Molecular determinants of voltage-dependent slow inactivation of the Ca2+ channel.

Ba(2+) current through the L-type Ca(2+) channel inactivates essentially by voltage-dependent mechanisms with fast and slow kinetics. Here we found that slow inactivation is mediated by an annular determinant composed of hydrophobic amino acids located near the cytoplasmic ends of transmembrane segments S6 of each repeat of the alpha(1C) subunit. We have determined the molecular requirements that completely obstruct slow inactivation. Critical interventions include simultaneous substitution of A752T in IIS6, V1165T in IIIS6, and I1475T in IVS6, each preventing in additive manner a considerable fraction of Ba(2+) current from inactivation. In addition, it requires the S405I mutation in segment IS6. The fractional inhibition of slow inactivation in tested mutants caused an acceleration of fast inactivation, suggesting that fast and slow inactivation mechanisms are linked. The channel lacking slow inactivation showed approximately 45% of the sustained Ba(2+) or Ca(2+) current with no indication of decay. The remaining fraction of the current was inactivated with a single-exponential decay (pi(f) approximately 10 ms), completely recovered from inactivation within 100 ms and did not exhibit Ca(2+)-dependent inactivation properties. No voltage-dependent characteristics were significantly changed, consistent with the C-type inactivation model suggesting constriction of the pore as the main mechanism possibly targeted by Ca(2+) sensors of inactivation.     

The congenital bicuspid aortic valve can experience high-frequency unsteady shear stresses on its leaflet surface

The bicuspid aortic valve (BAV) is a common congenital malformation of the aortic valve (AV) affecting 1% to 2% of the population. The BAV is predisposed to early degenerative calcification of valve leaflets, and BAV patients constitute 50% of AV stenosis patients. Although evidence shows that genetic defects can play a role in calcification of the BAV leaflets, we hypothesize that drastic changes in the mechanical environment of the BAV elicit pathological responses from the valve and might be concurrently responsible for early calcification. An in vitro model of the BAV was constructed by surgically manipulating a native trileaflet porcine AV. The BAV valve model and a trileaflet AV (TAV) model were tested in an in vitro pulsatile flow loop mimicking physiological hemodynamics. Laser Doppler velocimetry was used to make measurements of fluid shear stresses on the leaflet of the valve models using previously established methodologies. Furthermore, particle image velocimetry was used to visualize the flow fields downstream of the valves and in the sinuses. In the BAV model, flow near the leaflets and fluid shear stresses on the leaflets were much more unsteady than for the TAV model, most likely due to the moderate stenosis in the BAV and the skewed forward flow jet that collided with the aorta wall. This additional unsteadiness occurred during mid- to late-systole and was composed of cycle-to-cycle magnitude variability as well as high-frequency fluctuations about the mean shear stress. It has been demonstrated that the BAV geometry can lead to unsteady shear stresses under physiological flow and pressure conditions. Such altered shear stresses could play a role in accelerated calcification in BAVs.     

Structural insight into the molecular basis of polyextremophilicity of short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus

Biochemical analysis of enantioselective short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus (TsAdh319) revealed unique polyextremophilic properties of the enzyme – half-life of 1 h at 100 °C, tolerance to high salt (up to 4 M) and organic solvents (50% v/v) concentrations. To elucidate the molecular basis of TsAdh319 polyextremophilicity, we determined the crystal structure of the enzyme in a binary complex with 5-hydroxy-NADP at 1.68 Å resolution. TsAdh319 has a tetrameric structure both in the crystals and in solution with an intersubunit disulfide bond. The substrate-binding pocket is hydrophobic, spacious and open that is consistent with the observed promiscuity in substrate specificity of TsAdh319. The present study revealed an extraordinary number of charged residues on the surface of TsAdh319, 70% of which were involved in ion pair interactions. Further we compared the structure of TsAdh319 with the structures of other homologous short-chain dehydrogenases/reductases (SDRs) from thermophilic and mesophilic organisms. We found that TsAdh319 has the highest arginine and aspartate + glutamate contents compared to the counterparts. The frequency of occurrence of salt bridges on the surface of TsAdh319 is the highest among the SDRs under consideration. No differences in the proline, tryptophan, and phenylalanine contents are observed; the compactness of the protein core of TsAdh319, the monomer and tetramer organization do not differ from that of the counterparts. We suggest that the unique thermostability of TsAdh319 is associated with the rigidity and simultaneous “resilience” of the structure provided by a compact hydrophobic core and a large number of surface ion pairs. An extensive salt bridge network also might maintain the structural integrity of TsAdh319 in high salinity.     

