Mutations In Linker Research Articles

Temperature-Sensitive Mutants in the Influenza A Virus RNA Polymerase: Alterations in the PA Linker Reduce Nuclear Targeting of the PB1-PA Dimer and Result in Viral Attenuation.

The influenza virus RNA-dependent RNA polymerase catalyzes genome replication and transcription within the cell nucleus. Efficient nuclear import and assembly of the polymerase subunits PB1, PB2, and PA are critical steps in the virus life cycle. We investigated the structure and function of the PA linker (residues 197 to 256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichroism experiments revealed that the PA linker by itself is structurally disordered. A large series of PA linker mutants exhibited a temperature-sensitive (ts) phenotype (reduced viral growth at 39.5°C versus 37°C/33°C), suggesting an alteration of folding kinetic parameters. The ts phenotype was associated with a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that ts and lethal PA mutants did not efficiently recruit PB1 to reach the nucleus at 39.5°C. A protein complementation assay using PA mutants, PB1, and β-importin IPO5 tagged with fragments of the Gaussia princeps luciferase showed that increasing the temperature negatively modulated the PA-PB1 and the PA-PB1-IPO5 interactions or complex stability. The selection of revertant viruses allowed the identification of different types of compensatory mutations located in one or the other of the three polymerase subunits. Two ts mutants were shown to be attenuated and able to induce antibodies in mice. Taken together, our results identify a PA domain critical for PB1-PA nuclear import and that is a "hot spot" to engineer ts mutants that could be used to design novel attenuated vaccines. By targeting a discrete domain of the PA polymerase subunit of influenza virus, we were able to identify a series of 9 amino acid positions that are appropriate to engineer temperature-sensitive (ts) mutants. This is the first time that a large number of ts mutations were engineered in such a short domain, demonstrating that rational design of ts mutants can be achieved. We were able to associate this phenotype with a defect of transport of the PA-PB1 complex into the nucleus. Reversion substitutions restored the ability of the complex to move to the nucleus. Two of these ts mutants were shown to be attenuated and able to produce antibodies in mice. These results are of high interest for the design of novel attenuated vaccines and to develop new antiviral drugs.

Membrane anchoring of Epstein-Barr virus gp42 inhibits fusion with B cells even with increased flexibility allowed by engineered spacers.

ABSTRACTWe recently described the architecture of the Epstein-Barr virus (EBV) fusion-triggering complex consisting of the EBV B cell receptor human leukocyte antigen (HLA) class II and the EBV-encoded proteins gp42 and gH/gL. The architecture of this structure positioned the main body of gp42, comprising the C-type lectin domain (CTLD), away from the membrane and distant from where the membrane-bound form of gp42 might be tethered. gp42 is a type II membrane glycoprotein, with functional gp42 formed by cleavage near the gp42 amino-terminal transmembrane domain. This cleavage results in an approximately 50-amino-acid unstructured region that is responsible for binding gH/gL with nanomolar affinity. Our previous studies had shown that membrane-bound gp42 is not functional in B cell fusion. To investigate whether we could restore gp42 function by extending it from the membrane, we introduced one, two, and four structured immunoglobulin-like domains from muscle protein titin into a membrane-bound form of gp42 and tested function in binding to gHgL and HLA class II and function in fusion. We hypothesized that cleavage of gp42 generates a soluble functional form that relieves steric hindrance imposed on gHgL by membrane-bound gp42. All of the linker mutants had a dominant-negative effect on gp42 function, indicating that gp42 fusion function could not be restored simply by the addition of one to four titin domains.

Mad linker phosphorylations control the intensity and range of the BMP-activity gradient in developing Drosophila tissues.

The BMP ligand Dpp, operates as a long range morphogen to control many important functions during Drosophila development from tissue patterning to growth. The BMP signal is transduced intracellularly via C-terminal phosphorylation of the BMP transcription factor Mad, which forms an activity gradient in developing embryonic tissues. Here we show that Cyclin dependent kinase 8 and Shaggy phosphorylate three Mad linker serines. We demonstrate that linker phosphorylations control the peak intensity and range of the BMP signal across rapidly developing embryonic tissues. Shaggy knockdown broadened the range of the BMP-activity gradient and increased high threshold target gene expression in the early embryo, while expression of a Mad linker mutant in the wing disc resulted in enhanced levels of C-terminally phosphorylated Mad, a 30% increase in wing tissue, and elevated BMP target genes. In conclusion, our results describe how Mad linker phosphorylations work to control the peak intensity and range of the BMP signal in rapidly developing Drosophila tissues.

