Eukaryotic Counterparts Research Articles

Ushering in a new era of single-cell transcriptomics in bacteria.

Transcriptome analysis of individual cells by single-cell RNA-seq (scRNA-seq) has become routine for eukaryotic tissues, even being applied to whole multicellular organisms. In contrast, developing methods to read the transcriptome of single bacterial cells has proven more challenging, despite a general perception of bacteria as much simpler than eukaryotes. Bacterial cells are harder to lyse, their RNA content is about two orders of magnitude lower than that of eukaryotic cells, and bacterial mRNAs are less stable than their eukaryotic counterparts. Most importantly, bacterial transcripts lack functional poly(A) tails, precluding simple adaptation of popular standard eukaryotic scRNA-seq protocols that come with the double advantage of specific mRNA amplification and concomitant depletion of rRNA. However, thanks to very recent breakthroughs in methodology, bacterial scRNA-seq is now feasible. This short review will discuss recently published bacterial scRNA-seq approaches (MATQ-seq, microSPLiT, and PETRI-seq) and a spatial transcriptomics approach based on multiplexed in situ hybridization (par-seqFISH). Together, these novel approaches will not only enable a new understanding of cell-to-cell variation in bacterial gene expression, they also promise a new microbiology by enabling high-resolution profiling of gene activity in complex microbial consortia such as the microbiome or pathogens as they invade, replicate, and persist in host tissue.

Structural basis for the recognition of the bacterial tyrosine kinase Wzc by its cognate tyrosine phosphatase Wzb

Bacterial tyrosine kinases (BY-kinases) comprise a family of protein tyrosine kinases that are structurally distinct from their functional counterparts in eukaryotes and are highly conserved across the bacterial kingdom. BY-kinases act in concert with their counteracting phosphatases to regulate a variety of cellular processes, most notably the synthesis and export of polysaccharides involved in biofilm and capsule biogenesis. Biochemical data suggest that BY-kinase function involves the cyclic assembly and disassembly of oligomeric states coupled to the overall phosphorylation levels of a C-terminal tyrosine cluster. This process is driven by the opposing effects of intermolecular autophosphorylation, and dephosphorylation catalyzed by tyrosine phosphatases. In the absence of structural insight into the interactions between a BY-kinase and its phosphatase partner in atomic detail, the precise mechanism of this regulatory process has remained poorly defined. To address this gap in knowledge, we have determined the structure of the transiently assembled complex between the catalytic core of the Escherichia coli (K-12) BY-kinase Wzc and its counteracting low-molecular weight protein tyrosine phosphatase (LMW-PTP) Wzb using solution NMR techniques. Unambiguous distance restraints from paramagnetic relaxation effects were supplemented with ambiguous interaction restraints from static spectral perturbations and transient chemical shift changes inferred from relaxation dispersion measurements and used in a computational docking protocol for structure determination. This structurepresents an atomic picture of the mode of interaction between an LMW-PTP and its BY-kinase substrate, and provides mechanistic insight into the phosphorylation-coupled assembly/disassembly process proposed to drive BY-kinase function.

Comparative Analysis of Structural Features in SLiMs from Eukaryotes, Bacteria, and Viruses with Importance for Host-Pathogen Interactions.

Protein-protein interactions drive functions in eukaryotes that can be described by short linear motifs (SLiMs). Conservation of SLiMs help illuminate functional SLiMs in eukaryotic protein families. However, the simplicity of eukaryotic SLiMs makes them appear by chance due to mutational processes not only in eukaryotes but also in pathogenic bacteria and viruses. Further, functional eukaryotic SLiMs are often found in disordered regions. Although proteomes from pathogenic bacteria and viruses have less disorder than eukaryotic proteomes, their proteins can successfully mimic eukaryotic SLiMs and disrupt host cellular function. Identifying important SLiMs in pathogens is difficult but essential for understanding potential host-pathogen interactions. We performed a comparative analysis of structural features for experimentally verified SLiMs from the Eukaryotic Linear Motif (ELM) database across viruses, bacteria, and eukaryotes. Our results revealed that many viral SLiMs and specific motifs found across viruses and eukaryotes, such as some glycosylation motifs, have less disorder. Analyzing the disorder and coil properties of equivalent SLiMs from pathogens and eukaryotes revealed that some motifs are more structured in pathogens than their eukaryotic counterparts and vice versa. These results support a varying mechanism of interaction between pathogens and their eukaryotic hosts for some of the same motifs.

