The PA domain is crucial for determining optimum substrate length for soybean protease C1: Structure and kinetics correlate with molecular function

• Published in: Plant physiology and biochemistry Vol. 53; pp. 27 - 32
• Main Authors: Tan-Wilson, Anna, Bandak, Basel, Prabu-Jeyabalan, Moses
• Format: Journal Article
• Language: English
• Published: Paris Elsevier Masson SAS 01-04-2012; Elsevier
• Subjects: Active site; Amino Acids - metabolism; Antigens, Plant - metabolism; Biological and medical sciences; Endopeptidases - chemistry; Endopeptidases - metabolism; Fundamental and applied biological sciences. Psychology; Globulins - metabolism; Glycine max - enzymology; Homology modeling; Kinetics; Models, Molecular; PA domain; Plant physiology and development; Plant Proteins - metabolism; Protein Structure, Tertiary; Proteolytic enzyme; Seed Storage Proteins - metabolism; Solanum lycopersicum - enzymology; Soybean Proteins - metabolism; Storage protein mobilization; Substrate binding; Substrate Specificity; Subtilisin; Subtilisins - metabolism; PA; Ac; Storage protein mobilization; Proteolytic enzyme; Active site; Homology modeling; PA domain; Subtilisin; Substrate binding; Storage protein; Homology; Determination; Soybean; Modeling; Dicotyledones; Angiospermae; Structure; Serine endopeptidases; Enzyme; Glycine max; Substrate; Grain legume; Mobilization; Peptidases; Leguminosae; Plant physiology; Length; Hydrolases; Spermatophyta; Kinetics; Molecular domain
• ISSN: 0981-9428; 1873-2690
• DOI: 10.1016/j.plaphy.2012.01.005

A subtilisin-like enzyme, soybean protease C1 (EC 3.4.21.25), initiates the degradation of the β-conglycinin storage proteins in early seedling growth. Previous kinetic studies revealed a nine-residue (P5–P4′) length requirement for substrate peptides to attain optimum cleavage rates. This modeling study used the crystal structure of tomato subtilase (SBT3) as a starting model to explain the length requirement. The study also correlates structure to kinetic studies that elucidated the amino acid preferences of soybean protease C1 for P1, P1′ and P4′ locations of the cleavage sequence. The interactions of a number of protease C1 residues with P5, P4 and P4′ residues of its substrate elucidated by this analysis can explain why the enzyme only hydrolyzes peptide bonds outside of soybean storage protein's core double β-barrel cupin domains. The findings further correlate with the literature-reported hypothesis for the subtilisin-specific protease-associated (PA) domain to play a critical role. Residues of the SBT3 PA domain also interact with the P2′ residue on the substrate's carboxyl side of the scissile bond, while those on protease C1 interact with its substrate's P4′ residue. This stands in contrast with the subtilisin BPN′ that has no PA domain, and where the enzyme makes stronger interaction with residues on the amino side of the cleaved bond. The variable patterns of interactions between the substrate models and PA domains of tomato SBT3 and soybean protease C1 illustrate a crucial role for the PA domain in molecular recognition of their substrates.
[Display omitted] The PA domains in the plant subtilases soybean protease C1 and tomato SBT3 play crucial roles in determining optimum length of substrates required, which leads to functional diversity. Although five out seven loops in the substrate recognition site of subtilases are conserved, structural differences (red arrow) between BPN′ (yellow) and plant subtilases (protease C1 – cyan; SBT3 – magenta) lead to altered specificity with respect to substrate residues (stick figures) N-terminal to the cleavage site, which is between P1 and P1'. Differences in the plant-specific PA domains (black arrow) lead to altered interaction with the substrate residues C-terminal to the cleavage site. The structural models correlate with the published kinetic data of the two plant subtilases working on peptide substrates of different lengths.
► A homology model of soybean protease C1 is built using tomato subtilase SBT3. ► Molecular interactions correlate with previously published kinetic findings. ► The PA domains interact with the substrates C-terminal to the cleavage site. ► The plant-specific PA domains determine different optimum substrate lengths. ► Optimum substrate length is pertinent to the in vivo function of protease C1.