Comparison of a retroviral protease in monomeric and dimeric states

Abstract

Retroviral proteases (RPs) are of high interest owing to their crucial role in the maturation process of retroviral particles. RPs are obligatory homodimers, with a pepsin-like active site built around two aspartates (in DTG triads) that activate a water molecule, as the nucleophile, under two flap loops. Mason-Pfizer monkey virus (M-PMV) is unique among retroviruses as its protease is also stable in the monomeric form, as confirmed by an existing crystal structure of a 13 kDa variant of the protein (M-PMV PR) and its previous biochemical characterization. In the present work, two mutants of M-PMV PR, D26N and C7A/D26N/C106A, were crystallized in complex with a peptidomimetic inhibitor and one mutant (D26N) was crystallized without the inhibitor. The crystal structures were solved at resolutions of 1.6, 1.9 and 2.0 Å , respectively. At variance with the previous study, all of the new structures have the canonical dimeric form of retroviral proteases. The protomers within a dimer differ mainly in the flap-loop region, with the most extreme case observed in the apo structure, in which one flap loop is well defined while the other flap loop is not defined by electron density. The presence of the inhibitor molecules in the complex structures was assessed using polder maps, but some details of their conformations remain ambiguous. In all of the presented structures the active site contains a water molecule buried deeply between the Asn26-Thr27-Gly28 triads of the protomers. Such a water molecule is completely unique not only in retropepsins but also in aspartic proteases in general. The C7A and C106A mutations do not influence the conformation of the protein. The Cys106 residue is properly placed at the homodimer interface area for a disulfide cross-link, but the reducing conditions of the crystallization experiment prevented S-S bond formation. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Acta_Cryst_D:S2059798319011355.

Crystal structures of inhibitor complexes of M‐PMV protease with visible flap loops

“…The crystal structures of 3M1 and 3M2 were determined by molecular replacement in Phaser 14 using the coordinates of one subunit of dimeric M‐PMV PR (PDB ID 6s1v 6 ). Structural refinements were carried out in PHENIX 15 and Coot 16 was used for modeling in electron density maps, as well as to build the inhibitor molecules and to validate the solvent structure.…”

“…The orientation is preserved in a common D → N mutation, which suppresses the autoproteolytic activity. Mason‐Pfizer monkey virus (M‐PMV) PR is unique among retropepsins with a shift of the dimerization equilibrium toward the monomeric state, 3 as confirmed by a crystallographic study 4,5 and different active site conformation 6 . In all described M‐PMV PR structures (PDB IDs 6s1v, 6s1w, 6s1u, 3sqf) the active site is mutated.…”

“…The overall protein conformation agrees with the template retropepsin fold 1 and is similar to that of the dimers crystallized in space group P2 1 . 6 An important difference is the stabilization of flap loops of chains B and D, and the concomitant stabilization of the inhibitors. There is also a different active-site H-bond network.…”

“…The authors declare no conflict of interest. ORCID Stanislaw Wosicki https://orcid.org/0000-0002-4583-9024 Miroslaw Gilski https://orcid.org/0000-0002-3941-9947 Mariusz Jaskolski https://orcid.org/0000-0003-1587-6489 ENDNOTE * In the previous paper 6 we incorrectly reported that the active site of 1n49 is not mutated, while in fact it contains the same D25N mutation.…”

“…Mason-Pfizer monkey virus (M-PMV) PR is unique among retropepsins with a shift of the dimerization equilibrium toward the monomeric state, 3 as confirmed by a crystallographic study 4,5 and different active site conformation. 6 In all described M-PMV PR structures (PDB IDs 6s1v, 6s1w, 6s1u, 3sqf) the active site is mutated. The side chains of the Asn26 residues are moved apart and twisted, forming additional H-bonds with water molecules that are further coordinated by main-chain carbonyls.…”