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A Budding-Defective M2 Mutant Exhibits Reduced Membrane Interaction, Insensitivity to Cholesterol, and Perturbed Interdomain Coupling. Biochemistry 2017 Nov 07;56(44):5955-5963

Date

10/17/2017

Pubmed ID

29034683

Pubmed Central ID

PMC6112238

DOI

10.1021/acs.biochem.7b00924

Scopus ID

2-s2.0-85033361759 (requires institutional sign-in at Scopus site)   14 Citations

Abstract

Influenza A M2 is a membrane-associated protein with a C-terminal amphipathic helix that plays a cholesterol-dependent role in viral budding. An M2 mutant with alanine substitutions in the C-terminal amphipathic helix is deficient in viral scission. With the goal of providing atomic-level understanding of how the wild-type protein functions, we used a multipronged site-directed spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) approach to characterize the conformational properties of the alanine mutant. We spin-labeled sites in the transmembrane (TM) domain and the C-terminal amphipathic helix (AH) of wild-type (WT) and mutant M2, and collected information on line shapes, relaxation rates, membrane topology, and distances within the homotetramer in membranes with and without cholesterol. Our results identify marked differences in the conformation and dynamics between the WT and the alanine mutant. Compared to WT, the dominant population of the mutant AH is more dynamic, shallower in the membrane, and has altered quaternary arrangement of the C-terminal domain. While the AH becomes more dynamic, the dominant population of the TM domain of the mutant is immobilized. The presence of cholesterol changes the conformation and dynamics of the WT protein, while the alanine mutant is insensitive to cholesterol. These findings provide new insight into how M2 may facilitate budding. We propose the AH-membrane interaction modulates the arrangement of the TM helices, effectively stabilizing a conformational state that enables M2 to facilitate viral budding. Antagonizing the properties of the AH that enable interdomain coupling within M2 may therefore present a novel strategy for anti-influenza drug design.

Author List

Herneisen AL, Sahu ID, McCarrick RM, Feix JB, Lorigan GA, Howard KP

Author

Jimmy B. Feix PhD Professor in the Biophysics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Cell Membrane
Cholesterol
Electron Spin Resonance Spectroscopy
Humans
Influenza A virus
Mutation
Protein Conformation
Protein Domains
Protein Structural Elements
Viral Matrix Proteins
Virus Release