Volume 16, Issue 8, Pages 1226-1237 (August 2008) The Structure and Binding Behavior of the Bacterial Cell Surface Layer Protein SbsC  Tea Pavkov, Eva M. Egelseer, Manfred Tesarz, Dmitri I. Svergun, Uwe B. Sleytr, Walter Keller  Structure  Volume 16, Issue 8, Pages 1226-1237 (August 2008) DOI: 10.1016/j.str.2008.05.012 Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 1 Domain Structure of SbsC (A) rSbsC(31–844) exhibits a ring-like conformation where domain VI folds back onto domain I in the crystal structure. Domains I, II, and III are shown in ribbon, and domains IV, V and VI in surface representation, indicating a partial disorder. Flexible linkers are shown in yellow. (B) rSbsC(31–443) containing domains I, II and III is shown in ribbon presentation with domain I in the same orientation as in (A). (C) Schematic representation of the full-length SbsC protein. Structurally derived domains (I–VI) are colored consistently throughout Figure 1 and known domain borders are indicated by residue numbers. Sequence 1–31 represents the signal peptide, and the C-terminal part (gray) is of unknown domain structure. All structure figures were prepared with Pymol 2002 (DeLano Scientific; http://www.pymol.org). Structure 2008 16, 1226-1237DOI: (10.1016/j.str.2008.05.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 2 Domain Topology of rSbsC(31–443) Domain structure (right panel) and topology diagram (left panel) of domains I, II, and III in the crystal structure of rSbsC(31–443). (A) Domain I represents a novel, all α-helical fold consisting of three triple-helical bundels that are connected by two continuous helices α3 and α5. The bulged-out loop within helix α3 is colored magenta. (B) Domain II forms β sandwich resembling Ig-like h-type fold. The dashed line in the topology diagram indicates the separation of the two sheets; the additional β-ribbon is labeled F′G′. (C) Domain III represents a novel topology not resembling any of the structures deposited in the PDB. Structure 2008 16, 1226-1237DOI: (10.1016/j.str.2008.05.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 3 Interdomain Flexibility of SbsC (A) Superposition of domain I (32–158) indicating flexibility between the second and the third triple-helix bundle of domain I and between domains I and II. (B) Superposition of domain II (262–334). rSbsC(31–443) is colored orange, and the first three domains of rSbsC(31–844) are colored blue. Arrows indicate regions of high flexibility. Structure 2008 16, 1226-1237DOI: (10.1016/j.str.2008.05.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 4 Solution Structure of the N-Terminal Deletion Mutant rSbsC(447–1099) (A) P(r) function and (B) the most probable ab initio bead model of rSbsC(447–1099) obtained by averaging and filtering 10 GASBOR models. Structure 2008 16, 1226-1237DOI: (10.1016/j.str.2008.05.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 5 Putative SCWP Binding Site (A) The ribbon diagram represents the structure of the SCWP binding domain, including the residues of the putative ligand binding site (shown as stick model; Lys, yellow; Arg, green; Tyr, cyan; His, orange). (B) The electrostatic potential mapped onto the surface of domain I shows a positively charged trace extending along the putative SCWP binding site (marked in green), which is preferentially targeting the negatively charged SCWP. The electrostatic potential is colored from red (−16 kTe−1) to blue (+16 kTe−1). The orientation is the same as in Figure 5A. Structure 2008 16, 1226-1237DOI: (10.1016/j.str.2008.05.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 6 SCWP Binding Behavior of Soluble N-Terminal SbsC Mutants (A and B) Thermostability of the SbsC mutants and their complexes with SCWP integrated by CD spectroscopy. The normalized CD signal at various temperatures was used for fitting the thermal unfolding curve, and the transition temperatures, Tm, are indicated in the plots with Origin 5.0 (Microcal, Northampton, MA). (C and D) ITC of the binding reaction between SCWP and rSbsC(31-270) (C) and rSbsC(31–443) (D).The upper panels show the sequential titration of SCWP into the respective protein solutions. The lower panels show the binding enthalpies per injection step (closed red square), the calculated binding isotherm according to an independent site binding model (continuous red line), and the derived binding parameters (H = overall molar binding enthalpy; K = molar binding constant; N = molar ratio at the inflection point). Structure 2008 16, 1226-1237DOI: (10.1016/j.str.2008.05.012) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 7 Characterization of the rSbsC(31–443)-SCWP Complex (A) Complex formation with different rSbsC(31–443)-SCWP ratios was investigated by Superdex 200 gel filtration chromatography. The elution profile yielded three distinct peaks, corresponding to tetramer/trimer (1), dimer (2), and monomer (3). rSbsC(31–443)-SCWP ratios used are 2:1 (curve A), 1:1 (curve B), 1:2 (curve C). Peak 3 contains unbound protein. (B) An IEF gel showing the pI of unbound and complexed rSbsC(31–443). Lane M = IEF marker; lane P = rSbsC(31–443); lane 1 = peak 1; lane 2 = peak 2; and lane 3 = peak 3. Structure 2008 16, 1226-1237DOI: (10.1016/j.str.2008.05.012) Copyright © 2008 Elsevier Ltd Terms and Conditions