Structural basis of N-end rule substrate recognition in Escherichia coli by the ClpAP adaptor protein ClpS

Abstract

In Escherichia coli, the ClpAP protease, together with the adaptor protein ClpS, is responsible for the degradation of proteins bearing an amino-terminal destabilizing amino acid (N-degron). Here, we determined the three-dimensional structures of ClpS in complex with three peptides, each having a different destabilizing residue--Leu, Phe or Trp--at its N terminus. All peptides, regardless of the identity of their N-terminal residue, are bound in a surface pocket on ClpS in a stereo-specific manner. Several highly conserved residues in this binding pocket interact directly with the backbone of the N-degron peptide and hence are crucial for the binding of all N-degrons. By contrast, two hydrophobic residues define the volume of the binding pocket and influence the specificity of ClpS. Taken together, our data suggest that ClpS has been optimized for the binding and delivery of N-degrons containing an N-terminal Phe or Leu.

Figure 1

Binding and degradation of model N-degrons. (A–C) Isothermal titration calorimetry of ClpS (0.08 mM) binding to (A) Lpep, (B) Fpep and (C) Wpep. (A) Titration of 40 injections (4 μl) of Lpep (2 mM) were performed, revealing a dissociation constant of 4.8 μM. (B) Titration of 50 injections (3 μl) of Fpep (2 mM) was performed, showing a dissociation constant of 3.8 μM. (C) Titration of Wpep (0.6 mM) was performed with 60 injections (3 μl), showing a dissociation constant of 8.1 μM. (D) Schematic representation of all possible type 2 destabilizing amino acids: Leu, Phe, Trp, Tyr and Ile. (E) The ClpAP-mediated degradation of Lpep–GFP (green fluorescent protein; triangles), Fpep–GFP (circles) and Wpep–GFP (squares) was monitored by fluorescence in the absence (open symbols) or presence (filled symbols) of ClpS.

Figure 2

N-degron binding and ClpS structure. (A) The backbone of the ClpS–Lpep complex (Lpep) was superimposed onto four structures of ClpS in complex with the ClpA N-domain, two with wild-type ClpS (1mbu and 1r6o; Guo et al, 2002) and two with ClpS(H66A) mutant (1mg9 and 1lzw; Zeth et al, 2002). (B) Structure of ClpS in complex with Lpep at 1.7 Å resolution (peptide omitted for clarity). The N-terminal helix of ClpS (Glu 15–Lys 23) is boxed and the electrostatic surface shows the distribution of negatively charged (red) and positively charged (blue) regions. (C) Thermal melting curves of ClpS (0.02 mM) in the absence of peptide (open circles) or in the presence of Lpep (yellow circles), Fpep (blue squares) or Wpep (red triangles) were drawn at 222 nm.

Figure 3

Structures of ClpS in complex with various N-degron peptides. (A–C) The peptide (Lpep, grey) extends away from ClpS (orange). A water molecule (blue sphere) has an important function in complex formation. ClpS is represented as a ribbon (A) in complex with Lpep. Surface representation of ClpS binding to Lpep in a side (B) and top (C) view showing that the amino-terminal side chain is embedded into the surface of ClpS by approximately 8 Å. (D) Ribbon diagram showing the complex of ClpS (blue) with Fpep (grey). (E) Ribbon diagram showing the structure of ClpS (red) in complex with Wpep (grey). (F) A close-up of the backbone of all three complex structures superimposed shows the close alignment of the peptide residues.

Figure 4

The N-degron binding site of ClpS. (A) Peptide localization (Lpep) based on the ∣F_O−F_C∣ difference electron density calculated at 3 σ and a resolution of 1.7 Å. (B) Interaction matrix of Lpep seen from the same orientation as (A). Residues involved in salt bridges and polar interactions are marked using a black dotted line (<3 Å), whereas hydrophobic interactions (<3.5 Å) with the Leu side chain are indicated by green dotted lines. (C) The interaction matrix for Fpep is shown in the same orientation as (B) and the interactions are defined as described in (B). (D) Model showing the overall molecular interactions within the ClpS-binding pocket. Salt bridges and polar interactions common to both structures are indicated by black dotted lines, whereas hydrophobic interactions specific to Leu or Phe are represented by a light or dark grey dotted line, respectively. The stabilizing interaction between Asp 35 and Thr 38 is represented by a pink dotted line.

Figure 5

Met 40 and Met 62 modulate ClpS substrate specificity. (A) Sequence alignment of selected ClpS proteins together with the type 2 binding region from eukaryotic UBR1 and UBR2. (B,C) Surface representation of ClpS (orange) binding to Lpep (grey) showing the top view of (B) wild-type (WT) ClpS or (C) a model of M40A/M62A (MAMA). (D) The rate of ClpAP-mediated degradation of each N-degron–GFP (green fluorescent protein) fusion was monitored by fluorescence in the absence (white bar) or presence of wild-type ClpS (black bars), M40A (blue bars), M62A (yellow bars), MAMA (green bars) or H66A (red bars). Degradation rates were determined relative to the rate of degradation in the presence of wild-type ClpS. Error bars represent the standard error of the mean, from at least five separate experiments.