Unraveling the systematics and evolution of the 'Geophagus' brasiliensis (Cichliformes: Cichlidae) species complex.

The 'Geophagus' brasiliensis complex is one of the most abundant groups of cichlids from eastern coastal basins in South America. Traditionally, this fish group has been recognized as incertae sedis because of phylogenetic uncertainties and unclear taxonomy. In addition, the remarkable morphological, chromosomal, and DNA variation reported over recent years in several populations of these cichlids has increased the debate about their species richness and their distributional range. Here, we tested the presence of independent evolutionary lineages within the 'G.' brasiliensis complex, addressing their taxonomic status and evolutionary relationships, including a comparative analysis of genetic and morphological patterns, based on an extensive dataset, comprising 172 sampling sites along most of their known range using a mitochondrial marker, RADseq data and geometric morphometrics. The number of putative species in the present study varied from 9 to 11 depending on the molecular species delimitation methods used. Our results revealed at least two putative new taxa ('Geophagus' sp. Doce and 'Geophagus' sp. Upper Contas). Morphometric analyses, particularly those based on Canonical Variate Analysis (CVA), revealed significant morphological differentiation between species within the main clades. On the other hand, analyses of morphological phylogenetic signal and phylomorphospace provided no evidence of adaptive differentiation among these species. Thus, diversification in the 'G.' brasiliensis complex seems to have been influenced by hydrogeological events that promoted allopatry, such as the presence of paleodrainages and distributional reconfiguration through river captures. We propose major changes in the known distribution of some species within the complex and conservatively suggest the recognition of 10 species within the 'Geophagus' brasiliensis complex, with the potential for further dividing 'G.' rufomarginatus after additional taxonomic evaluation.

Rivers of Indochina as potential drivers of lineage diversification in the spotted flying lizard (Draco maculatus) species complex.

Southeast Asia hosts a rich concentration of biodiversity within multiple biodiversity hotspots. Indochina, a region with remarkably high levels of in situ diversification, possesses five major rivers (Ayeyarwady, Chiang Mai, Mekong, Red, and Salween), several of which coincide with phylogenetic breaks of terrestrial taxa. Draco maculatus possesses a range that stretches across Indochina, which widespread geographic distribution along with potential discrete variation within subspecies alludes to the possibility of this taxon constituting multiple divergent lineages. Using sequence data from three mitochondrial (12S, 16S, and ND2) and three nuclear (BDNF, CMOS, and PNN) genes, we provide the first estimated phylogeny of this hypothesized species complex and examine its phylogeographic architecture with maximum likelihood and Bayes factor delimitation (BFD) approaches. Our results support multiple divergent lineages with phylogenetic breaks coincident with rivers, indicating that river barriers may be contributing to the elevated levels of in situ diversification of Indochina.

Cytochrome C Oxidase Subunit 1, Internal Transcribed Spacer 1, Nicotinamide Adenine Dinucleotide Hydrogen Dehydrogenase Subunits 2 and 5 of Clonorchis sinensis Ancient DNA Retrieved from Joseon Dynasty Mummy Specimens.

We analyzed Clonorchis sinensis ancient DNA (aDNA) acquired from the specimens of the Joseon mummies. The target regions were cytochrome C oxidase subunit 1 (CO1), internal transcribed spacer 1 (ITS1), nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase subunits 2 (NAD2) and 5 (NAD5). The sequences of C. sinensis aDNA was completely or almost identical to modern C. sinensis sequences in GenBank. We also found that ITS1, NAD2 and NAD5 could be good markers for molecular diagnosis between C. sinensis and the other trematode parasite species. The current result could improve our knowledge about genetic history of C. sinensis.

Photosynthetic Trichomes Contain a Specific Rubisco with a Modified pH-Dependent Activity.

Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme in plants and is responsible for CO2 fixation during photosynthesis. This enzyme is assembled from eight large subunits (RbcL) encoded by a single chloroplast gene and eight small subunits (RbcS) encoded by a nuclear gene family. Rubisco is primarily found in the chloroplasts of mesophyll (C3 plants), bundle-sheath (C4 plants), and guard cells. In certain species, photosynthesis also takes place in the secretory cells of glandular trichomes, which are epidermal outgrowths (hairs) involved in the secretion of specialized metabolites. However, photosynthesis and, in particular, Rubisco have not been characterized in trichomes. Here, we show that tobacco (Nicotiana tabacum) trichomes contain a specific Rubisco small subunit, NtRbcS-T, which belongs to an uncharacterized phylogenetic cluster (T). This cluster contains RbcS from at least 33 species, including monocots, many of which are known to possess glandular trichomes. Cluster T is distinct from the cluster M, which includes the abundant, functionally characterized RbcS isoforms expressed in mesophyll or bundle-sheath cells. Expression of NtRbcS-T in Chlamydomonas reinhardtii and purification of the full Rubisco complex showed that this isoform conferred higher Vmax and Km values as well as higher acidic pH-dependent activity than NtRbcS-M, an isoform expressed in the mesophyll. This observation was confirmed with trichome extracts. These data show that an ancient divergence allowed for the emergence of a so-far-uncharacterized RbcS cluster. We propose that secretory trichomes have a particular Rubisco uniquely adapted to secretory cells where CO2 is released by the active specialized metabolism.

Protein flexibility facilitates quaternary structure assembly and evolution.

The intrinsic flexibility of proteins allows them to undergo large conformational fluctuations in solution or upon interaction with other molecules. Proteins also commonly assemble into complexes with diverse quaternary structure arrangements. Here we investigate how the flexibility of individual protein chains influences the assembly and evolution of protein complexes. We find that flexibility appears to be particularly conducive to the formation of heterologous (i.e., asymmetric) intersubunit interfaces. This leads to a strong association between subunit flexibility and homomeric complexes with cyclic and asymmetric quaternary structure topologies. Similarly, we also observe that the more nonhomologous subunits that assemble together within a complex, the more flexible those subunits tend to be. Importantly, these findings suggest that subunit flexibility should be closely related to the evolutionary history of a complex. We confirm this by showing that evolutionarily more recent subunits are generally more flexible than evolutionarily older subunits. Finally, we investigate the very different explorations of quaternary structure space that have occurred in different evolutionary lineages. In particular, the increased flexibility of eukaryotic proteins appears to enable the assembly of heteromeric complexes with more unique components.

PCI complexes: Beyond the proteasome, CSN, and eIF3 Troika.

The bipartite PCI domain serves as the principal scaffold for proteasome lid, CSN, and eIF3, complexes that influence protein life span. PCI domains are also found in newly identified complexes directing nucleic acid regulation. The breadth of functions associated with the extended PCI family is a factor of shared subunits, among them a common factor Sem1/DSS1 that facilitates complex assembly.

Comparative analyses of quaternary arrangements in homo-oligomeric proteins in superfamilies: Functional implications.

A comprehensive analysis of the quaternary features of distantly related homo-oligomeric proteins is the focus of the current study. This study has been performed at the levels of quaternary state, symmetry, and quaternary structure. Quaternary state and quaternary structure refers to the number of subunits and spatial arrangements of subunits, respectively. Using a large dataset of available 3D structures of biologically relevant assemblies, we show that only 53% of the distantly related homo-oligomeric proteins have the same quaternary state. Considering these homologous homo-oligomers with the same quaternary state, conservation of quaternary structures is observed only in 38% of the pairs. In 36% of the pairs of distantly related homo-oligomers with different quaternary states the larger assembly in a pair shows high structural similarity with the entire quaternary structure of the related protein with lower quaternary state and it is referred as "Russian doll effect." The differences in quaternary state and structure have been suggested to contribute to the functional diversity. Detailed investigations show that even though the gross functions of many distantly related homo-oligomers are the same, finer level differences in molecular functions are manifested by differences in quaternary states and structures. Comparison of structures of biological assemblies in distantly and closely related homo-oligomeric proteins throughout the study differentiates the effects of sequence divergence on the quaternary structures and function. Knowledge inferred from this study can provide insights for improved protein structure classification and function prediction of homo-oligomers. Proteins 2016; 84:1190-1202. © 2016 Wiley Periodicals, Inc.

Classes of phosphoinositide 3-kinases at a glance.

The phosphoinositide 3-kinase (PI3K) family is important to nearly all aspects of cell and tissue biology and central to human cancer, diabetes and aging. PI3Ks are spatially regulated and multifunctional, and together, act at nearly all membranes in the cell to regulate a wide range of signaling, membrane trafficking and metabolic processes. There is a broadening recognition of the importance of distinct roles for each of the three different PI3K classes (I, II and III), as well as for the different isoforms within each class. Ongoing issues include the need for a better understanding of the in vivo complexity of PI3K regulation and cellular functions. This Cell Science at a Glance article and the accompanying poster summarize the biochemical activities, cellular roles and functional requirements for the three classes of PI3Ks. In doing so, we aim to provide an overview of the parallels, the key differences and crucial interplays between the regulation and roles of the three PI3K classes.

