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Helicobacter pylori infection is associated with the development of gastric and duodenal ulcers as well as gastric cancer. GroES of H. pylori (HpGroES) was previously identified as a gastric cancer-associated virulence factor. Our group showed that HpGroES induces interleukin-8 (IL-8) cytokine release via a Toll-like receptor 4 (TLR4)-dependent mechanism and domain B of the protein is crucial for interactions with TLR4. In the present study, we investigated the importance of the histidine residues in domain B. To this end, a series of point mutants were expressed in Escherichia coli, and the corresponding proteins purified. Interestingly, H96, H104 and H115 were not essential, whereas H100, H102, H108, H113 and H118 were crucial for IL-8 production and TLR4 interactions in KATO-III cells. These residues were involved in nickel binding. Four of five residues, H102, H108, H113 and H118 induced certain conformation changes in extended domain B structure, which is essential for interactions with TLR4 and consequent IL-8 production. We conclude that interactions of nickel ions with histidine residues in domain B help to maintain the conformation of the C-terminal region to conserve the integrity of the HpGroES structure and modulate IL-8 release.

Hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS) is a severe salt-losing renal tubular disorder and results from the mutation of renal outer medullary K(+) (ROMK1) channels. The aberrant ROMK1 function induces alterations in intracellular pH (pH(i)) gating under physiological conditions. We investigate the role of protein kinase A (PKA) in the pH(i) gating of ROMK1 channels. Using giant patch clamp with Xenopus oocytes expressing wild-type and mutant ROMK1 channels, PKA-mediated phosphorylation decreased the sensitivity of ROMK1 channels to pH(i). A homology model of ROMK1 reveals that a PKA phosphorylation site (S219) is spatially juxtaposed to the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding residues (R188, R217, and K218). Molecular dynamics simulations suggest a stable transition state, in which the shortening of distance between S219 and R217 and the movement of K218 towards the membrane after the PKA-phosphorylation can be observed. Such conformational change may bring the PIP(2) binding residues (K218) more accessible to the membrane-bound PIP(2). In addition, PIP(2) dose-dependently reactivates the acidification-induced rundown channels only when ROMK1 channels have been phosphorylated by PKA. This implies a sequence regulatory episode reflecting the role of PIP(2) in the pH(i) gating of ROMK1 channels by PKA-mediated phosphorylation. Our results provide new insights into the molecular mechanisms underlying the ROMK1 channel regulation associated with HPS/aBS.

Background/Purpose: Inward rectifying potassium channel 6.2 (Kir6.2 Δ C26 channel) is closely related to ATP-sensitive potassium channels. Whether sodium azide, barium ion, d-amphetamine or procaine acts directly on the Kir6.2 Δ C26 channel remains unclear. We studied the effects of these compounds on Kir6.2 Δ C26 channel expressed in Xenopus oocytes. Methods: The coding sequence of a truncated form of mouse Kir6.2 (GenBank accession number NP_034732.1), Kir6.21-364 (i.e. Kir6.2 Δ C26), was subcloned into the pET20b(+) vector. Plasmid containing the correct T7 promoter-Kir6.21-364 cDNA fragment [Kir6.2/pET20b(+)] was then subject to Not I digestion to generate the templates for in vitro run-off transcriptions. The channel was expressed in Xenopus laevis oocytes. Two-electrode voltage clamping was used to measure the effects of sodium azide, barium ion, d-amphetamine and procaine on Kir6.2 Δ C26 channel current. Results: Sodium azide activated and barium ion and d-amphetamine inhibited the Kir6.2 Δ C26 channel. Procaine did not have any significant effect on the Kir6.2 Δ C26 channel. Conclusion: Kir6.2 Δ C26 channel expressed in Xenopus oocytes can be used as a pharmacological tool for the study of inward rectifying potassium channels.

Rundown is a generally encountered problem while recording KATP channel activity with inside-out patches. No assigned structural fragment related to this mechanism has yet been derived from any of the functional analyses performed. Therefore, based on a combined sequence and secondary structure alignment against known crystal structure of segments from closely related proteins, we propose here the three-dimensional structural model of an intracellular C-terminal domain of the Kir6.2 subunit in KATP channels. An E. coli CMP-kinase was suggested as template for the model building. The subdomain arrangement of this novel kinase domain and the structural correlation for UDP-docking are described. With structural-functional interpretation, we conclude that the reactivation of KATP channel rundown by MgATP or UDP is very possibly regulated by this intracellular kinase domain at the C-terminus of Kir6.2 subunit in KATP channels.

