Journal:IUCrJ:S2052252522007497
From Proteopedia
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 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. Biological representation of Mpro. 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). The close-up view of substrate binding pocket 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). 7k3t anomalous density map. 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. Overview of the local conformational shifts connecting the active site, interdomain interface, and dimer interface. The 240 K dataset is in cyan and the 100 K dataset is in dark blue. Ligands from cocrystal structures are shown at the active site (salmon, 6lu7), interdomain interface (violet, 5ree; yellow, 5rec), and dimer interface (orange, 7lfp; pink, 5rf0). Conformational shifts at the dimer interface. At the dimer interface Glu290 switches from one side-chain rotamer at 100 K to two alternate rotamers at 240 K. Glu290 is spatially adjacent to Cys128, which switches from two alternate rotamers at 100 and 240 K 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 two ligands from separate crystallographic screens (7lfp and 5rf0), as well as many ordered PEG molecules from the crystallization cocktails of various structures (7kvr, 7kvl, 7kfi, and 7lfe). Remember to drag the structures with the mouse to rotate them. These two ligands bind at the dimer interface 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. Two ligands are shown at the interdomain interface (violet, 5ree; yellow, 5rec). 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
|
| ||||||||||
