Chemical ligation of the influenza <scp>M</scp>2 protein for solid‐state <scp>NMR</scp> characterization of the cytoplasmic domain

Kwon, Byungsu; Tietze, Daniel; White, Paul B.; Liao, Shu-Yu; Hong, Mei

doi:10.1002/pro.2690

Cited by 36 publications

(34 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An amphipathic helix C-terminal to the TM helix mediates ESCRT-independent membrane scission during virus budding [23–25]. Finally a disordered cytoplasmic tail [26] is involved in M1 recognition and virus assembly [27]. The proton channel function is fully encapsulated in the TM domain based on proton-current measurements in vivo and in vitro .…”

Section: Introductionmentioning

confidence: 99%

Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

Mandala

Liao

Kwon

et al. 2017

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

The influenza M2 protein forms an acid-activated proton channel that is essential for virus replication. The transmembrane H37 selects for protons under low external pH (pHout) while W41 ensures proton conduction only from the N-terminus to the C-terminus and prevents reverse current under low internal pH (pHin). Here we address the molecular basis for this asymmetric conduction by investigating the structure and dynamics of a mutant channel, W41F, which permits reverse current under low pHin. Solid-state NMR experiments show that W41F M2 retains the pH-dependent α-helical conformations and tetrameric structure of the wild-type channel, but has significantly altered protonation and tautomeric equilibria at H37. At high pH, the H37 structure is shifted towards the π tautomer and less cationic tetrads, consistent with faster forward deprotonation to the C-terminus. At low pH, the mutant channel contains more cationic tetrads than the wild-type channel, consistent with faster reverse protonation from the C-terminus. 15N NMR spectra allow the extraction of four H37 pKa’s and show that the pKa’s are more clustered in the mutant channel compared to wild-type M2. Moreover, binding of the antiviral drug, amantadine, at the N-terminal pore at low pH did not convert all histidines to the neutral state, as seen in wild-type M2, but left half of all histidines cationic, unambiguously demonstrating C-terminal protonation of H37 in the mutant. These results indicate that asymmetric conduction in wild-type M2 is due to W41 inhibition of C-terminal acid activation by H37. When Trp is replaced by Phe, protons can be transferred to H37 bidirectionally with distinct rate constants.

show abstract

Section: Introductionmentioning

confidence: 99%

Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

Mandala

Liao

Kwon

et al. 2017

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

show abstract

“…), then adding di- tert -butyl dicarbonate (0.5 mmol, 5 eq.) in DMF (1.5 mL) to the solution and stirring for 2 h. After Boc protection, the resin-bound M2(1–18) was methylated at the sulfamyl group 42 . The resin-bound peptide was swelled for 2 h in 2 mL tetrahydrofuran (THF), then a 5.3 mL solution (1 : 1 v/v THF : hexane) of 1 M (trimethylsilyl)-diazomethane was added and stirred for 2 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

The Influenza M2 Ectodomain Regulates the Conformational Equilibria of the Transmembrane Proton Channel: Insights from Solid-State Nuclear Magnetic Resonance

Kwon

Hong

2016

Biochemistry

Self Cite

View full text Add to dashboard Cite

The influenza M2 protein is the target of the amantadine family of antiviral drugs, and its transmembrane (TM) domain structure and dynamics have been extensively studied. However, little is known about the structure of the highly conserved N-terminal ectodomain, which contains epitopes targeted by influenza vaccines. In this study, we synthesized an M2 construct containing the N-terminal ectodomain and the TM domain, to understand the site-specific conformation and dynamics of the ectodomain and to investigate the ectodomain effect on the TM structure. We incorporated 13C, 15N-labeled residues into both domains and measured their chemical shifts and linewidths using solid-state NMR. The data indicate that the entire ectodomain is unstructured and dynamic, but the motion is slower for residues closer to the TM domain. 13C lineshapes indicate that this ecto-TM construct undergoes fast uniaxial rotational diffusion, similar to the isolated TM peptide, but drug binding increases the motional rates of the TM helix while slowing down the local motion of the ectodomain residues that are close to the TM domain. Moreover, 13C and 15N chemical shifts indicate that the ectodomain shifts the conformational equilibria of the TM residues towards the drug-bound state even in the absence of amantadine, thus providing a molecular structural basis for the lower inhibitory concentration of full-length M2 compared to the ectodomain-truncated M2. We propose that this conformational selection may result from electrostatic repulsion between negatively charged ectodomain residues in the tetrameric protein. Together with the recent study of the M2 cytoplasmic domain, these results show that intrinsically disordered extramembrane domains in membrane proteins can regulate functionally relevant conformation and dynamics of the structurally ordered transmembrane domains.

show abstract

“…NCL is also compatible with protein denaturants or detergents allowing the construction of aggregation prone polypeptide chains or even polytopic membrane proteins (Hejjaoui et al . 2012; Kwon et al . 2015; Valiyaveetil et al .…”

Section: Molecular Engineering Toolbox For Complex Biological Samplesmentioning

confidence: 99%

A molecular engineering toolbox for the structural biologist

Debelouchina

Muir

2017

Quart. Rev. Biophys.

View full text Add to dashboard Cite

Exciting new technological developments have pushed the boundaries of structural biology, and have enabled studies of biological macromolecules and assemblies that would have been unthinkable not long ago. Yet, the enhanced capabilities of structural biologists to pry into the complex molecular world have also placed new demands on the abilities of protein engineers to reproduce this complexity into the test tube. With this challenge in mind, we review the contents of the modern molecular engineering toolbox that allow the manipulation of proteins in a site-specific and chemically well-defined fashion. Thus, we cover concepts related to the modification of cysteines and other natural amino acids, native chemical ligation, intein and sortase-based approaches, amber suppression, as well as chemical and enzymatic bio-conjugation strategies. We also describe how these tools can be used to aid methodology development in X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy and in the studies of dynamic interactions. It is our hope that this monograph will inspire structural biologists and protein engineers alike to apply these tools to novel systems, and to enhance and broaden their scope to meet the outstanding challenges in understanding the molecular basis of cellular processes and disease.

show abstract

Chemical ligation of the influenza M2 protein for solid‐state NMR characterization of the cytoplasmic domain

Cited by 36 publications

References 68 publications

Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

The Influenza M2 Ectodomain Regulates the Conformational Equilibria of the Transmembrane Proton Channel: Insights from Solid-State Nuclear Magnetic Resonance

A molecular engineering toolbox for the structural biologist

Contact Info

Product

Resources

About