Atomic structures of closed and open influenza B M2 proton channel reveal the conduction mechanism

Mandala, Venkata S.; Loftis, Alexander R.; Shcherbakov, A.; Pentelute, Bradley L.; Hong, Mei

doi:10.1038/s41594-019-0371-2

Cited by 60 publications

(110 citation statements)

References 60 publications

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“…Understanding these features as they relate to drug binding gives further insight into the specific interactions that stabilize the adamantyl amine inhibitors in wild-type M2, and these results suggests that this approach could be used to aid future drug-design efforts to methodically create new inhibitors for S31N mutants. With the recent publication of high resolution influenza B M2 (BM2) structures, 91 it is possible to conduct similar studies to elucidate the detailed PT mechanism in BM2 to guide drug design in this functionally similar protein.…”

Section: Discussionmentioning

confidence: 99%

Influenza A M2 Inhibitor Binding Understood through Mechanisms of Excess Proton Stabilization and Channel Dynamics

2020

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Prevalent resistance to inhibitors that target the influenza A M2 proton channel has necessitated a continued drug design effort, supported by a sustained study of the mechanism of channel function and inhibition. Recent high-resolution X-ray crystal structures present the first opportunity to see how the adamantyl amine class of inhibitors bind to M2 and disrupt and interact with the channel’s water network, providing insight into the critical properties that enable their effective inhibition in wild-type M2. In this work, we examine the hypothesis that these drugs act primarily as mechanism-based inhibitors by comparing hydrated excess proton stabilization during proton transport in M2 with the interactions revealed in the crystal structures, using the Multiscale Reactive Molecular Dynamics (MS-RMD) methodology. MS-RMD, unlike classical molecular dynamics, models the hydrated proton (hydronium-like cation) as a dynamic excess charge defect and allows bonds to break and form, capturing the intricate interactions between the hydrated excess proton, protein atoms, and water. Through this, we show that the ammonium group of the inhibitors is effectively positioned to take advantage of the channel’s natural ability to stabilize an excess protonic charge and act as a hydronium mimic. Additionally, we show that the channel is especially stable in the drug binding region, highlighting the importance of this property for binding the adamantane group. Finally, we characterize an additional hinge point near Val27, which dynamically responds to charge and inhibitor binding. Altogether, this work further illuminates a dynamic understanding of the mechanism of drug inhibition in M2, grounded in the fundamental properties that enable the channel to transport and stabilize excess protons, with critical implications for future drug design efforts.

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Section: Discussionmentioning

confidence: 99%

Influenza A M2 Inhibitor Binding Understood through Mechanisms of Excess Proton Stabilization and Channel Dynamics

2020

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“…Additional information was gained from the structures of the drug-resistant AM2 mutant S31N [ 137 , 138 ], showing that amantadine resistance of mutant S31N is caused by steric hindrance. More recently, the solid-state NMR structures of the BM2 channel in its open and closed conformation were solved in a phospholipid environment [ 130 ] ( Figure 7 b), revealing that the channel is more accessible to water at a low pH and that side chain dynamics are the essential driver of proton shuttling. Solid-state NMR structures were all solved in lipid bilayers, whereas most X-ray structures, e.g., of the transmembrane domain of AM2 in 2008 [ 139 ], were solved in detergents.…”

Section: Examples Of Solid-state Nmr Studies On Viral Proteinsmentioning

confidence: 99%

“… Solid-state NMR structures of ( a ) Vpu from HIV-1 (PDB: 2N28 [ 127 ]), and ( b ) closed and open influenza BM2 channels in lipid membranes (PDB: 6pvr (pH 7.5) and 6pvt (pH 4.5) [ 130 ]). Panel ( a ) reprinted from reference [ 127 ] Copyright (2015), with permission from Elsevier.…”

Section: Figurementioning

confidence: 99%

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Solid-State NMR for Studying the Structure and Dynamics of Viral Assemblies

Lecoq

Fogeron

Meier

et al. 2020

Viruses

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Structural virology reveals the architecture underlying infection. While notably electron microscopy images have provided an atomic view on viruses which profoundly changed our understanding of these assemblies incapable of independent life, spectroscopic techniques like NMR enter the field with their strengths in detailed conformational analysis and investigation of dynamic behavior. Typically, the large assemblies represented by viral particles fall in the regime of biological high-resolution solid-state NMR, able to follow with high sensitivity the path of the viral proteins through their interactions and maturation steps during the viral life cycle. We here trace the way from first solid-state NMR investigations to the state-of-the-art approaches currently developing, including applications focused on HIV, HBV, HCV and influenza, and an outlook to the possibilities opening in the coming years.