The effects on the channel characteristics of four single amino acid substitutions in OmpF porin and of a deletion mutant in the constriction loop L3 have been studied. These mutations are all located in the narrow section of the channel of the protein that forms pores across the outer membrane of Escherichia coli. The single channel conductance of the deletion mutant (Delta109-114) is decreased by one third, whereas the point mutations do not exhibit significant deviations from that of the wild-type protein. The mutants exhibit drastic changes in ion selectivities. In the wild-type protein, the critical threshold potential (Vc), above which channels close reversibly, exhibits a strong pH dependence, with a titration point of approximately pH 7.7, which is abolished in all mutants studied here. Diffusion of six monosaccharides is little affected in the point mutants, while four disaccharides are taken up at highly increased rates by the deletion mutant. The functional results, presented here, are correlated to the x-ray structures of the mutants (Lou, K.-L., Saint, N., Prilipov, A., Rummel, G., Benson, S.A., Rosenbusch, J.P., and Schirmer, T. (1996) J. Biol. Chem. 271, 20669-20675). In most, but not all, cases, the structural changes explain the functional alterations observed.

OmpF porin is a nonspecific pore protein from the outer membrane of Escherichia coli. Previously, a set of mutants was selected that allow the passage of long maltodextrins that do not translocate through the wild-type pore. Here, we describe the crystal structures of four point mutants and one deletion mutant from this set; their functional characterization is reported in the accompanying paper (Saint, N., Lou, K.-L., Widmer, C., Luckey, M., Schirmer, T., Rosenbusch, J. P. (1996) J. Biol. Chem. 271, 20676-20680). All mutations have a local effect on the structure of the pore constriction and result in a larger pore cross-section. Substitution of each of the three closely packed arginine residues at the pore constriction (Arg-42, Arg-82, and Arg-132) by shorter uncharged residues causes rearrangement of the adjacent basic residues. This demonstrates mutual stabilization of these residues in the wild-type porin. Deletion of six residues from the internal loop (Delta109-114) results in disorder of seven adjacent residues but does not alter the structure of the beta-barrel framework. Thus, the large hollow beta-barrel motif can be regarded as an autonomous structure.

Chemokines are a large group of proteins implicated in migration, activation, and differentiation of leukocytes. They are well-surveyed in mammals, but less is known in lower vertebrates about their spatiotemporal expressions and functions. From an evolutionary point of view, comparative analyses may provide some fundamental insights into these molecules. In mammals, CCL21 and CCL25 are crucial for thymocyte homing. Herein, we identified and cloned the zebrafish orthologues of CCL21 and CCL25, and analyzed their expression in embryos and adult fish by in situ hybridization. We found that CCL21 was expressed in the craniofacial region, pharynx, and blood vessels in embryos. In adult fish, CCL21 transcripts were located in the kidney, spinal cord, and blood cells. In contrast, expression of CCL25 was only detected in the thymus primordia in embryos. In adult fish, transcripts of CCL25 were maintained in the thymus, and they were also found in the brain and oocytes. Furthermore, we performed an antisense oligonucleotide experiment to evaluate the biological function of CCL25. Results showed that the recruitment of thymocytes was impeded by morpholino-mediated knockdown of CCL25, suggesting that CCL25 is essential for colonization of T-cells in the thymus in early development. Together, our results demonstrate the basic profiles of two CCL chemokines in zebrafish. The tissue-specific expression patterns may pave the way for further genetic dissection in this model organism.

The protein kinase C (PKC) pathway is important for the regulation of K+ transport. The renal outer medullar K+ (ROMK1) channels show an exquisite sensitivity to intracellular protons (pH i ) (effective pK a approximately 6.8) and play a key role in K+ homeostasis during metabolic acidosis. Our molecular dynamic simulation results suggest that PKC-mediated phosphorylation on Thr-193 may disrupt the PIP2-channel interaction via a charge–charge interaction between Thr-193 and Arg-188. Therefore, we investigated the role of PKC and pH i in regulation of ROMK1 channel activity using a giant patch clamp with Xenopus oocytes expressing wild-type and mutant ROMK1 channels. ROMK1 channels pre-incubated with the PKC activator phorbol-12-myristate-13-acetate exhibited increased sensitivity to pH i (effective pK a shifted to pH approximately 7.0). In the presence of GF109203X—a PKC selective inhibitor—the effective pK a for inhibition of ROMK1 channels by pH i decreased (effective pK a shifted to pH approximately 6.5). The pH i sensitivity of ROMK1 channels mediated by PKC appeared to be dependent of PIP2 depletion. The giant patch clamp together with site direct mutagenesis revealed that Thr-193 is the phosphorylation site on PKC that regulates the pH i sensitivity of ROMK1 channels. Mutation of PKC-induced phosphorylation sites (T193A) decreases the pH i sensitivity and increases the interaction of channel-PIP2. Taken together, these results provide new insights into the molecular mechanisms underlying the pH i gating of ROMK1 channel regulation by PKC.

