Cryo-EM structure of acylpeptide hydrolase reveals substrate selection by multimerization and a multi-state serine-protease triad

Abstract

The structure of tetrameric mammalian acylaminoacyl peptidase – a key upstream regulator of the proteasome – was determined by cryo-EM (and elucidated by MD), showing a “shutters-and-channels” substrate selection apparatus created by oligomerization.

A carbapenem antibiotic inhibiting a mammalian serine protease: structure of the acylaminoacyl peptidase–meropenem complex

“…B) The cryo-EM map (side view, semi-transparent) with the fitted model of the AAP tetramer and the meropenem molecules bound to the active sites (magenta). C) A cross section of the tetramer shows the large inner cavity: the four side openings of the monomers (black arrows) -the second frontier of the "double-gated channels-and-shutters" system created by the tetrameric assembly [33]. The active site residues (C  atoms of the catalytic triad are shown as orange spheres) are located between the propeller domain (dark colors) and the hydrolase domain (light colors) of the monomer units.…”

“…(C) A cross section of the tetramer shows the large inner cavity: the four side openings of the monomers (black arrows)the second frontier of the "doublegated channel and shutter" system created by the tetrameric assembly. 33 The active site residues (C a atoms of the catalytic triad are shown as orange spheres) are located between the propeller domain (dark colors) and the hydrolase domain (light colors) of the monomer units. Meropenem (magenta) is covalently bound to catalytic Ser587.…”

“…pAAP (and its human counterpart sharing a 92% of sequence identity) is unique among serine-proteases of the S9 oligopeptidase family from this respect -with a Pro (Pro506) inserted into the central -sheet of its hydrolase domain, an unusual degree of conformational variability is granted to the Ser-loop. In the latent conformations, the catalytic Ser587 is too far from His707 to establish an H-bond with it [33], thus the flip of His707 is made considerably easier in mammalian AAP than in other serine-proteases where the Ser-His-Asp triad is intact (Fig. 2C).…”

“…The preparation and purification of the mammalian AAP sample (from porcine liver) was based on a previous method [58] with an additional size exclusion chromatographic step using AKTA FPLC system, Superose 6 30/100 column (GE Healthcare, 20 mM TRIS, pH=8, 0.15M NaCl, 1mM EDTA, 1mM DTT). Tetrameric composition was verified by size exclusion chromatography [33]. To monitor that the catalytically competent form of the enzyme was preserved during the purification process, concentrated samples of pAAP were incubated with N-acetyl-alanine p-nitroanilide (AANA, eNovation Chemicals LLC) as a substrate [49](1.6 µM in 5% DMF/water) in buffer (50 mM phosphate, pH=8, 0.3 M NaCl, 1mM EDTA, 5mM mercaptoethanol) at 37°C (reaction mixture: 10 µl of AANA solution, 985 µl buffer, 5 µl protein sample).…”

“…The many functions and wide substrate range of AAP is served by a classical serine protease catalytic apparatus [32,33], which prompts two important questions: why -out of all the serine protease enzymes of the human physiology -has AAP proved to be the partner of the carbapenems, and why do only the carbapenems of the β-lactam family of antibiotics [31] participate in this interaction?…”