Minimal Yet Powerful: The Role of Archaeal Small Heat Shock Proteins in Maintaining Protein Homeostasis.

Small heat shock proteins (sHsp) are a ubiquitous group of ATP-independent chaperones found in all three domains of life. Although sHsps in bacteria and eukaryotes have been studied extensively, little information was available on their archaeal homologs until recently. Interestingly, archaeal heat shock machinery is strikingly simplified, offering a minimal repertoire of heat shock proteins to mitigate heat stress. sHsps play a crucial role in preventing protein aggregation and holding unfolded protein substrates in a folding-competent form. Besides protein aggregation protection, archaeal sHsps have been shown recently to stabilize membranes and contribute to transferring captured substrate proteins to chaperonin for refolding. Furthermore, recent studies on archaeal sHsps have shown that environment-induced oligomeric plasticity plays a crucial role in maintaining their functional form. Despite being prokaryotes, the archaeal heat shock protein repository shares several features with its highly sophisticated eukaryotic counterpart. The minimal nature of the archaeal heat shock protein repository offers ample scope to explore the function and regulation of heat shock protein(s) to shed light on their evolution. Moreover, similar structural dynamics of archaeal and human sHsps have made the former an excellent system to study different chaperonopathies since archaeal sHsps are more stable under in vitro experiments.

Trimeric photosystem I facilitates energy transfer from phycobilisomes in Synechocystis sp. PCC 6803.

In cyanobacteria, phycobilisomes (PBS) serve as peripheral light-harvesting complexes of the two photosystems, extending their antenna size and the wavelength range of photons available for photosynthesis. The abundance of PBS, the number of phycobiliproteins they contain, and their light-harvesting function are dynamically adjusted in response to the physiological conditions. PBS are also thought to be involved in state transitions that maintain the excitation balance between the two photosystems. Unlike its eukaryotic counterpart, PSI is trimeric in many cyanobacterial species and the physiological significance of this is not well understood. Here, we compared the composition and light-harvesting function of PBS in cells of Synechocystis sp. PCC 6803, which has primarily trimeric PSI, and the ΔpsaL mutant, which lacks the PsaL subunit of PSI and is unable to form trimers. We also investigated a mutant additionally lacking the PsaJ and PsaF subunits of PSI. Both strains with monomeric PSI accumulated significantly more allophycocyanin per chlorophyll, indicating higher abundance of PBS. On the other hand, a higher phycocyanin:allophycocyanin ratio in the wild type suggests larger PBS or the presence of APC-less PBS (CpcL-type) that are not assembled in cells with monomeric PSI. Steady-state and time-resolved fluorescence spectroscopy at room temperature and 77 K revealed that PSII receives more energy from the PBS at the expense of PSI in cells with monomeric PSI, regardless of the presence of PsaF. Taken together, these results show that the oligomeric state of PSI impacts the excitation energy flow in Synechocystis.

Regulation of the catalytic activity of by-kinases

BY-kinases (for Bacterial tYrosine kinase) comprise a unique family of protein tyrosine kinases that highly conserved in bacteria but that have no eukaryotic counterparts. A unique feature thatsets the BY-kinases apart from eukaryotic protein kinases is their deployment of a P-loop NTPase structural scaffold, noted by the presence of nucleotide-binding Walker-A and B motifs, in lieu of the dual-lobed fold characteristic of eukaryotic tyrosine kinases. Unlike many eukaryotic protein kinases, the BY-kinases cannot be classified as active or inactive based on the quantitative phosphorylation state of any particular residue or residues.