The carboxyl terminus of the S3 segment (S3C) in voltage-gated potassium channels was suggested to be the binding site of gating modifier toxins like hanatoxin. It has also been proposed to have a helical secondary structural arrangement. The currently available structures in high resolution for such channel molecules are restricted to regions illustrating the pore function. Therefore no further direct experimental data to elucidate the detailed mechanism for such toxin binding can be derived. In order to examine the putative three-dimensional structure of S3C and to analyze the residues required for hanatoxin binding, molecular simulation and docking were performed, based on the solution structure of hanatoxin and the structural information from mutational scanning data for the S3C fragment in Kv2.1. Our results indicate that hydrophobic and electrostatic interactions are both utilized to stabilize the toxin binding. Precise docking residues and the appropriate orientation for binding regarding amphipathic environments are also described. Compared with the functional data proposed by previous studies, the helical structural arrangement for the C-terminus of the S3 segment in voltage-gated potassium channels can therefore be further emphasized and analyzed. The possible location/orientation for toxin binding with respect to membrane distribution around the S3C segment is also discussed in this paper.

Glucosyltransferases (GtfB/C/D) in Streptococcus mutans are responsible for synthesizing water-insoluble and water-soluble glucans from sucrose and play very crucial roles in the formation of dental plaque. A monoclonal antibody against a 19-mer peptide fragment named Gtf-P1 was found in GtfC to reduce the enzyme activity to 50%. However, a similar experiment suggested almost unchanged activity in GtfD, despite of the very high sequence homology between the two enzymes. No further details are yet available to elucidate the biochemical mechanism responsible for such discrimination. For a better understanding of the catalytic behavior of these glucosyltransferases, structural and functional analyses were performed. First, the exact epitope was identified to specify the residue(s) required for monoclonal antibody recognition. The results suggest that the discrimination is determined solely by single residue substitution. Second, based on a combined sequence and secondary structure alignment against known crystal structure of segments from closely related proteins, a three-dimensional homology model for GtfC was built. Structural analysis for the region communicating between Gtf-P1 and the catalytic triad revealed the possibility for an "en bloc" movement of hydrophobic residues, which may transduce the functional influence on enzyme activity from the surface of molecule into the proximity of the active site. Figure Side chain interactions between Gtf-P1 and catalytic Asp-477 in GtfC. Calpha-tracing of GtfC with the two crucial peptides (Gtf-P1, orange; Gtf-P2, blue) and the catalytic triad residues ( red) highlighted to show their relative spatial organization. Side chains for the residues are also depicted according to their atom types. The structure is viewed with the barrel opening facing down

Glucosyltransferases (GtfB/C/D) of Streptococcus mutans, a pathogen for human dental caries, synthesize water-insoluble glucan through the hydrolysis of sucrose. Genetic and biochemical approaches have identified several active sites of these enzymes, but no three-dimensional (3D) structural evidence is yet available to elucidate the subdomain arrangement and molecular mechanism of catalysis. Based on a combined sequence and secondary structure alignment against known crystal structures of segments from closely related proteins, we propose here the 3D model of an N-terminal domain essential for the sucrose binding and splitting in GtfB. A Tim-barrel of (alpha/beta)(8) structural characteristics is revealed and the structural correlation for two peptides is described.

In the homotrimeric OmpF porin from Escherichia coli , each channel is constricted by a loop protruding into the β-barrel of the monomer about halfway through the membrane. The water-filled channels exist in open or closed states, depending on the transmembrane potential. For the transition between these conformations, two fundamentally different mechanisms may be envisaged: a bulk movement of the constriction loop L3 or a redistribution of charges in the channel lumen. To distinguish between these hypotheses, nine mutant proteins were constructed on the basis of the high-resolution x-ray structure of the wild-type protein. Functional changes were monitored by measuring single-channel conductance and critical voltage of channel closing. Structural alterations were determined by x-ray analysis to resolutions between 3.1 and 2.1 Å. Tethering the tip of L3 to the barrel wall by a disulfide bridge (E117C/A333C), mobilizing L3 by perturbing its interaction with the barrel wall (D312N, S272A, E296L), or deleting residues at the tip of the loop (Δ116–120) did not alter appreciably the sensitivity of the channels to an external potential. A physical occlusion, due to a gross movement of L3, which would cause the channels to assume a closed conformation, can therefore be excluded.

Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are retained indicating that the helix B-Met loop-helix C is the minimal functional “domain” found to date for the matrixin family. Conclusions The helix B-Met loop-helix C folding conserved in metalloprotease metzincin super family is able to facilitate proteolytic catalysis for specific substrate and inhibitor recognition. The autolysis processing and producing 6 kDa mini MMP-7 is the smallest metalloprotease in living world.