Substrate access to the active sites in aminopeptidase T, a representative of a new metallopeptidase clan.
Aminopeptidase T (AmpT) from Thermus thermophilus is a metalloexopeptidase with no similarity to prototypical metallopeptidases with an HExxH or HxxEH motif. The crystal structure of the Staphylococcus aureus homologue of AmpT, which is known as aminopeptidase S (AmpS), has been reported recently. This structure revealed a dimeric protein with a very unusual, elongated shape and a large internal cavity. The active sites were found on the inner walls of the cavity and were entirely shielded from the environment, which suggested either that the dimer in the crystals was not physiologically relevant, or that an inactive conformation had been crystallized. Here, we show by gel-filtration and analytical ultracentrifugation that AmpT, like AmpS, forms dimers in solution, and we present the structure of AmpT in a crystal form with five protomers in the asymmetric unit. The five protomers take conformations that range from fully closed, as in the AmpS structure, to nearly open, so that the active site is almost directly accessible. The different conformations indicate flexibility between the AmpT N and C-domains, and explain how AmpT can be active, although the unusual AmpS dimerization mode applies to AmpT as well.
Odintsov
Sergey G
SG
International Institute of Molecular and Cell Biology, ul. Trojdena 4, 02-109 Warsaw, Poland.
Sabała
Izabela
I
Bourenkov
Gleb
G
Rybin
Vladimir
V
Bochtler
Matthias
M
D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D004744:Enkephalin, Methionine / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology*
D001426:Bacterial Proteins; D004744:Enkephalin, Methionine; D000626:Aminopeptidases; C067298:aminopeptidase T protein, Thermus aquaticus