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A new K+-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K+ channels and the voltage-dependent K+ channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an α-helix and a 310-helix at N-terminal Gly4-Lys10 and a double-stranded β-sheet at Gly13-Ile16 and Arg19-Tyr23, with a type I′ β-turn at Asn17-Gly18. Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 ± 0.05 Å for the backbone atoms and of 1.40 ± 0.23 Å for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K+-channel blockers. We also found that some of the residues in the larger scorpion K+-channel blockers (31 to 40 amino acids) are not involved in K+-channel blocking activity. [ABSTRACT FROM AUTHOR]

The small α-domain of Lon protease is thought to carry the substrate-recognition, nucleotide-binding, and DNA-binding sites. Here we report the complete resonance assignment of the α-domain for Bacillus subtilis Lon protease (Bs-Lon α-domain). [ABSTRACT FROM AUTHOR]

Human sialic acid-- binding immunoglobulin-like lectin 14 (Siglec-14) is a glycan-recognition protein that is expressed on myeloid cells, recognizes bacterial pathogens, and elicits pro-inflammatory responses. Although Siglec-14 is a transmembrane protein, a soluble form of Siglec-14 is also present in human blood. However, the mechanism that generates soluble Siglec-14 and what role this protein form may play remain unknown. Here, investigating the generation and function of soluble Siglec-14, we found that soluble Siglec-14 is derived from an alternatively spliced mRNA that retains intron 5, containing a termination codon and thus preventing the translation of exon 6, which encodes Siglec-14's transmembrane domain. We also note that the translated segment in intron 5 encodes a unique C-terminal 7-amino acid extension, which allowed the specific antibody-mediated detection of this isoform in human blood. Moreover, soluble Siglec-14 dose-dependently suppressed pro-inflammatory responses of myeloid cells that expressed membrane-bound Siglec-14, likely by interfering with the interaction between membrane-bound Siglec-14 and Toll-like receptor 2 on the cell surface. We also found that intron 5 contains a G-rich segment that assumes an RNA tertiary structure called a G-quadruplex, which may regulate the efficiency of intron 5 splicing. Taken together, we propose that soluble Siglec-14 suppresses pro-inflammatory responses triggered by membrane-bound Siglec-14. [ABSTRACT FROM AUTHOR]

Lon protease has been well studied in many aspects; however, the DNA-binding specificity of Lon in prokaryotes has not been clearly identified. Here we examined the DNA-binding activity of Lon protease alpha-domains from Brevibacillus thermoruber (Bt), Bacillus subtilis (Bs), and Escherichia coli (Ec). MALDI-TOF mass spectroscopy showed that the alpha-domain from Bt-Lon binds to the duplex nucleotide sequence 5'-CTGTTAGCGGGC-3' (ms1) and protected it from DNase I digestion. Surface plasmon resonance showed that the Bt-Lon alpha-domain binds with ms1 double-stranded DNA tighter than Bs- and Ec-Lon alpha-domains, whereas the Bt-Lon alpha-domain has dramatically lower affinity for double-stranded DNA with 0 and 50% identity to the ms1 binding sequence. Our results indicated that Bt-Lon alpha-domain plays a critical role with ms1 sequence in the DNA-binding specificity.

A number of beta-microseminoproteins (MSPs) have been identified from different species. MSPs are all non-glycosylated and disulfide bond-rich, but show a relatively low level of conservation. Although all Cys residues are conserved, our previous study showed that the disulfide bond pairings differ in porcine and ostrich MSPs. Despite the variety of biological functions that have been suggested for MSPs, their real function is still poorly understood. Furthermore, no 3D structure has been reported for any MSP, so the determination of the structure and function of MSPs is an interesting and important task. In the present study, we determined the 3D solution structure of porcine MSP on the basis of 1018 restraints. The ensemble of 20 NMR structures was well defined, with average root-mean-square deviations of 0.83(+/-0.16) A for the backbone atoms and 1.37(+/-0.17) A for heavy-atoms in residues 2-90. The 3D structure showed that porcine MSP is clearly composed of two domains, an N-terminal domain consisting of one double-stranded and one four-stranded antiparallel beta-sheet, and a C-terminal domain consisting of two double-stranded antiparallel beta-sheet. The orientation of the two domains was derived mainly on the basis of long-range NOEs and verified using residual dipolar coupling data. No inter-domain hydrophobic interaction or H-bonding was detected. However, a number of charged residues were found in close proximity between the domains, indicating that electrostatic interaction may be the key factor for the orientation of the two domains. This is the first report of a 3D structure for any MSP. In addition, structural comparison based on distance matrix alignment (DALI), class architecture topology and homologous superfamily (CATH) and combinatorial extension (CE) methods revealed that porcine MSP has a novel structure with a new fold providing valuable information for future structural studies on other MSPs and for understanding their biological functions.

A sperm motility inhibitor isolated from porcine seminal plasma is identical to porcine beta-microseminoprotein (MSP). Circular dichroism (CD) and nuclear magnetic resonance (NMR) data showed that the native and recombinant porcine MSPs exhibit very similar structure. The five disulfide pairings on porcine MSP were unambiguously assigned based on NMR data and further confirmed using structural calculations. Surprisingly, our derived pairings differ from those recently reported for ostrich MSP based on matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis. Furthermore, the secondary structure was determined to comprise one four-stranded and two double-stranded antiparallel beta-sheets. As we know, this is the first detailed secondary structure reported among several types of MSPs.

A new K(+)-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K(+) channels and the voltage-dependent K(+) channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an alpha-helix and a 3(10)-helix at N-terminal Gly(4)-Lys(10) and a double-stranded beta-sheet at Gly(13)-Ile(16) and Arg(19)-Tyr(23), with a type I' beta-turn at Asn(17)-Gly(18). Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 +/- 0.05 A for the backbone atoms and of 1.40 +/- 0.23 A for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K(+)-channel blockers. We also found that some of the residues in the larger scorpion K(+)-channel blockers (31 to 40 amino acids) are not involved in K(+)-channel blocking activity.