Prokaryotic Domains Research Articles

The RCK1 domain of the human BKCa channel transduces Ca2+ binding into structural rearrangements

Large-conductance voltage- and Ca2+-activated K+ (BKCa) channels play a fundamental role in cellular function by integrating information from their voltage and Ca2+ sensors to control membrane potential and Ca2+ homeostasis. The molecular mechanism of Ca2+-dependent regulation of BKCa channels is unknown, but likely relies on the operation of two cytosolic domains, regulator of K+ conductance (RCK)1 and RCK2. Using solution-based investigations, we demonstrate that the purified BKCa RCK1 domain adopts an α/β fold, binds Ca2+, and assembles into an octameric superstructure similar to prokaryotic RCK domains. Results from steady-state and time-resolved spectroscopy reveal Ca2+-induced conformational changes in physiologically relevant [Ca2+]. The neutralization of residues known to be involved in high-affinity Ca2+ sensing (D362 and D367) prevented Ca2+-induced structural transitions in RCK1 but did not abolish Ca2+ binding. We provide evidence that the RCK1 domain is a high-affinity Ca2+ sensor that transduces Ca2+ binding into structural rearrangements, likely representing elementary steps in the Ca2+-dependent activation of human BKCa channels.

Deletion of Switch 3 Results in an Archaeal RNA Polymerase That Is Defective in Transcript Elongation

Switch 3 is a polypeptide loop conserved in all multisubunit DNA-dependent RNA polymerases (RNAPs) that extends into the main cleft of the RNAP and contacts each base in a nascent transcript as that base is released from the internal DNA-RNA hybrid. Plasmids have been constructed and transformed into Thermococcus kodakaraensis, which direct the constitutive synthesis of the archaeal RNAP subunit RpoB with an N-terminal His(6) tag and the Switch 3 loop either intact (wild-type) or deleted (DeltaS3). RNAPs containing these plasmid-encoded RpoB subunits were purified, and, in vitro, the absence of Switch 3 had no negative effects on transcription initiation or elongation complex stability but reduced the rate of transcript elongation. The defect in elongation occurred at every template position and increased the sensitivity of the archaeal RNAP to intrinsic termination. Comparing these properties and those reported for a bacterial RNAP lacking Switch 3 argues that this loop functions differently in the RNAPs from the two prokaryotic domains. The close structural homology of archaeal and eukaryotic RNAPs would predict that eukaryotic Switch 3 loops likely conform to the archaeal rather than bacterial functional paradigm.

Tantalizing evidence for caspase‐like protein expression and activity in the cellular stress response of Archaea

An enigmatic feature of microbial evolution is the emergence of programmed cell death (PCD), a genetically controlled form of cell suicide triggered by environmental stimuli. Archaea, the second major prokaryotic domain of life, have been notably absent from the PCD inheritance discussion, due to a lack of genetic homologues. Using the model haloarchaeon Haloferax volcanii, we document extremely high caspase-specific activity and expression of immunoreactive proteins to human caspase 8 antisera, both of which were induced by salt stress and death and were abolished by in vivo addition of a broad-spectrum caspase inhibitor. Caspase inhibition severely impaired cell growth under low and high salt stress, demonstrating a critical role in the cellular stress response. In silico analysis of the H. volcanii proteome identified a subset of 18 potential target proteins containing a signature tetrapeptide caspase cleavage motif (IETD), some with putative roles in allosteric regulation, signal transduction, osmotic stress and cell communication. Detection of similarly high activity and expression in other haloarchaea (Halorubrum and Haloarcula) and in diverse members of Euryarchaeota (the methanogen Methanosarcina acetivorans and the hyperthermophile Pyrococcus furiosus) and Crenarchaeota (the acidophile Sulfolobus solfataricus) argue for a broad representation within the archaeal domain. By playing a role in normal cell function, caspase-like proteases in Archaea appear to have co-evolved with other metabolic pathways, broadening their biological roles beyond apoptosis and cell death.

