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

The structure of porcine AAP (pAAP) in a covalently bound complex with meropenem was determined by cryo-EM to 2.1 Å resolution, showing the mammalian serine-protease inhibited by a carbapenem antibiotic. AAP is a modulator of the ubiquitin-proteasome degradation system and the site of a drug–drug interaction between the widely used antipsychotic, valproate and carbapenems. The active form of pAAP – a toroidal tetramer – binds four meropenem molecules covalently linked to the catalytic Ser587 of the serine-protease triad, in an acyl–enzyme state. AAP is hindered from fully processing the antibiotic by the displacement and protonation of His707 of the catalytic triad. We show that AAP is made susceptible to the association by its unusually sheltered active pockets and flexible catalytic triads, while the carbapenems possess sufficiently small substituents on their β-lactam rings to fit into the shallow substrate-specificity pocket of the enzyme.

. Workflow of cryo-EM data processing. A) Data processing workflow using cryoSPARC, showing 2D classes (left panel) and a typical micrograph for AAP-MEPM complex (right panel, particles circled in red). B) Maps obtained applying C1 and C2 point group symmetries in 3D refinement C) Local resolution maps contoured at a 0.12 threshold level (left), calculated using ResMap. Local resolution maps with the fitted models (right, derived using Phenix Dock) D) Local resolution of cryo-EM map (C2 symmetry, 0.12 threshold level) around meropenem (MEPM).

Figure S3. Validation of the cryo-EM data processing. A)
The calculated map to map FSC curves (from cryoSPARC). B) The half-maps FSC curves were calculated using Phenix and C) the calculated model to map FSC curves.     In EMRinger model-to-map agreement method the Cγ atom is rotated around the χ1 angle (upper panels) of a side chain, interpolating the density value in the map as it rotates. Properly modeled side chains should have χ1 density peaks at or near rotameric positions (60°, 180° and 300°). The angle of peak density is interpreted as the correct position of the side chain in dihedral space. The peak in the distribution represents the most likely position of the Cγ atom of the side chain. a) AAP-MEPM tetramer fitted into cryo-EM map refined in C1 symmetry and b) in C2 symmetry calculated by phenix.emringer. Upper panels show the χ1 angle, the lower panels show the χ2 angle of His707 residues in chains A-D. Based on this His707 appears to be in an on-rotameric, low energy state and the side chain only rotates around its χ2 angle.

Figure S7. Determining both the tautomerization and the protonation states of meropenem within the experimentally determined structure of the pAAP-meropenem complex using QM/MM calculations. a)
Overview of the QM/MM system (green: QM region (meropenem shown in spacefilling representation): MEPM, Ser587, His707, Phe274, Ser512, 2 water molecules; lilac: freely moving MM region; gold: frozen MM region). b) Selecting the most likely state based on RMSD calculated for the non-hydrogen atoms of meropenem (2 nd column), the distance between His707-N2 atom and the carbonyl oxygen of the amide substituent of the pyrrolidine ring of meropenem (3 rd column) and the calculated conformational energy (4 th column). c-h) The QM region of the optimized structure of the considered states (shown in dark green, overlaid on the experimental structure that is coloured according to a). The dihydro-pyrrole ring (indicated with cyan star) can assume imine (c-e) or enamine (f-h) form. Depending on the protonation state of its N-atom, the pyrrolidine ring (magenta star) can be neutral (single proton pointing toward Ser512 (c, f) or toward His707 (d, g)), or positively charged (doubly protonated: e, h).

Figure S8. Monte Carlo Multiple Minimum conformational search conducted for intact and hydrolyzed (open) meropenem in water. a)
The ensemble of low energy conformers of water solvated unhydrolyzed meropenem (global minimum arrangement and all those within a 3 kcal/mol energy window) as derived by MCMM calculation. b) Comparing the MCMM derived binding conformation of unhydrolyzed meropenem within the pAAP-meropenem complex derived by MCMM (pink) and a low energy conformer (+0.3 kcal/mol) from the water solvated ensemble, with high structure similarity (grey). c) The ensemble of low energy conformers of water solvated hydrolysed /open meropenem (global minimum arrangement and all those within a 3 kcal/mol energy window) as derived by MCMM calculation. d) Comparing the cryo-EM determined binding conformation of covalently bound meropenem within the pAAP-meropenem complex (pink) and the structurally most similar conformer from the low energy ensemble (+1.4 kcal/mol) of the water solvated system (grey).

Figure S9. MCMM calculations concerning the non-covalent (pre-reaction) pAAP/VPA-G and pAAP/MEPM complexes.
Overview of the minimum-energy conformers (a-b) and details of the local interactions (c-d). On all panels the hydrolase domain of pAAP is colored light green, the propeller dark green, while the residues of the catalytic triad in orange. VPA-G is shown in cyan, unhydrolyzed meropenem in pink. Figure S10. The proposed mechanism for VPA-G hydrolysis by AAP: nucleophilic attack of the carbonyl moiety of VPA-G, stabilization of the acyl-enzyme intermediate and subsequent deacylation by water (activated by His707) leading to the release of the products. The nucleophilic nature of Ser587 side chain OH is enhanced by the intact H-bond toward His707. Figure S11. Sequence alignment of porcine and human AAP. (Uniprot proteinBLAST, codes P19205 and P13798) The sequence identity is 91.9% with 673 identical, 45 similar and 14 different amino acids (full length of the sequence is 732 amino acids). The active site (Ser587, Asp625, His707) is highlighted with red dots and rectangles. Blue dotted lines mark the borders of the hydrolase and the propeller domain, purple dotted lines mark the sequence of blade 3 insertion (Insert2).

Figure S12 Structure of various human serine proteases showing well-accessible active sites.
Chains are rainbow coloured from blue (N-term) to red (C-term). Catalytic serines are shown as black spheres.
(PDB and AlphaFold codes are in parenthesis) Figure S13. The ring structure of penicillin, carbapenem and cephalosporin type antibiotics.
Carbapenems are notably different from the other -lactams in the moderate size of their R 1 substituent (marked in blue) which is able to fit into the shallow S1 pocket of AAP. Figure S14. Interaction of meropenem with dithiothreitol monitored by mass spectrometry. a) LC-UV-MS analysis of meropenem solution in water, b) 10 mM meropenem in buffer solution containing 5% dithiotheritol (5 mM NaOAc, pH=5, 44 mM EDTA, 0.8 w/w% PEG4000, 6.6 mM DTT) and c) 10 mM meropenem solution in buffer (5 mM NaOAc, pH=5, 44 mM EDTA, 0.8 w/w% PEG4000) used for crystallization (peaks corresponding to intact, unhydrolized meropenem are shown in green).