Structural comparison of mutant and wild‐type acidic leucine aminopeptidase suggests the basis for secondary chaperone activity (567.11)

Abstract

The acidic leucine aminopeptidase (LAP‐A) of tomato is involved in wound signaling and insect defense. Observation of chaperone activity by LAP‐A suggests new mechanisms of defense against environmental stresses. To evaluate how this chaperone activity is facilitated, the crystal structures of wild‐type and a loss of function mutant of LAP‐A were determined to 2.20 and 2.30 Å, respectively. LAP‐A is a dimer of trimers composed of 6 bilobal monomers. Six substrate entry channels formed as a result of the hexameric structure connect in a central cavity in which all six active sites are localized. Coordination of two active‐site magnesium ions required for catalysis is facilitated by a highly conserved network of amino acid residues. Mutation of select residues within this network can result in loss of activity and/or breakdown of hexameric structure. The structure of the K354E mutant shows disruption of a loop from residue 350 through 361, resulting in the loss of intra‐subunit contacts that stabilize binding of the catalytic metal ions, thus resulting in loss of peptidase activity. These residues also provide inter‐subunit contacts at the trimerization interface, presumably stabilizing the hexameric form of the enzyme. However, breakdown of the hexamer into a tetrameric form induced by the K354E mutation results in increased chaperone activity, likely through heightened exposure of hydrophobic surfaces. These data reveal insights into the structural basis for LAP‐A's secondary function as a molecular chaperone.Grant Funding Source: Partially supported by a National Science Foundation grant (IOS 0725093) to LLW.