Structure of a Eukaryotic Nonribosomal Peptide Synthetase Adenylation Domain That Activates a Large Hydroxamate Amino Acid in Siderophore Biosynthesis

Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N(delta)-cis-anhydromevalonyl-N(delta)-hydroxy-L-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically, and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes that play important and diverse roles in the biology of fungi.

Genome Networks Root the Tree of Life between Prokaryotic Domains

Eukaryotes arose from prokaryotes, hence the root in the tree of life resides among the prokaryotic domains. The position of the root is still debated, although pinpointing it would aid our understanding of the early evolution of life. Because prokaryote evolution was long viewed as a tree-like process of lineage bifurcations, efforts to identify the most ancient microbial lineage split have traditionally focused on positioning a root on a phylogenetic tree constructed from one or several genes. Such studies have delivered widely conflicting results on the position of the root, this being mainly due to methodological problems inherent to deep gene phylogeny and the workings of lateral gene transfer among prokaryotes over evolutionary time. Here, we report the position of the root determined with whole genome data using network-based procedures that take into account both gene presence or absence and the level of sequence similarity among all individual gene families that are shared across genomes. On the basis of 562,321 protein-coding gene families distributed across 191 genomes, we find that the deepest divide in the prokaryotic world is interdomain, that is, separating the archaebacteria from the eubacteria. This result resonates with some older views but conflicts with the results of most studies over the last decade that have addressed the issue. In particular, several studies have suggested that the molecular distinctness of archaebacteria is not evidence for their antiquity relative to eubacteria but instead stems from some kind of inherently elevated rate of archaebacterial sequence change. Here, we specifically test for such a rate elevation across all prokaryotic lineages through the analysis of all possible quartets among eight genes duplicated in all prokaryotes, hence the last common ancestor thereof. The results show that neither the archaebacteria as a group nor the eubacteria as a group harbor evidence for elevated evolutionary rates in the sampled genes, either in the recent evolutionary past or in their common ancestor. The interdomain prokaryotic position of the root is thus not attributable to lineage-specific rate variation.

The Na+-Activated Potassium Channel Slack Shares a Similar Na+ Coordination Site with Kir3 Channels

Characteristics of Na+ activated potassium channel (Slack or Slo2.2) currents, including high conductance, rundown, regulation by Na+, Cl- and phosphorylation have long been reported but underlying mechanisms remain unknown. Here we report identification of a sodium regulatory site in the RCK2 domain of Slack channels by screening the C-terminus with the conserved sodium coordination motif of Kir channels. While the charge preserving D818E mutation exhibited similar Na+ sensitivity as the wild-type Slack channel, both a neutralization mutant D818N and a charge reversal D818R mutant within this site dramatically decreased Na+ sensitivity. Thus, D818 is engaged in an important electrostatic interaction with Na+. Similarly, the H823N mutant within this site also greatly decreased Na+ sensitivity of Slack channels. Simulations of the Slack RCK2 domain based on the crystallized structure of a prokaryotic RCK domain structure (Jiang et al., 2001, Neuron 29:593) provided a model of the Na+ coordination site in Slack channels. Moreover, simulations of the Na+ coordination site in Slo2.2 channels predicted a 5∼7 fold selectivity for Na+ over Li+ that were confirmed by electrophysiological data. Our results suggest that the Slack channel shares a similar Na+ regulatory mechanism with Kir channels but with important differences, such as an intricate coupling mechanism to Cl- co-regulation and possibly additional Na+ sensitive sites.

Molecular Determinants in Glycine and GABA-A Receptors that Sense Intracellular Chloride Concentration

