Comparing the binding properties of peptides mimicking the Envelope protein of SARS-CoV and SARS-CoV-2 to the PDZ domain of the tight junction-associated PALS1 protein

Abstract

The Envelope protein (E) is one of the four structural proteins encoded by the genome of SARS-CoV and SARS-CoV-2 Coronaviruses. It is an integral membrane protein, highly expressed in the host cell, which is known to have an important role in Coronaviruses maturation, assembly and virulence. The E protein presents a PDZ-binding motif at its C-terminus. One of the key interactors of the E protein in the intracellular environment is the PDZ containing protein PALS1. This interaction is known to play a key role in the SARS-CoV pathology and suspected to affect the integrity of the lung epithelia. In this paper we measured and compared the affinity of peptides mimicking the E protein from SARS-CoV and SARS-CoV-2 for the PDZ domain of PALS1, through equilibrium and kinetic binding experiments. Our results support the hypothesis that the increased virulence of SARS-CoV-2 compared to SARS-CoV may rely on the increased affinity of its Envelope protein for PALS1.

Keywords: PALS1; PDZ; SARS-CoV-2; binding; envelope protein; kinetics.

FIGURE 1

Structural features of the interaction between the E protein and PALS1. (a) Three‐dimensional structure of the PDZ domain of PALS1 (PDB: 4UU5); (b) comparison of the sequences of peptides mimicking the Envelope protein of SARS‐CoV and SARS‐CoV‐2. Residues not conserved are highlighted in red. The PDZ binding motifs are highlighted in green

FIGURE 2

Comparing the binding of peptides mimicking the Envelope protein of SARS‐CoV and SARS‐CoV‐2 to the PDZ domain of PALS1. (a) Equilibrium binding titration monitored by FRET between PALS1 PDZ domain and dansylated peptides mimicking the Envelope protein of SARS‐CoV (in gray) and SARS‐CoV‐2 (in black). Lines are the best fit to a hyperbolic function. (b) Observed rate constants calculated from T‐jump kinetics at different concentrations of dansylated SARS‐CoV (gray empty circles) and SARS‐CoV‐2 (black empty circles) peptides. Lines represent the best fit to a linear function. Full gray and black circles represent the microscopic dissociation rate constant (k _off) calculated from stopped‐flow displacement experiments (displacement traces fitted to a single exponential equation are reported in (d)—gray SARS‐CoV, black SARS‐CoV‐2). (c) Representative T‐jump experiment carried out in the presence of PALS1 PDZ and dansylated peptide mimicking the Envelope protein from SARS‐CoV‐2. The monitored reaction appears to be complete within ~4 ms. Fluorescence emission before and after the temperature jump is shown (in gray). Black line represents the best fit to a single exponential function