Structure of aminopeptidase N from Escherichia coli suggests a compartmentalized, gated active site

Abstract

Aminopeptidase N from Escherichia coli is a major metalloprotease that participates in the controlled hydrolysis of peptides in the proteolytic pathway. Determination of the 870-aa structure reveals that it has four domains similar to the tricorn-interacting factor F3. The thermolysin-like active site is enclosed within a large cavity with a volume of 2,200 A(3), which is inaccessible to substrates except for a small opening of approximately 8-10 A. The substrate-based inhibitor bestatin binds to the protein with minimal changes, suggesting that this is the active form of the enzyme. The previously described structure of F3 had three distinct conformations that were described as "closed," "intermediate," and "open." The structure of aminopeptidase N from E. coli, however, is substantially more closed than any of these. Taken together, the results suggest that these proteases, which are involved in intracellular peptide degradation, prevent inadvertent hydrolysis of inappropriate substrates by enclosing the active site within a large cavity. There is also some evidence that the open form of the enzyme, which admits substrates, remains inactive until it adopts the closed form.

Fig. 1.

Sturcture of ePepN. (A) Stereodiagram showing the overall structure of ePepN. Individual domains are shown in different colors: blue, domain I; red, domain II; gold, domain III; green, domain IV. The N and C termini are indicated by arrows, and the red ovals indicate the location of the small opening in domain IV. The catalytic zinc ion is blue. (B) Electrostatic surface diagram of the catalytic domain (domain II) of ePepN. A bestatin molecule is shown in the active site cleft, with the arrows indicating its N and C termini. The cleft is narrower on the left where the N terminus of the substrate binds and widens on the right to accommodate an extended polypeptide substrate.

Fig. 2.

Binding of bestatin to ePepN. (A) Bird’s-eye view of the stereodiagram of bestatin bound in the active site. The electron density is a 2.3-Å resolution omit map drawn at 3.9 σ. Bestatin is shown in yellow. Residues from the catalytic domain are pink, those from domain I are blue, and those from domain IV are green. Four water molecules are represented as red spheres, and Zn^II is represented as a blue sphere. (B) LIGPLOT diagram (36) of bestatin (yellow bonds) bound to ePepN. Residues from the catalytic domain are represented in gray, and Glu-121 from domain I is blue. Zn^II is represented as a cyan sphere. The dashed lines indicate hydrogen bonds, and the spikes represent hydrophobic interactions.

Fig. 3.

Interaction surfaces between the catalytic domain (domain II) and other domains of the ePepN structure. Two views of the catalytic domain are shown, related by a 180° rotation. Bestatin is in the active site. The part of the surface colored blue is in contact with domain I, the part in green contacts domain IV, and the part in yellow contacts domain III. The remainder of domain II contacts bulk solvent and is red.

Fig. 4.

Stereodiagram showing the superposition of ePepN and the F3 open structure (Protein Data Bank ID code 1Z5H, molecule A). The ePepN structure is color-coded blue, red, gold, and green for domains I, II, III, and IV, respectively. The F3 structure is gray. The C-terminal half of the α4-helix and the N terminus of α5 and the connecting loop are represented as solid lines along with a loop that undergoes a movement between the two structures. The catalytic zinc ion is blue. There are five lines in red, four of which are numbered 1–4 (also in red). These lines show the shifts in the α-helical pairs listed in Table 1. The blue double-headed arrow labeled 5 indicates a long loop in domain III that has a different conformation in F3 relative to ePepN.

Fig. 5.

Close-up stereoview of the superposition of the closed ePepN (red ribbons) and open F3 (gray) active sites. Helices α4 and α5 of the F3 structure bend away from the active site, and, in so doing, Tyr-351 of F3 (black) rotates away from the active site represented by bestatin (yellow) and zinc ion (blue). In the closed ePepN–bestatin complex, the analogous residue, Tyr-381, points into the active site and interacts with the inhibitor.