GABA-A and glycine receptors are ligand-gated ion channels belonging to the Cys-loop superfamily. They are permeable to anions and mediate fast synaptic inhibition in the central nervous system. We recently showed that intracellular chloride can affect the time course of deactivation of these channels (1). The aim of the present work was to identify the residues that are involved in “sensing” the intracellular chloride concentration.We used the fast concentration jump technique to apply brief pulses (1-3 ms) of saturating glycine or GABA to outside-out patches from transiently-transfected HEK cells expressing alpha1 or alpha1 beta glycine receptors or alpha1 beta2 gamma2L GABA-A receptors. The effect of intracellular chloride could be mediated either by residues in intracellular loops, such as M1-M2 and M3-M4, or by the residues lining the channel itself. We used a combination of deletion and Ala/Cys-scanning mutagenesis approaches to address this question.In other nicotinic channels, the long M3-M4 intracellular loop can be replaced by the equivalent domain (7 amino acids) of the orthologous prokaryotic channel from Gloeobacter violaceus without losing channel function (2). We found that chimeric constructs of glycine and GABA-A receptors containing the prokaryotic M3-M4 domain retained modulation by intracellular chloride, suggesting that the major cytoplasmic domain is unlikely to mediate this effect. The results of mutagenesis of residues facing the ion permeation pathway on the contrary strongly suggests that chloride ions affect channel kinetic by binding to a site along the pore; we have identified conserved residues that reduce the differences between the deactivation rates measured at different chloride concentrations.(1) Pitt, Sivilotti and Beato (2008) J.Neurosci 28, 11454-11467(2) Jansen, Bali and Akabas (2008) J.Gen.Physiol. 131, 137-146

NMR assignments of the DNA binding domain of Ml4 protein from Mesorhizobium loti

Ml4 protein from Mesorhizobium loti has a 58% sequence identity with the Ros protein from Agrobacterium tumefaciens that contains a prokaryotic Cys(2)His(2) zinc finger domain. Interestingly, Ml4 is a zinc-lacking protein that does not contain the Cys(2)His(2) motif and is able to bind the Ros DNA target sequence with high affinity. Here we report the (1)H, (15)N and (13)C NMR assignments of the Ml4 protein DNA binding domain (residue 52-151), as an important step toward elucidating at a molecular level how this prokaryotic domain can overcome the metal requirement for proper folding and DNA-binding activity.

Hydrothermal Focusing of Chemical and Chemiosmotic Energy, Supported by Delivery of Catalytic Fe, Ni, Mo/W, Co, S and Se, Forced Life to Emerge

Energised by the protonmotive force and with the intervention of inorganic catalysts, at base Life reacts hydrogen from a variety of sources with atmospheric carbon dioxide. It seems inescapable that life emerged to fulfil the same role (i.e., to hydrogenate CO(2)) on the early Earth, thus outcompeting the slow geochemical reduction to methane. Life would have done so where hydrothermal hydrogen interfaced a carbonic ocean through inorganic precipitate membranes. Thus we argue that the first carbon-fixing reaction was the molybdenum-dependent, proton-translocating formate hydrogenlyase system described by Andrews et al. (Microbiology 143:3633-3647, 1997), but driven in reverse. Alkaline on the inside and acidic and carbonic on the outside - a submarine chambered hydrothermal mound built above an alkaline hydrothermal spring of long duration - offered just the conditions for such a reverse reaction imposed by the ambient protonmotive force. Assisted by the same inorganic catalysts and potential energy stores that were to evolve into the active centres of enzymes supplied variously from ocean or hydrothermal system, the formate reaction enabled the rest of the acetyl coenzyme-A pathway to be followed exergonically, first to acetate, then separately to methane. Thus the two prokaryotic domains both emerged within the hydrothermal mound-the acetogens were the forerunners of the Bacteria and the methanogens were the forerunners of the Archaea.

Structural Basis of Novel Interactions Between the Small-GTPase and GDI-like Domains in Prokaryotic FeoB Iron Transporter

The FeoB family proteins are widely distributed prokaryotic membrane proteins involved in Fe(2+) uptake. FeoB consists of N-terminal cytosolic and C-terminal transmembrane domains. The N-terminal region of the cytosolic domain is homologous to small GTPase (G) proteins and is considered to regulate Fe(2+) uptake. The spacer region connecting the G and TM domains reportedly functions as a GDP dissociation inhibitor (GDI)-like domain that stabilizes the GDP-binding state. However, the function of the G and GDI-like domains in iron uptake remains unclear. Here, we report the structural and functional analyses of the FeoB cytosolic domain from Thermotoga maritima. The structure-based mutational analysis indicated that the interaction between the G and GDI-like domains is important for both the GDI and Fe(2+) uptake activities. On the basis of these results, we propose a regulatory mechanism of Fe(2+) uptake.