Characterization of ESCRTs proteins encoded by Asgard archaea highlights evolutionary conserved features of the ESCRT-III complex

Conserved from archaea to mammals, the Endosomal Sorting Complex Required for Transport (ESCRT) constitutes one of the most versatile cellular machinery for membrane remodeling across evolution. Recently, genes encoding for ESCRT proteins have been found in Asgard archaea: a newly discovered superphylum that, according to current evolutionary theories, is the closest archaeal relative to eukaryotes. Interestingly, Asgard-ESCRT homologs were recently found to partly compensate for the loss of the equivalent proteins in yeast raising the possibility that these ancient Asgard-ESCRT proteins can interact with their eukaryotic counterparts.

ChIP-Seq Occupancy Mapping of the Archaeal Transcription Machinery.

Genome-wide occupancy studies for RNA polymerases and their basal transcription factors deliver information about transcription dynamics and the recruitment of transcription elongation and termination factors in eukaryotes and prokaryotes. The primary method to determine genome-wide occupancies is chromatin immunoprecipitation combined with deep sequencing (ChIP-seq). Archaea possess a transcription machinery that is evolutionarily closer related to its eukaryotic counterpart but it operates in a prokaryotic cellular context. Studies on archaeal transcription brought insight into the evolution of transcription machineries and the universality of transcription mechanisms. Because of the limited resolution of ChIP-seq, the close spacing of promoters and transcription units found in archaeal genomes pose a challenge for ChIP-seq and the ensuing data analysis. The extreme growth temperature of many established archaeal model organisms necessitates further adaptations. This chapter describes a version of ChIP-seq adapted for the basal transcription machinery of thermophilic archaea and some modifications to the data analysis.

Discovery and Characterization of the Phospholemman/SIMP/Viroporin Superfamily

Using bioinformatic approaches, we present evidence of distant relatedness among the Ephemerovirus Viroporin family, the Rhabdoviridae Putative Viroporin U5 family, the Phospholemman family, and the Small Integral Membrane Protein family. Our approach is based on the transitivity property of homology complemented with five validation criteria: (1) significant sequence similarity and alignment coverage, (2) compatibility of topology of transmembrane segments, (3) overlap of hydropathy profiles, (4) conservation of protein domains, and (5) conservation of sequence motifs. Our results indicate that Pfam protein domains PF02038 and PF15831 can be found in or projected onto members of all four families. In addition, we identified a 26-residue motif conserved across the superfamily. This motif is characterized by hydrophobic residues that help anchor the protein to the membrane and charged residues that constitute phosphorylation sites. In addition, all members of the four families with annotated function are either responsible for or affect the transport of ions into and/or out of the cell. Taken together, these results justify the creation of the novel Phospholemman/SIMP/Viroporin superfamily. Given that transport proteins can be found not just in cells, but also in viruses, the ability to relate viroporin protein families with their eukaryotic and bacterial counterparts is an important development in this superfamily.

Implications of divergence of methionine adenosyltransferase in archaea.

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S‐adenosyl methionine from l‐methionine and ATP. MAT enzymes are ancient, believed to share a common ancestor, and are highly conserved in all three domains of life. However, the sequences of archaeal MATs show considerable divergence compared with their bacterial and eukaryotic counterparts. Furthermore, the structural significance and functional significance of this sequence divergence are not well understood. In the present study, we employed structural analysis and ancestral sequence reconstruction to investigate archaeal MAT divergence. We observed that the dimer interface containing the active site (which is usually well conserved) diverged considerably between the bacterial/eukaryotic MATs and archaeal MAT. A detailed investigation of the available structures supports the sequence analysis outcome: The protein domains and subdomains of bacterial and eukaryotic MAT are more similar than those of archaea. Finally, we resurrected archaeal MAT ancestors. Interestingly, archaeal MAT ancestors show substrate specificity, which is lost during evolution. This observation supports the hypothesis of a common MAT ancestor for the three domains of life. In conclusion, we have demonstrated that archaeal MAT is an ideal system for studying an enzyme family that evolved differently in one domain compared with others while maintaining the same catalytic activity.