Ligation-based assembly for constructing mouse synthetic scFv libraries by chain shuffling with in vivo-amplified VH and VL fragments

In vitro assembly of two or three PCR fragments using primers is a common method of constructing scFv fragments for display on the surface of phage. However, mismatch annealing often occurs during in this step, leading to cloning and display of incomplete Fab or scFv fragments. To overcome this limitation, we developed a ligation-based two-fragment assembly (LTFA) protocol that involved separately cloning VH and Vκ fragments into the high-copy-number plasmid pUC18. The VH and Vκ fragments had randomized complementarity-determining region 3 (CDR3) and were joined with a peptidyl linker composed of (G4S)3. Using this approach, complete sequences of scFv fragments were successfully constructed, and the sequencing of 83 scFv clones revealed that none of the sequences, including the linker region, contained deletions or mutations. In contrast, linker sequences generated using a conventional two-fragment PCR assembly (TFPA) protocol often contained sequence anomalies, including large truncations. Using the LTFA protocol, a final library size of 1.0×108cfu was achieved. Examination of the amino acid profiles of the generated scFv fragments within the randomized regions introduced using degenerate codons did not detect any bias from that expected based on stochastic distribution. After several cycles of panning with this library, antigen-specific scFvs against two reference antigens, hen egg lysozyme and streptavidin were detected. In addition, scFvs with specificity against peptidyl antigens in the loop region of the Medaka ortholog of human C6orf89, which encodes a histone deacetylase enhancer that interacts with the bombesin receptor, were also obtained. The LTFA protocol developed here is robust and allows for the easy construction of integral scFv fragments compared with conventional TFPA. Utilizing LTFA, other CDRs can be readily combined. This approach also allows for the in vitro maturation of scFv fragments by separately introducing randomization in CDRs or using error-prone PCR for the amplification of pre-selected sequences as a template scaffold.

Investigation of the juxtamembrane region of neuronal-Synaptobrevin in synaptic transmission at the Drosophila neuromuscular junction.

In this study, the juxtamembrane region of the Drosophila SNARE protein neuronal-Synaptobrevin (n-Syb) was tested for its role in synaptic transmission. A transgenic approach was used to express n-Syb mutant genes. The transgenes carried engineered point mutations that alter the amino acid sequence of the conserved tryptophan residues in the juxtamembrane sequence. Such transgenes were expressed in an n-syb hypomorphic background, which produces little endogenous protein. On their own, hypomorphic flies displayed severe motor inhibition, limited life span, reduced evoked junctional potentials (EJPs), decreased synchronicity in EJP time to peak, and potentiation of EJPs with 10-Hz stimulation. All of these deficits were restored to wild-type levels with the expression of wild-type transgenic n-syb, regulated by the endogenous promoter (n-syb(WT)). We created transgenic mutants with one additional tryptophan (n-syb(WW)) or one less tryptophan (n-syb(AA)) than the wild-type sequence. While n-syb(WW) resembled n-syb(WT) in all variables listed, n-syb(AA) exhibited decreased EJP amplitude, synchronicity, and quantal content. To determine whether the n-syb juxtamembrane region is important for transduction of force arising from SNARE complex assembly during membrane fusion, we introduced short 6-amino acid (n-syb(L6)) or long 24-amino acid (n-syb(L24)) flexible linkers into the n-syb transgene. We observed a reduced EJP amplitude in n-syb(L6) but not n-syb(L24), while both linker mutants showed a decreased quantal content and an effect on the readily releasable and recycling vesicle pools. In conclusion, mutation of the juxtamembrane region of n-syb deleteriously affected synaptic transmission at the Drosophila neuromuscular junction.

The role of intracellular linkers in gating and desensitization of human pentameric ligand-gated ion channels.