The Anabaena sensory rhodopsin transducer defines a novel superfamily of prokaryotic small-molecule binding domains

The Anabaena sensory rhodopsin transducer (ASRT) is a small protein that has been claimed to function as a signaling molecule downstream of the cyanobacterial sensory rhodopsin. However, orthologs of ASRT have been detected in several bacteria that lack rhodopsin, raising questions about the generality of this function. Using sequence profile searches we show that ASRT defines a novel superfamily of β-sandwich fold domains. Through contextual inference based on domain architectures and predicted operons and structural analysis we present strong evidence that these domains bind small molecules, most probably sugars. We propose that the intracellular versions like ASRT probably participate as sensors that regulate a diverse range of sugar metabolism operons or even the light sensory behavior in Anabaena by binding sugars or related metabolites. We also show that one of the extracellular versions define a predicted sugar-binding structure in a novel cell-surface lipoprotein found across actinobacteria, including several pathogens such as Tropheryma, Actinomyces and Thermobifida. The analysis of this superfamily also provides new data to investigate the evolution of carbohydrate binding modes in β-sandwich domains with very different topologies.Reviewers: This article was reviewed by M. Madan Babu and Mark A. Ragan.

The structural role of the zinc ion can be dispensable in prokaryotic zinc-finger domains

The recent characterization of the prokaryotic Cys(2)His(2) zinc-finger domain, identified in Ros protein from Agrobacterium tumefaciens, has demonstrated that, although possessing a similar zinc coordination sphere, this domain is structurally very different from its eukaryotic counterpart. A search in the databases has identified approximately 300 homologues with a high sequence identity to the Ros protein, including the amino acids that form the extensive hydrophobic core in Ros. Surprisingly, the Cys(2)His(2) zinc coordination sphere is generally poorly conserved in the Ros homologues, raising the question of whether the zinc ion is always preserved in these proteins. Here, we present a functional and structural study of a point mutant of Ros protein, Ros(56-142)C82D, in which the second coordinating cysteine is replaced by an aspartate, 5 previously-uncharacterized representative Ros homologues from Mesorhizobium loti, and 2 mutants of the homologues. Our results indicate that the prokaryotic zinc-finger domain, which in Ros protein tetrahedrally coordinates Zn(II) through the typical Cys(2)His(2) coordination, in Ros homologues can either exploit a CysAspHis(2) coordination sphere, previously never described in DNA binding zinc finger domains to our knowledge, or lose the metal, while still preserving the DNA-binding activity. We demonstrate that this class of prokaryotic zinc-finger domains is structurally very adaptable, and surprisingly single mutations can transform a zinc-binding domain into a nonzinc-binding domain and vice versa, without affecting the DNA-binding ability. In light of our findings an evolutionary link between the prokaryotic and eukaryotic zinc-finger domains, based on bacteria-to-eukaryota horizontal gene transfer, is discussed.

Catalytic Mechanism of Eukaryotic Selenocysteine Synthase

The biosynthetic pathway for Sec has been established in the three domains of life, prokaryotes, archaea and eukaryotes. Mammalian Sec synthase (SecS) uses O‐phosphoseryl‐tRNA[Ser]Sec as a substrate. We have found that SecS does not dephosphorylate free O‐phospho‐L‐serine. Based on the crystal structure, we have made several mutants of this enzyme, SecSQ105E, SecSR313S, and SecSR313E, and they all showed corrupted SecS activity. These residues are strictly conserved in SecS orthologs and function to coordinate the phosphate but are lacking or variant in related enzymes. The data suggested that a phosphate loop accommodates the alpha‐phosphate moiety of O‐phospho‐L‐seryl‐tRNA[Ser]Sec and, after phosphate elimination, binds selenophosphate to initiate attack on the proposed aminoacrylyl‐tRNA[Ser]Sec intermediate. The activity profiles of mechanism‐based inhibitors were also obtained and provided additional insights into the reaction mechanism of SecS. This work was supported by Grant WA1126‐2, the Max‐Planck‐Society, the Intramural Research Program of the NIH, NCI, CCR and by NIH grants to VNG.,

