Figure 5. The ClpS NTE contacts ClpA near the axial pore.

A) Top: FeBABE was attached to residue 12 of ^Q12CClpS variant for cleavage studies. Bottom: As assayed by SDS-PAGE, cleavage of ClpA required FeBABE-modified ^Q12CClpS and ATPγS.

B) ClpA residues 259-268 are highlighted in blue in a top view of a model of the hexameric D1 ring (Guo et al., 2002). In one ClpA subunit, blue-wire shading shows regions within 12 Å of residues 259-268, which represents the approximate reach of the tethered FeBABE.

C) A substrate consisting of residues 2-26 of ClpS fused to GFP was efficiently degraded by ClpAP, as shown by Michaelis-Menten analysis (K_M = 16.4 μM; V_max = 0.62 min⁻¹ enz⁻¹).

D) Assays monitored by SDS-PAGE showed that ClpAP only partially degraded the H₆-Sumo-ClpS and H₆-Sumo-ClpS^core fusion proteins, resulting in truncated products of a lower molecular weight (marked by red arrowheads in lanes 2 & 4).

E) ClpAP partially degraded the ylfvqela-GFP-ClpS fusion protein, resulting in a lower molecular weight truncation product (marked by a red arrowhead in lane 2).

F) Depiction of the ClpS fusion proteins used to test degradation by ClpAP (left) and the corresponding truncation products produced by degradation (right). N-terminal sequencing of the truncation products revealed that the new N-termini corresponded to an internal sequence in the protein fused to ClpS (either Sumo or GFP). The truncation products consisted of the ClpS core and an additional N-terminal tail of 45-50 amino acids.