Journal:IUCrJ:S2052252522007497

The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (Mpro)

Ali Ebrahim, Blake T. Riley, Desigan Kumaran, Babak Andi, Martin R. Fuchs, Sean McSweeneyd, and Daniel A. Keedy ^[1]

Molecular Tour
The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or Mpro, is part of a polyprotein encoded by the viral RNA genome. After being excized from the polyprotein by its own proteolytic activity, Mpro cleaves at no fewer than 11 sites in the polyprotein to generate individual functional proteins that help the virus replicate. Halting the function of Mpro may therefore disrupt the SARS-CoV-2 viral lifecycle, making it a promising target for the development of novel antiviral therapeutics.

Previous X-ray crystal structures of Mpro were obtained at cryogenic temperature or room temperature only. Here we report a series of high-resolution crystal structures of unliganded Mpro across multiple temperatures from cryogenic to physiological, and another at high humidity. This allows us to perturb the crystal structures in an attempt to reveal how structural movements may relate to function. To do this, we interrogate these data sets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps.

Here we show a perturbation-dependent conformational landscape for Mpro, including a mobile zinc ion interleaved between the catalytic dyad, mercurial conformational heterogeneity at various sites including a key substrate-binding loop, and a far-reaching intramolecular network bridging the active site and dimer interface. Our results may inspire new strategies for antiviral drug development to aid preparation for future coronavirus pandemics.

. Our 310 K Mpro model (7mhk) is represented here as the biologically relevant dimer. Here we show one monomer as cartoon only (red), including bound zinc (pale purple, sphere) between the catalytic dyad of Cys145 and His41 (red, sticks). The second monomer is shown in spacefill representation (white smoke, semi-transparent). is shown in dark yellow surface representation, while including bound zinc (pale purple, sphere) and catalytic dyad (red, sticks). We also highlight a fragment (competitive inhibitor N3) bound to the substrate binding pocket from PDB 6lu7 (dark gray).

. When collecting X-ray diffraction data, heavy atoms have a property called anomalous scattering which helps us pinpoint their location within a crystal structure. This gives rise to anomalous electron density, which is present in the asymmetric unit for data collected for PDB entry 7kt3, above 4 σ in the vicinity of the active site (as shown here). This strong anomalous peak at the position in question for 7k3t is critical, as these data were collected from the same batch of crystals as our reported multitemperature data sets, despite also having used an off-edge wavelength for Zn2+ during data collection. This not only allowed the identification of Zn2+ alternate conformations modeled in 7k3t, displaying tetrahedral coordination geometry (white dotted lines), but is extremely important to demonstrate definitive placement of Zn2+ in our multitemperature models.

Fo - Fo difference maps reveal local conformational shifts

Isomorphous Fo - Fo difference maps are calculated by subtracting of one set of observed experimental data from another, in turn showing where these data disagree. This allows the visualization of changes or motions between two datasets. Here we show an overview of the isomorphous Fo - Fo difference electron density map at +/- 3 σ (green/red volume) for the 240 K dataset (cyan) minus the 100 K dataset (dark blue). Ligands from cocrystal structures are shown at the active site (pale orange, 6lu7), interdomain interface (purple, 5ree; yellow, 5rec), and dimer interface (orange, 7lfp; pink, 5rf0). . At the dimer interface Glu290 switches from to . Glu290 is spatially adjacent to Cys128, which switches from to a single rotamer at 277 K and above in our multiconformer models; the alternate rotamer occupancy is lower at 240 K, consistent with its positive Fo - Fo peak. These residues are near (7lfp and 5rf0), as well as many ordered PEG molecules from the crystallization cocktails of various structures (7kvr, 7kvl, 7kfi, and 7lfe). of the biological monomer, constituted in the crystal from a symmetry-related protomer (gray surface). This interface also includes the Asp197 region.

At the interdomain interface, Thr198 switches from two alternate side-chain rotamers at 100 K to a single rotamer at 240 K, while Glu240 – located across the interdomain interface – changes side-chain rotamer, with additional effects on the adjacent backbone of Pro241. Meanwhile, an interacting water molecule at 100 K (blue sphere) becomes displaced at 240 K, and is correspondingly absent in that model. In all, this highlights how a series of conformational changes may link the dimer interface to the substrate binding pocket, as well as how conformational changes may cascade through the interdomain interface toward the substrate binding pocket and active site, highlighting the possibility of allostery in this enzyme.

References

1. ↑ Ebrahim A, Riley BT, Kumaran D, Andi B, Fuchs MR, McSweeney S, Keedy DA. The tem-per-ature-dependent conformational ensemble of SARS-CoV-2 main protease (M(pro)). IUCrJ. 2022 Aug 17;9(Pt 5):682-694. doi: 10.1107/S2052252522007497. eCollection, 2022 Sep 1. PMID:36071812 doi:http://dx.doi.org/10.1107/S2052252522007497