It has recently been proposed that post-translational modification of not only the M3-M4 linker but also the M1-M2 linker of pentameric ligand-gated ion channels modulates function in vivo. To estimate the involvement of the M1-M2 linker in gating and desensitization, we engineered a series of mutations to this linker of the human adult-muscle acetylcholine receptor (AChR), the α3β4 AChR and the homomeric α1 glycine receptor (GlyR). All tested M1-M2 linker mutations had little effect on the kinetics of deactivation or desensitization compared with the effects of mutations to the M2 α-helix or the extracellular M2-M3 linker. However, when the effects of mutations were assessed with 50 Hz trains of ∼1 ms pulses of saturating neurotransmitter, some mutations led to much more, and others to much less, peak-current depression than observed for the wild-type channels, suggesting that these mutations could affect the fidelity of fast synaptic transmission. Nevertheless, no mutation to this linker could mimic the irreversible loss of responsiveness reported to result from the oxidation of the M1-M2 linker cysteines of the α3 AChR subunit. We also replaced the M3-M4 linker of the α1 GlyR with much shorter peptides and found that none of these extensive changes affects channel deactivation strongly or reduces the marked variability in desensitization kinetics that characterizes the wild-type channel. However, we found that these large mutations to the M3-M4 linker can have pronounced effects on desensitization kinetics, supporting the notion that its post-translational modification could indeed modulate α1 GlyR behavior.

Linker mutations reveal the complexity of synaptotagmin 1 action during synaptic transmission

The Ca2+ sensor for rapid synaptic vesicle exocytosis, synaptotagmin I (syt), is largely composed of two Ca2+-sensing C2-domains, C2A and C2B. We have investigated the apparent synergy between the tandem C2 domains by altering the length and rigidity of the linker that connects them. The behavior of the linker mutants revealed a correlation between the ability of the C2-domains to penetrate membranes in response to Ca2+ and to drive evoked neurotransmitter release in cultured mouse neurons, uncovering a step in excitation-secretion coupling. Atomic force microscopy experiments indicate that the synergy between these C2-domains involves intra-molecular interactions between them. Thus, syt function is profoundly affected by changes in the physical nature of the linker that connects its tandem C2-domains. Moreover, the linker mutations uncoupled syt-mediated regulation of evoked and spontaneous release, revealing that syt also acts as a fusion clamp prior to the Ca2+ trigger.

Regional flexibility in the S4–S5 linker regulates hERG channel closed-state stabilization

hERG K(+) channel function is vital for normal cardiac rhythm, yet the mechanisms underlying the unique biophysical characteristics of the channel, such as slow activation and deactivation gating, are incompletely understood. The S4-S5 linker is thought to transduce voltage sensor movement to opening of the pore gate, but may also integrate signals from cytoplasmic domains. Previously, we showed that substitutions of G546 within the S4-S5 linker destabilize the closed state of the channel. Here, we present results of a glycine-scan in the background of 546L. We demonstrate site-specific restoration of WT-like activation which suggests that flexibility in the N-terminal portion of the S4-S5 linker is critical for the voltage dependence of hERG channel activation. In addition, we show that the voltage dependence of deactivation, which was recently shown to be left-shifted from that of activation due to voltage sensor mode-shift, is also modulated by the S4-S5 linker. The G546L mutation greatly attenuated the coupling of voltage sensor mode-shift to the pore gate without altering the mode-shift itself. Indeed, all of the S4-S5 linker mutations tested similarly reduced coupling of the mode-shift to the pore gate. These data demonstrate a key role for S4-S5 linker in the unique activation and deactivation gating of hERG channels. Furthermore, uncoupling of the mode-shift to the pore by S4-S5 linker mutations parallels the effects of mutations in the N-terminus suggestive of functional interactions between the two regions.

Probing the relation between protein–protein interactions and DNA binding for a linker mutant of the bacterial nucleoid protein H-NS

We have investigated the relationship between oligomerization in solution and DNA binding for the bacterial nucleoid protein H-NS. This was done by comparing oligomerization and DNA binding of H-NS with that of a H-NS D68V-D71V linker mutant. The double linker mutation D68V-D71V, that makes the linker significantly more hydrophobic, leads to a dramatically enhanced and strongly temperature-dependent H-NS oligomerization in solution, as detected by dynamic light scattering. The DNA binding affinity of H-NS D68V-D71V for the hns promoter region is lower and has stronger temperature dependence than that of H-NS. DNase I footprinting experiments show that at high concentrations, regions protected by H-NS D68V-D71V are larger and less defined than for H-NS. In vitro transcription assays show that the enhanced protection also leads to enhanced transcriptional repression. Whereas the lower affinity of the H-NS D68V-D71V for DNA could be caused by competition between oligomerization in solution and oligomerization on DNA, the larger size of protected regions clearly confirms the notion that cooperative binding of H-NS to DNA is related to protein–protein interactions. These results emphasize the relative contributions of protein–protein interactions and substrate-dependent oligomerization in the control of gene repression operated by H-NS.

