Downstream Domain Research Articles

Structural characteristics of the Relict Gull (Larus relictus) mitochondrial DNA control region and its comparison to other Laridae

The structure of the mitochondrial DNA control region in the Relict Gull (Larus relictus) was predicted and compared with data from the National Center for Biotechnology Information (NCBI) on five other gulls. The results showed that the control regions of the six gulls comprise three domains. Sequences of CSB-1-like (domain I) and CSB-1 (domain III) in L. relictus have the highest similarity with those in the other five gulls. The insertion fragments are located in downstream domain I of L. ridibundus, L. brunnicephalus, and L. saundersi. Seven conserved sequence boxes (additional box, F-box, E-box, D-box, C-box, bird-similarity-box, and B-box) are located in domain II in all six gulls. It is suggested that the CSB-2/3 sequence, the origin of H-strand replication, and bidirectional light- and heavy-strand transcription promoters in domain III of L. relictus have some distinguishing features to those of other gulls. Some repeat units are contained in the 3′ end of the control region in the five gulls; however, no repeat units are found in the sequence CAAACAACAAA in L. relictus. The distribution of nucleotide diversity analysis will provide the useful information on the selected DNA fragment within the control region for genetic analyses among gulls.

A Universal Stress Protein (USP) in Mycobacteria Binds cAMP

Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as "sinks" for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell.

Integration of the Gila River drainage system through the Basin and Range province of southern Arizona and southwestern New Mexico (USA)

The Gila River and its tributaries in southern Arizona and adjoining states incorporate several dozen individual extensional basins of the central Basin and Range province into a single integrated drainage network. Forty basins in the Gila domain contain more than 1000m (maximum ~3500m) of post-12Ma basin fill. Subsurface evaporites in many basins document internal drainages terminating in isolated playa lakes during early phases of basin history. The nature of intrabasinal and interbasinal divides and of eroded or sedimented stream passages through mountain ranges intervening between the basins reveal the geomorphic mechanisms that achieved drainage integration over late Miocene to early Pleistocene time. Drainage integration accompanied by headward erosion eastward toward Gila headwaters was a response to Miocene opening of the Gulf of California, into which the Gila River debouched directly before the Pliocene (<5Ma) lower course of the Colorado River was established. Residual basins of internal drainage where headward erosion has not yet penetrated into basin fill are most common in the easternmost Gila domain but also persist locally farther west. Most basin fill was dissected during drainage integration within the upstream Gila domain but continued accumulation of undissected basin fill by sediment aggradation is dominant in the downstream Gila domain. Basin dissection was initiated by Pliocene time in the central Gila domain but was delayed until Pleistocene time farther east. In the westernmost Gila domain, interaction with erosional and depositional episodes along the Colorado River influenced the development of Quaternary landscapes along the tributary Gila River. The sedimentary history of the Gila drainage network illustrates the means by which trunk rivers can establish courses across corrugated topography produced by the extensional rupture of continental blocks.

Neuroinflammation triggered by β-glucan/dectin-1 signaling enables CNS axon regeneration

Innate immunity can facilitate nervous system regeneration, yet the underlying cellular and molecular mechanisms are not well understood. Here we show that intraocular injection of lipopolysaccharide (LPS), a bacterial cell wall component, or the fungal cell wall extract zymosan both lead to rapid and comparable intravitreal accumulation of blood-derived myeloid cells. However, when combined with retro-orbital optic nerve crush injury, lengthy growth of severed retinal ganglion cell (RGC) axons occurs only in zymosan-injected mice, and not in LPS-injected mice. In mice deficient for the pattern recognition receptor dectin-1 but not Toll-like receptor-2 (TLR2), zymosan-mediated RGC regeneration is greatly reduced. The combined loss of dectin-1 and TLR2 completely blocks the proregenerative effects of zymosan. In the retina, dectin-1 is expressed by microglia and dendritic cells, but not by RGCs. Dectin-1 is also present on blood-derived myeloid cells that accumulate in the vitreous. Intraocular injection of the dectin-1 ligand curdlan [a particulate form of β(1, 3)-glucan] promotes optic nerve regeneration comparable to zymosan in WT mice, but not in dectin-1(-/-) mice. Particulate β(1, 3)-glucan leads to increased Erk1/2 MAP-kinase signaling and cAMP response element-binding protein (CREB) activation in myeloid cells in vivo. Loss of the dectin-1 downstream effector caspase recruitment domain 9 (CARD9) blocks CREB activation and attenuates the axon-regenerative effects of β(1, 3)-glucan. Studies with dectin-1(-/-)/WT reciprocal bone marrow chimeric mice revealed a requirement for dectin-1 in both retina-resident immune cells and bone marrow-derived cells for β(1, 3)-glucan-elicited optic nerve regeneration. Collectively, these studies identify a molecular framework of how innate immunity enables repair of injured central nervous system neurons.

