The N-linker region of hERG1a upregulates hERG1b potassium channels

A major physiological role of hERG1 (human Ether-a-go-go-Related Gene) potassium channels is to repolarize cardiac action potentials. Two isoforms, hERG1a and hERG1b, associate to form the native cardiac IKr current in vivo. Inherited mutations in hERG1a or hERG1b cause prolonged cardiac repolarization, Long QT Syndrome and sudden death arrhythmia. hERG1a subunits assemble with and enhance the number of hERG1b subunits at the plasma membrane, but the mechanism for the increase in hERG1b by hERG1a is not well understood. Here, we report that the hERG1a N-terminal PAS (Per-Arnt-Sim) domain-N-linker region expressed in trans with hERG1b markedly increased hERG1b currents and increased biotin-labelled hERG1b protein at the membrane surface. hERG1b channels with a deletion of the 1b domain did not have a measurable increase in current or biotinylated protein when co-expressed with hERG1a PAS domain-N-linker regions indicating that the 1b domain was required for the increase in hERG1b. Using a biochemical pull-down interaction assay and a FRET hybridization experiment, we detected a direct interaction between the hERG1a PAS domain-N-linker region and the hERG1b N-terminal 1b domain. Using engineered deletions and alanine mutagenesis, we identified a short span of amino acids at positions 216-220 within the hERG1a N-linker region that were necessary for the upregulation of hERG1b. Taken together, we propose that direct structural interactions between the hERG1a N-linker region and the hERG1b N-terminal 1b domain increase hERG1b at the plasma membrane. Mechanisms that enhance hERG1b current would be anticipated to shorten action potentials, which could be anti-arrhythmic, and may point toward hERG1b or the hERG1a N-linker as molecular targets for therapy for Long QT syndrome.

Exploring the Two Coupled Conformational Changes That Activate the Munc18-1/Syntaxin-1 Complex

Calcium-dependent synaptic vesicle exocytosis is mediated by SNARE complex formation. The transition from the Munc18-1/syntaxin-1 complex to the SNARE complex is catalyzed by the Munc13-1 MUN domain and involves at least two conformational changes: opening of the syntaxin-1 linker region and extension of Munc18-1 domain 3a. However, the relationship and the action order of the two conformational changes remain not fully understood. Here, our data show that an open conformation in the syntaxin-1 linker region can bypass the requirement of the MUN NF sequence. In addition, an extended state of Munc18-1 domain 3a can compensate the role of the syntaxin-1 RI sequence. Altogether, the current data strongly support our previous notion that opening of the syntaxin-1 linker region by Munc13-1 is a key step to initiate SNARE complex assembly, and consequently, Munc18-1 domain 3a can extend its conformation to serve as a template for association of synaptobrevin-2 and syntaxin-1.

A shape-shifting nuclease unravels structured RNA

RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts that may otherwise interfere with cellular programs. The enzyme Dis3-like protein 2 (Dis3L2) is a 3′-5′ exoribonuclease that, through its RNA turnover activity, plays a critical role in human development1. Dis3L2 can independently degrade structured substrates and its targets include many coding and non-coding 3′-uridylated RNAs1-5. While the basis for Dis3L2 substrate recognition has been well-characterized6, the mechanism of structured RNA degradation by this family of enzymes is unknown. We characterized the discrete steps of the degradation cycle by determining electron cryo-microscopy structures representing snapshots along the RNA turnover pathway and measuring kinetic parameters for single-stranded (ss) and double-stranded (ds) RNA processing. We discovered a dramatic conformational change that is triggered by the dsRNA, involving repositioning of two cold shock domains by 70 Å. This movement exposes a trihelix-linker region, which acts as a wedge to separate the two RNA strands. Furthermore, we show that the trihelix linker is critical for dsRNA, but not ssRNA, degradation. These findings reveal the conformational plasticity of this enzyme, and detail a novel mechanism of structured RNA degradation.