Direct interaction of eag domains and cyclic nucleotide–binding homology domains regulate deactivation gating in hERG channels

Human ether-á-go-go (eag)-related gene (hERG) potassium channels play a critical role in cardiac repolarization and are characterized by unusually slow closing (deactivation) kinetics. The N-terminal “eag” domain and a C-terminal C-linker/cyclic nucleotide–binding homology domain (CNBHD) are required for regulation of slow deactivation. The region between the S4 and S5 transmembrane domains (S4–S5 linker) is also implicated in this process, but the mechanism for regulation of slow deactivation is unclear. Here, using an eag domain–deleted channel (hERG Δeag) fused to Citrine fluorescent protein, we found that most channels bearing individual alanine mutations in the S4–S5 linker were directly regulated by recombinant eag domains fused to a cyan fluorescent protein (N-eag-CFP) and had robust Förster resonance energy transfer (FRET). Additionally, a channel bearing a group of eight alanine residues in the S4–S5 linker was not measurably regulated by N-eag-CFP domains, but robust FRET was measured. These findings demonstrate that the eag domain associated with all of the S4–S5 linker mutant channels. In contrast, channels that also lacked the CNBHD (hERG Δeag ΔCNBHD-Citrine) were not measurably regulated by N-eag-CFP nor was FRET detected, suggesting that the C-linker/CNBHD was required for eag domains to directly associate with the channel. In a FRET hybridization assay, N-eag-CFP had robust FRET with a C-linker/CNBHD-Citrine, suggesting a direct and specific interaction between the eag domain and the C-linker/CNBHD. Lastly, coexpression of a hERG subunit lacking the CNBHD and the distal C-terminal region (hERG ΔpCT-Citrine) with hERG Δeag-CFP subunits had FRET and partial restoration of slow deactivation. Collectively, these findings reveal that the C-linker/CNBHD, but not the S4–S5 linker, was necessary for the eag domain to associate with the channel, that the eag domain and the C-linker/CNBHD were sufficient for a direct interaction, and that an intersubunit interaction between the eag domain and the C-linker/CNBHD regulated slow deactivation in hERG channels at the plasma membrane.

Global Shapes of F-actin Depolymerization-competent Minimal Gelsolins: INSIGHT INTO THE ROLE OF g2-g3 LINKER IN pH/Ca2+ INSENSITIVITY OF THE FIRST HALF

Because of its ability to rapidly depolymerize F-actin, plasma gelsolin has emerged as a therapeutic molecule in different disease conditions. High amounts of exogenous gelsolin are, however, required to treat animal models of different diseases. Knowing that the F-actin depolymerizing property of gelsolin resides in its N terminus, we made several truncated versions of plasma gelsolin. The smaller versions, particularly the one composed of the first 28-161 residues, depolymerized the F-actin much faster than the native gelsolin and other truncates at the same molar ratios. Although G1-G3 loses its dependence on Ca(2+) or low pH for the actin depolymerization function, interestingly, G1-G2 and its smaller versions were found to regain this requirement. Small angle x-ray scattering-based shape reconstructions revealed that G1-G3 adopts an open shape in both the presence and the absence of Ca(2+) as well as low pH, whereas G1-G2 and residues 28-161 prefer collapsed states in Ca(2+)-free conditions at pH 8. The mutations in the g2-g3 linker resulted in the calcium sensitivity of the mutant G1-G3 for F-actin depolymerization activity, although the F-actin-binding sites remained exposed in the mutant G1-G3 as well as in the smaller truncates even in the Ca(2+)-free conditions at pH 8. Furthermore, unlike wild type G1-G3, calcium-sensitive mutants of G1-G3 acquired closed shapes in the absence of free calcium, implying a role of g2-g3 linker in determining the open F-actin depolymerizing-competent shape of G1-G3 in this condition. We demonstrate that the mobility of the G1 domain, essential for F-actin depolymerization, is indirectly regulated by the gelsolin-like sequence of g2-g3 linker.