Identification of Major FKBP12 Binding Determinants in RyR1

We used site-directed mutagenesis and labeling of the type 1 ryanodine receptor (RyR1) recombinantly expressed in HEK-293T cells to identify RyR1 residues that interact with FKBP12, a constitutively-bound protein that promotes the closed state of the RyR1 calcium channel. While the location of bound FKBP on the RyR1 cryo electron microscopic (EM) map is known, the RyR1 sequence elements forming the FKBP binding site are poorly understood. Previously, we have shown that a His10 tag inserted at RyR1 position 620, adjacent to the N-terminal “ABC” domains, abolished FKBP binding. Here, we used structural modeling to predict loop regions in downstream domains, and then used these loops for His10 insertion sites. Fluorescent FKBP (F-FKBP) binding was then assessed in functional, full-length RyR1 containing these His10 tags. We determined that insertion of His10 tags in specific loops could completely abolish F-FKBP binding, even though RyR1 calcium release activity was unaffected. Scrambling the amino acid sequence of the loops identified using His10 tags also eliminated F-FKBP binding. The effects of disrupting these loops on F-FKBP binding were far more pronounced compared to previously established FKBP binding motifs, such as the VP dipeptide at RyR1 position 2461 and the PKA phosphorylation site at position 2843 within the PKA/CaMKII phosphorylation loop of RyR1. Thus, our results identify novel major FKBP binding determinants in RyR1. Supported by NIH and Canadian grants R01AR059126 (to JDF) R01HL092097 (to RLC), and CIHR 259009 (to FVP).

A magnesium-induced RNA conformational switch at the internal ribosome entry site of hepatitis C virus genome visualized by atomic force microscopy

The 5′ untranslated region of hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) element, composed of domains II–IV, which is required for cap-independent translation initiation. Little information on the 3D structure of the whole functional HCV IRES is still available. Here, we use atomic force microscopy to visualize the HCV IRES conformation in its natural sequence context, which includes the upstream domain I and the essential, downstream domains V and VI. The 574 nt-long molecule analyzed underwent an unexpected, Mg2+-induced switch between two alternative conformations: from ‘open’, elongated morphologies at 0–2 mM Mg2+ concentration to a ‘closed’, comma-shaped conformation at 4–6 mM Mg2+. This sharp transition, confirmed by gel-shift analysis and partial RNase T1 cleavage, was hindered by the microRNA miR-122. The comma-shaped IRES-574 molecules visualized at 4–6 mM Mg2+ in the absence of miR-122 showed two arms. Our data support that the first arm would contain domain III, while the second one would be composed of domains (I–II)+(V–VI) thanks to a long-range RNA interaction between the I-II spacer and the basal region of domain VI. This reinforces the previously described structural continuity between the HCV IRES and its flanking domains I, V and VI.

Structure and Mechanism of Cysteine Peptidase Gingipain K (Kgp), a Major Virulence Factor of Porphyromonas gingivalis in Periodontitis

Cysteine peptidases are key proteolytic virulence factors of the periodontopathogen Porphyromonas gingivalis, which causes chronic periodontitis, the most prevalent dysbiosis-driven disease in humans. Two peptidases, gingipain K (Kgp) and R (RgpA and RgpB), which differ in their selectivity after lysines and arginines, respectively, collectively account for 85% of the extracellular proteolytic activity of P. gingivalis at the site of infection. Therefore, they are promising targets for the design of specific inhibitors. Although the structure of the catalytic domain of RgpB is known, little is known about Kgp, which shares only 27% sequence identity. We report the high resolution crystal structure of a competent fragment of Kgp encompassing the catalytic cysteine peptidase domain and a downstream immunoglobulin superfamily-like domain, which is required for folding and secretion of Kgp in vivo. The structure, which strikingly resembles a tooth, was serendipitously trapped with a fragment of a covalent inhibitor targeting the catalytic cysteine. This provided accurate insight into the active site and suggested that catalysis may require a catalytic triad, Cys(477)-His(444)-Asp(388), rather than the cysteine-histidine dyad normally found in cysteine peptidases. In addition, a 20-Å-long solvent-filled interior channel traverses the molecule and links the bottom of the specificity pocket with the molecular surface opposite the active site cleft. This channel, absent in RgpB, may enhance the plasticity of the enzyme, which would explain the much lower activity in vitro toward comparable specific synthetic substrates. Overall, the present results report the architecture and molecular determinants of the working mechanism of Kgp, including interaction with its substrates.

