Structural and mutational analyses of the interaction between the barley alpha-amylase/subtilisin inhibitor and the subtilisin savinase reveal a novel mode of inhibition.
Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley alpha-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone.
Micheelsen
Pernille Ollendorff
PO
Research and Development, Novozymes A/S, Krogshøjvej 36, 2880 Bagsvaerd, Denmark. pnmi@novozymes.com
Vévodová
Jitka
J
De Maria
Leonardo
L
Ostergaard
Peter Rahbek
PR
Friis
Esben Peter
EP
Wilson
Keith
K
Skjøt
Michael
M
D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D003902:Detergents / Q000737:chemistry; D019286:Endopeptidase K / Q000037:antagonists & inhibitors; D001467:Hordeum / Q000378:metabolism*; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020860:Subtilisin / Q000037:antagonists & inhibitors*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
D003902:Detergents; D008670:Metals; D010940:Plant Proteins; C092772:barley alpha-amylase-subtilisin inhibitor; D014360:Trypsin Inhibitor, Kunitz Soybean; D000516:alpha-Amylases; D012697:Serine Endopeptidases; D020860:Subtilisin; C032923:microbial serine proteinases; D019286:Endopeptidase K