Structural basis for the inhibition of M1 family aminopeptidases by the natural product actinonin: Crystal structure in complex with E. coli aminopeptidase N

Abstract

Actinonin is a pseudotripeptide that displays a high affinity towards metalloproteases including peptide deformylases (PDFs) and M1 family aminopeptidases. PDF and M1 family aminopeptidases belong to thermolysin-metzincin superfamily. One of the major differences in terms of substrate binding pockets between these families is presence (in M1 aminopeptidases) or absence (in PDFs) of an S1 substrate pocket. The binding mode of actinonin to PDFs has been established previously; however, it is not clear how the actinonin, without a P1 residue, would bind to the M1 aminopeptidases. Here we describe the crystal structure of Escherichia coli aminopeptidase N (ePepN), a model protein of the M1 family aminopeptidases in complex with actinonin. For comparison we have also determined the structure of ePepN in complex with a well-known tetrapeptide inhibitor, amastatin. From the comparison of the actinonin and amastatin ePepN complexes, it is clear that the P1 residue is not critical as long as strong metal chelating head groups, like hydroxamic acid or α-hydroxy ketone, are present. Results from this study will be useful for the design of selective and efficient hydroxamate inhibitors against M1 family aminopeptidases.

Keywords: M1 aminopeptidase; actinonin; amastatin; matrix metalloproteinase; peptide deformylase.

Figure 1

Schematic representation of chemical composition of actinonin and amastatin. The side chains are labeled as per the sub-site occupancy in the enzyme pocket.

Figure 2

Crystal structure of actinonin in complex with ePepN at 1.9 Å. a) 2Fo-Fc = 1.1 σ electron density map representation around the zinc bound actinonin. Molecule binds in the extended conformation making two strong interactions with a zinc ion. A cover radius of 2.0 Å was used during the map generation. b) Stereo representation of the actinonin bound in the enzyme pocket with surrounding residues. Note that hydroxamate head group makes a series of hydrogen bonds in the active site. c). A two-dimensional schematic representation of all residues surrounding the actinonin in LigPlot diagram. Apart from hydrogen bonds, several hydrophobic interactions are noticed between the ligand and the protein. An interactive view is available in the electronic version of the article

Figure 3

Crystal structure of amastatin in complex with ePepN at 2.3 Å resolution. a). 2Fo-Fc Electron density map surrounding the amastatin and the zinc in the active site at 1.1 σ. Similar to actinonin, amastatin forms two coordinate bonds with the active site zinc ion. A cover radius of 2.0 Å was used during the map generation. b). Extended conformation of the amastatin is represented in the stereo diagram. Amastatin makes several hydrogen bonds with residues that surround it. c). LigPlot diagram of amastatin (green sticks) in the active site of ePepN. Note that the orientation of the amastatin in this panel is rotated by 180° compared with that in (b). Large network of hydrogen bonds were noticed between the ligand and the protein. An interactive view is available in the electronic version of the article

Figure 4

Superposition of actinonin (brown sticks) and amastatin (green sticks) bound ePepN structures near the active site are depicted (0.25 Å rmsd between two structures). Except for the P2' residue (valine in both structures), most parts of ligand align well. The presence or absence of P1 residue side chain does not seem to affect the mode of binding of ligands in this case.

Figure 5

Structural alignment of catalytic domain of ePepN (200–444 residues) and ePDF in complex with actinonin. a). Cartoon representation of ePepN (green) and ePDF (white). The actinonin is shown in the sticks (brown in the ePepN complex while it is white in ePDF structure). Note that the core of the protein structure aligns well. b). Stick representation of actinonin from both the structure in the active site. Molecular trajectory deviates from P2' residue and becomes maximal at P3'.