scholarly journals Probing allosteric coupling in a constitutively open mutant of the ion channel KcsA using solid-state NMR

2020 ◽  
Vol 117 (13) ◽  
pp. 7171-7175
Author(s):  
Zhiyu Sun ◽  
Yunyao Xu ◽  
Dongyu Zhang ◽  
Ann E. McDermott
Keyword(s):  
Solid State ◽  
Binding Affinity ◽  
Solid State Nmr ◽  
Selectivity Filter ◽  
Wild Type ◽  
Proton Binding ◽  
Allosteric Coupling ◽  
Neutral Ph ◽  
Potassium Channel Kcsa ◽  
Potassium Binding

Transmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the potassium binding affinity of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch, leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about four orders of magnitude weaker than that of wild-type KcsA at neutral pH and is comparable to the value for wild-type KcsA at low pH (pH ≈ 3.5). This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant, the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.

scholarly journals Probing Allosteric Coupling of a Constitutively Open Mutant of the Ion Channel KcsA using Solid State NMR

2019 ◽  
Author(s):  
Zhiyu Sun ◽  
Yunyao Xu ◽  
Dongyu Zhang ◽  
Ann E McDermott
Keyword(s):  
Solid State ◽  
Binding Affinity ◽  
Solid State Nmr ◽  
Selectivity Filter ◽  
Wild Type ◽  
Allosteric Coupling ◽  
Channel Inactivation ◽  
Activation Gate ◽  
Inactivation Process ◽  
Potassium Binding

AbstractTransmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the mean open time of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that has the pH gate open across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about 4 orders of magnitude weaker than that of wild type KcsA at neutral pH and is comparable to the value for wild type KcsA at low pH (pH ∼ 3.5). This result strongly supports our assertion that the open pH gate allosterically effects the potassium binding affinity of the selectivity filter. In this mutant the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.Significance statementInactivation of potassium channels controls mean open times and provides exquisite control over biological processes. In the highly conserved C-type inactivation process, opening of the activation gate causes subsequent inactivation. We test whether the open state of the channel simply has a poor ability to bind the K+ ion. Previously, activated and inactivated states were stabilized using truncations or a significant pH drop. Here, we use the H25R/E118A constitutively open mutant of KcsA and also observe a large drop in potassium binding affinity. This provides strong evidence that channel opening causes an allosteric loss of ion affinity, and that the central feature of this universal channel inactivation process is loss of ion affinity at the selectivity filter.

scholarly journals Solid-State NMR Reveals Structural Differences between Fibrils of Wild-Type and Disease-Related A53T Mutant α-Synuclein

2008 ◽  
Vol 380 (3) ◽  
pp. 444-450 ◽  
Author(s):  
Henrike Heise ◽  
M. Soledad Celej ◽  
Stefan Becker ◽  
Dietmar Riedel ◽  
Avishay Pelah ◽  
...  
Keyword(s):  
Solid State ◽  
Solid State Nmr ◽  
Wild Type ◽  
Structural Differences

scholarly journals Structural characteristics of oligomers formed by pyroglutamate-modified amyloid β peptides studied by solid-state NMR

2020 ◽  
Vol 22 (29) ◽  
pp. 16887-16895 ◽  
Author(s):  
Holger A. Scheidt ◽  
Anirban Das ◽  
Alexander Korn ◽  
Martin Krueger ◽  
Sudipta Maiti ◽  
...  
Keyword(s):  
Solid State ◽  
Solid State Nmr ◽  
Structural Characteristics ◽  
Amyloid Β ◽  
Wild Type

Structure of oligomers of truncated and pyroglutamate modified amyloid β variants is similar to the wild type.

scholarly journals Transmembrane allosteric energetics characterization for strong coupling between proton and potassium ion binding in the KcsA channel

2017 ◽  
Vol 114 (33) ◽  
pp. 8788-8793 ◽  
Author(s):  
Yunyao Xu ◽  
Manasi P. Bhate ◽  
Ann E. McDermott
Keyword(s):  
Ion Channel ◽  
Binding Site ◽  
Ph Sensor ◽  
Selectivity Filter ◽  
Ion Binding ◽  
Wild Type ◽  
Channel Activation ◽  
Site Specific ◽  
Allosteric Coupling ◽  
Allosteric Mechanism

The slow spontaneous inactivation of potassium channels exhibits classic signatures of transmembrane allostery. A variety of data support a model in which the loss of K+ ions from the selectivity filter is a major factor in promoting inactivation, which defeats transmission, and is allosterically coupled to protonation of key channel activation residues, more than 30 Å from the K+ ion binding site. We show that proton binding at the intracellular pH sensor perturbs the potassium affinity at the extracellular selectivity filter by more than three orders of magnitude for the full-length wild-type KcsA, a pH-gated bacterial channel, in membrane bilayers. Studies of F103 in the hinge of the inner helix suggest an important role for its bulky sidechain in the allosteric mechanism; we show that the energetic strength of coupling of the gates is strongly altered when this residue is mutated to alanine. These results provide quantitative site-specific measurements of allostery in a bilayer environment, and highlight the power of describing ion channel gating through the lens of allosteric coupling.

scholarly journals [K+] Induced Conformational Dynamics of the Selectivity Filter of KcsA Monitored by Solid-State NMR

2010 ◽  
Vol 98 (3) ◽  
pp. 331a
Author(s):  
Manasi Bhate ◽  
Benjamin Wylie ◽  
Lin Tian ◽  
Ann McDermott
Keyword(s):  
Solid State ◽  
Solid State Nmr ◽  
Conformational Dynamics ◽  
Selectivity Filter

scholarly journals Conformational changes upon gating of KirBac1.1 into an open-activated state revealed by solid-state NMR and functional assays

2020 ◽  
Vol 117 (6) ◽  
pp. 2938-2947 ◽  
Author(s):  
Reza Amani ◽  
Collin G. Borcik ◽  
Nazmul H. Khan ◽  
Derek B. Versteeg ◽  
Maryam Yekefallah ◽  
...  
Keyword(s):  
Solid State ◽  
Conformational Changes ◽  
Solid State Nmr ◽  
Lipid Binding ◽  
Selectivity Filter ◽  
Conformational Exchange ◽  
Lipid Mixture ◽  
Kir Channels ◽  
Activated State ◽  
Activation Gate

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.