Autoreactive T cells induce necrosis and not BCL-2-regulated or death receptor-mediated apoptosis or RIPK3-dependent necroptosis of transplanted islets in a mouse model of type 1 diabetes.

Type 1 diabetes results from T cell-mediated destruction of pancreatic beta cells. The mechanisms of beta cell destruction in vivo, however, remain unclear. We aimed to test the relative roles of the main cell death pathways: apoptosis, necrosis and necroptosis, in beta cell death in the development of CD4(+) T cell-mediated autoimmune diabetes. We altered expression levels of critical cell death proteins in mouse islets and tested their ability to survive CD4(+) T cell-mediated attack using an in vivo graft model. Loss of the B cell leukaemia/lymphoma 2 (BCL-2) homology domain 3-only proteins BIM, PUMA or BID did not protect beta cells from this death. Overexpression of the anti-apoptotic protein BCL-2 or combined deficiency of the pro-apoptotic multi-BCL2 homology domain proteins BAX and BAK also failed to prevent beta cell destruction. Furthermore, loss of function of the death receptor Fas or its essential downstream signalling molecule Fas-associated death domain (FADD) in islets was also not protective. Using electron microscopy we observed that dying beta cells showed features of necrosis. However, islets deficient in receptor-interacting serine/threonine protein kinase 3 (RIPK3), a critical initiator of necroptosis, were still normally susceptible to CD4(+) T cell-mediated destruction. Remarkably, simultaneous inhibition of apoptosis and necroptosis by combining loss of RIPK3 and overexpression of BCL-2 in islets did not protect them against immune attack either. Collectively, our data indicate that beta cells die by necrosis in autoimmune diabetes and that the programmed cell death pathways apoptosis and necroptosis are both dispensable for this process.

Re-analysis of cryoEM data on HCV IRES bound to 40S subunit of human ribosome integrated with recent structural information suggests new contact regions between ribosomal proteins and HCV RNA

In this study, we combine available high resolution structural information on eukaryotic ribosomes with low resolution cryo-EM data on the Hepatitis C Viral RNA (IRES) human ribosome complex. Aided further by the prediction of RNA-protein interactions and restrained docking studies, we gain insights on their interaction at the residue level. We identified the components involved at the major and minor contact regions, and propose that there are energetically favorable local interactions between 40S ribosomal proteins and IRES domains. Domain II of the IRES interacts with ribosomal proteins S5 and S25 while the pseudoknot and the downstream domain IV region bind to ribosomal proteins S26, S28 and S5. We also provide support using UV cross-linking studies to validate our proposition of interaction between the S5 and IRES domains II and IV. We found that domain IIIe makes contact with the ribosomal protein S3a (S1e). Our model also suggests that the ribosomal protein S27 interacts with domain IIIc while S7 has a weak contact with a single base RNA bulge between junction IIIabc and IIId. The interacting residues are highly conserved among mammalian homologs while IRES RNA bases involved in contact do not show strict conservation. IRES RNA binding sites for S25 and S3a show the best conservation among related viral IRESs. The new contacts identified between ribosomal proteins and RNA are consistent with previous independent studies on RNA-binding properties of ribosomal proteins reported in literature, though information at the residue level is not available in previous studies.

On the Global Existence and Stability of a Multi-Dimensional Supersonic Conic Shock Wave

We establish the global existence and stability of a three-dimensional supersonic conic shock wave for a compactly perturbed steady supersonic flow past an infinitely long circular cone with a sharp angle. The flow is described by a 3-D steady potential equation, which is multi-dimensional, quasilinear, and hyperbolic with respect to the supersonic direction. Making use of the geometric properties of the pointed shock surface together with the Rankine–Hugoniot conditions on the conic shock surface and the boundary condition on the surface of the cone, we obtain a global uniform weighted energy estimate for the nonlinear problem by finding an appropriate multiplier and establishing a new Hardy-type inequality on the shock surface. Based on this, we prove that a multi-dimensional conic shock attached to the vertex of the cone exists globally when the Mach number of the incoming supersonic flow is sufficiently large. Moreover, the asymptotic behavior of the 3-D supersonic conic shock solution, which is shown to approach the corresponding background shock solution in the downstream domain for the uniform supersonic constant flow past the sharp cone, is also explicitly given.