Conserved L464 in p97 D1-D2 linker is critical for p97 cofactor regulated ATPase activity

p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1-D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1-D2 linker and the D1 domain, we produced p97 ND1L mutants and demonstrated that this 22-residue linker region is essential for D1 ATPase activity. The conserved amino acid leucine 464 (L464) is critical for regulating D1 and D2 ATPase activity by p97 cofactors p37, p47, and Npl4-Ufd1 (NU). Changing leucine to alanine, proline, or glutamate increased the maximum rate of ATP turnover (kcat) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the kcat of the proline substituted linker, suggesting that they induced linker conformations facilitating ATP hydrolysis. NU inhibited D1 ATPase activities of WT and mutant ND1L proteins, but activated D2 ATPase activity of full-length p97. To further understand the mutant mechanism, we used single-particle cryo-EM to visualize the full-length p97L464P and revealed the conformational change of the D1-D2 linker, resulting in a movement of the helix-turn-helix motif (543-569). Taken together with the biochemical and structural results we conclude that the linker helps maintain D1 in a competent conformation and relays the communication to/from the N-domain to the D1 and D2 ATPase domains, which are ~50 Å away.

Osteoarthritis-Related Inflammation Blocks TGF-β’s Protective Effect on Chondrocyte Hypertrophy via (De)Phosphorylation of the SMAD2/3 Linker Region

Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-β (TGF-β) signaling via SMAD2/3 is crucial for blocking hypertrophy. The post-translational modifications of these SMAD proteins in the linker domain regulate their function and these can be triggered by inflammation through the activation of kinases or phosphatases. Therefore, we investigated if OA-related inflammation affects TGF-β signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1β and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-β signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1β was less if the SMAD3 linker could not be phosphorylated at Ser204. Our study shows evidence that inflammation inhibits SMAD2/3 signaling in chondrocytes via SMAD linker (de)-phosphorylation. The involvement of linker region modifications may represent a new therapeutic target for OA.

Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

Hsp90β is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90β for proper folding and/or activity. Regulation of Hsp90β is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90β, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90β is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses (e.g., heat shock, inhibition with drugs) that impact Hsp90β activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90β globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90β and its secretion, through changes in the conformation of the chaperone.

Conversion of the Sensor Kinase DcuS to the Fumarate Sensitive State by Interaction of the Bifunctional Transporter DctA at the TM2/PASC-Linker Region

The membrane-bound C4-dicarboxylate (C4DC) sensor kinase DcuS of Escherichia coli typically forms a protein complex with the C4DC transporter DctA. The DctA × DcuS complex is able to respond to C4DCs, whereas DcuS without DctA is in the permanent ON state. In DctA, the C-terminal helix 8b (H8b) serves as the site for interaction with DcuS. Here the interaction site in DcuS and the related structural and functional adaptation in DcuS were determined. The Linker connecting transmembrane helix 2 (TM2) and the cytosolic PASC (Per-ARNT-SIM) domain of DcuS, was identified as the major site for interaction with DctA-H8b by in vivo interaction studies. The Linker is known to convert the piston-type transmembrane signaling of TM2 to a tilting motion which relies on a resolution of the Linker-Linker’ homodimer in the presence of C4DCs. Absence of DctA caused decreased cross-linking in the Linker, as identified by oxidative Cys-cross-linking. This response resembled structurally and functionally that of fumarate activation in the DctA × DcuS complex. Overall, formation of the DctA × DcuS complex is based on the interaction of the DcuS Linker with DctA H8b; the interaction is required to set DcuS in the C4DC-responsive state by stabilizing the linker-linker’ homodimer in DcuS. This work identifies DctA as a structural co-regulator of DcuS sensor kinase.

