Structure of puromycin-sensitive aminopeptidase and polyglutamine binding

Abstract

Puromycin-sensitive aminopeptidase (E.C. 3.4.11.14, UniProt P55786), a zinc metallopeptidase belonging to the M1 family, degrades a number of bioactive peptides as well as peptides released from the proteasome, including polyglutamine. We report the crystal structure of PSA at 2.3 Ǻ. Overall, the enzyme adopts a V-shaped architecture with four domains characteristic of the M1 family aminopeptidases, but it is in a less compact conformation compared to most M1 enzymes of known structure. A microtubule binding sequence is present in a C-terminal HEAT repeat domain of the enzyme in a position where it might serve to mediate interaction with tubulin. In the catalytic metallopeptidase domain, an elongated active site groove lined with aromatic and hydrophobic residues and a large S1 subsite may play a role in broad substrate recognition. The structure with bound polyglutamine shows a possible interacting mode of this peptide, which is supported by mutation.

Modulation of endogenous opioid signaling by inhibitors of puromycin sensitive aminopeptidase

“…A salt bridge between the cationic α-ammonium group of the tyrosine residue in puromycin ( 3 ) and the anionic carboxylate of Asp403 also contributed significantly toward binding energy (docking score -8.390 kcal/mol). These results are consistent with the docking results of puromycin reported previously [23, 47].…”

“…The active site of PSA appears to be a long cleft with a catalytic site present at the closed end that contains the negatively charged residues Glu319 and Glu180, which correctly position the N-terminal end of the substrate. The long groove formed by hydrophobic and aromatic residues from helices α5 and α6 presents a rather large surface that likely is the reason for the broad substrate specificity of this enzyme [23]. All three low energy docked conformers of puromycin positioned the nucleoside portion in the proximity of the active site zinc(II) and the coordinating residues Glu375 at 1.96 Å, His352 at 2.12 Å, and His356 at 2.11 Å [23].…”

“…To gain a better understanding of the PSA inhibitory results, molecular modeling analysis was performed by docking the lead compounds 19 and 22 into the active site of PSA. The X-ray crystal structure of human PSA (PDB code 8SW0) was recently reported by Madabushi et al [23] and was imported for our ligand-receptor analysis. Validation of the receptor grid generated for docking studies was performed by docking the known ligand, puromycin (Figure 9A).…”

“…The long groove formed by hydrophobic and aromatic residues from helices α5 and α6 presents a rather large surface that likely is the reason for the broad substrate specificity of this enzyme [23]. All three low energy docked conformers of puromycin positioned the nucleoside portion in the proximity of the active site zinc(II) and the coordinating residues Glu375 at 1.96 Å, His352 at 2.12 Å, and His356 at 2.11 Å [23]. When docked, the dimethylamino (as ammonium) group of puromycin lies in proximity to the negatively charged pocket of the active site (Glu180 at 5.27 Å, Glu319 at 5.73 Å) and also interacts with the side chains of amino acids located in helix 11, helix 5, and helix 6. Notable π -π interaction occurs between the purine nucleobase and His352 (helix 5) and Tyr383 (helix 6).…”

“…It plays a neuroprotective role in amyotrophic lateral sclerosis by providing a degradation pathway for neurotoxic proteins such as SOD1 [22]. Recently, a crystal structure of PSA was reported at 2.3 Å resolution, which explains the broad substrate tolerance of this enzyme [23]. The canonical inhibitor puromycin binds with the nucleoside portion interacting with the active site zinc and coordinating residues.…”