Identification of RNF114 as a novel positive regulatory protein for T cell activation

RNF114 [RING (really interesting new gene) finger protein 114] has been shown to be a novel psoriasis susceptibility gene, with a putative role in the regulation of immune responses, though the underlying mechanism was not fully identified. In the present study, to investigate whether RNF114 is involved in T cell activation, a series of fluorescence activated cell sortings (FACS) were performed. The analysis confirmed that RNF114 over-expression had a promotion effect on T cell activation with an average 43.97% increment and the upregulatory roles showed a dose-dependent effect with 18.44% increment. Interestingly, the two C2H2 domains were shown to play important but opposite roles in T cell activation. The deficiency of upstream C2H2 domain increased the efficiency of T cell activation by 12.81%, while the downstream C2H2 domain alone promoted it with an average level 25.12% higher than intact RNF114 protein. Combined with tandem affinity purification (TAP) and mass spectrometry, our investigations found 23 RNF114-interacting proteins that have distinct physiological roles in transcription, translation, DNA repair and signaling pathways. These findings, including recognition of RNF114 as a positive regulatory protein and identification of its interacting proteins, widen the understanding for investigating functions of RNF114 involved in T cell activation.

Association of Epha2 Receptor with the Membrane through its Second Fibronectin Domain: A Biophysical and Computational Study

Eph receptors are cell surface type I transmembrane receptors involved in numerous cellular processes. Ligand-binding causes Eph clustering, a process that is, to a large extent, driven by the extracellular region of the receptor. The structural mechanisms that underly Eph clustering have recently been elucidated by crystal structure data for complete Eph ectodomains and their ligands. While it is understood that the clustering depends largely on the N-terminal domains of the extracellular region, little is known about the functions of the downstream domains that lead into the transmembrane helix. To address this issue, we have combined multiscale molecular dynamics (MD) simulations with biophysical assays to study the extracellular region of EphA2 upstream of the transmembrane helix, the fibronectin domain 2 (FN2). A number of novel insights emerged: (i) the FN2 domain can interact with the membrane, (ii) this domain preferentially interacts with anionic lipids, (iii) this preference is maintained through a positively charged surface of the protein, including K441 and R443. The study allows us, for the first time, to combine the recently emerged X-ray crystallography with NMR models of the transmembrane region. We present a full atomistic model of an Eph signalling cluster (extracellular regions and transmembrane regions) assembled at a model cell membrane composed of a lipid mixture. This work is a significant step in understanding the formation of Eph signalling clusters and paves the way towards understanding the assembly of full length Eph receptor platforms at the surface of cells.

Positional Effects of AAN Motifs in rpoS Regulation by sRNAs and Hfq

The Escherichia coli stationary phase transcription factor RpoS is translated in response to small noncoding RNAs (sRNAs), which base pair with the rpoS mRNA leader. The bacterial Sm-like protein Hfq anneals sRNAs with their mRNA targets by simultaneously binding the mRNA and sRNA. Intriguingly, Hfq is recruited to the rpoS leader via AAN motifs far upstream of the sRNA. SHAPE (selective 2′-hydroxyl acylation and primer extension) chemical footprinting showed that the rpoS leader is divided into a far upstream domain, an Hfq binding domain, and a downstream inhibitory stem–loop containing the sRNA and ribosome binding sites. To investigate how Hfq promotes sRNA–mRNA base pairing from a distance, we deleted the natural AAN Hfq binding site, and we inserted artificial AAN binding sites at various positions in the rpoS leader. All the relocated AAN motifs restored tight Hfq binding in vitro, but only insertion at the natural position restored Hfq-dependent sRNA annealing in vitro and sRNA regulation of rpoS translation in vivo. Furthermore, U-rich motifs in the downstream inhibitory domain stabilized the rpoS mRNA–Hfq complex and contributed to regulation of rpoS expression. We propose that the natural Hfq binding domain is optimal for positive regulation because it recruits Hfq to the mRNA and allows it to act on incoming sRNAs without opening the inhibitory stem–loop when sRNA is absent.

