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 a-hydroxy ketone, are present. Results from this study will be useful for the design of selective and efficient hydroxamate inhibitors against M1 family aminopeptidases.

Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens

“…First, Tg FtsH1 localizes to the apicoplast ( Karnataki et al, 2007 ). Second, actinonin is a peptide mimetic containing a metal-binding hydroxamic acid, a class of molecules that typically binds metalloproteases in their active site ( Figure 1a ) ( Chen et al, 2000 ; Ganji et al, 2015 ). Third, the N805S variant of Tg FtsH1 is within the metalloprotease domain ( Figure 2d ), raising the possibility that actinonin binding to the Tg FtsH1 metalloprotease active site may be prevented by this variant.…”

Inhibitors of BMP‐1/tolloid‐like proteinases: efficacy, selectivity and cellular toxicity

“…Noteworthy, FG-2575, S33A, UK383,367, RXP-1001 and sizzled have been tested on representative panels of MMPs and found to have no significant effects [7,13,16]. In contrast, the natural antibiotic actinonin was previously shown to inhibit aminopeptidases or peptide deformylases [36] and GM6001 is a broad-spectrum inhibitor of several metalloproteases [37].…”

Aminobenzosuberone Scaffold as a Modular Chemical Tool for the Inhibition of Therapeutically Relevant M1 Aminopeptidases

“…The determined structures from various species indicate that the active site of a M1 aminopeptidase is buried inside the protein and its zinc binding site may be located in a spacious cavity with wide openings, or located in a small and gated compartment as it is the case for bacterial/parasite orthologues [ 71 ]: this was in agreement with the active site’s cavity volume of different M1 aminopeptidases analysed by KVFinder ( Table S2 and Figure S2 ) [ 72 ]. Local or intradomain/interdomain movements can cause enlargement or contraction of the active site as encountered for Ec PepN or mammalian APN [ 20 , 25 , 71 , 73 ].…”

“…Data were plotted as 1/rate versus inhibitor concentration for each substrate concentration and a linear fit was calculated by non-linear regression using SigmaPlot12.5. All the reactions were performed in triplicates, SD values were reported [ 73 ].…”