title: Structural insights into chaperone-activity enhancement by a K354E mutation in tomato acidic leucine aminopeptidase.
abstract: Tomato plants express acidic leucine aminopeptidase (LAP-A) in response to various environmental stressors. LAP-A not only functions as a peptidase for diverse peptide substrates, but also displays chaperone activity. A K354E mutation has been shown to abolish the peptidase activity but to enhance the chaperone activity of LAP-A. To better understand this moonlighting function of LAP-A, the crystal structure of the K354E mutant was determined at 2.15 Å resolution. The structure reveals that the K354E mutation destabilizes an active-site loop and causes significant rearrangement of active-site residues, leading to loss of the catalytic metal-ion coordination required for the peptidase activity. Although the mutant was crystallized in the same hexameric form as wild-type LAP-A, gel-filtration chromatography revealed an apparent shift from the hexamer to lower-order oligomers for the K354E mutant, showing a mixture of monomers to trimers in solution. In addition, surface-probing assays indicated that the K354E mutant has more accessible hydrophobic areas than wild-type LAP-A. Consistently, computational thermodynamic estimations of the interfaces between LAP-A monomers suggest that increased exposure of hydrophobic surfaces occurs upon hexamer breakdown. These results suggest that the K354E mutation disrupts the active-site loop, which also contributes to the hexameric assembly, and destabilizes the hexamers, resulting in much greater hydrophobic areas accessible for efficient chaperone activity than in the wild-type LAP-A.
authors.affiliation: Department of Biochemistry, University of California Riverside, Riverside, CA 92521, USA.
authors.forename: Kevin T
authors.initials: KT
authors.lastname: DuPrez
authors.affiliation: Department of Botany and Plant Sciences and Center for Plant Cell Biology, University of California Riverside, Riverside, CA 92521, USA.
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Scranton
authors.affiliation: Department of Botany and Plant Sciences and Center for Plant Cell Biology, University of California Riverside, Riverside, CA 92521, USA.
authors.forename: Linda L
authors.initials: LL
authors.lastname: Walling
authors.affiliation: Department of Biochemistry, University of California Riverside, Riverside, CA 92521, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Fan
mesh_terms: D020134:Catalytic Domain; D004795:Enzyme Stability; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018551:Solanum lycopersicum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D055503:Protein Multimerization
chemical_list: D007931:Leucyl Aminopeptidase
keywords: chaperone; leucine aminopeptidase; moonlighting enzyme; peptidase