An alternative domain-swapped structure of the Pyrococcus horikoshii PolII mini-intein

Protein splicing is a post-translational process by which an intein catalyzes its own excision from flanking polypeptides, or exteins, concomitant with extein ligation. Many inteins have nested homing endonuclease domains that facilitate their propagation into intein-less alleles, whereas other inteins lack the homing endonuclease (HEN) and are called mini-inteins. The mini-intein that interrupts the DNA PolII of Pyrococcus horikoshii has a linker region in place of the HEN domain that is shorter than the linker in a closely related intein from Pyrococcus abyssi. The P. horikoshii PolII intein requires a higher temperature for catalytic activity and is more stable to digestion by the thermostable protease thermolysin, suggesting that it is more rigid than the P. abyssi intein. We solved a crystal structure of the intein precursor that revealed a domain-swapped dimer. Inteins found as domain swapped dimers have been shown to promote intein-mediated protein alternative splicing, but the solved P. horikoshii PolII intein structure has an active site unlikely to be catalytically competent.

Identification of an intramolecular switch that controls the interaction of the helicase nsp10 with the membrane-associated nsp12 of porcine reproductive and respiratory syndrome virus

A critical step in replication of positive-stranded RNA viruses is the assembly of replication and transcription complexes (RTC). We have recently mapped the nonstructural protein (nsp) interaction network of porcine reproductive and respiratory syndrome virus (PRRSV) and provided evidence by truncation mutagenesis that the recruitment of viral core replicase enzymes (nsp9 and nsp10) to membrane proteins (nsp2, nsp3, nsp5 and nsp12) is subject to regulation. Here, we went further to discover an intramolecular switch within the helicase nsp10 that controls its interaction with the membrane-associated protein nsp12. Deletion of nsp10 linker region aa.124-133 connecting the domain 1B to 1A led to complete re-localization and co-localization in the cells co-expressing nsp12. Moreover, single amino acid substitutions (e.g., nsp10 E131A and I132A) were sufficient to enable the nsp10-nsp12 interaction. Further proof came from membrane floatation assays that revealed a clear movement of nsp10 mutants, but not WT nsp10, towards the top of sucrose gradients in the presence of nsp12. Interestingly, the same mutations were not able to activate the nsp10-nsp2/3 interaction, suggesting a differential requirement for conformation. Reverse genetics analysis showed that PRRSV mutants carrying the single substitutions were not viable and defective of subgenomic RNA (sg RNA) accumulation. Together, our results provide strong evidence for a regulated interaction between nsp10 and nsp12 and suggest an essential role for an orchestrated RTC assembly in sg RNA synthesis. IMPORTANCE Assembly of replication and transcription complexes (RTC) is a limiting step for viral RNA synthesis. The PRRSV RTC macromolecular complexes are comprised of mainly viral nonstructural replicase proteins (nsps), but how they come together remains elusive. We previously showed that viral helicase nsp10 interacts nsp12 in a regulated manner by truncation mutagenesis. Here we revealed that the interaction is controlled by single residues within the domain linker region of nsp10. Moreover, the activation mutations leads to defect in viral sg RNA synthesis. Our results provide important insight into the mechanisms of PRRSV RTC assembly and regulation of viral sg RNA synthesis.

Acid-Sensing Histidine Kinase With a Redox Switch

The EvgS/EvgA two-component signal transduction system in Escherichia coli is activated under mildly acidic pH conditions. Upon activation, this system induces the expression of a number of genes that confer acid resistance. The EvgS histidine kinase sensor has a large periplasmic domain that is required for perceiving acidic signals. In addition, we have previously proposed that the cytoplasmic linker region of EvgS is also involved in the activation of this sensor. The cytoplasmic linker region resembles a Per-ARNT-Sim (PAS) domain, which is known to act as a molecular sensor that is responsive to chemical and physical stimuli and regulates the activity of diverse effector domains. Our EvgS/EvgA reporter assays revealed that under EvgS-activating mildly acidic pH conditions, EvgS was activated only during aerobic growth conditions, and not during anaerobic growth. Studies using EvgS mutants revealed that C671A and C683A mutations in the cytoplasmic PAS domain activated EvgS even under anaerobic conditions. Furthermore, among the electron carriers of the electron transport chain, ubiquinone was required for EvgS activation. The present study proposes a model of EvgS activation by oxidation and suggests that the cytoplasmic PAS domain serves as an intermediate redox switch for this sensor.