The Role of an Amphiphilic Helix and Transmembrane Region in the Efficient Acylation of the M2 Protein from Influenza Virus

Overview

Journal Sci Rep

Specialty Science

Date 2023 Nov 3

PMID 37919373

Authors

Xiaorong Meng

Clark Templeton

Cecilia Clementi

Michael Veit

Affiliations

Soon will be listed here.

Abstract

Protein palmitoylation, a cellular process occurring at the membrane-cytosol interface, is orchestrated by members of the DHHC enzyme family and plays a pivotal role in regulating various cellular functions. The M2 protein of the influenza virus, which is acylated at a membrane-near amphiphilic helix serves as a model for studying the intricate signals governing acylation and its interaction with the cognate enzyme, DHHC20. We investigate it here using both experimental and computational assays. We report that altering the biophysical properties of the amphiphilic helix, particularly by shortening or disrupting it, results in a substantial reduction in M2 palmitoylation, but does not entirely abolish the process. Intriguingly, DHHC20 exhibits an augmented affinity for some M2 mutants compared to the wildtype M2. Molecular dynamics simulations unveil interactions between amino acids of the helix and the catalytically significant DHHC and TTXE motifs of DHHC20. Our findings suggest that the binding of M2 to DHHC20, while not highly specific, is mediated by requisite contacts, possibly instigating the transfer of fatty acids. A comprehensive comprehension of protein palmitoylation mechanisms is imperative for the development of DHHC-specific inhibitors, holding promise for the treatment of diverse human diseases.

References

Brett K, Kordyukova L, Serebryakova M, Mintaev R, Alexeevski A, Veit M . Site-specific S-acylation of influenza virus hemagglutinin: the location of the acylation site relative to the membrane border is the decisive factor for attachment of stearate. J Biol Chem. 2014; 289(50):34978-89. PMC: 4263894. DOI: 10.1074/jbc.M114.586180. View

Philippe J, Jenkins P . Spatial organization of palmitoyl acyl transferases governs substrate localization and function. Mol Membr Biol. 2020; 35(1):60-75. PMC: 7031816. DOI: 10.1080/09687688.2019.1710274. View

Gottlieb C, Zhang S, Linder M . The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc. J Biol Chem. 2015; 290(49):29259-69. PMC: 4705932. DOI: 10.1074/jbc.M115.691147. View

Zaballa M, van der Goot F . The molecular era of protein S-acylation: spotlight on structure, mechanisms, and dynamics. Crit Rev Biochem Mol Biol. 2018; 53(4):420-451. DOI: 10.1080/10409238.2018.1488804. View

Chamberlain L, Shipston M . The physiology of protein S-acylation. Physiol Rev. 2015; 95(2):341-76. PMC: 4551212. DOI: 10.1152/physrev.00032.2014. View

Fraser N, Howie J, Wypijewski K, Fuller W . Therapeutic targeting of protein S-acylation for the treatment of disease. Biochem Soc Trans. 2019; 48(1):281-290. DOI: 10.1042/BST20190707. View

Holsinger L, Lamb R . Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds. Virology. 1991; 183(1):32-43. DOI: 10.1016/0042-6822(91)90115-r. View

Gadalla M, Abrami L, van der Goot F, Veit M . Hemagglutinin of Influenza A, but not of Influenza B and C viruses is acylated by ZDHHC2, 8, 15 and 20. Biochem J. 2019; 477(1):285-303. DOI: 10.1042/BCJ20190752. View

Castrucci M, Hughes M, Calzoletti L, Donatelli I, Wells K, Takada A . The cysteine residues of the M2 protein are not required for influenza A virus replication. Virology. 1997; 238(1):128-34. DOI: 10.1006/viro.1997.8809. View

10.

Lamb R, Zebedee S, Richardson C . Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface. Cell. 1985; 40(3):627-33. DOI: 10.1016/0092-8674(85)90211-9. View

11.

Thaa B, Tielesch C, Moller L, Schmitt A, Wolff T, Bannert N . Growth of influenza A virus is not impeded by simultaneous removal of the cholesterol-binding and acylation sites in the M2 protein. J Gen Virol. 2011; 93(Pt 2):282-292. DOI: 10.1099/vir.0.038554-0. View

12.

Mitchell D, Vasudevan A, Linder M, Deschenes R . Protein palmitoylation by a family of DHHC protein S-acyltransferases. J Lipid Res. 2006; 47(6):1118-27. DOI: 10.1194/jlr.R600007-JLR200. View

13.

Ellgaard L, Helenius A . Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol. 2003; 4(3):181-91. DOI: 10.1038/nrm1052. View

14.

Malgapo M, Linder M . Substrate recruitment by zDHHC protein acyltransferases. Open Biol. 2021; 11(4):210026. PMC: 8059564. DOI: 10.1098/rsob.210026. View

15.

Gadalla M, Veit M . Toward the identification of ZDHHC enzymes required for palmitoylation of viral protein as potential drug targets. Expert Opin Drug Discov. 2019; 15(2):159-177. DOI: 10.1080/17460441.2020.1696306. View

16.

Abdulrahman D, Meng X, Veit M . S-Acylation of Proteins of Coronavirus and Influenza Virus: Conservation of Acylation Sites in Animal Viruses and DHHC Acyltransferases in Their Animal Reservoirs. Pathogens. 2021; 10(6). PMC: 8227752. DOI: 10.3390/pathogens10060669. View

17.

Fukata M, Fukata Y, Adesnik H, Nicoll R, Bredt D . Identification of PSD-95 palmitoylating enzymes. Neuron. 2004; 44(6):987-96. DOI: 10.1016/j.neuron.2004.12.005. View

18.

Rana M, Lee C, Banerjee A . The molecular mechanism of DHHC protein acyltransferases. Biochem Soc Trans. 2018; 47(1):157-167. DOI: 10.1042/BST20180429. View

19.

Thaa B, Levental I, Herrmann A, Veit M . Intrinsic membrane association of the cytoplasmic tail of influenza virus M2 protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation. Biochem J. 2011; 437(3):389-97. DOI: 10.1042/BJ20110706. View

20.

Veit M, Sachs K, Heckelmann M, Maretzki D, Hofmann K, Schmidt M . Palmitoylation of rhodopsin with S-protein acyltransferase: enzyme catalyzed reaction versus autocatalytic acylation. Biochim Biophys Acta. 1998; 1394(1):90-8. DOI: 10.1016/s0005-2760(98)00097-6. View