Distinct affinity of binding sites for S-layer homologous domains in Clostridium thermocellum and Bacillus anthracis cell envelopes

Abstract

Binding parameters were determined for the SLH (S-layer homologous) domains from the Clostridium thermocellum outer layer protein OlpB, from the C. thermocellum S-layer protein SlpA, and from the Bacillus anthracis S-layer proteins EA1 and Sap, using cell walls from C. thermocellum and B. anthracis. Each SLH domain bound to C. thermocellum and B. anthracis cell walls with a different KD, ranging between 7.1 x 10(-7) and 1.8 x 10(-8) M. Cell wall binding sites for SLH domains displayed different binding specificities in C. thermocellum and B. anthracis. SLH-binding sites were not detected in cell walls of Bacillus subtilis. Cell walls of C. thermocellum lost their affinity for SLH domains after treatment with 48% hydrofluoric acid but not after treatment with formamide or dilute acid. A soluble component, extracted from C. thermocellum cells by sodium dodecyl sulfate treatment, bound the SLH domains from C. thermocellum but not those from B. anthracis proteins. A corresponding component was not found in B. anthracis.

FIG. 1

Schematic representation of SLH polypeptides purified from E. coli. The triplicated segments of SLH domains extending between amino acids indicated by the numbers are shown by boxes. Amino acids encoded by the expression vector are indicated. The SLH domain of OlpB carries a circular permutation (6), resulting in the location of the N-terminal part of the first SLH segment at the C terminus of the third SLH segment.

FIG. 2

Binding of SLH domains to cell walls of C. thermocellum containing 34.6 μM meso-diaminopimelate (A) and to cell walls of B. anthracis containing 97.3 μM meso-diaminopimelate (B). Curve 1, OlpB_1391-1664 (▾); curve 2, SlpA_27-235 (⧫); curve 3, EA1_32-213 (▴); curve 4, Sap_32-211 (■).

FIG. 3

Binding of MalE-OlpB_1391-1664 to C. thermocellum cell walls subjected to various extraction procedures. Native cell walls (a) were treated with formamide at 100°C (b) or 150°C (c), with 25 mM glycine-HCl (pH 2.5) (d), with 0.1 M HCl (e), or with 48% HF (f). Lanes: S, soluble fraction; W, wash fraction; I, insoluble fraction. The leftmost lane shows positions of molecular size markers (in kilodaltons).

FIG. 4

Binding of OlpB_1391-1664 (A), SlpA_27-235 (B), EA1_32-213 (C), or Sap_32-211 (D) polypeptides labeled with ¹²⁵I to the SDS-soluble component extracted from C. thermocellum (lane 1) or B. anthracis (lane 2). The masses (in kilodaltons) and positions of migration of molecular size markers are indicated on the left.