title: Tryptophan-dependent membrane interaction and heteromerization with the internal fusion peptide by the membrane proximal external region of SARS-CoV spike protein.
abstract: The spike (S) protein of severe acute respiratory syndrome-associated CoV (SARS-CoV) mediates membrane fusion and viral entry. These events involve structural rearrangements, including heteromerization between two heptad repeats (HR1 and HR2) to form a trimer of dimers as a six-helix bundle (6-HB), a quaternary protein structure that brings two distant clusters of hydrophobic sequences into the proximity of each other, the internal fusion peptide (IFP) preceding HR1, and the highly conserved tryptophan (Trp)-rich membrane proximal external region (MPER) following HR2. Here, we show that MPER can undergo self-oligomerization and heteromerization with IFP, events that are Trp-dependent. To delineate the roles of Trp residues of MPER in forming these quaternary structures and interacting with membranes, we employed a panel of synthetic peptides: MPER peptide (M-wt) and its alanine (Ala) and phenylalanine (Phe) analogues. Ala substitutions of Trp inhibited its association with cellular membranes. Chemical cross-linking experiments showed that M-wt can self-interact to form oligomers and cross-interact with IFP23, a synthetic IFP peptide, to form a heterohexamer. In comparison, little high-order oligomer was formed between M-wt and fusion peptide. The specific interaction between M-wt and IFP23 was confirmed by immunofluorescence staining experiments. In aqueous solutions, both M-wt and IFP23 displayed random secondary structures that became helical in hydrophobic solvents. Triple-Ala substitutions of Trp in M-wt, but not the corresponding triple-Phe analogue, disrupted oligomerization of M-wt and hetero-oligomerization of M-wt with IFP23. Overall, our results show that Trp residues of MPER play a key role in maintaining the structure and functions of MPER, allowing it to interact with IFP to form a MPER-IFP heteromer, a putative quaternary structure extending from the 6-HB, and function in membrane fusion. Finally, we showed that a MPER peptide could serve as an inhibitor in the entry process.
authors: School of Biological Sciences, Nanyang Technological University , 60 Nanyang Drive, Singapore 637551.
authors: Ying
authors: Y
authors: Liao
authors: Si Min
authors: SM
authors: Zhang
authors: Tuan Ling
authors: TL
authors: Neo
authors: James P
authors: JP
authors: Tam
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism* / Q000821:virology; D002522:Chlorocebus aethiops; D010446:Peptide Fragments / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D055503:Protein Multimerization / Q000235:genetics; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000378:metabolism; D064370:Spike Glycoprotein, Coronavirus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000502:physiology*; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D064370:Spike Glycoprotein, Coronavirus; D014760:Viral Fusion Proteins; D010649:Phenylalanine; D014364:Tryptophan; D000409:Alanine