CBL Linker Region and RING Finger Mutations Lead to Enhanced Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) Signaling via Elevated Levels of JAK2 and LYN

Juvenile myelomonocytic leukemia (JMML) is characterized by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). SHP2, NF-1, KRAS, and NRAS are mutated in JMML patients, leading to aberrant regulation of RAS signaling. A subset of JMML patients harbor CBL mutations associated with 11q acquired uniparental disomy. Many of these mutations are in the linker region and the RING finger of CBL, leading to a loss of E3 ligase activity. We investigated the mechanism by which CBL-Y371H, a linker region mutant, and CBL-C384R, a RING finger mutant, lead to enhanced GM-CSF signaling. Expression of CBL mutants in the TF-1 cell line resulted in enhanced survival in the absence of GM-CSF. Cells expressing CBL mutations displayed increased phosphorylation of GM-CSF receptor βc subunit in response to stimulation, although expression of total GM-CSFR βc was lower. This suggested enhanced kinase activity downstream of GM-CSFR. JAK2 and LYN kinase expression is elevated in CBL-Y371H and CBL-C384R mutant cells, resulting in enhanced phosphorylation of CBL and S6 in response to GM-CSF stimulation. Incubation with the JAK2 inhibitor, TG101348, abolished the increased phosphorylation of GM-CSFR βc in cells expressing CBL mutants, whereas treatment with the SRC kinase inhibitor dasatinib resulted in equalization of GM-CSFR βc phosphorylation signal between wild type CBL and CBL mutant samples. Dasatinib treatment inhibited the elevated phosphorylation of CBL-Y371H and CBL-C384R mutants. Our study indicates that CBL linker and RING finger mutants lead to enhanced GM-CSF signaling due to elevated kinase expression, which can be blocked using small molecule inhibitors targeting specific downstream pathways.

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors*

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.

Determining the role of the S4-S5 Linker in hERG eag Domain Regulation of Channel Gating

Human ether-a-go-go-related gene (hERG) potassium channels exhibit unique kinetic properties, including unusually slow deactivation kinetics, which help to specialize them for their role in the heart. Previously, we demonstrated that an N-terminal eag domain is essential in regulating channel deactivation kinetics. The mechanism by which the eag domain regulates gating remains unclear. Recent evidence suggests the intracellular loop between the S4 and S5 transmembrane domains (S4-S5 linker) may be important in regulating both activation and deactivation, and that modulation of gating by the eag domain may act via the S4-S5 linker. Here we sought to investigate the role of the S4-S5 linker using site-directed mutagenesis and a combination of electrophysiology and Forster Resonance Energy Transfer (FRET). We found that channels with alanine mutations in the S4-S5 linker exhibited altered gating. All the S4-S5 mutant channels caused an acceleration of deactivation kinetics, except for S543A which had significantly slowed deactivation. Co-expressing an eag domain gene fragment (N-eag) with S4-S5 mutant channels which additionally lacked a native eag domain (Δeag) failed to restore slow deactivation kinetics to the mutant channels. FRET analysis revealed that eag domains tagged with a CFP were in close proximity to each of the S4-S5 mutant channels tagged with a Citrine. Replacement of the entire S4-S5 linker with alanines (hERG[S4-S5]Ala) produced channels with altered gating, including fast deactivation and a far left-shifted steady-state activation curve. Co-expression of hERG Δeag[S4-S5]Ala channels with N-eag did not alter channel gating; however, FRET analysis revealed that N-eag was in close proximity to the mutant channels. Together, these findings suggest that an intact S4-S5 linker is necessary to transduce eag domain-dependent regulation of gating, but it is not required for the eag domain to bind to the channel.

Oxa1-Ribosome Complexes Coordinate the Assembly of Cytochrome c Oxidase in Mitochondria

