH) or the residual part of SbsB were used. Independent of the setup, the data showed that the SLH domain was exclusively responsible for SCWP binding. The interaction was found to be highly specific, since neither the peptidoglycan nor SCWPs from other organisms nor other polysaccharides were recognized. Data analysis from that setup in which 3SLH was immobilized on a sensor chip and SCWP represented the soluble analyte was done in accordance with a model that describes binding of a bivalent analyte to a fixed ligand in terms of an overall affinity for all binding sites. The measured data revealed the presence of at least two binding sites on a single SCWP molecule with a distance of about 14 nm and an overall K d of 7.7 ؋ 10 ؊7 M. Analysis of data from the inverted setup in which the SCWP was immobilized on a sensor chip was done in accordance with an extension of the heterogeneous-ligand model, which indicated the existence of three binding sites with low (K d ‫؍‬ 2.6 ؋ 10 ؊5 M), medium (K d ‫؍‬ 6.1 ؋ 10 ؊8 M), and high (K d ‫؍‬ 6.7 ؋ 10 ؊11 M) affinities. Since in this setup 3SLH was the soluble analyte and the presence of small amounts of oligomers in even monomeric protein solutions cannot be excluded, the high-affinity binding site may result from avidity effects caused by binding of at least dimeric 3SLH. Solution competition assays performed with both setups confirmed the specificity of the protein-carbohydrate interaction investigated.In many bacteria and archaea, crystalline cell surface layers (S-layers) represent the outermost cell envelope component (37, 42). S-layers are composed of identical species of protein or glycoprotein subunits that are arranged in monomolecular crystalline arrays and cover the entire cell surface during all stages of cell growth and division. S-layer lattices show oblique (p1, p2), square (p4), or hexagonal (p3, p6) symmetry and possess pores of uniform size and morphology. Individual Slayer subunits interact with each other and with the supporting cell envelope components through noncovalent forces. After removal of the disrupting agent used for their isolation, extracted S-layer (glyco)proteins frequently maintain the ability to recrystallize in suspension or on various phase interfaces. This first-order self-assembly process leads to lattices that are identical to those observed on intact cells (33,36,41).On the basis of sequence comparisons, the existence of a cell wall-targeting domain on surface proteins, including S-layer proteins and cell-associated exoenzymes of gram-positive bacteria, or outer membrane proteins of g...

Interactions between TonB from Escherichia coli and the Periplasmic Protein FhuD

“…SPR data were fit to the heterogeneous ligand model, a model previously used to distinguish independent binding sites on biological macromolecules (32,33). Similar to fluorescence, fits of the data to this model quantified two binding sites as follows: a high affinity site with K D values in the low nanomolar range and a weak affinity site with K D values in the low micromolar range.…”

High-Affinity Interaction between the S-Layer Protein SbsC and the Secondary Cell Wall Polymer ofGeobacillus stearothermophilusATCC 12980 Determined by Surface Plasmon Resonance Technology

“…Molecular masses in kilodaltons are indicated on the left. Ϫ8 M), and high (K d ϭ 6.7 ϫ 10 Ϫ11 M) affinities have been described previously (15). Furthermore, for rSbpA , the minimal SCWP-binding form of the S-layer protein SbpA, three K d values corresponding to low (K d ϭ 2.4 ϫ 10 Ϫ2 ), medium (K d ϭ 3.0 ϫ 10 Ϫ8 ), and high (K d ϭ 1.2 ϫ 10 Ϫ11 ) affinities could be determined.…”

“…In Bacillaceae, at least two types of mechanisms for the binding of S-layer proteins and SCWPs have been identified. The first one involves so-called S-layer homologous (SLH) domains and pyruvylated SCWPs (3,4,8,9,(14)(15)(16)(17)(19)(20)(21)(22) and was previously found to be widespread among prokaryotes (16). By using surface plasmon resonance (SPR) spectroscopy, it has been demonstrated that the SLH domain of the S-layer protein SbsB of Geobacillus stearothermophilus PV72/p2 shows specific affinity for the pyruvylated SCWP of this organism but that it does not bind to any other type of SCWP or to peptidoglycan (PG) or other heteropolysaccharides (15).…”

“…Kinetic data were analyzed in terms of the "heterogeneous ligand-parallel reactions" model as described in a previous study (15). To avoid the effects of sample dispersion, the first 10 s of the wash-on and wash-out phases was omitted from the fits (13).…”

“…For quantitative analyses of the binding between rSbsC and rSbsC , which represented the soluble analytes, and the immobilized SCWP, the SCWP of G. stearothermophilus ATCC 12980 was immobilized on the CM5 sensor chip after the introduction of carbohydrazide at its reducing end (15) by applying the standard amine coupling FIG. 1.…”