Lights, Camera, Interaction: Studying Protein-Protein Interactions of the ER Protein Translocase in Living Cells.

Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein–protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α–Sec61β, TRAPα–TRAPβ) or protein complexes in juxtaposition (inter-complex interactions: Sec61α–TRAPα, Sec61α–Sec63, and Sec61β–Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein–protein interactions in response to changing cellular conditions in living cells.

Surface Modification of E. coli Outer Membrane Vesicles with Glycosylphosphatidylinositol-Anchored Proteins: Generating Pro/Eukaryote Chimera Constructs.

Extracellular vesicles produced by different types of cells have recently attracted great attention, not only for their role in physiology and pathology, but also because of the emerging applications in gene therapy, vaccine production and diagnostics. Less well known than their eukaryotic counterpart, also bacteria produce extracellular vesicles, in the case of the Gram-negative E. coli the main species is termed outer membrane vesicles (OMVs). In this study, we show for the first time the functional surface modification of E. coli OMVs with glycosylphosphatidylinositol (GPI)-anchored protein, exploiting a process variably described as molecular painting or protein engineering in eukaryotic membranes, whereby the lipid part of the GPI anchor inserts in cell membranes. By transferring the process to bacterial vesicles, we can generate a hybrid of perfectly eukaryotic proteins (in terms of folding and post-translational modifications) on a prokaryotic platform. We could demonstrate that two different GPI proteins can be displayed on the same OMV. In addition to fluorescent marker proteins, cytokines, growth factors and antigens canb be potentially transferred, generating a versatile modular platform for a novel vaccine strategy.

Structural analysis of the SRP Alu domain from Plasmodium falciparum reveals a non-canonical open conformation

The eukaryotic signal recognition particle (SRP) contains an Alu domain, which docks into the factor binding site of translating ribosomes and confers translation retardation. The canonical Alu domain consists of the SRP9/14 protein heterodimer and a tRNA-like folded Alu RNA that adopts a strictly ‘closed’ conformation involving a loop-loop pseudoknot. Here, we study the structure of the Alu domain from Plasmodium falciparum (PfAlu), a divergent apicomplexan protozoan that causes human malaria. Using NMR, SAXS and cryo-EM analyses, we show that, in contrast to its prokaryotic and eukaryotic counterparts, the PfAlu domain adopts an ‘open’ Y-shaped conformation. We show that cytoplasmic P. falciparum ribosomes are non-discriminative and recognize both the open PfAlu and closed human Alu domains with nanomolar affinity. In contrast, human ribosomes do not provide high affinity binding sites for either of the Alu domains. Our analyses extend the structural database of Alu domains to the protozoan species and reveal species-specific differences in the recognition of SRP Alu domains by ribosomes.

Enigmatic Evolutionary History of Porphobilinogen Deaminase in Eukaryotic Phototrophs.

Simple SummaryThe heme pathway is essential for most of cellular life. In eukaryotic phototrophs, the entire pathway is plastid localized. Despite that, the enzyme responsible for the synthesis of hydroxymethylbilane, porphobilinogen deaminase, shows α-proteobacterial instead of expected cyanobacterial origins in rhodophytes, chlorophytes, plants, and most algae with complex plastid. However, no such enzyme has been found in the supposed partners of plastid endosymbioses, the heterotrophic eukaryotes, and cyanobacteria. I propose two scenarios explaining this phenomenon by either endosymbiotic gene transfer from the ancestor of mitochondria or a non-endosymbiotic lateral gene transfer from unspecified α-proteobacterium. Phylogenetic analysis of porphobilinogen deaminases does not reject any of the two proposed evolutionary scenarios.In most eukaryotic phototrophs, the entire heme synthesis is localized to the plastid, and enzymes of cyanobacterial origin dominate the pathway. Despite that, porphobilinogen deaminase (PBGD), the enzyme responsible for the synthesis of hydroxymethybilane in the plastid, shows phylogenetic affiliation to α-proteobacteria, the supposed ancestor of mitochondria. Surprisingly, no PBGD of such origin is found in the heme pathway of the supposed partners of the primary plastid endosymbiosis, a primarily heterotrophic eukaryote, and a cyanobacterium. It appears that α-proteobacterial PBGD is absent from glaucophytes but is present in rhodophytes, chlorophytes, plants, and most algae with complex plastids. This may suggest that in eukaryotic phototrophs, except for glaucophytes, either the gene from the mitochondrial ancestor was retained while the cyanobacterial and eukaryotic pseudoparalogs were lost in evolution, or the gene was acquired by non-endosymbiotic gene transfer from an unspecified α-proteobacterium and functionally replaced its cyanobacterial and eukaryotic counterparts.

