Inhibitory properties and solution structure of a potent Bowman-Birk protease inhibitor from lentil (Lens culinaris, L) seeds.
Bowman-Birk serine protease inhibitors are a family of small plant proteins, whose physiological role has not been ascertained as yet, while chemopreventive anticarcinogenic properties have repeatedly been claimed. In this work we present data on the isolation of a lentil (Lens culinaris, L., var. Macrosperma) seed trypsin inhibitor (LCTI) and its functional and structural characterization. LCTI is a 7448 Da double-headed trypsin/chymotrypsin inhibitor with dissociation constants equal to 0.54 nM and 7.25 nM for the two proteases, respectively. The inhibitor is, however, hydrolysed by trypsin in a few minutes timescale, leading to a dramatic loss of its affinity for the enzyme. This is due to a substantial difference in the kon and k*on values (1.1 microM-1.s-1 vs. 0.002 microM-1.s-1), respectively, for the intact and modified inhibitor. A similar behaviour was not observed with chymotrypsin. The twenty best NMR structures concurrently showed a canonical Bowman-Birk inhibitor (BBI) conformation with two antipodal beta-hairpins containing the inhibitory domains. The tertiary structure is stabilized by ion pairs and hydrogen bonds involving the side chain and backbone of Asp10-Asp26-Arg28 and Asp36-Asp52 residues. At physiological pH, the final structure results in an asymmetric distribution of opposite charges with a negative electrostatic potential, centred on the C-terminus, and a highly positive potential, surrounding the antitryptic domain. The segment 53-55 lacks the anchoring capacity found in analogous BBIs, thus rendering the protein susceptible to hydrolysis. The inhibitory properties of LCTI, related to the simultaneous presence of two key amino acids (Gln18 and His54), render the molecule unusual within the natural Bowman-Birk inhibitor family.
Ragg
Enzio M
EM
Department of Agri-Food Molecular Sciences, Università degli Studi, Milano, Italy. enzio.ragg@unimi.it
Galbusera
Valerio
V
Scarafoni
Alessio
A
Negri
Armando
A
Tedeschi
Gabriella
G
Consonni
Alessandro
A
Sessa
Fabio
F
Duranti
Marcello
M
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D027806:Lens Plant / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012639:Seeds / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D012996:Solutions; D014358:Trypsin Inhibitor, Bowman-Birk Soybean