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 Å 3 , which is inaccessible to substrates except for a small opening of approximately 8 -10 Å. The substratebased 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.bestatin ͉ closed active site ͉ thermolysin-like protease

Glu121‐Lys319 salt bridge between catalytic and N‐terminal domains is pivotal for the activity and stability of Escherichia coli aminopeptidase N

“…One of the sulfates occupies the active site probably, where the carboxylate of a P 3 0 or P 4 0 residue in a tetra-or pentapeptide substrate would bind. This sulfate connects Domains II and IV by making strong interactions with K274, Y275, R783, and R825.…”

“…1(b,c)]. For example, Q136 in LTA 4 H, E183 in hErPepN, E101 in F3, E117 in NmPepN, and E319 in PfPepN play analogous role. Another interesting aspect of this residue in all the structures is that its main chain peptide adopts a cis-configuration.…”

“…2,3 M1 class aminopeptidases comprise large group of metalloproteases classified as gluzincins with a consensus zinc-binding motif (HEXXH-(X18)-E) and an exopeptidase motif (GXMEN) in their active site. Crystal structures of ePepN, hErPepN [not published, deposited in protein data bank (PDB) with ID: 2XDT], F3, LTA 4 H, PepN from Neisseria meningitidis (NmPepN) and from Plasmodium falciparum (PfPepN) have been reported. [4][5][6][7][8][9][10] Within the M1 class peptidases, all enzymes have four domains except, LTA 4 H which has only three domains.…”

“…Crystal structures of ePepN, hErPepN [not published, deposited in protein data bank (PDB) with ID: 2XDT], F3, LTA 4 H, PepN from Neisseria meningitidis (NmPepN) and from Plasmodium falciparum (PfPepN) have been reported. [4][5][6][7][8][9][10] Within the M1 class peptidases, all enzymes have four domains except, LTA 4 H which has only three domains. First two domains (Domains I and II) share highest sequence and structural homology among all these structures including the LTA 4 H. The LTA 4 H has a unique C-terminal domain instead of Domains III and IV, probably required to perform the leukotriene hydrolase activity.…”

“…[4][5][6][7][8][9][10] Within the M1 class peptidases, all enzymes have four domains except, LTA 4 H which has only three domains. First two domains (Domains I and II) share highest sequence and structural homology among all these structures including the LTA 4 H. The LTA 4 H has a unique C-terminal domain instead of Domains III and IV, probably required to perform the leukotriene hydrolase activity. All the M1 family proteins have thermolysin (bacterial endopeptidase) like catalytic domain (Domain II) 11 [ Fig.…”