Homing endonuclease mediated gene targeting in Anopheles gambiae cells and embryos

Homing endonuclease genes (HEGs) are ‘selfish’ genetic elements that combine the capability to selectively disrupt specific gene sequences with the ability to rapidly spread from a few individuals to an entire population through homologous recombination repair events. Because of these properties, HEGs are regarded as promising candidates to transfer genetic modifications from engineered laboratory mosquitoes to wild-type populations including Anopheles gambiae the vector of human malaria. Here we show that I-SceI and I-PpoI homing endonucleases cleave their recognition sites with high efficiency in A. gambiae cells and embryos and we demonstrate HEG-induced homologous and non-homologous repair events in a variety of functional assays. We also propose a gene drive system for mosquitoes that is based on our finding that I-PpoI cuts genomic rDNA located on the X chromosome in A. gambiae, which could be used to selectively incapacitate X-carrying spermatozoa thereby imposing a severe male-biased sex ratio.     

Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.     

Mapping of functional domains in F plasmid partition proteins reveals a bipartite SopB-recognition domain in SopA

Active partition of the F plasmid to dividing daughter cells is assured by interactions between proteins SopA and SopB, and a centromere, sopC. A close homologue of the sop operon is present in the linear prophage N15 and, together with sopC-like sequences, it ensures stability of this replicon. We have exploited this sequence similarity to construct hybrid sop operons with the aim of locating specific interaction determinants within the SopA and SopB proteins that are needed for partition function and for autoregulation of sopAB expression. Centromere binding was found to be specified entirely by a central 25 residue region of SopB strongly predicted to form a helix-turn-helix structure. SopB protein also carries a species-specific SopA-interaction determinant within its N-terminal 45 amino acids, and, as shown by Escherichia coli two-hybrid analysis, a dimerization domain within its C-terminal 75 (F) or 97 (N15) residues. Promoter-operator binding specificity was located within an N-terminal 66 residue region of SopA, which is predicted to contain a helix-turn-helix motif. Two other regions of SopA protein, one next to the ATPase Walker A-box, the other C-terminal, specify interaction with SopB. Yeast two-hybrid analysis indicated that these regions contact SopB directly. Evidence for the involvement of the SopA N terminus in autoinhibition of SopA function was obtained, revealing a possible new aspect of the role of SopB in SopA activation.     

Identification of new human cadherin genes using a combination of protein motif search and gene finding methods

We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains.     

NMR structures of loop B RNAs from the stem-loop IV domain of the enterovirus internal ribosome entry site: a single C to U substitution drastically changes the shape and flexibility of RNA

The 5'-untranslated region of positive-strand RNA viruses harbors many cis-acting RNA structural elements that are important for various viral processes such as replication, translation, and packaging of new virions. Among these is loop B RNA of the stem-loop IV domain within the internal ribosomal entry site (IRES) of enteroviruses, including Poliovirus type 1 (PV1). Studies on PV1 have shown that specific recognition of loop B by the first KH (hnRNP K homology) domain of cellular poly(rC)-binding protein 2 (PCBP2) is essential for efficient translation of the viral mRNA. Here we report the NMR solution structures of two representative sequence variants of enteroviral loop B RNA. The two RNA variants differ at only one position (C vs U) within a six-nucleotide asymmetric internal loop sequence that is the binding site for the PCBP2 KH1 domain. Surprisingly, the two RNAs are drastically different in the overall shape and local dynamics of the bulge region. The RNA with the 5'-AUCCCU bulge sequence adopts an overall L shape. Its bulge nucleotides, especially the last four, are highly flexible and not very well defined by NMR. The RNA with the 5'-AUUCCU bulge sequence adopts an overall U shape, and its bulge sequence exhibits only limited flexibility. A detailed analysis of the two RNA structures and their dynamic properties, as well as available sequence data and known KH domain-RNA complex structures, not only provides insights into how loop B RNA might be recognized by the PCBP2 KH1 domain but also suggests a possible correlation between structural flexibility and pre-existing structural features for protein recognition.