Journal:Acta Cryst D:S2059798319011355

Comparison of a retroviral protease crystallized as a monomer and a dimer

Stanislaw Wosicki , Miroslaw Gilski, Helena Zabranska, Iva Pichova, Mariusz Jaskolski ^[1]

Molecular Tour
Mason-Pfizer Monkey Virus (M-PMV) is a D-type retrovirus that causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its aspartic protease (PR) processes the retroviral polyproteins to release the final protein products necessary for virion maturation. Functionally, M-PMV PR is a homodimer but in the absence of inhibitors the equilibrium is shifted toward the monomeric state. Following our earlier crystallographic studies of the monomeric form of the protein, we have now determined three crystal structures of M-PMV PR in its dimeric form, using two protein variants crystallized with or without a substrate-based active-site inhibitor, Pro-Tyr(OMet)-Val-PSA-Ala-Met-Thr (where Tyr(OMet) is Oη-methylated tyrosine and PSA is a non-standard non-hydrolyzable residue, 3-hydroxy-4-amino-5-phenylpentanoic acid, used for inhibition of pepsin-like proteases). Both proteins are inactive by virtue of the D26N active site mutation. One of the proteins (C7A/D26N/C106A) has in addition its Cys residues replaced by Ala. Both proteins were crystallized in complex with the inhibitor. In addition, the D26N mutant was also crystallized in the apo form.

All presented crystal forms of dimeric M-PMV PR, i.e.: single mutant with the inhibitor (6s1v), triple mutant with the inhibitor (6s1u), and single mutant without inhibitor (6s1w), are isomorphous and different from the crystals of the monomeric protein, despite the fact that one of the present crystals of dimeric M-PMV PR was obtained using nearly identical crystallization conditions as those that yielded the monomeric form. The crystal structures were solved at resolutions of 1.6 Å, 1.9 Å, and 2.0 Å, respectively.

The structures of M-PMV PR contain an unusual water molecule buried deep in the active site structure, hydrogen-bonded in a tetrahedral fashion between the two active-site loops of the dimer, with the G28 N-H groups acting as donors and the side-chain O atoms of the N26 residues acting as acceptors. This water molecule is different from the nucleophilic molecule found in the active site of active aspartic proteases or from the one found at the inhibitor-flap interface of inhibitor complexes of retropepsins. Such a coordination of a water molecule has not been seen in any (retro)pepsin structures before. The side chains of the N26 residues take part in a wider H-bond network with water molecules coordinated also by V31 and L92. A similar pattern can be partially found in HIV-1 PR (5kr1, 1n49). In the apo structure of the protein (6s1w), one of the flaps (long loops gating access to the active site) is curled into the binding pocket and very well defined in the electron density, while the electron density of the other flap loop is invisible. While flap disorder is not surprising in apo structures of reropepsins, the perfect order of the other flap is an unusual feature, confirming again the unusual structural properties of the M-PMV enzyme. Also intriguingly, the tips of all the flap loops in the inhibitor complexes (6s1v, 6s1u) are disordered and not present in the electron density, although their visible stems seem to trace the direction found in inhibitor complexes of other dimeric retropepsins. The C/A mutations (C7A, C106A) have no effect on the M-PMV PR structure.

References

1. ↑ Wosicki S, Gilski M, Zabranska H, Pichova I, Jaskolski M. Comparison of a retroviral protease in monomeric and dimeric states. Acta Crystallogr D Struct Biol. 2019 Oct 1;75(Pt 10):904-917. doi:, 10.1107/S2059798319011355. Epub 2019 Sep 20. PMID:31588922 doi:http://dx.doi.org/10.1107/S2059798319011355