Rapid Determination of the Topology of Oligomeric & alpha;-Helical Membrane Proteins by Water- and Lipid-Edited Methyl NMR

Single-span oligomeric & alpha;-helical transmembraneproteins arecommon in virus ion channels, which are targets of antiviral drugs.Knowledge about the high-resolution structures of these oligomeric & alpha;-helical bundles is so far scarce. Structure determinationof these membrane proteins by solid-state NMR traditionally requiresresolving and assigning protein chemical shifts and measuring manyinterhelical distances, which are time-consuming. To accelerate experimentalstructure determination, here we introduce a simple solid-state NMRapproach that uses magnetization transfer from water and lipid protonsto the protein. By detecting the water- and lipid-transferred intensitiesof the high-sensitivity methyl C-13 signals of Leu, Val,and Ile residues, which are highly enriched in these membrane proteins,we can derive models of the topology of these homo-oligomeric helicalbundles. The topology is specified by the positions of amino acidresidues in heptad repeats and the orientations of residues relativeto the channel pore, lipids, and the helical interface. We demonstratethis water- and lipid-edited methyl NMR approach on the envelope (E)protein of SARS-CoV-2, the causative agent of the COVID-19 pandemic.We show that water-edited and lipid-edited 2D C-13-C-13 correlation spectra can be measured with sufficient sensitivity.Even without resolving multiple residues of the same type in the NMRspectra, we can obtain the helical bundle topology. We apply theseexperiments to the structurally unknown E proteins of the MERS coronavirusand the human coronavirus NL63. The resulting structural topologiesshow interesting differences in the positions of the aromatic residuesin these three E proteins, suggesting that these viroporins may havedifferent mechanisms of activation and ion conduction.

Automated summary

Single-span oligomeric & alpha;-helical transmembrane proteins are common targets of antiviral drugs, but their high-resolution structures are not well understood. This study introduces a simple solid-state NMR approach using magnetization transfer from water and lipid protons to accelerate the experimental structure determination of these proteins. Results demonstrate successful application of this approach on the envelope protein of SARS-CoV-2, revealing the topology of the helical bundles. The experiments also show interesting differences in the positions of aromatic residues in the envelope proteins of different coronaviruses.