The Archaeal Elongation Factor EF-2 Induces the Release of aIF6 From 50S Ribosomal Subunit.

The translation factor IF6 is a protein of about 25 kDa shared by the Archaea and the Eukarya but absent in Bacteria. It acts as a ribosome anti-association factor that binds to the large subunit preventing the joining to the small subunit. It must be released from the large ribosomal subunit to permit its entry to the translation cycle. In Eukarya, this process occurs by the coordinated action of the GTPase Efl1 and the docking protein SBDS. Archaea do not possess a homolog of the former factor while they have a homolog of SBDS. In the past, we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homolog (aIF6) highlighting its similarity to the eukaryotic counterpart. Here, we analyzed the mechanism of aIF6 release from the large ribosomal subunit. We found that, similarly to the Eukarya, the detachment of aIF6 from the 50S subunit requires a GTPase activity which involves the archaeal elongation factor 2 (aEF-2). However, the release of aIF6 from the 50S subunits does not require the archaeal homolog of SBDS, being on the contrary inhibited by its presence. Molecular modeling, using published structural data of closely related homologous proteins, elucidated the mechanistic interplay between the aIF6, aSBDS, and aEF2 on the ribosome surface. The results suggest that a conformational rearrangement of aEF2, upon GTP hydrolysis, promotes aIF6 ejection. On the other hand, aSBDS and aEF2 share the same binding site, whose occupation by SBDS prevents aEF2 binding, thereby inhibiting aIF6 release.

How sequence populations persist inside bacterial genomes.

Compared to their eukaryotic counterparts, bacterial genomes are small and contain extremely tightly packed genes. Repetitive sequences are rare but not completely absent. One of the most common repeat families is REPINs. REPINs can replicate in the host genome and form populations that persist for millions of years. Here, we model the interactions of these intragenomic sequence populations with the bacterial host. We first confirm well-established results, in the presence and absence of horizontal gene transfer (hgt) sequence populations either expand until they drive the host to extinction or the sequence population gets purged from the genome. We then show that a sequence population can be stably maintained, when each individual sequence provides a benefit that decreases with increasing sequence population size. Maintaining a sequence population of stable size also requires the replication of the sequence population to be costly to the host, otherwise the sequence population size will increase indefinitely. Surprisingly, in regimes with high hgt rates, the benefit conferred by the sequence population does not have to exceed the damage it causes to its host. Our analyses provide a plausible scenario for the persistence of sequence populations in bacterial genomes. We also hypothesize a limited biologically relevant parameter range for the provided benefit, which can be tested in future experiments.

H/ACA Small Ribonucleoproteins: Structural and Functional Comparison Between Archaea and Eukaryotes.

