Impaired Binding to Junctophilin2 and Nanostructural Alteration in CPVT Mutation

Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare disease, manifested by syncope or sudden death in children or young adults under stress conditions. Mutations in the Ca 2+ release channel/ryanodine receptor (RyR2) gene account for about 60% of the identified mutations. Recently, we found and described a mutation in RyR2 N-terminal domain, RyR2 R420Q . Objective: To determine the arrhythmogenic mechanisms of this mutation. Methods and Results: Ventricular tachycardias under stress conditions were observed in both CPVT patients and KI mice. During action potential recording (by patch-clamp in KI mouse cardiomyocytes and by microelectrodes in mutant hiPSC-CM) we observed an increased occurrence of delayed after-depolarizations (DADs) under isoproterenol stimulation, associated with increased Ca 2+ waves during confocal Ca 2+ recording in both mouse and human RyR2 R420Q cardiomyocytes. In addition, Ca 2+ -induced Ca 2+ -release, as well as a rough indicator of fractional Ca 2+ release, were higher and Ca 2+ sparks longer in the RyR2 R420Q expressing cells. At the ultrastructural nanodomain level, we observed smaller RyR2 clusters and widened junctional sarcoplasmic reticulum (jSR) measured by g-STED super-resolution and electronic microscopy, respectively. The increase in jSR width might be due to the impairment of RyR2 R420Q binding to junctophilin-2, as there were less junctophilin-2 co-immunoprecipitated with RyR2 R420Q . At the single current level, the RyR2R420Q channel dwells longer in the open state at low [Ca 2+ ] i , but there is predominance of a subconductance state. The latter might be correlated with an enhanced interaction between the N-terminus and the core solenoid, a RyR2 inter-domain association that has not been previously implicated in the pathogenesis of arrhythmias and sudden cardiac death. Conclusions: The RyR2 R420Q CPVT mutation modifies the interdomain interaction of the channel and weaken its association with junctophillin-2. These defects may underlie both nanoscale disarrangement of the dyad and channel dysfunction.

Coiled-coil 1-mediated fastening of the neck and motor domains for kinesin-3 autoinhibition

In kinesin-3, the coiled-coil 1 (CC1) can sequester the preceding neck coil (NC) for autoinhibition, but the underlying mechanism is poorly understood. Here, we determined the structures of the uninhibited motor domain (MD)-NC dimer and inhibited MD-NC-CC1 monomer of kinesin-3 KIF13B. In the MD-NC-CC1 monomer, CC1 is broken into two short helices that unexpectedly interact with both the NC and the MD. Compared with the MD-NC dimer, the CC1-mediated integration of NC and MD not only blocks the NC dimer formation, but also prevents the neck linker (NL) undocking and the ADP release from the MD. Mutations of the essential residues in the interdomain interaction interface in the MD-NC-CC1 monomer restored the MD activity. Thus, CC1 fastens the neck domain and MD and inhibits both NC and NL. This CC1-mediated lockdown of the entire neck domain may represent a paradigm for kinesin autoinhibition that could be applicable to other kinesin-3 motors.

Crystallization and preliminary X-ray analysis of Rv1674c fromMycobacterium tuberculosis

Adaptations to hypoxia play an important role inMycobacterium tuberculosispathogenesis. Rv0324, which contains an HTH DNA-binding domain and a rhodanese domain, is one of the key transcription regulators in response to hypoxia.M. tuberculosisRv1674c is a homologue of Rv0324. To understand the interdomain interaction and regulation of the HTH domain and the rhodanese domain, recombinant Rv1674c protein was purified and crystallized by the vapour-diffusion method. The crystals diffracted to 2.25 Å resolution. Preliminary diffraction analysis suggests that the crystals belonged to space groupP3121 orP3221, with unit-cell parametersa=b= 67.8,c= 174.5 Å, α = β = 90, γ = 120°. The Matthews coefficient was calculated to be 2.44 Å3 Da−1, assuming that the crystallographic asymmetric unit contains two protein molecules.

