Figure 3. ClpS binds ClpA₆ more tightly in the presence of N-end-rule peptides.

A) As assayed by anisotropy, ClpA₆ bound 200 nM fluorescent ClpS*^F tightly in the presence 20 μM Trp-conh₂, LLYVQRDSKEC, or FV N-end-rule peptides (K_app < 20 nM) and more weakly in the absence of peptide or with 20 μM MLYVQRDSKEC peptide (K_app ~ 180 nM).

B) The H66 residue of ClpS is one of the side chains involved in the formation of one of the three hydrogen bonds that the adaptor forms with the α-amino group of the N-end degron. Overlay of the apo (green, PDB 3O1F) and the peptide-bound (blue, 2W9R) crystal structures of ClpS reveal no major global changes occur upon peptide binding. The most substantial change is the rotation of the H66 side chain, which appear to need to move away from the pocket in the apo form to accommodate the N-degron in the peptide binding site.

C) ClpA₆ bound ClpS*^F (K_D = 200 ± 6 nM) and ^H66AClpS*^F (K_D = 345 ± 3 nM) with similar affinities. Addition of 20 μM LLYVQRDSKEC N-end-rule peptide enhanced ClpA₆ affinity for ClpS*^F substantially (K_app = 20 ± 10 nM) but increased affinity for ^H66AClpS*^F only modestly (K_app = 178 ± 4 nM).

D) ^H66AClpS-ClpA₆ complex binds more weakly to an N-end-rule fluorescent peptide (LLYVQRDSKEC-fl) when compared to ClpS-ClpA₆ (K_app = 560 nM vs K_app = 42 ± 6 nM for wild type).

E) An N-end-rule peptide (LLYVQRDSKEC; 20 μM) enhanced binding of ClpS*^F to ClpA₆ but not to ^ΔLClpA₆, which has shorter linker between the N and D1 domains (Cranz-Mileva et al., 2008).

F) Michaelis-Menten plots showed that substituting ^ΔLClpA₆ (4-residue linker) for ClpA₆ (26-residue linker) decreased K_M and V_max for ClpAPS degradation (100 nM ClpA₆ or ^ΔLClpA₆; 200 nm ClpP₁₄; 600 nm ClpS) of the N-end-rule substrate ylfvqela-GFP.