Sequence requirements of the FFAT‐like motif for specific binding to VAP‐A are revealed by NMR

Abstract

The endoplasmic reticulum transmembrane protein vesicle‐associated membrane protein‐associated protein (VAP) plays a central role in the formation and function of membrane contact sites (MCS) through its interactions with proteins. The major sperm protein (MSP) domain of VAP binds to a variety of sequences which are referred to as FFAT‐like motifs. In this study, we investigated the interactions of eight peptides containing FFAT‐like motifs with the VAP‐A MSP domain (VAP‐AMSP) by solution NMR. Six of eight peptides are specifically bound to VAP‐A. Furthermore, we found that the RNA‐dependent RNA polymerase of severe acute respiratory syndrome coronavirus 2 has an FFAT‐like motif which specifically binds to VAP‐AMSP as well as other FFAT‐like motifs. Our results will contribute to the discovery of new VAP interactors.

XopM, a FFAT motif containing type-III effector protein fromXanthomonas, suppresses PTI responses at the plant plasma membrane

“…Intriguingly, both XopM FFAT motifs can substitute for each other for VAP binding suggesting that the presence of two FFAT motifs in XopM is an evolutionary adaptation to increase the interaction with VAPs or represents a possibility to interact with two VAPs simultaneously. The sequence of the XopM cFFAT (Q-F-Y-D-E-A-D) closely matches the consensus FFAT motif and key residues that have been shown to mediate the FFAT/VAP interaction, such as the aromatic amino acid residue at position 2, are conserved (Loewen & Levine, 2005, Furuita et al ., 2021). Structural predictions of the XopM/VAP interaction suggest that the phenylalanine at position 2 of the XopM cFFAT is positioned in close proximity of critical FFAT interaction residues with the VAP MSP.…”

“…However, it also carries an aromatic residue at position 2 (Y) and position 4 harbors a threonine residue that could be phosphorylated and therefore mimic the aspartate residue present in the canonical sequence (Di Mattia et al ., 2020). Furthermore, both XopM FFAT motifs are directly preceded by multiple serine residues, which, if phosphorylated could impart additional negative charges to the motif, which would increase VAP binding (Furuita et al, 2021).…”

A snapshot of protein trafficking in SARS‐CoV‐2 infection

“…Using the identified characteristics of these interactions, the authors searched for potential FFAT‐like motifs in SARS‐CoV‐2 proteins and detected an EFYEA sequence at the C‐terminus of nsp12 (the RNA‐dependent RNA polymerase) as potential VAP interactor. NMR analysis with a peptide containing the nsp12 EFYEA sequence confirmed its interaction with VAP with an affinity comparable to the one of known interacting peptides (Furuita et al., 2021 ), raising the possibility of a role of nsp12‐VAP interaction at ER‐derived membranes during viral replication. Since nsp12 is the viral RNA‐dependent RNA polymerase replicating viral RNA in the ER‐derived DMVs, nsp12‐VAP interaction might enrich viral and cellular co‐factors required for viral replication.…”

“…VAP interactors usually associate with other cellular membranes, hence creating molecular bridges and contact sites between ER and these organelles. A recent NMR‐based study determined the interaction of six peptides containing the FFAT‐like motifs of known VAP interactors (Furuita et al., 2021 ). Using the identified characteristics of these interactions, the authors searched for potential FFAT‐like motifs in SARS‐CoV‐2 proteins and detected an EFYEA sequence at the C‐terminus of nsp12 (the RNA‐dependent RNA polymerase) as potential VAP interactor.…”

Two years into COVID‐19 – Lessons in SARS‐CoV‐2 and a perspective from papers in FEBS Letters

“…OSBP1 shuttles cholesterol and PI4P between ER and Golgi at membrane contact sites and drives a counter‐current lipid flux for membrane growth. Using in‐solution NMR, Furuita and colleagues identified an FFAT‐like motif in the SARS‐CoV‐2 RNA‐dependent RNA polymerase (RdRP), which specifically binds to the major sperm protein domain of VAP‐A [30]. Such mechanism might ensure appropriate localization of RdRP on the viral replication membrane.…”