Solution Structure and Characterization of the DNA-Binding Activity of the B3BP–Smr Domain

The MutS1 protein recognizes unpaired bases and initiates mismatch repair, which are essential for high-fidelity DNA replication. The homologous MutS2 protein does not contribute to mismatch repair, but suppresses homologous recombination. MutS2 lacks the damage-recognition domain of MutS1, but contains an additional C-terminal extension: the small MutS-related (Smr) domain. This domain, which is present in both prokaryotes and eukaryotes, has previously been reported to bind to DNA and to possess nicking endonuclease activity. We determine here the solution structure of the functionally active Smr domain of the Bcl3-binding protein (also known as Nedd4-binding protein 2), a protein with unknown function that lacks other domains present in MutS proteins. The Smr domain adopts a two-layer α–β sandwich fold, which has a structural similarity to the C-terminal domain of IF3, the R3H domain, and the N-terminal domain of DNase I. The most conserved residues are located in three loops that form a contiguous, exposed, and positively charged surface with distinct sequence identity for prokaryotic and eukaryotic Smr domains. NMR titration experiments and DNA binding studies using Bcl3-binding protein–Smr domain mutants suggested that these most conserved loop regions participate in DNA binding to single-stranded/double-stranded DNA junctions. Based on the observed DNA-binding-induced multimerization, the structural similarity with both subdomains of DNase I, and the experimentally identified DNA-binding surface, we propose a model for DNA recognition by the Smr domain.

Autoactivation Is the Key

BIOCHEMISTRY Protein tyrosine kinases (PTKs) regulate cellular activities by transferring phosphate groups from ATP to other proteins. PTKs must first be activated by autophosphorylation of their own specific tyrosine residues. The structural basis for eukaryotic PTK activation involves displacement of an amino acid loop, which initially blocks access to the active site but shifts out of the way upon autophosphorylation. Much less is known about prokaryotic PTKs, which do not have significant sequence homology to eukaryotic PTKs. Lee et al. have determined the crystal structure of a prokaryotic PTK domain from the Escherichia coli tyrosine kinase Etk, alone and with ADP bound. The side chain of tyrosine 574 (Y574), which must be autophosphorylated in order to activate Etk, is positioned facing ADP in the active site and blocks access by peptide substrates. Although Y574 is not part of a flexible loop, a phosphorylated Y574 side chain could rotate away from the active site into an alternate conformation that would be stabilized by a salt bridge to a nearby arginine and multiple hydrogen bonds to surrounding amino acids. The Etk structure therefore suggests a new mechanism of activation of PTKs, in which phosphorylation-triggered displacement of only a single amino acid side chain suffices to unlock the door to the active site. — NM [*][1] EMBO J . 27 , 10.1038/emboj.2008.97 (2008). [1]: #fn-1

Functional analysis of mammalian selenocysteine synthase

Selenocysteine (Sec) is co‐translationally inserted into protein in response to UGA codons as the 21st amino acid in the genetic code. The biosynthetic pathway for Sec has been established in the three domains of life, prokaryotes, archaea and eukaryotes. Mammalian Sec synthase (SecS) uses O‐phosphoseryl‐tRNA as a substrate and accepts monoselenophosphate as the active selenium donor to yield Sec‐tRNA. We have found, in addition, that the Sec biosynthetic factors, SECp43 and SPS1, bind to SecS in vivo and that SECp43 binds to SecS in vitro as well. These bindings did not affect the activity of SecS in vitro. The in vitro reactions which showed that these factors are not essential for the activity of SecS indicate that the in vivo binding of SECp43 and SPS1 to SecS is likely involved in other functions.

