Figure 4. A minimal NTE length is required for ClpS function.

A) ClpS variants (1 μM) with N-terminal truncations were assayed for delivery of ylfvqela-GFP (1 μM) for ClpAP degradation (gray curve) and for effects on ClpAP ATP hydrolysis (blue curve) using 100 nM ClpA₆ and 270 nM ClpP₁₄ for both assays. Data points represent averages (n=3) ± 1 SD. Each ClpS variant is named by the first wild-type residue in the construct. Those marked with an asterisk contain an additional N-terminal methionine and are therefore one residue longer than the labels indicate; these mutants were expressed as SUMO-fusion proteins and cleaved in vitro (see Experimental Procedures) or were expressed as standard non-fusion proteins but retained the initiator Met (verified by mass spectrometry). The T4 and W7 variants were also expressed as standard non-fusion proteins but mass spectrometry and/or N-terminal sequencing showed that the initiator Met was removed from both of these proteins. Note the sharp activity transitions between *L13 ClpS (starting Met¹²Leu¹³) and *A14 ClpS (starting Met¹³Ala¹⁴). The processing of the W7 variant is inconsistent with canonical methionine aminopeptidase activity and generates a good N-end-rule residue, which may be responsible for the poor activity of this ClpS variant in delivering other N-end-rule substrates.

B) Michaelis-Menten plots of ylfvqela-GFP degradation by ClpAP and ClpS or variants (100 nM ClpA₆; 200 nm ClpP₁₄; 600 nm ClpS or variants). Wild-type ClpS and *L13 ClpS (beginning Met¹²Leu¹³Ala¹⁴) supported roughly similar steady-state degradation kinetics, but delivery by *A14 ClpS (beginning Met¹³Ala¹⁴Glu¹⁵) resulted in a substantial decrease in V_max. Thus, the NTE must have a critical minimal length to support efficient substrate delivery. The solid lines are a global fit to a model in which the ClpS-substrate complex binds ClpA in an initial bimolecular step (K₁ = 1.1 μM) and then is engaged for degradation in a second unimolecular step (K₂), which depends on NTE length. In this model, apparent V_max = E_total•k_deg/(1+K₂) and apparent K_M = K₁•K₂/(1+K₂). For the fits shown, the k_deg value was 2.1 min⁻¹ and the K₂ values were 0.37 (wild-type ClpS), 0.74 (*L13 ClpS), and 9.2 (*A14 ClpS).

C) Binding to an N-end-rule peptide (LLYVQRDSKEC-fl; 150 nM) by complexes of ClpA₆ with ClpS variants (1 ClpS per ClpA₆) showed that ClpS junction residues are important for formation of the high-affinity delivery complex. Variants marked * have an additional N-terminal methionine. Apparent affinity constants were 43 nM (wild-type ClpS), 100 nM (*L13), 130 nM (*A14), 83 nM (*V18), 130 nM (D20), 290 nM (L22), and 1500 nM (S26).