Fig. 4: Clientization alters the S tail conformation and strengthens binding to WD40 domains.
From: A single C-terminal residue controls SARS-CoV-2 spike trafficking and incorporation into VLPs
A Two-dimensional 1H-15N HSQC spectrum of wild-type S tail 21-mer peptide at 25oC with backbone amide assignments. Assignments in italics are putatively due to a small population ( ~ 10%) of peptide with cis-Pro1263. B Plot of 13C secondary chemical shifts (experiment-calculated random coil) in the wild-type (top) and Cys1253Ala-Thr1273Glu clientized (bottom) S tail peptides for Cα (black) and CO (red) resonances. Weak consensus β-propensity is observed in residues Glu1258-Asp1260 for both the wild-type and clientized S-tails and in the dibasic motif-containing Val1268-His1271 for the wild-type S tail but not the clientized S tail. The error bars represent an estimate for the error in measuring the 13C chemical shifts based on two independent experiments with wild-type S tail samples. C, D BLI assays showing αWD40 interactions of, C wild-type and, D clientized S tails, the latter being substantially stronger but slower. The concentration of WD40 domains used in BLI assays are shown on the right. E PRE plots of Iox/Ired versus residue for Cys1253Ala S tail 21-mer peptide, Cys1253Ala-Thr1273Glu clientized S tail, and a control for intermolecular effects. PRE experiments were performed in duplicate for the Cys1253Ala A tail peptide and intermolecular control, and in triplicate for the Cys1253Ala-Thr1273Glu clientized S tail. The plots show the mean values (bars, points) and error bars are for ±1 standard deviation for n = 2 (SARS-CoV-2 wild-type S tail), n = 3 (SARS-CoV-2 Cys1253Ala-Thr1273Glu clientized S tail), and n = 2 (inter-peptide PRE) biologically independent samples. The position of the MTSL spin label is indicated (orange arrow).