Structural and Functional Properties of Platelet-Derived Growth Factor and Stem Cell Factor Receptors

The receptors for platelet-derived growth factor (PDGF) and stem cell factor (SCF) are members of the type III class of PTK receptors, which are characterized by five Ig-like domains extracellularly and a split kinase domain intracellularly. The receptors are activated by ligand-induced dimerization, leading to autophosphorylation on specific tyrosine residues. Thereby the kinase activities of the receptors are activated and docking sites for downstream SH2 domain signal transduction molecules are created; activation of these pathways promotes cell growth, survival, and migration. These receptors mediate important signals during the embryonal development, and control tissue homeostasis in the adult. Their overactivity is seen in malignancies and other diseases involving excessive cell proliferation, such as atherosclerosis and fibrotic diseases. In cancer, mutations of PDGF and SCF receptors-including gene fusions, point mutations, and amplifications-drive subpopulations of certain malignancies, such as gastrointestinal stromal tumors, chronic myelomonocytic leukemia, hypereosinophilic syndrome, glioblastoma, acute myeloid leukemia, mastocytosis, and melanoma.

Effects of wind speed changes on wake instability of a wind turbine in a virtual wind tunnel using large eddy simulation

Large Eddy Simulation (LES) of the National Renewable Energy Laboratory (NREL) Phase VI wind turbine inside a virtual wind tunnel, with the same test section as that of NASA Ames 24.4m×36.6m, was carried out in order to analyze and better understand the wake instability and its breakdown behind the wind turbine. LES was performed using the commercial CFD software, ANSYS FLUENT, based on the dynamic Smagorinsky–Lilly model. The wind turbine was placed at a distance of two rotor diameters from the upstream boundary with a downstream domain of 20 rotor diameters in length. The results of the simulation were compared with the experimental data published by the NREL and a good agreement was found between the two. Furthermore, the average turbulence intensities from the LES were compared with a semi-empirical model and very good agreement was observed, except for the regions of on-going wake instability and vortex breakdown. It was observed that the wake behind the wind turbine consists of a system of intense and stable rotating helical vortices. These vortices persisted for some distance downstream of the wind turbine and finally become unstable producing a sinuous shape. The downstream distance at which wake instability and vortex breakdown occur, was observed to be a function of the upstream wind speed. For example, for an upstream wind speed of 7m/s, it was observed that the primary vortex structure became unstable at a downstream distance of four rotor diameters and complete breakdown occurred at approximately six rotor diameters. On the other hand, when the upstream wind speed was 15.1m/s, wake instability occurred at approximately 11 rotor diameters downstream of the wind turbine and complete breakdown was observed at 13 rotor diameters downstream of the wind turbine. Furthermore, it was observed that the turbulence intensity rapidly decreased during the process of wake instability and vortex breakdown; the location of the decrease is a function of the upstream wind speed. It is suggested that the distinction between the near and far wake can be identified as the average location between the start of the wake instability and the end of the process, at complete breakdown. Therefore the average location of this boundary is a function of the upstream wind speed. Hence for upstream wind speeds of 7m/s, 10m/s, 13.1m/s and 15.1m/s, the boundary between the near and far wake lies at five, seven, ten and twelve rotor diameters downstream respectively.

IsoPlotter(+): A Tool for Studying the Compositional Architecture of Genomes.

Eukaryotic genomes, particularly animal genomes, have a complex, nonuniform, and nonrandom internal compositional organization. The compositional organization of animal genomes can be described as a mosaic of discrete genomic regions, called “compositional domains,” each with a distinct GC content that significantly differs from those of its upstream and downstream neighboring domains. A typical animal genome consists of a mixture of compositionally homogeneous and nonhomogeneous domains of varying lengths and nucleotide compositions that are interspersed with one another. We have devised IsoPlotter, an unbiased segmentation algorithm for inferring the compositional organization of genomes. IsoPlotter has become an indispensable tool for describing genomic composition and has been used in the analysis of more than a dozen genomes. Applications include describing new genomes, correlating domain composition with gene composition and their density, studying the evolution of genomes, testing phylogenomic hypotheses, and detect regions of potential interbreeding between human and extinct hominines. To extend the use of IsoPlotter, we designed a completely automated pipeline, called IsoPlotter+ to carry out all segmentation analyses, including graphical display, and built a repository for compositional domain maps of all fully sequenced vertebrate and invertebrate genomes. The IsoPlotter+ pipeline and repository offer a comprehensive solution to the study of genome compositional architecture. Here, we demonstrate IsoPlotter+ by applying it to human and insect genomes. The computational tools and data repository are available online.