Phylogenomic Analysis of Type 1 NADH:Quinone Oxidoreductase.

We performed phylogenomic analysis of the catalytic core of NADH:quinone oxidoreductases of type 1 (NDH-1). Analysis of phylogenetic trees, as constructed for the core subunits of NDH-1, revealed fundamental differences in their topologies. In the case of four putatively homologous ion-carrying membrane subunits, the trees for the NuoH and NuoN subunits contained separate archaeal clades, whereas subunits NuoL and NuoM were characterized by multiple archaeal clades spread among bacterial branches. Large, separate clades, which united sequences belonging to different archaeal subdomains, were also found for cytoplasmic subunits NuoD and NuoB, homologous to the large and small subunits of nickel-iron hydrogenases. A smaller such clade was also shown for subunit NuoC. Based on these data, we suggest that the ancestral NDH-1 complex could be present already at the stage of the Last Universal Cellular Ancestor (LUCA). Ancestral forms of membrane subunits NuoN and NuoH and cytoplasmic subunits NuoD, NuoB, and, perhaps NuoC, may have formed a membrane complex that operated as an ion-translocating membrane hydrogenase. After the complex attained the ability to reduce membrane quinones, gene duplications could yield the subunits NuoL and NuoM, which enabled translocation of additional ions.

Developmental and Regulatory Functions of Na(+) Channel Non-pore-forming ß Subunits.

Voltage-gated Na(+) channels (VGSCs) isolated from mammalian neurons are heterotrimeric complexes containing one pore-forming α subunit and two non-pore-forming ß subunits. In excitable cells, VGSCs are responsible for the initiation of action potentials. VGSC ß subunits are type I topology glycoproteins, containing an extracellular amino-terminal immunoglobulin (Ig) domain with homology to many neural cell adhesion molecules (CAMs), a single transmembrane segment, and an intracellular carboxyl-terminal domain. VGSC ß subunits are encoded by a gene family that is distinct from the α subunits. While α subunits are expressed in prokaryotes, ß subunit orthologs did not arise until after the emergence of vertebrates. ß subunits regulate the cell surface expression, subcellular localization, and gating properties of their associated α subunits. In addition, like many other Ig-CAMs, ß subunits are involved in cell migration, neurite outgrowth, and axon pathfinding and may function in these roles in the absence of associated α subunits. In sum, these multifunctional proteins are critical for both channel regulation and central nervous system development.

Unique subunit packing in mycobacterial nanoRNase leads to alternate substrate recognitions in DHH phosphodiesterases.

DHH superfamily includes RecJ, nanoRNases (NrnA), cyclic nucleotide phosphodiesterases and pyrophosphatases. In this study, we have carried out in vitro and in vivo investigations on the bifunctional NrnA-homolog from Mycobacterium smegmatis, MSMEG_2630. The crystal structure of MSMEG_2630 was determined to 2.2-Å resolution and reveals a dimer consisting of two identical subunits with each subunit folding into an N-terminal DHH domain and a C-terminal DHHA1 domain. The overall structure and fold of the individual domains is similar to other members of DHH superfamily. However, MSMEG_2630 exhibits a distinct quaternary structure in contrast to other DHH phosphodiesterases. This novel mode of subunit packing and variations in the linker region that enlarge the domain interface are responsible for alternate recognitions of substrates in the bifunctional nanoRNases. MSMEG_2630 exhibits bifunctional 3'-5' exonuclease [on both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) substrates] as well as CysQ-like phosphatase activity (on pAp) in vitro with a preference for nanoRNA substrates over single-stranded DNA of equivalent lengths. A transposon disruption of MSMEG_2630 in M. smegmatis causes growth impairment in the presence of various DNA-damaging agents. Further phylogenetic analysis and genome organization reveals clustering of bacterial nanoRNases into two distinct subfamilies with possible role in transcriptional and translational events during stress.

Genetic analysis of the Replication Protein A large subunit family in Arabidopsis reveals unique and overlapping roles in DNA repair, meiosis and DNA replication.