The terminal enzyme of the respiratory chain, cytochrome c oxidase, consists of a hydrophobic reaction center formed by three mitochondrially encoded subunits with which 9-10 nuclear encoded subunits are associated. The three core subunits are synthesized on mitochondrial ribosomes and inserted into the inner membrane in a co-translational reaction facilitated by the Oxa1 insertase. Oxa1 consists of an N-terminal insertase domain and a C-terminal ribosome-binding region. Mutants lacking the C-terminal region show specific defects in co-translational insertion, suggesting that the close contact of the ribosome with the insertase promotes co-translational insertion of nascent chains. In this study, we inserted flexible linkers of 100 or 200 amino acid residues between the insertase domain and ribosome-binding region of Oxa1 of Saccharomyces cerevisiae. In the absence of the ribosome receptor Mba1, these linkers caused a length-dependent decrease in mitochondrial respiratory activity caused by diminished levels of cytochrome c oxidase. Interestingly, considerable amounts of mitochondrial translation products were still integrated into the inner membrane in these linker mutants. However, they showed severe defects in later stages of the biogenesis process, presumably during assembly into functional complexes. Our observations suggest that the close proximity of Oxa1 to ribosomes is not only used to improve membrane insertion but is also critical for the productive assembly of the subunits of the cytochrome c oxidase. This points to a role for Oxa1 in the spatial coordination of the ribosome with assembly factors that are critical for enzyme biogenesis.

Modulation of beta-amyloid aggregation by engineering the sequence connecting beta-strand forming domains

Aggregation of beta-amyloid (Aβ) into oligomers and fibrils is associated with the pathology of Alzheimer's disease. The major structural characteristics of Aβ fibrils include the presence of β sheet-loop-β sheet conformations. Several lines of study suggested a potentially important role of the Aβ loop forming sequence (referred to as the Aβ linker region) in Aβ aggregation. Effects of mutations in several charged residues within the Aβ linker region on aggregation have been extensively studied. However, little is known about oligomerization effects of sequence variation in other residues within the Aβ linker region. Moreover, modulation effects of the Aβ linker mutants on Aβ aggregation have yet to be characterized. Here, we created and characterized Aβ linker variants containing sequences preferentially found in specific β turn conformations. Our results indicate that a propensity to form oligomers may be changed by local sequence variation in the Aβ linker region without mutating the charged residues. Strikingly, one Aβ linker variant rapidly formed protofibrillar oligomers, which did not convert to fibrillar aggregates in contrast to Aβ aggregating to fibrils under similar incubation conditions. Moreover, our results suggest that molecular forces critical in oligomerization and fibrillization may differ at least for those involved in the linker region. When co-incubated with Aβ, some Aβ linker variants were found to induce accumulation of Aβ oligomers. Our results suggest that engineering of the Aβ linker region as described in this paper may represent a novel approach to control Aβ oligomerization and create Aβ oligomerization modulators.

Mutagenesis of the DI/DIII Linker in Dengue Virus Envelope Protein Impairs Viral Particle Assembly

The dengue virus (DV) envelope (E) protein is important in mediating viral entry and assembly of progeny virus during cellular infection. Domains I and III (DI and DIII, respectively) of the DV E protein are connected by a highly conserved but poorly ordered region, the DI/DIII linker. Although the flexibility of the DI/DIII linker is thought to be important for accommodating the structural rearrangements undergone by the E protein during viral entry, the function of the linker in the DV infectious cycle is not well understood. In this study, we performed site-directed mutagenesis on conserved residues in the DI/DIII linker of the DV2 E protein and showed that the resulting mutations had little or no effect on the entry process but greatly affected virus assembly. Biochemical fractionation and immunofluorescence microscopy experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the DI/DIII linker mutants express the DV structural proteins at the sites of particle assembly near the ER but fail to form infectious particles. This defect is not due to disruption of E's interaction with prM and pr in immature and mature virions, respectively. Serial passaging of the DV2 mutant E-Y299F led to the identification of a mutation in the membrane-proximal stem region of E that fully compensates for the assembly defect of this DI/DIII linker mutant. Together, our results suggest a critical and previously unidentified role for the E protein DI/DIII linker region during the DV2 assembly process.