HAMP domain conformers that propagate opposite signals in bacterial chemoreceptors.

Author SummaryA central question in biological signal transduction is how cell-surface receptors transmit signals from the outside world across cell membranes and into the cells themselves. In bacteria and lower eukaryotes such receptors are composed of individual modules responsible for specific functions (e.g., sensing, relay, or output). HAMP domains act as the signal relay modules in many receptors, physically bridging input and output components and transferring signals between them. Through a combination of crystallographic, biophysical, spectroscopic, and functional studies we are able to associate two structurally defined HAMP conformational states with functional “on” and “off” signals in bacterial chemoreceptors, and thereby resolve the mechanism by which HAMPs can relay information. The two states differ in both their structure and dynamics and appear to enforce their properties on downstream output modules. Chemoreceptors allow bacteria to track chemical gradients with exquisite sensitivity and dynamic range; we further show that the response to chemoattractant depends critically on specific HAMP residues close to the membrane. Finally, based on the switching mechanism, we design and generate an inverse signaling HAMP domain that provides a new tool to engineer bacterial responses and may be especially advantageous in remediation efforts for directing bacteria towards chemicals that are normally repellants.

Architecture of the Soluble Receptor Aer2 Indicates an In-Line Mechanism for PAS and HAMP Domain Signaling

Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here, we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS (Per–Arnt–Sim) and poly-HAMP (histidine kinase–adenylyl cyclase–methyl-accepting chemotaxis protein–phosphatase) domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state, identifies conformational changes induced by ligand binding that are likely essential for the signaling mechanism. Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS but propagate to the Iβ strand in a manner predicted to alter PAS–PAS associations and the downstream HAMP junction within full-length Aer2. Small-angle X-ray scattering of PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initio small-angle X-ray scattering molecular envelopes, and pulsed dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the Escherichia coli Aer paradigm, where side-on PAS–HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains.

C-Terminal Membrane Association of Bestrophin 3 and Its Activation as a Chloride Channel

Bestrophin 3 (Best3), a member of the bestrophin Cl(-) channel family, is a candidate of cGMP-sensitive, Ca(2+)-activated Cl(-) channel in vascular smooth muscle cells. The Best3 channel was recently found to play an important role in vasomotion. However, the mechanism for its activation has not been clarified. In previous studies, we found that a Best3 C-terminal sequence (amino acids 353-404) was associated with the cellular membrane. The sequence includes an autoinhibitory domain ((356)IPSFLGS(362)) and a downstream basic residue domain (amino acids 384-397). In this study, we found that the sequence (368-383) between the two domains is actually a determinant for Best3 C-terminal membrane associability. Deletion of the sequence almost abolished the membrane association but did not activate the Best3 channel. Treatment of Best3-expressing HEK293 cells with the PI3Kα inhibitor IV (a Best3 activator) could not abolish but weakened the Best3 membrane association. The result supports the assumption that the positively charged basic residues in the Best3 C terminus are likely associated with the membranous negatively charged phospholipids, which plays a role in the regulation of Best3 activation. But the relationship between membrane associability and Best3 activation seems more complicated than expected.

B12 cofactors directly stabilize an mRNA regulatory switch

Structures of riboswitch receptor domains bound to their effector have revealed how mRNAs recognize diverse small molecules, but mechanistic details into its linkage with regulation of gene expression remain elusive1,2. To address this, we solved crystal structures of two different classes of cobalamin (vitamin B12) binding riboswitches that include the structural switch of the downstream regulatory domain. These classes share a common cobalamin-binding core, but use distinct peripheral extensions to recognize different B12 derivatives. In each case, recognition is accomplished through shape complementarity between the RNA and cobalamin with relatively few hydrogen bonding interactions that typically govern RNA-small molecule recognition. We show that a composite cobalamin/RNA scaffold stabilizes an unusual long-range intramolecular kissing-loop interaction that controls mRNA expression. This is the first riboswitch crystal structure detailing how the receptor and regulatory domains communicate in a ligand-dependent fashion to regulate mRNA expression.