Prediction of conformational states in a coronavirus channel using Alphafold-2 and DeepMSA2: Strengths and limitations

Abstract

The envelope (E) protein is present in all coronavirus genera. This protein can form pentameric oligomers with ion channel activity which have been proposed as a possible therapeutic target. However, high resolution structures of E channels are limited to those of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for the recent COVID-19 pandemic. In the present work, we used Alphafold-2 (AF2), in ColabFold without templates, to predict the transmembrane domain (TMD) structure of six E-channels representative of genera alpha-, beta- and gamma-coronaviruses in the Coronaviridae family. High-confidence models were produced in all cases when combining multiple sequence alignments (MSAs) obtained from DeepMSA2. Overall, AF2 predicted at least two possible orientations of the α-helices in E-TMD channels: one where a conserved polar residue (Asn-15 in the SARS sequence) is oriented towards the center of the channel, 'polar-in', and one where this residue is in an interhelical orientation 'polar-inter'. For the SARS models, the comparison with the two experimental models 'closed' (PDB: 7K3G) and 'open' (PDB: 8SUZ) is described, and suggests a ∼60˚ α-helix rotation mechanism involving either the full TMD or only its N-terminal half, to allow the passage of ions. While the results obtained are not identical to the two high resolution models available, they suggest various conformational states with striking similarities to those models. We believe these results can be further optimized by means of MSA subsampling, and guide future high resolution structural studies in these and other viral channels.

Keywords: AlphaFold-2; Coronavirus; Deep MSA; Envelope protein; IBV; Ion channel; SARS.

Graphical abstract

Fig. 1

Representative CoV E TMD sequences used for AF2 prediction. Acidic residues (red), basic residues (blue) and a conserved polar residue (highlighted yellow) are indicated, to visualize the similarity between the sequences: E proteins tend to have negatively charged residues at the N-terminal side, and basic residues on the C-terminal side at juxtamembrane positions. It is also noted that one of the two mutations that completely inactivate the channel is Asn-15 in SARS, and a polar residue at this or equivalent position is conserved in most E proteins. This residue has been used as indicator to preliminarily visualise the orientation of the bundles (see next sections).

Fig. 2

Number of sequences in MSAs obtained by three different methods. MSA-1 (blue), MSA-2 (green) and MSA-3 (red).

Fig. 3

Scores results obtained using various MSAs. (A) individual MSA-1; (B) individual MSA-2; (C) local AF2; (D) combined MSA-1; (E) individual MSA-3; (F) combined all MSA-1 and MSA-3 (MSA-4).

Fig. 4

Prediction for increasing number of sequences in the combined MSA-4. Scores corresponding to the 20 models obtained using MSA-4 that contained an increasing number of sequences; from 71 (group 1, including only E sequences), and after addition of branches of the evolutionary tree that were progressively farther from the E sequences, from group 2 (101 sequences) to group 6 (585 sequences) for the TMDs of the representative viruses indicated in each panel. Red cross and bar represent average and median scores, respectively.

Fig. 5

Representative best models obtained for each of the TMD sequences. The pentameric models are represented in a side view (top row) and a view from the N-terminus (bottom row). To guide the eye, the side chain of the fully conserved polar residue in each sequence is shown as sticks. The models are colored according to pLDDT in AF2; (C) schematic representation of the way in which the rotational orientation per residue was calculated; one of the monomers in the pentamer shows two vectors, both coming from the centre the helix. One is directed towards the centre of the channel and the other towards the α-carbon (CA) of a residue. The angle between these two vectors, ω, was obtained and represented; (D) rotational orientation ω of the residues in the four ‘polar in’ orientations indicated; (E) residues directly exposed to the lumen of the channel (yellow) according to HOLE.

Fig. 6

Comparison of AF2-predicted and experimental SARS channels. (A) AF2-predicted ‘polar_in’ and ‘polar-inter’ models colored according to the pLDDT score; (B) top view from the N-terminus of the models in (A), showing the orientation of the polar residue Asn15; (C) inner volume of the channel for the models in (A) where the most constricted region is indicated by a red rectangle; (D-E) experimental models in side (D) and top (E) views; (F) discrepancy between the AF2-predicted and experimental models; (G) calculated difference between ω angles of SARS ‘polar-in’ minus ‘SARS-inter’ or minus 8SUZ. The horizontal dotted line shows the average ω difference between the two predicted SARS models (−62.6°); (H) same as (G) for the differences respect to model 7K3G; (I) residues in these four models directly in contact with the lumen of the channel (highlighted in yellow). A more complete description of the luminal orientation in these and other models is shown in Fig. S6.

Fig. 7

Representative three clusters of models above the cut-off for MHV ETM pentamers. (A) Side view, (B) top view from the N-terminus, showing the orientation of the polar residue Q15; (C) lumenal space obtained with HOLE . Models in (A-B) are colored according to the pLDDT score. Residues exposed to the channel are shown in Supplementary Fig. S6.