Replication Protein A (RPA) is a heterotrimeric protein complex that binds single-stranded DNA. In plants, multiple genes encode the three RPA subunits (RPA1, RPA2 and RPA3), including five RPA1-like genes in Arabidopsis. Phylogenetic analysis suggests two distinct groups composed of RPA1A, RPA1C, RPA1E (ACE group) and RPA1B, RPA1D (BD group). ACE-group members are transcriptionally induced by ionizing radiation, while BD-group members show higher basal transcription and are not induced by ionizing radiation. Analysis of rpa1 T-DNA insertion mutants demonstrates that although each mutant line is likely null, all mutant lines are viable and display normal vegetative growth. The rpa1c and rpa1e single mutants however display hypersensitivity to ionizing radiation, and combination of rpa1c and rpa1e results in additive hypersensitivity to a variety of DNA damaging agents. Combination of the partially sterile rpa1a with rpa1c results in complete sterility, incomplete synapsis and meiotic chromosome fragmentation, suggesting an early role for RPA1C in promoting homologous recombination. Combination of either rpa1c and/or rpa1e with atr revealed additive hypersensitivity phenotypes consistent with each functioning in unique repair pathways. In contrast, rpa1b rpa1d double mutant plants display slow growth and developmental defects under non-damaging conditions. We show these defects in the rpa1b rpa1d mutant are likely the result of defective DNA replication leading to reduction in cell division.

KCa and Ca(2+) channels: the complex thought.

Potassium channels belong to the largest and the most diverse super-families of ion channels. Among them, Ca(2+)-activated K(+) channels (KCa) comprise many members. Based on their single channel conductance they are divided into three subfamilies: big conductance (BKCa), intermediate conductance (IKCa) and small conductance (SKCa; SK1, SK2 and SK3). Ca(2+) channels are divided into two main families, voltage gated/voltage dependent Ca(2+) channels and non-voltage gated/voltage independent Ca(2+) channels. Based on their electrophysiological and pharmacological properties and on the tissue where there are expressed, voltage gated Ca(2+) channels (Cav) are divided into 5 families: T-type, L-type, N-type, P/Q-type and R-type Ca(2+). Non-voltage gated Ca(2+) channels comprise the TRP (TRPC, TRPV, TRPM, TRPA, TRPP, TRPML and TRPN) and Orai (Orai1 to Orai3) families and their partners STIM (STIM1 to STIM2). A depolarization is needed to activate voltage-gated Ca(2+) channels while non-voltage gated Ca(2+) channels are activated by Ca(2+) depletion of the endoplasmic reticulum stores (SOCs) or by receptors (ROCs). These two Ca(2+) channel families also control constitutive Ca(2+) entries. For reducing the energy consumption and for the fine regulation of Ca(2+), KCa and Ca(2+) channels appear associated as complexes in excitable and non-excitable cells. Interestingly, there is now evidence that KCa-Ca(2+) channel complexes are also found in cancer cells and contribute to cancer-associated functions such as cell proliferation, cell migration and the capacity to develop metastases. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Definition and classification of evaluation units for CASP10.

For the 10th experiment on Critical Assessment of the techniques of protein Structure Prediction (CASP), the prediction target proteins were broken into independent evaluation units (EUs), which were then classified into template-based modeling (TBM) or free modeling (FM) categories. We describe here how the EUs were defined and classified, what issues arose in the process, and how we resolved them. EUs are frequently not the whole target proteins but the constituting structural domains. However, the assessors from CASP7 on combined more than one domain into 1 EU for some targets, which implied that the assessment also included evaluation of the prediction of the relative position and orientation of these domains. In CASP10, we followed and expanded this notion by defining multidomain EUs for a number of targets. These included 3 EUs, each made of two domains of familiar fold but arranged in a novel manner and for which the focus of evaluation was the interdomain arrangement. An EU was classified to the TBM category if a template could be found by sequence similarity searches and to FM if a structural template could not be found by structural similarity searches. The EUs that did not fall cleanly in either of these cases were classified case-by-case, often including consideration of the overall quality and characteristics of the predictions.

Subunit stoichiometry, evolution, and functional implications of an asymmetric plant plastid ClpP/R protease complex in Arabidopsis.

