Crystal Structure and Biochemical Characterization of an Aminopeptidase LapB from Legionella pneumophila

Abstract

Aminopeptidases are a group of exopeptidases that catalyze the removal of a wide range of N-terminal amino acid residues from peptides and proteins. They have many important commercial applications in the food industry. We determined the crystal structure of an aminopeptidase LapB from Legionella pneumophila. The overall structure reveals that the N-terminal protease-associated (PA) domain presents a new fold and shields the active site cavity of the conserved C-terminal peptidase domain. The steady-state kinetic analysis of LapB and the PA domain deletion mutant indicate that the PA domain inhibited enzyme activity of the peptidase domain. Interestingly, the activity of LapB was largely increased by various organic solvents such as ethanol, propanol, and methanol at the concentration of 60% (v/v). CD analysis provided evidence that organic solvents induce the PA domain conformational changes that eliminate the inhibition role. The unique properties indicate the application potential of LapB in the food processing industry.

Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition during Legionella pneumophila Intracellular Infection of Amoebas

“…LapA was predicted to contain an N-terminal, “protease-associated” (PA) domain, which in some M28 peptidases is necessarily excised in order to release a C-terminal peptidase domain ( 40 , 41 ). That LapA might be processed is suggested from our prior two-dimensional (2-D) PAGE analysis of supernatants, which had detected truncated forms of LapA ( 28 ).…”

“…To determine how LapA provides its broad specificity, we began structural studies of recombinant LapA. Crystals were readily obtained, and its structure was determined by molecular replacement, using the coordinates of LapB (PDB code: 5GNE) ( 41 ) as the search model, and electron density maps were refined to 1.9 Å. The structure of LapA contains two molecules in the asymmetrical unit, and all residues could be built except for the N-terminal His tags and residues within the interdomain linkers (residues 82 to 101) that connect the PA domain (residues 1 to 81) and aminopeptidase domains (residues 102 to 378).…”

Peripheral Routes to Neurodegeneration: Passing Through the Blood–Brain Barrier

“…As inflammation comprises BBB dysfunction, the inhibition of IL-1 as proposed is a strategy enabling cerebrovascular protection (Marchi et al, 2009(Marchi et al, , 2011Vezzani et al, 2011;Kenney-Jung et al, 2016). Recent strategies include the development of IL-1Ra molecules fused with a cell-penetrating peptide to enhance brain access (Zhang et al, 2017). After transient middle cerebral artery occlusion in rats, IL-1Ra-PEP reduced neuro-inflammation and ischemia (Zhang et al, 2017).…”

“…Recent strategies include the development of IL-1Ra molecules fused with a cell-penetrating peptide to enhance brain access (Zhang et al, 2017). After transient middle cerebral artery occlusion in rats, IL-1Ra-PEP reduced neuro-inflammation and ischemia (Zhang et al, 2017). The fourth option is IPW-5371, a small molecule blocking the transforming growth factor β receptor (TGFβR) signaling.…”

Structural Modelling of the Lumenal Domain of Human GPAA1, the Metallo-Peptide Synthetase Subunit of the Transamidase Complex, Reveals Zinc-Binding Mode and Two Flaps Surrounding the Active Site

“…For each leave of the phylogenetic tree, we list (i) a color code, (ii) the taxonomic abbreviation, (iii) the accession number from the protein data bank RCSB or UniProt, (iv) the sequence segment from the original sequence used for the alignment behind the tree, (v) the sequence length of the sequence segment and (vi) the molecular function code (AM aminopeptidase, CP carboxypeptidase, CT cyclotransferase, CTI cyclotransferase type I, CTII cyclotransferase type II). The sequence groups presented in the phylogenetic tree are (i) known two zinc-binding 3D structures 3fec [66], 1zbl [64], 4twe [69], 5ib9, 6hc6, 3iib, 1tkj, 1amp [28], 5gne [70], and 6esl [71] color-coded red; (ii) known single zincbinding 3D structures 49fu [30], 4mhn, 4fai [30], 3pb4 [67], 2afo [65], 3si1 [68], 6qql, 3tc8, and 4fuu colorcoded green as well as (iii) six sequences from the GPAA1 family color-coded blue. We show also the position of 3gux in the tree although it is not known how many zinc ions it does bind in the catalytic cleft (we predict one) as it was part of the alignment in Figure 1 of reference [23].…”