Differentiation Increases the Resistance of Neuronal Cells to Amyloid Toxicity

A substantial lack of information is recognized on the features underlying the variable susceptibility to amyloid aggregate toxicity of cells with different phenotypes. Recently, we showed that different cell types are variously affected by early aggregates of a prokaryotic hydrogenase domain (HypF-N). In the present study we investigated whether differentiation affects cell susceptibility to amyloid injury using a human neurotypic SH-SY5Y cell differentiation model. We found that retinoic acid-differentiated cells were significantly more resistant against Abeta1-40, Abeta1-42 and HypF-N prefibrillar aggregate toxicity respect to undifferentiated cells treated similarly. Earlier and sharper increases in cytosolic Ca(2+) and ROS with marked lipid peroxidation and mitochondrial dysfunction were also detected in exposed undifferentiated cells resulting in apoptosis activation. The reduced vulnerability of differentiated cells matched a more efficient Ca(2+)-ATPase equipment and a higher total antioxidant capacity. Finally, increasing the content of membrane cholesterol resulted in a remarkable reduction of vulnerability and ability to bind the aggregates in either undifferentiated and differentiated cells.

The RCK2 domain of the human BKCa channel is a calcium sensor.

Large conductance voltage and Ca(2+)-dependent K(+) channels (BK(Ca)) are activated by both membrane depolarization and intracellular Ca(2+). Recent studies on bacterial channels have proposed that a Ca(2+)-induced conformational change within specialized regulators of K(+) conductance (RCK) domains is responsible for channel gating. Each pore-forming alpha subunit of the homotetrameric BK(Ca) channel is expected to contain two intracellular RCK domains. The first RCK domain in BK(Ca) channels (RCK1) has been shown to contain residues critical for Ca(2+) sensitivity, possibly participating in the formation of a Ca(2+)-binding site. The location and structure of the second RCK domain in the BK(Ca) channel (RCK2) is still being examined, and the presence of a high-affinity Ca(2+)-binding site within this region is not yet established. Here, we present a structure-based alignment of the C terminus of BK(Ca) and prokaryotic RCK domains that reveal the location of a second RCK domain in human BK(Ca) channels (hSloRCK2). hSloRCK2 includes a high-affinity Ca(2+)-binding site (Ca bowl) and contains similar secondary structural elements as the bacterial RCK domains. Using CD spectroscopy, we provide evidence that hSloRCK2 undergoes a Ca(2+)-induced change in conformation, associated with an alpha-to-beta structural transition. We also show that the Ca bowl is an essential element for the Ca(2+)-induced rearrangement of hSloRCK2. We speculate that the molecular rearrangements of RCK2 likely underlie the Ca(2+)-dependent gating mechanism of BK(Ca) channels. A structural model of the heterodimeric complex of hSloRCK1 and hSloRCK2 domains is discussed.

The prokaryotic V4R domain is the likely ancestor of a key component of the eukaryotic vesicle transport system

Intracellular vesicle traffic that enables delivery of proteins between the endoplasmic reticulum, Golgi and various endosomal subcompartments is one of the hallmarks of the eukaryotic cell. Its evolutionary history is not well understood but the process itself and the core vesicle traffic machinery are believed to be ancient. We show here that the 4-vinyl reductase (V4R) protein domain present in bacteria and archaea is homologous to the Bet3 subunit of the TRAPP1 vesicle-tethering complex that is conserved in all eukaryotes. This suggests, for the first time, a prokaryotic origin for one of the key eukaryotic trafficking proteins.This article was reviewed by Gaspar Jekely and Mark A. Ragan

Characterization of a Novel Prokaryotic GDP Dissociation Inhibitor Domain from the G Protein Coupled Membrane Protein FeoB

The FeoB family of membrane embedded G proteins are involved with high affinity Fe(II) uptake in prokaryotes. Here, we report that FeoB harbors a novel GDP dissociation inhibitor-like domain that specifically stabilizes GDP-binding through an interaction with the switch I region of the G protein. We show that the stabilization of GDP binding is conserved between species despite a high degree of sequence variability in their guanine nucleotide dissociation inhibitor (GDI)-like domains, and demonstrate that the presence of the membrane embedded domain increases GDP-binding affinity roughly 150-fold over the level accomplished by action of the GDI-like domain alone. To our knowledge, this is the first example for a prokaryotic GDI, targeting a bacterial G protein-coupled membrane process. Our findings suggest that Fe(II) uptake in bacteria involves a G protein regulatory pathway reminiscent of signaling mechanisms found in higher-order organisms.