Figure 5.
Distinct Dimer States of Smc Head Domains
(A) Mapping of Smc(Cys) cross-linking onto the Bs Smc head (PDB: 3ZGX). The N-terminal ScpA fragments is omitted from the view. Color coding as in Figure 2B. Selected residues displaying efficient cross-linking when mutated to cysteine are marked. In addition, residues of the ABC signature motif and the Walker box motifs are displayed in light and dark green colors, respectively.
(B) Crystal structure of BsSmcHd-CC30:ATPγS–ScpAC in surface representation in front (left), top, and bottom views (right). The C-terminal ScpA fragments are omitted from all views. The Cα-Cα distance between pairs of selected residues (in red colors) across the ATP-engaged dimer is given. General color coding as in Figure 2B. ATPase motif residues are colored as in (A). ATPγS is displayed in spheres in black colors. For size reference, the scale of the cross-linker BMOE is displayed.
(C) A model of the Bs Smc head domain (PDB: 3ZGX) in the rod dimer configuration. The head dimer is constructed by superimposition onto the Py Smc rod structure (Figure 4B) and reflects the cysteine cross-linking data (Figure 2A) (see also Figure S5D). Display as in (B).
(D) In vivo cysteine cross-linking of Smc(Cys) proteins with wild-type and mutant ATPase domains. Cross-linking of S152C, D193C, and K1151C residues in Smc-HaloTag proteins bearing ATP binding (K37I), head engagement (S1090R), ATP hydrolysis (E1118Q), and hinge dimerization interface (G657A, G658A, G662A, G663A; “mH”) mutations. In all strains, four endogenous cysteines were substituted for serines (Hirano and Hirano, 2006). Cell extracts were labeled with HaloTag-TMR substrate. Smc-HaloTag species were separated by SDS-PAGE and quantified by in-gel fluorescence scanning. Data are represented as mean values and standard deviation from biological replicates (duplicates containing technical triplicates).