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“…Magic-angle-spinning (MAS) solid-state nuclear magnetic resonance (SSNMR) spectroscopy produces high-resolution spectra by averaging orientation-dependent nuclear spin interactions such as chemical shift anisotropy (CSA), dipolar couplings, and quadrupolar couplings. MAS SSNMR is uniquely suited among spectroscopic techniques to provide atomic-resolution structural information of insoluble, non-crystalline biological macromolecules such as membrane proteins [1][2][3][4] , amyloid fibrils [5][6][7][8][9][10][11][12][13] , and cell walls [14][15][16][17] . Once chemical shift assignment is complete, three-dimensional structure determination predominantly relies on inter-atomic distance restraints.…”

Section: Introductionmentioning

confidence: 99%

Pulsed Third-Spin-Assisted Recoupling NMR for Obtaining Long-Range¹³C-¹³C and¹⁵N-¹³C Distance Restraints

Gelenter

Dregni

Hong

2020

Preprint

Self Cite

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We introduce a class of pulsed third-spin-assisted recoupling (P-TSAR) magic-angle-spinning (MAS) solid-state NMR techniques that achieve efficient polarization transfer over long distances to provide important restraints for structure determination. These experiments operate with the same principle as continuous-wave (CW) TSAR experiments, by utilizing second-order cross terms between strong 1 H-13 C and 1 H-15 N dipolar couplings to achieve 13 C-13 C and 13 C-15 N polarization transfer. However, in contrast to the CW-TSAR experiments, these pulsed P-TSAR experiments require much less radiofrequency (rf) energy and allow a much simpler routine for optimizing the rf field strength. We call the techniques PULSAR (PULSed proton Asissted Recoupling) for homonuclear spin pairs and PERSPIRATION CP (Proton-Enhanced Rotor-echo Short Pulse IRradiATION Cross-Polarization) for heteronuclear spin pairs. We demonstrate these techniques on the model protein GB1, and found cross peaks for distances as long as 10 and 8 Å for 13 C-13 C and 15 N-13 C spin pairs, respectively. We also apply these methods to the amyloid fibrils formed by the peptide hormone glucagon, and show that longrange correlation peaks are readily observed to constrain intermolecular packing in this cross-b fibril.We provide an analytical model for the PULSAR and PERSPIRATION CP experiments to explain the measured and simulated chemical shift dependence and pulse flip angle dependence of polarization transfer. These two techniques are useful for measuring long-range distance restraints to determine the three-dimensional structures of proteins and other biological macromolecules.Third-Spin-Assisted Recoupling (TSAR) experiments are a class of techniques that transfer polarization between low-g nuclei A and B 27 . This transfer occurs via cross terms in the second-order average Hamiltonian between the H-A and H-B dipolar couplings, which are generally much stronger than the direct A-B dipolar coupling. In continuous-wave TSAR (CW-TSAR) experiments, the homonuclear variant is known as Proton-Asisted Recoupling (PAR) 28 whereas the heteronuclear variant is called Proton-Asissted Insensitive Nuclei Cross Polarization ( PAIN CP) [29][30] . The CW irradiation in these experiments requires carefully optimized rf field strengths on both the 1 H channel and the heteronuclear channels. This is usually achieved with an extensive multidimensional search of the rf field strengths, without which the polarization transfer efficiency can easily fall below the theoretical maximum 31 . Moreover, mixing times of 10-30 ms are usually required to observe long-range 13 C-13 C and 15 N-13 C correlation peaks. The extended high-power CW irradiation on two to three channels can damage the NMR probe and heat-sensitive biological samples. These two practical drawbacks have limited the use of the PAR and PAIN CP experiments, despite their theoretical ability for long-range spin polarization transfer, compared to the simpler spin diffusion experiments.Here we introduce pulsed TSAR (P-TSAR...

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Atomic structures of closed and open influenza B M2 proton channel reveal the conduction mechanism

Cited by 60 publications

References 60 publications

Influenza A M2 Inhibitor Binding Understood through Mechanisms of Excess Proton Stabilization and Channel Dynamics

Influenza A M2 Inhibitor Binding Understood through Mechanisms of Excess Proton Stabilization and Channel Dynamics

Solid-State NMR for Studying the Structure and Dynamics of Viral Assemblies

Pulsed Third-Spin-Assisted Recoupling NMR for Obtaining Long-Range¹³C-¹³C and¹⁵N-¹³C Distance Restraints

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Atomic structures of closed and open influenza B M2 proton channel reveal the conduction mechanism

Cited by 60 publications

References 60 publications

Influenza A M2 Inhibitor Binding Understood through Mechanisms of Excess Proton Stabilization and Channel Dynamics

Influenza A M2 Inhibitor Binding Understood through Mechanisms of Excess Proton Stabilization and Channel Dynamics

Solid-State NMR for Studying the Structure and Dynamics of Viral Assemblies

Pulsed Third-Spin-Assisted Recoupling NMR for Obtaining Long-Range13C-13C and15N-13C Distance Restraints

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Product

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About

Pulsed Third-Spin-Assisted Recoupling NMR for Obtaining Long-Range¹³C-¹³C and¹⁵N-¹³C Distance Restraints