Conformational changes upon gating of KirBac1.1 into an open-activated state revealed by solid-state NMR and functional assays
| Autor: | Nazmul H. Khan, Collin G. Borcik, Heather R. Coats, Benjamin J. Wylie, Maryam Yekefallah, Hoa Quynh Do, Reza Amani, Derek B. Versteeg |
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| Rok vydání: | 2020 |
| Předmět: |
Models
Molecular Magnetic Resonance Spectroscopy Protein Conformation Allosteric regulation Gating Protein Structure Secondary 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Bacterial Proteins Protein Domains Catalytic Domain Fluorescence Resonance Energy Transfer lipid activation membrane protein Potassium Channels Inwardly Rectifying POPC Conformational isomerism 030304 developmental biology 0303 health sciences allostery Multidisciplinary Phosphatidylglycerols Biological Sciences Transmembrane protein Biophysics and Computational Biology Kinetics Transmembrane domain Förster resonance energy transfer chemistry Helix Phosphatidylcholines Potassium Biophysics solid-state NMR 030217 neurology & neurosurgery potassium channel |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | Significance Inward rectifier K+ (Kir) channels play an important role in reestablishing the resting membrane state of the action potential of excitable cells in humans. KirBac1.1 is a prokaryotic Kir channel with a high degree of homology to human Kir channels and can be isotopically labeled in NMR quantities for structural studies. Functional assays and NMR assignments confirm that KirBac1.1 is in a constitutively conductive state. Solid-state NMR assignments further reveal alternate conformations at key sites in the protein that are well conserved through human Kir channels, hinting at a possible allosteric network between channels. These underlying sequential and structural motifs could explain abnormal conductive properties of these channels fundamental to their native gating processes. The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, we examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (wt/wt). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. We establish that KirBac1.1 is in a constitutively active state in POPC:POPG bilayers through the use of real-time fluorescence quenching assays and Förster resonance energy transfer (FRET) distance measurements. Multidimensional solid-state NMR (SSNMR) spectroscopy experiments reveal two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlight conformational changes associated with an open activation gate and suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. We also identify specific residues involved in this conformational exchange that are highly conserved among human Kir channels. |
| Databáze: | OpenAIRE |
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