Structure of mouse muskelin discoidin domain and biochemical characterization of its self-association

Muskelin is an intracellular kelch-repeat protein comprised of discoidin, LisH, CTLH and kelch-repeat domains. It is involved in cell adhesion and the regulation of cytoskeleton dynamics as well as being a component of a putative E3 ligase complex. Here, the first crystal structure of mouse muskelin discoidin domain (MK-DD) is reported at 1.55 Å resolution, which reveals a distorted eight-stranded β-barrel with two short α-helices at one end of the barrel. Interestingly, the N- and C-termini are not linked by the disulfide bonds found in other eukaryotic discoidin structures. A highly conserved MIND motif appears to be the determinant for MK-DD specific interaction together with the spike loops. Analysis of interdomain interaction shows that MK-DD binds the kelch-repeat domain directly and that this interaction depends on the presence of the LisH domain.

Structure determination of human Fas apoptosis inhibitory molecule and identification of the critical residues linking the interdomain interaction to the anti-apoptotic activity

Fas apoptosis inhibitory molecule (FAIM) is a highly conserved anti-apoptotic protein which plays important roles in cells. There are two isoforms of FAIM, of which the short isoform FAIM-S is broadly expressed in all tissues, whereas the long isoform FAIM-L is exclusively expressed in the nervous system. No structure of human FAIM has been reported to date and the detailed molecular mechanisms underlying the anti-apoptotic function of FAIM remain unknown. Here, the crystal structure of the human FAIM-S N-terminal domain (NTD) and the NMR solution structure of the human FAIM-S C-terminal domain (CTD) were determined. The structures revealed that the NTD and CTD adopt a similar protein fold containing eight antiparallel β-strands which form two sheets. Both structural and biochemical analyses implied that the NTD exists as a dimer and the CTD as a monomer and that they can interact with each other. Several critical residues were identified to be involved in this interaction. Moreover, mutations of these critical residues also interfered in the anti-apoptotic activity of FAIM-S. Thus, the structural and functional data presented here will provide insight into the anti-apoptotic mechanism of FAIM-S.

CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions

Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositides (PIPn) such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIPn regulation. We determined the functional consequences of this mutation using electrophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIPn inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIPn sensitivity depended on an intact amino (NH2) terminal PIPn regulation module. These observations led us to hypothesize that L633P enhances PIPn inhibition by altering the coupling between NH2- and COOH-terminal regions of CNGA3. A recombinant COOH-terminal fragment partially restored normal PIPn sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the L633P mutation.

Mechanism for modulation of gating of connexin26-containing channels by taurine

The mechanisms of action of endogenous modulatory ligands of connexin channels are largely unknown. Previous work showed that protonated aminosulfonates (AS), notably taurine, directly and reversibly inhibit homomeric and heteromeric channels that contain Cx26, a widely distributed connexin, but not homomeric Cx32 channels. The present study investigated the molecular mechanisms of connexin channel modulation by taurine, using hemichannels and junctional channels composed of Cx26 (homomeric) and Cx26/Cx32 (heteromeric). The addition of a 28–amino acid “tag” to the carboxyl-terminal domain (CT) of Cx26 (Cx26T) eliminated taurine sensitivity of homomeric and heteromeric hemichannels in cells and liposomes. Cleavage of all but four residues of the tag (Cx26Tc) resulted in taurine-induced pore narrowing in homomeric hemichannels, and restored taurine inhibition of heteromeric hemichannels (Cx26Tc/Cx32). Taurine actions on junctional channels were fully consistent with those on hemichannels. Taurine-induced inhibition of Cx26/Cx32T and nontagged Cx26 junctional channels was blocked by extracellular HEPES, a blocker of the taurine transporter, confirming that the taurine-sensitive site of Cx26 is cytoplasmic. Nuclear magnetic resonance of peptides corresponding to Cx26 cytoplasmic domains showed that taurine binds to the cytoplasmic loop (CL) and not the CT, and that the CT and CL directly interact. ELISA showed that taurine disrupts a pH-dependent interaction between the CT and the CT-proximal half of the CL. These studies reveal that AS disrupt a pH-driven cytoplasmic interdomain interaction in Cx26-containing channels, causing closure, and that the Cx26CT has a modulatory role in Cx26 function.