Tuning the Strength of a Bacterial N-end Rule Degradation Signal

Kevin H Wang,Elizabeth S.C Oakes,Robert T Sauer,Tania A Baker

doi:10.1074/jbc.m802213200

Abstract

The N-end rule is a degradation pathway conserved from bacteria to mammals that links a protein's stability in vivo to the identity of its N-terminal residue. In Escherichia coli, the components of this pathway directly responsible for protein degradation are the ClpAP protease and its adaptor ClpS. We recently demonstrated that ClpAP is able to recognize N-end motifs in the absence of ClpS although with significantly reduced substrate affinity. In this study, a systematic sequence analysis reveals new features of N-end rule degradation signals. To achieve specificity, recognition of an N-end motif by the protease-adaptor complex uses both the identity of the N-terminal residue and a free alpha-amino group. Acidic residues near the first residue decrease substrate affinity, demonstrating that the identity of adjacent residues can affect recognition although significant flexibility is tolerated. However, shortening the distance between the N-end residue and the stably folded portion of a protein prevents degradation entirely, indicating that an N-end signal alone is not always sufficient for degradation. Together, these data define in vitro the sequence and structural requirements for the function of bacterial N-end signals.

Highlights

Energy-dependent proteases are composed of an oligomeric ATP-dependent unfolding enzyme and an enclosed proteolytic chamber [7]
The presence of several acidic residues (YLEEEEL-GFP) near the N-end residue slowed substrate degradation by ClpAP. These results indicate that acidic N-end signals affect ClpAPS and ClpAP recognition differently
The results presented here further define the molecular basis for N-end rule sequence selectivity and the roles of ClpA and ClpS in recognition

Summary

EXPERIMENTAL PROCEDURES

Plasmids and Proteins—GFP variants (GFPuv with serine at position 65 changed to threonine) and Y-titin were cloned into a pET23b.smt vector using AgeI and NotI sites [22]. Degradation reactions of unfolded GFP were performed using ClpA6 (800 nM), (ClpP-His6) (1.6 ␮M), and ClpS (4.8 ␮M). Unfolded YA3-GFP (1.5 ␮M) was added at time 0 to initiate the reaction. ␤-Galactosidase peptides were synthesized by the MIT Biopolymers facility and contained the first 21 residues of ␤-galactosidase fused to different N-terminal residues These peptides were added to a final concentration of 50 ␮M in reactions containing 50/100/450 nM ClpA/P/S and 500 nM YLFVQ-GFP; degradation was started by adding ATP. Acetyl-YLFVQR or unmodified YLFVQR peptide was added to degradation reactions containing 50/100/450 nM ClpA/P/S, 35 nM YLFVQ-GFP, and 3.3 or 6.6 ␮M peptide. YLFVQR peptide with a free or acetylated ␣-amino group (AcYLFVQR) was added before initiation of degradation, and rates were normalized to that of a reaction lacking peptide.

RESULTS

B ClpS binding to N-end peptides

DISCUSSION