CombLabel: rational design of optimized sequence-specific combinatorial labeling schemes. Application to backbone assignment of membrane proteins with low stability

Myshkin, Mikhail Yu.; Dubinnyi, Maxim A.; Kulbatskii, Dmitrii S.; Lyukmanova, Ekaterina N.; Kirpichnikov, Mikhaǐl P.; Шенкарев, Захар О.

doi:10.1007/s10858-019-00259-z

Cited by 4 publications

(4 citation statements)

References 34 publications

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“…The identification of starting and anchor points is key in the de novo sequential assignment process, and amino‐acid selective labeling, as can be obtained from cell‐free synthesis, [77–81] was in this context central for success. We prepared three different selectively labelled samples (Figure S2, Table S3 and Table S4) on which both 3D hCANH and hCONH experiments were recorded (Figure 3).…”

Section: Resultsmentioning

confidence: 99%

Dimer Organization of Membrane‐Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive ¹H‐Detected Solid‐State NMR

et al. 2021

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The Hepatitis Cv irus nonstructural protein 5A (NS5A) is amembrane-associated protein involved in multiple steps of the viral life cycle.D irect-acting antivirals (DAAs) targeting NS5A are ac ornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure.Herein, we present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes.W e use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches.S pectra analyses disclose that both the AH membrane anchor and the linker are highly flexible.P aramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires an ew type of NS5A selfinteraction not reflected in previous crystal structures.I n conclusion, we providethe first characterization of NS5A AHlinker-D1 in al ipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors.

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Section: Resultsmentioning

confidence: 99%

Dimer Organization of Membrane‐Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive ¹H‐Detected Solid‐State NMR

et al. 2021

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“…To use the CSL method, a scheme of isotope incorporation is required, e.g., one needs to specify the number of labeled samples and the labeling pattern for each residue type in each of the samples. For calculation of the optimal CSL scheme, we used the recently developed CombLabel program (; Myshkin et al, 2019). This program takes into account the available AA stock, minimizes the number of required selectively labeled samples and their price, and maximizes assignment information.…”

Section: Resultsmentioning

confidence: 99%

Cell-Free Expression of Sodium Channel Domains for Pharmacology Studies. Noncanonical Spider Toxin Binding Site in the Second Voltage-Sensing Domain of Human Nav1.4 Channel

et al. 2019

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Voltage-gated sodium (NaV) channels are essential for the normal functioning of cardiovascular, muscular, and nervous systems. These channels have modular organization; the central pore domain allows current flow and provides ion selectivity, whereas four peripherally located voltage-sensing domains (VSDs-I/IV) are needed for voltage-dependent gating. Mutations in the S4 voltage-sensing segments of VSDs in the skeletal muscle channel NaV1.4 trigger leak (gating pore) currents and cause hypokalemic and normokalemic periodic paralyses. Previously, we have shown that the gating modifier toxin Hm-3 from the crab spider Heriaeus melloteei binds to the S3-S4 extracellular loop in VSD-I of NaV1.4 channel and inhibits gating pore currents through the channel with mutations in VSD-I. Here, we report that Hm-3 also inhibits gating pore currents through the same channel with the R675G mutation in VSD-II. To investigate the molecular basis of Hm-3 interaction with VSD-II, we produced the corresponding 554-696 fragment of NaV1.4 in a continuous exchange cell-free expression system based on the Escherichia coli S30 extract. We then performed a combined nuclear magnetic resonance (NMR) and electron paramagnetic resonance spectroscopy study of isolated VSD-II in zwitterionic dodecylphosphocholine/lauryldimethylamine-N-oxide or dodecylphosphocholine micelles. To speed up the assignment of backbone resonances, five selectively 13C,15N-labeled VSD-II samples were produced in accordance with specially calculated combinatorial scheme. This labeling approach provides assignment for ∼50% of the backbone. Obtained NMR and electron paramagnetic resonance data revealed correct secondary structure, quasi-native VSD-II fold, and enhanced ps–ns timescale dynamics in the micelle-solubilized domain. We modeled the structure of the VSD-II/Hm-3 complex by protein–protein docking involving binding surfaces mapped by NMR. Hm-3 binds to VSDs I and II using different modes. In VSD-II, the protruding ß-hairpin of Hm-3 interacts with the S1-S2 extracellular loop, and the complex is stabilized by ionic interactions between the positively charged toxin residue K24 and the negatively charged channel residues E604 or D607. We suggest that Hm-3 binding to these charged groups inhibits voltage sensor transition to the activated state and blocks the depolarization-activated gating pore currents. Our results indicate that spider toxins represent a useful hit for periodic paralyses therapy development and may have multiple structurally different binding sites within one NaV molecule.

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“…In the hCONH (Figure 2c), nearly as many peaks as in the hCANH are present, indicating that inter-residue connections can be obtained for most signals.A dditional inter-residue information can be obtained from the hCAcoNH,h COcaNH and hncaCBcaNH 3D spectra, which show however as maller number of resonances. [75] Thei dentification of starting and anchor points is key in the de novo sequential assignment process,a nd amino-acid selective labeling,a sc an be obtained from cell-free synthesis, [77][78][79][80][81] was in this context central for success.W e prepared three different selectively labelled samples (Figure S2, Table S3 and Table S4) on which both 3D hCANH and hCONH experiments were recorded (Figure 3). The hCONH should show only signals for labelled pairs,a nd indeed for the ten G/T/Y pairs in the sequence,n ine signals are observed in the hCONH spectrum (Figure 3g), meaning that only one signal remained unobserved.…”

Section: Resonance Assignments By Combining 3d Spectroscopy and Selec...mentioning

confidence: 99%

Dimer Organization of Membrane‐Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive ¹H‐Detected Solid‐State NMR

et al. 2021

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CombLabel: rational design of optimized sequence-specific combinatorial labeling schemes. Application to backbone assignment of membrane proteins with low stability

Cited by 4 publications

References 34 publications

Dimer Organization of Membrane‐Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive ¹H‐Detected Solid‐State NMR

Dimer Organization of Membrane‐Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive ¹H‐Detected Solid‐State NMR

Cell-Free Expression of Sodium Channel Domains for Pharmacology Studies. Noncanonical Spider Toxin Binding Site in the Second Voltage-Sensing Domain of Human Nav1.4 Channel

Dimer Organization of Membrane‐Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive ¹H‐Detected Solid‐State NMR

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CombLabel: rational design of optimized sequence-specific combinatorial labeling schemes. Application to backbone assignment of membrane proteins with low stability

Cited by 4 publications

References 34 publications

Dimer Organization of Membrane‐Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive 1H‐Detected Solid‐State NMR

Dimer Organization of Membrane‐Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive 1H‐Detected Solid‐State NMR

Cell-Free Expression of Sodium Channel Domains for Pharmacology Studies. Noncanonical Spider Toxin Binding Site in the Second Voltage-Sensing Domain of Human Nav1.4 Channel

Dimer Organization of Membrane‐Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive 1H‐Detected Solid‐State NMR

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Dimer Organization of Membrane‐Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive ¹H‐Detected Solid‐State NMR

Dimer Organization of Membrane‐Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive ¹H‐Detected Solid‐State NMR

Dimer Organization of Membrane‐Associated NS5A of Hepatitis C Virus as Determined by Highly Sensitive ¹H‐Detected Solid‐State NMR