The Interaction of Voltage-Sensor and Gate in BK Channels

To study the molecular basis and mechanism of voltage sensor/gate interaction in BK channels we performed an alanine-scan of the S4-S5 linker and the C-terminal end of S6 in mSlo1. Potassium conductance was measured over a wide range of voltage and calcium and fit to the Horrigan-Aldrich (HA) gating model. Open probability was measured at extreme negative voltages to determine if mutations altered the stability of the gate when voltage sensors are not activated (L in the HA model). Mutations in both linker and S6 were identified that alter L and therefore could be involved in voltage-sensor/gate interaction. The largest inhibitory effects were observed at the ends of S4-S5 linker (Q222A, I233A and K234A) and P319A in S6, each of which decreased L by 10- to 100-fold and shifted V0.5 to more positive voltages. I233A, unlike Q222A or K234A, also produced a decrease in voltage-dependence that is consistent with a decrease in voltage-sensor/gate coupling (D in the HA model). A structural model, based on Kv1.2, suggests inter- or intra-subunit interactions could potentially exist between these sites. Similarly, mutations near the end of S6 (R329A, K330A) that produce an approximate 3 fold increase in L without changing V0.5, may decrease voltage-sensor/gate coupling and could potentially interact with the N-terminal end of the linker where mutations F223A, L227A also produced a moderate increase in L. On the other hand, the largest increases in L were observed with mutations in S6 (P320A, E321A, E324A) that have no obvious interaction partner in S4-S5 and potentially alter the stability of the gate directly. In summary, we have identified several candidates that may be involved in voltage-sensor/gate interaction and will provide a basis for future experiments involving double mutant cycle analysis.

Determining the Role of the hERG Intracellular Loop Regions in Regulating Channel Gating

Human ether-a-go-go-related gene (hERG) potassium channels exhibit unique kinetic properties, including unusually slow deactivation kinetics, which help to specialize them for their role in the heart. Previously, we demonstrated that an N-terminal Per-Arnt-Sim (PAS) domain is essential in regulating channel deactivation kinetics, and that this regulation occurs through a direct interaction with a cyclic nucleotide binding domain (CNBD) in the C-terminal tail. Recent evidence suggests the intracellular loop between the S4 and S5 transmembrane domains (S4-S5 linker) may also be of importance in regulating both activation and deactivation gating and that modulation of gating by the N-terminal PAS domain may act via the S4-S5 linker; however, the mechanism for this remains unclear. Here, we sought to investigate the role of the S4-S5 linker, together with the PAS domain, in regulating channel gating by performing site-directed mutagenesis within the S4-S5 linker region. We found that mutations in the S4-S5 linker produced channels with altered gating properties, including accelerated deactivation kinetics and shifts in the steady-state activation curves, supporting the notion that this region may play an important role in gating. For example, hERG E544R mutant channels exhibited faster deactivation kinetics and a rightward shift in the steady-state activation curve to more positive potentials. On the other hand, hERG Y545A mutant channels also exhibited accelerated deactivation kinetics, but instead showed a leftward shift in the steady-state activation curve to more negative potentials. Together, these experiments work towards elucidating the role of the S4-S5 linker region in regulating hERG channel gating.

Modification of Kv4.2 Channel Complexes by the Diphenylurea Compound NS5806

NS5806 (N-[3,5-bis(trifluoromethyl)phenyl]-N-[2,4-dibromo-6-(1H-tetrazol-5-yl)phenyl]-urea) can increase the amplitude and slow the decay of currents mediated by voltage-gated potassium (Kv) channels of the Kv4 subfamily if Kv Channel Interacting Proteins (KChIPs) participate in channel complex formation. We studied the effects of NS5806 on the somatodendritic A-type current (Isa) in hippocampal neurons and on the currents mediated by Kv4.2 channels coexpressed with different auxilliary beta-subunits in HEK 293 cells with the whole-cell patch-clamp technique. The effects of NS5806 on the inactivation gating of Kv4.2 mutants were studied under two-electrode voltage-clamp in Xenopus oocytes. Notably, the Isa component in hippocampal neurons was reduced rather than potentiated, but showed a pronounced slowing of inactivation in the presence of NS5806. The peak amplitudes of currents mediated by ternary Kv4.2 channels, coexpressed with different KChIPs and dipeptidyl aminopeptidase-related protein (DPP) 6 were potentiated and their macroscopic inactivation slowed by NS5806. The currents mediated by binary Kv4.2 channels, coexpressed only with DPP6, were suppressed by NS5806 and the effects on macroscopic inactivation were less pronounced. Similar to the Isa component in hippocampal neurons, the midpoint voltage of inactivation of recombinant Kv4.2 channel complexes was shifted negative by NS5806. However, the recovery from inactivation was accelerated in hippocampal neurons and slowed in recombinant channels. Functional analysis of Kv4.2 S4S5 linker and S6 mutants support the notion that NS5806 influences the dynamic coupling between voltage sensor and gate. Supported by the Deutsche Forschungsgemeinschaft (DFG)