The caseinolytic protease (Clp) protease system has been expanded in plant plastids compared with its prokaryotic progenitors. The plastid Clp core protease consists of five different proteolytic ClpP proteins and four different noncatalytic ClpR proteins, with each present in one or more copies and organized in two heptameric rings. We determined the exact subunit composition and stoichiometry for the intact core and each ring. The chloroplast ClpP/R protease was affinity purified from clpr4 and clpp3 Arabidopsis thaliana null mutants complemented with C-terminal StrepII-tagged versions of CLPR4 and CLPP3, respectively. The subunit stoichiometry was determined by mass spectrometry-based absolute quantification using stable isotope-labeled proteotypic peptides generated from a synthetic gene. One heptameric ring contained ClpP3,4,5,6 in a 1:2:3:1 ratio. The other ring contained ClpP1 and ClpR1,2,3,4 in a 3:1:1:1:1 ratio, resulting in only three catalytic sites. These ClpP1/R1-4 proteins are most closely related to the two subunits of the cyanobacterial P3/R complex and the identical P:R ratio suggests conserved adaptation. Furthermore, the plant-specific C-terminal extensions of the ClpP/R subunits were not proteolytically removed upon assembly, suggesting a regulatory role in Clp chaperone interaction. These results will now allow testing ClpP/R structure-function relationships using rationale design. The quantification workflow we have designed is applicable to other protein complexes.

Evolution of the ABPA subunit of androgen-binding protein expressed in the submaxillary glands in New and Old World rodent taxa.

The salivary androgen-binding proteins (ABPs) are members of the secretoglobin gene family present in mammals. Each ABP is a heterodimer assembled as an ABPA subunit encoded by an Abpa gene and linked by disulfide bridges to an ABPBG subunit encoded by an Abpbg gene. The ABP dimers are secreted into the saliva of mice and then transferred to the pelage after grooming and subsequently to the environment allowing an animal to mark territory with a biochemical signal. The putative role of the mouse salivary ABPs is that of pheromones mediating mate selection resulting in assortative mating in the Mus musculus species complex. We focused on comparing patterns of molecular evolution between the Abpa genes expressed in the submaxillary glands of species of New World and Old World muroids. We found that in both sets of rodents the Abpa genes expressed in the submaxillary glands appear to be evolving under a similar evolutionary regime, with relatively high nonsynonymous substitution rates, suggesting that ABP might play a similar biological role in both systems. Thus, ABP could be involved with mate recognition and species isolation in New World as well as Old World muroids.

In vitro and in vivo characterization of a novel insect decaprenyl diphosphate synthase: a two-major step catalytic mechanism is proposed.

Medium- and long-chain polyprenyl diphosphate synthases (PDDSs) catalyze the synthesis of the side-chain prenyl tails of ubiquinones, which play critical physiological roles in all organisms. This class of enzymes has been extensively studied in bacteria, yeast, plants and mammals, but very little information about such enzymes is available in insects. Here we cloned the cDNAs encoding the two subunits of an aphid long-chain PDDS (designated as AgDPPS1 and AgDPPS2). AgDPPS1 and AgDPPS2 had an open reading frame of 1230 bp and 1275 bp, with a calculated isoelectric point of 8.13 and 6.28, respectively. Sequence alignment and phylogenetic analysis showed that the enzyme was a candidate decaprenyl diphosphate (DPP) synthase with two heterologous subunits. Recombinant expression and in vitro enzymatic assay revealed that the two subunits were essential for the activity of the enzyme that catalyzed the formation of a major intermediate product geranylgeranyl diphosphate. In vivo analysis of ubiquinone (UQ) by expressing the insect enzyme in Escherichia coli identified UQ-10. Our data suggested that the insect enzyme is a novel DPP synthase with a two-major step catalytic mechanism, which catalyzes the formation of DPP as the final product, with geranylgeranyl diphosphate as the major intermediate product. This is the first characterization of an insect long-chain DPPS that synthesizes the side-chain of coenzyme Q-10.

Functional prediction and physiological characterization of a novel short trans-membrane protein 1 as a subunit of mitochondrial respiratory complexes.

