Structural analysis of a group III Glu62-phospholipase A2 from the scorpion, Mesobuthus tamulus: Targeting and reversible inhibition by native peptides.
Group III phospholipase A(2) enzyme transcript from the Mesobuthus tamulus (Indian red scorpion) codes for three distinct products that include a large enzymatic subunit, a pentameric peptide and a small non-enzymatic subunit. The structures of these two subunits were modeled based on their sequence identity to bee venom PLA(2) and the partial sequence of MU2 adaptin subunit of AP2 clathrin adaptor, respectively. The enzymatic subunit comprises of three helices, the calcium binding loop and a substrate binding hydrophobic channel where the structure is stabilized by four disulfide bonds. The active site of the enzyme shows a catalytic histidine residue. Interestingly, there is a conservative mutation of the conserved aspartic acid, a classical participant of catalysis in this enzyme family, to glutamic acid. However, the side chain oxygen atoms of this glutamate are oriented away from the catalytic histidine implicating the non-participation of this residue in stabilizing the tautomeric conformation of the histidine. The acidic non-enzymatic subunit comprises of extensive hydrophobic residues with a conformation of an anti-parallel β-sheets making it ideal for tissue specific targeting. The native pentapeptide with the sequence Alanine-Arginine-Serine-Alanine-Arginine was docked to the enzymatic subunit. The peptide ligand occupies the hydrophobic cavity and makes a plethora of interactions with the residues in the channel, including a hydrogen bond with the crucial catalytic histidine and coordinate bond with the calcium ion. This ligand has a binding constant (K(D)) of 1.5μM. This makes the ligand a potential reversible inhibitor, ideal to prevent the enzyme from interacting with non-specific molecules enroute to the target. The enzyme-ligand complex also provides a model to understand the stereochemistry required for the design of more potent drug molecules against such enzyme drug targets.
Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India. g.hariprasad@rediffmail.com
Gururao
G
Hariprasad
Manoj
M
Kumar
Alagiri
A
Srinivasan
Punit
P
Kaur
Tej Pal
TP
Singh
Othayoth
O
Jithesh
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D054520:Group III Phospholipases A2 / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry; D012605:Scorpions / Q000201:enzymology*; D016415:Sequence Alignment
D010455:Peptides; D021122:Protein Subunits; D054520:Group III Phospholipases A2; D002118:Calcium