The closed structure of presequence protease PreP forms a unique 10,000 Angstroms3 chamber for proteolysis

Abstract

Presequence protease PreP is a novel protease that degrades targeting peptides as well as other unstructured peptides in both mitochondria and chloroplasts. The first structure of PreP from Arabidopsis thaliana refined at 2.1 Angstroms resolution shows how the 995-residue polypeptide forms a unique proteolytic chamber of more than 10,000 Angstroms(3) in which the active site resides. Although there is no visible opening to the chamber, a peptide is bound to the active site. The closed conformation places previously unidentified residues from the C-terminal domain at the active site, separated by almost 800 residues in sequence to active site residues located in the N-terminal domain. Based on the structure, a novel mechanism for proteolysis is proposed involving hinge-bending motions that cause the protease to open and close in response to substrate binding. In support of this model, cysteine double mutants designed to keep the chamber covalently locked show no activity under oxidizing conditions. The manner in which substrates are processed inside the chamber is reminiscent of the proteasome; therefore, we refer to this protein as a peptidasome.

Figure 1

Structure of the presequence protease PreP. (A) Ribbon presentation of AtPreP E80Q with the four domains, hinge, and connecting linkers distinguished by color. The 10 000 ų proteolytic chamber is shown as a gray surface representation with the zinc displayed as a purple sphere and the two magnesium binding sites, active site and substrate peptide (orange) drawn as sticks. (B) Detailed view of the magnesium atoms coordinated by acidic residues. (C) The isolated first domain indicating the topology of the secondary structure and the position of the active site and the large insertion in helix 4.

Figure 2

The active site. (A) Details of the substrate binding to the D strand (drawn as sticks) and active site residues from domains 1 (blue) and 4 (green). Selected hydrogen bonds are shown as black dashes. (B) The S1′ pocket with the two acidic residues Glu94 and Glu155 forming salt bridges to the basic P1′ Arg in the substrate. The inner surface of the chamber is shown in gray. (C) Stereo representation of an early 2F_o–F_c electron density map calculated before the peptide was built into the model. The whole map is contoured at 1σ (blue) and the density for the peptide is also shown for 0.5σ (red). (D–E) Proteolytic activity of native (wt) AtPreP1 and active site mutants measured as the degradation of (D) the presequence N_5.7pF₁β(2–54) and (E) the P1 peptide. Substrate in the absence of protease is shown in lanes 1, 4 and 7. Degradation by native AtPreP1 is shown in lanes 2, 3 and 8 and by different active site mutants in lanes 5–6 and 9–13. For comparison, the inhibitory effect on proteolysis by ortho-phenanthroline is shown in lanes 3 and 6.

Figure 3

Effect of divalent cations on the proteolytic activity of AtPreP1. Degradation of presequence N_5.7pF₁β(2–54) by native AtPreP1 in absence or presence of different concentrations of MgCl₂ and CaCl₂. The results are derived from three independent experiments (standard deviation is indicated).

Figure 4

Structure of different pitrilysin family members. (A) E. coli pitrilysin (PTRA) from subfamily M16A (Maskos K, Jozic D, Fernandez-Catalan C, unpublished) and (B) Yeast MPP from subfamily M16B (Taylor et al, 2001). The position of the substrate peptide from the AtPreP1 structure is shown for clarity. (C) Model of the open conformation of AtPreP1 from subfamily M16C created by superimposing the two enzyme halves onto the subunits of MPP. (D) Surface representation of the closed and proposed open form of AtPreP1 colored according to the electrostatic potential (negative red/positive blue). (E) Positions of the introduced cysteine pairs designed to form disulfide bonds that lock AtPreP1 in a closed conformation: K171C-G852C (C1), K179C-Q810C (C2), E345C-S682C (C3) and A331C-N615C (C4).

Figure 5

Proposed mechanism for the PreP peptidasome substrate binding and release.

Figure 6

AtPreP1 is inactive if locked in a closed conformation. (A) Schematic representation of the AtPreP1 cysteine double mutants K171C-G852C (C1), K179C-Q810C (C2), E345C-S682C (C3) and A331C-N615C (C4) under reducing and oxidizing conditions. Proteolytic activity of native (wt) AtPreP1 and the cysteine double mutants measured as the degradation of N_5.7pF₁β(2–54) under reducing (B) and oxidizing conditions (C) and degradation of the P1 peptide under reducing (D) and oxidizing conditions (E).

Figure 7

Blocking of free SH-groups restores the activity of the AtPreP1 cysteine double mutants. (A) Covalent modification of free SH-groups by NEM. Degradation of the presequence N_5.7pF₁β(2–54) (B) and the P1 peptide (C) by native (wt) AtPreP1 and the cysteine double mutants K171C-G852C (C1), K179C-Q810C (C2), E345C-S682C (C3) and A331C-N615C (C4) under oxidizing conditions after modification by NEM.