Identification of a novel pyruvyltransferase using 13 C solid-state NMR to analyze rhizobial exopolysaccharides

2021 ◽  
Author(s):  
Derek H. Wells ◽  
Nicolette F. Goularte ◽  
Melanie J. Barnett ◽  
Lynette Cegelski ◽  
Sharon R. Long
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Solid State ◽  
Magnetic Resonance ◽  
Solid State Nmr ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Nitrogen Fixing ◽  
Nmr Analysis ◽  
Wild Type ◽  
Nuclear Magnetic

The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPS), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially enable it to adapt to a changing environment. Succinoglycan is essential for invasion of plant hosts, and thus for formation of nitrogen-fixing root nodules. Galactoglucan is critical for population-based behaviors such as swarming and biofilm formation, and can facilitate invasion in the absence of succinoglycan on some host plants. Biosynthesis of galactoglucan is not as completely understood as that of succinoglycan. We devised a pipeline to: identify putative pyruvyltransferase and acetyltransferase genes; construct genomic deletions in strains engineered to produce either succinoglycan or galactoglucan; and analyze EPS from mutant bacterial strains. EPS samples were examined by 13 C cross-polarization magic-angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). CPMAS NMR is uniquely suited to defining chemical composition in complex samples and enable detection and quantification of distinct EPS functional groups. Galactoglucan was isolated from mutant strains, with deletions in five candidate acyl/acetyltransferase genes ( exoZ , exoH , SMb20810 , SMb21188 , SMa1016 ) and a putative pyruvyltransferase ( wgaE or SMb21322). Most samples were similar in composition to wild-type EPSII by CPMAS NMR analysis. However, galactoglucan produced from a strain lacking wgaE exhibited a significant reduction in pyruvylation. Pyruvylation was restored through ectopic expression of plasmid-encoded wgaE . Our work has thus identified WgaE as a galactoglucan pyruvyltransferase. This exemplifies how the systematic combination of genetic analyses and solid-state NMR detection is a rapid means to identify genes responsible for modification of rhizobial exopolysaccharides. IMPORTANCE Nitrogen-fixing bacteria are crucial for geochemical cycles and global nitrogen nutrition. Symbioses between legumes and rhizobial bacteria establish root nodules, where bacteria convert dinitrogen to ammonia for plant utilization. Secreted exopolysaccharides (EPS) produced by Sinorhizobium meliloti (succinoglycan and galactoglucan) play important roles in soil and plant environments. Biosynthesis of galactoglucan is not as well characterized as succinoglycan. We employed solid-state nuclear magnetic resonance (NMR) to examine intact EPS from wild type and mutant S. meliloti strains. NMR analysis of EPS isolated from a wgaE gene mutant revealed a novel pyruvyltransferase that modifies galactoglucan. Few EPS pyruvyltransferases have been characterized. Our work provides insight into biosynthesis of an important S. meliloti EPS and expands knowledge of enzymes that modify polysaccharides.

scholarly journals Ion Interactions in the High-Affinity Binding Locus of a Voltage-Gated Ca2+ Channel

2000 ◽  
Vol 116 (4) ◽  
pp. 569-586 ◽  
Author(s):  
Robin K. Cloues ◽  
Susan M. Cibulsky ◽  
William A. Sather
Keyword(s):  
Binding Affinity ◽  
Single Channel ◽  
Ion Selectivity ◽  
Ion Pairs ◽  
Selectivity Filter ◽  
Ion Binding ◽  
Wild Type ◽  
Filter Function ◽  
Voltage Gated ◽  
Ca2 Channels

The selectivity filter of voltage-gated Ca2+ channels is in part composed of four Glu residues, termed the EEEE locus. Ion selectivity in Ca2+ channels is based on interactions between permeant ions and the EEEE locus: in a mixture of ions, all of which can pass through the pore when present alone, those ions that bind weakly are impermeant, those that bind more strongly are permeant, and those that bind more strongly yet act as pore blockers as a consequence of their low rate of unbinding from the EEEE locus. Thus, competition among ion species is a determining feature of selectivity filter function in Ca2+ channels. Previous work has shown that Asp and Ala substitutions in the EEEE locus reduce ion selectivity by weakening ion binding affinity. Here we describe for wild-type and EEEE locus mutants an analysis at the single channel level of competition between Cd2+, which binds very tightly within the EEEE locus, and Ba2+ or Li+, which bind less tightly and hence exhibit high flux rates: Cd2+ binds to the EEEE locus ∼104× more tightly than does Ba2+, and ∼108× more tightly than does Li+. For wild-type channels, Cd2+ entry into the EEEE locus was 400× faster when Li+ rather than Ba2+ was the current carrier, reflecting the large difference between Ba2+ and Li+ in affinity for the EEEE locus. For the substitution mutants, analysis of Cd2+ block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate. Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs. Ala), but was instead determined by the valence and affinity of the current-carrying ion (Ba2+ vs. Li+). The dependence of Cd2+ block kinetics upon properties of the current-carrying ion can be understood by considering the number of EEEE locus oxygen atoms available to interact with the different ion pairs.

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