During ribosome synthesis, ribosomal RNA is modified through the formation of many pseudouridines and methylations which contribute to ribosome function across all domains of life. In archaea and eukaryotes, pseudouridylation of rRNA is catalyzed by H/ACA small ribonucleoproteins (sRNPs) utilizing different H/ACA guide RNAs to identify target uridines for modification. H/ACA sRNPs are conserved in archaea and eukaryotes, as they share a common general architecture and function, but there are also several notable differences between archaeal and eukaryotic H/ACA sRNPs. Due to the higher protein stability in archaea, we have more information on the structure of archaeal H/ACA sRNPs compared to eukaryotic counterparts. However, based on the long history of yeast genetic and other cellular studies, the biological role of H/ACA sRNPs during ribosome biogenesis is better understood in eukaryotes than archaea. Therefore, this review provides an overview of the current knowledge on H/ACA sRNPs from archaea, in particular their structure and function, and relates it to our understanding of the roles of eukaryotic H/ACA sRNP during eukaryotic ribosome synthesis and beyond. Based on this comparison of our current insights into archaeal and eukaryotic H/ACA sRNPs, we discuss what role archaeal H/ACA sRNPs may play in the formation of ribosomes.

Mapping the Conformational States of the Outer Vestibule of Kcsa, a Prokaryote Potassium Channel, using the Long Chain Quaternary Ammonium Blocker, Tetraoctylammonium

KcsA is a homotetrameric prokaryotic potassium channel, homologous to its eukaryotic counterparts. In this study, we have used a KcsA mutant channel bearing a single tryptophan residue per monomer (W67 KcsA) to investigate the selectivity filter (SF) and pore-helix dynamics using fluorescence spectroscopy. Our steady-state and time-resolved fluorescence measurements indicate that the W67 residues undergo an efficient homo-FRET process among the four channel subunits. An analytical solution describing homo-FRET among a homo-tetramer in a square geometry allowed retrieving W67-W67 lateral distances (R) from the W67 KcsA fluorescence anisotropy decays. Comparison between the distances determined for the closed/conductive (pH 7.0, 200 mM K+, R=15.4 Å), closed/non-conductive (pH 7.0, 0.1 mM K+, R=16.5 Å) and open/inactivated (pH4.0, 200 mM K+, R=15.3 Å) of the detergent-solubilized W67 KcsA, ruled out the hypothesis that the inactivated state is structurally similar to its collapsed state. To further study the inactivated conformation, we added tetraoctylammonium (TOA+), which is a potassium channel blocker that binds to the internal cavity, beneath the SF. TOA+ allosterically triggers a structural rearrangement characterized by a decrease in the affinity for K+ at pH 7 and longer W67-W67 lateral distances, while the shorter alkyl chain salt tetrabutylammonium (TBA+) does not affect the SF conformation of KcsA. Such loss of affinity is similar to that seen for the open/inactivated state at pH 4. This allosteric modification is also linked to a decrease of the thermal stability of KcsA monitored by a thermal denaturation assay. These results reinforce the argument that long chain tetraalkyl ammonium salts, such as TOA+, stabilize an inactivated-like conformation of the SF, which has a lower affinity for K+ and a different conformation than the low-K+ (collapsed) structure.

Architecture of bacterial respiratory chains.

Bacteria power their energy metabolism using membrane-bound respiratory enzymes that capture chemical energy and transduce it by pumping protons or Na+ ions across their cell membranes. Recent breakthroughs in molecular bioenergetics have elucidated the architecture and function of many bacterial respiratory enzymes, although key mechanistic principles remain debated. In this Review, we present an overview of the structure, function and bioenergetic principles of modular bacterial respiratory chains and discuss their differences from the eukaryotic counterparts. We also discuss bacterial supercomplexes, which provide central energy transduction systems in several bacteria, including important pathogens, and which could open up possible avenues for treatment of disease.

Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA.

Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA+) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA+ binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, TOA+ bound at the cavity allosterically shifts the equilibrium of the channel’s selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA+–KcsA complex exhibits a loss in the affinity towards permeant K+ at pH 7.0, when the channel is in its closed state, but maintains the two sets of K+ binding sites and the W67–W67 intersubunit distances characteristic of the selectivity filter in the channel resting state. Thus, the TOA+–bound state differs clearly from the collapsed channel state described by X-ray crystallography and claimed to represent the inactivated form of KcsA.