Mitochondrial respiration is mediated by a set of multisubunit assemblies of proteins that are embedded in the mitochondrial inner membranes. Respiratory complexes do not only contain central catalytic subunits essential for the bioenergetic transformation, but also many short trans-membrane subunits (sTMs) that are implicated in the proper assembly of complexes. Defects in sTMs have been discovered in some human neurodegenerative diseases. Here we identify a new subunit that we named Stmp1 and have characterized its function using both computational and experimental approaches. Stmp1 is a short trans-membrane protein, and sequence/structure analysis revealed that it shares common features like the small size, presence of a single or two TM region, and a COOH-terminal charged region, as many typical sTMs of respiratory complexes. In situ hybridization and RT-PCR assays showed that the Stmp1 expression is ubiquitous throughout zebrafish embryogenesis. In adults, Stmp1 expression was highest in the brain compared with muscle and liver. In zebrafish larvae (3-5 days postfertilization), antisense morpholino oligonucleotide-mediated knockdown of the Stmp1 gene (Stmp1-MO) resulted in a series of mild morphological defects, including abnormal shape of head and jaw and cardiac edema. Larvae injected with the Stmp1-MO had negligible responses to touch stimuli. By ventilation frequency analysis we found that Stmp1-MO-injected zebrafish displayed a severe dysfunction of ventilatory activities when exposed to hypoxic conditions, suggesting a defective mitochondrial activity induced by the loss of Stmp1. Phylogenetic profiling of known respiratory sTMs compared with Stmp1 revealed that all defined sTMs from four respiratory complexes have restricted or variable phyletic distribution, indicating that they are products of evolutionary innovations to fulfill lineage-related functional requirements for respiratory complexes. Thus, being present in animals, filasterea, choanoflagellida, amoebozoa, and plants, Stmp1 may have evolved to confer a new or complementary regulation of respiratory activities.

A single subunit MCM6 from pea promotes salinity stress tolerance without affecting yield.

The eukaryotic pre-replicative complex (Pre-RC), including heterohexameric minichromosome maintenance (MCM2-7) proteins, ensures that the DNA in genome is replicated only once per cell division cycle. The MCMs provide DNA unwinding function during the DNA replication. Since MCM proteins play essential role in cell division and most likely are affected during stress conditions therefore their overexpression in plants may help in stress tolerance. But the role of MCMs in abiotic stress tolerance in plants has not been reported so far. In this study we report that: a) the MCM6 transcript is upregulated in pea plant in response to high salinity and cold stress and not with ABA, drought and heat stress; b) MCM6 overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in tobacco plants confers salinity tolerance. The T(1) transgenics plants were able to grow to maturity and set normal viable seeds under continuous salinity stress, without yield penalty. It was observed that in salt-grown T(1) transgenic plants the Na(+) ions is mostly accumulated in mature leaves and not in seeds of T(1) transgenic lines as compared with the wild-type (WT) plants. T(1) transgenic plants exhibited better growth status under salinity stress conditions in comparison to WT plants. Furthermore, the T(1) transgenic plants maintained significantly higher levels of leaf chlorophyll content, net photosynthetic rate and therefore higher dry matter accumulation and yield with 200 mM NaCl as compared to the WT plants. Tolerance index data showed better salt tolerance potential of T(1) transgenic plants in comparison to WT. These findings provide first direct evidence that overexpression of single subunit MCM6 confers salinity stress tolerance without yield loss. The possible mechanism of salinity tolerance is discussed. These findings suggest that DNA replication machinery can be exploited for promoting stress tolerance in crop plants.

Functional interactions between residues in the S1, S4, and S5 domains of Kv2.1.

The voltage-gated potassium channel subunit Kv2.1 forms heterotetrameric channels with the silent subunit Kv6.4. Chimeric Kv2.1 channels containing a single transmembrane segment from Kv6.4 have been shown to be functional. However, a Kv2.1 chimera containing both S1 and S5 from Kv6.4 was not functional. Back mutation of individual residues in this chimera (to the Kv2.1 counterpart) identified four positions that were critical for functionality: A200V and A203T in S1, and T343M and P347S in S5. To test for possible interactions in Kv2.1, we used substitutions with charged residues and tryptophan for the outermost pair 203/347. Combinations of substitutions with opposite charges at both T203 and S347 were tolerated but resulted in channels with altered gating kinetics, as did the combination of negatively charged aspartate substitutions. Double mutant cycle analysis with these mutants indicated that both residues are energetically coupled. In contrast, replacing both residues with a positively charged lysine together (T203K + S347K) was not tolerated and resulted in a folding or trafficking deficiency. The nonfunctionality of the T203K + S347K mutation could be restored by introducing the R300E mutation in the S4 segment of the voltage sensor. These results indicate that these specific S1, S4, and S5 residues are in close proximity and interact with each other in the functional channel, but are also important determinants for Kv2.1 channel maturation. These data support the view of an anchoring interaction between S1 and S5, but indicate that this interaction surface is more extensive than previously proposed.