Purification and structural characterization of bovine cathelicidins, precursors of antimicrobial peptides

Cathelicidins are a novel family of antimicrobial peptide precursors from mammalian myeloid cells. They are characterized by a conserved N-terminal region while the C-terminal antimicrobial domain can vary considerably in both primary sequence and length. Four cathelicidins, proBac5, proBac7, prododecapeptide and proBMAP-28, have been concurrently purified from bovine neutrophils, using simple and rapid methodologies. The correlation of ES-MS data from the purified proteins with their cDNA-deduced sequences has revealed several common features of their primary sequence, such as the presence of N-terminal 5-oxoproline (pyroglutamate) residues and two disulfide bridges in a 1-2, 3-4 arrangement. The N-terminal domains of the cathelicidins present one or two Asp-Pro bonds, which are particularly acid-labile in proBac5 and proBac7, but stable in prododecapeptide. This suggests that the spatial organization around these bonds may vary in different cathelicidins, and favour hydrolysis in some cases. An unexpected feature of the prododecapeptide is that it exists as dimers formed by three possible combinations of its two isoforms. The isolation of a truncated, monomeric form of this protein, lacking the cysteine-containing antimicrobial dodecapeptide, indicates that dimerization occurs via disulfide bridge formation at the level of the C-terminal domain and that the dodecapeptide is likely released as a dimer from its precursor. Sequence-based secondary structure predictions and CD results indicate for cathelicidins a 30-50% content of extended conformation and <20% content of alpha-helical conformation, with the alpha-helical segment placed near the N-terminus. Finally, similarity searching and topology-based structure prediction underline a significant sequential and structural similarity between the conserved N-terminal domain of cathelicidins and cystatin-like domains, placing this family within the cystatin superfamily. When assayed against cathepsin L, unlike the potent cystatin inhibitors, three of the four cathelicidins show only a poor inhibitory activity (Ki = 0.6-3 microM).     

Deletion mapping of N-terminal domains of surfactant protein A. The N-terminal segment is required for phospholipid aggregation and specific inhibition of surfactant secretion

The objective of the current study was to examine the functional importance of the N-terminal domains of surfactant protein A (SP-A) including the N-terminal segment from Asn1 to Ala7 (denoted domain 1), the N-terminal portion of the collagen domain from Gly8 to Gly44 (domain 2), and the C-terminal portion of the collagen-like domain from Gly45 to Pro80 (domain 3). Wild type recombinant SP-A (SP-Ahyp; where hyp indicates hydroxyproline-deficient) and truncated mutant (TM) SP-As containing deletions of domain(s) 1 (TM1), 2 (TM2), 1 and 2 (TM1-2), and 1, 2, and 3 (TM1-2-3) were synthesized in insect cells and purified by mannose-Sepharose affinity chromatography. N-terminal disulfide-dependent dimerization was preserved at near wild type levels in the TM1-2 (at Cys-1) and TM2 proteins (at Cys-1 and Cys6), and to a lesser extent in TM1 (at Cys-1), but not in TM1-2-3. Cross-linking analyses demonstrated that the neck + CRD was sufficient for assembly of monomers into noncovalent trimers and that the N-terminal segment was required for the association of trimers to form higher oligomers. All TM proteins except TM1-2-3 bound to phospholipid, but only the N-terminal segment containing TM proteins aggregated phospholipid vesicles. The TM1, TM1-2, and TM2 but not the TM1-2-3 inhibited the secretion of surfactant from type II cells as effectively as SP-Ahyp, but the inhibitory activity of each mutant was blocked by excess alpha-methylmannoside and therefore nonspecific. TM1 and TM1-2-3 did not enhance the uptake of phospholipids by isolated type II cells, but the TM1-2 and TM2 had activities that were 72 and 83% of SP-Ahyp, respectively. We conclude the following for SP-A: 1) trimerization does not require the collagen-like region or interchain disulfide linkage; 2) the N-terminal portion of the collagen-like domain is required for specific inhibition of surfactant secretion but not for binding to liposomes or for enhanced uptake of phospholipids into type II cells; 3) N-terminal interchain disulfide linkage can functionally replace the N-terminal segment for lipid binding, receptor binding, and enhancement of lipid uptake; 4) the N-terminal segment is required for the association of trimeric subunits into higher oligomers, for phospholipid aggregation, and for specific inhibition of surfactant secretion and cannot be functionally replaced by disulfide linkage alone for these activities.     

Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication

Interactions of the DnaK (Hsp70) chaperone from Escherichia coli with substrates are controlled by ATP. Nucleotide-induced changes in DnaK conformation were investigated by monitoring changes in tryptic digestion pattern and tryptophan fluorescence. Using nucleotide-free DnaK preparations, not only the known ATP-induced major changes in kinetics and pattern of proteolysis but also minor ADP-induced changes were detected. Similar ATP-induced conformational changes occurred in the DnaK-T199A mutant protein defective in ATPase activity, demonstrating that they result from binding, not hydrolysis, of ATP. N-terminal sequencing and immunological mapping of tryptic fragments of DnaK identified cleavage sites that, upon ATP addition, appeared within the proposed C-terminal substrate binding region and disappeared in the N-terminal ATPase domain. They hence reflect structural alterations in DnaK correlated to substrate release and indicate ATP-dependent domain interactions. Domain interactions are a prerequisite for efficient tryptic degradation as fragments of DnaK comprising the ATPase and C-terminal domains were highly protease-resistant. Fluorescence analysis of the N-terminally located single tryptophan residue of DnaK revealed that the known ATP-induced alteration of the emission spectrum, proposed to result directly from conformational changes in the ATPase domain, requires the presence of the C-terminal domain and therefore mainly results from altered domain interaction. Analyses of the C-terminally truncated DnaK163 mutant protein revealed that nucleotide-dependent interdomain communication requires a 15-kDa segment assumed to constitute the substrate binding site.     

Plasmodium vivax: epitope mapping of monoclonal antibodies against the N-terminal region of the merozoite surface protein 1

Plasmodium vivax is the most widely distributed human malaria with an estimate of 35 million cases per year. The deduced amino acid sequence comparisons of the Merozoite Surface Protein 1 (MSP1) from several plasmodial species, including that of P. vivax (PvMSP1), revealed the existence of highly conserved blocks and polymorphic blocks. We had previously shown that sequences within conserved blocks from the N-terminal region of the PvMSP1 were poorly immunogenic in natural human infections. These results suggest that these regions code for important and unknown structural and/or functional features and thus they could potentially be tested as a sub-unit PvMSP1 vaccine. In the present study, a battery of monoclonal antibodies (Mabs) was produced against the N-terminal region of the PvMSP1 in an attempt to determine whether these N-terminal ICBs contained all the epitopes exposed on the native molecule. The results suggest that the most terminal ICB2 and ICB3 blocks are not exposed on the surface of the PvMSP1 native molecule and clearly eliminate the possibility of considering the N-terminal domains as unique components of a sub-unit PvMSP1 vaccine candidate.     

Mutant huntingtin expression in clonal striatal cells: dissociation of inclusion formation and neuronal survival by caspase inhibition

Neuronal intranuclear inclusions are found in the brains of patients with Huntington's disease and form from the polyglutamine-expanded N-terminal region of mutant huntingtin. To explore the properties of inclusions and their involvement in cell death, mouse clonal striatal cells were transiently transfected with truncated and full-length human wild-type and mutant huntingtin cDNAs. Both normal and mutant proteins localized in the cytoplasm, and infrequently, in dispersed and perinuclear vacuoles. Only mutant huntingtin formed nuclear and cytoplasmic inclusions, which increased with polyglutamine expansion and with time after transfection. Nuclear inclusions contained primarily cleaved N-terminal products, whereas cytoplasmic inclusions contained cleaved and larger intact proteins. Cells with wild-type or mutant protein had distinct apoptotic features (membrane blebbing, shrinkage, cellular fragmentation), but those with mutant huntingtin generated the most cell fragments (apoptotic bodies). The caspase inhibitor Z-VAD-FMK significantly increased cell survival but did not diminish nuclear and cytoplasmic inclusions. In contrast, Z-DEVD-FMK significantly reduced nuclear and cytoplasmic inclusions but did not increase survival. A series of N-terminal products was formed from truncated normal and mutant proteins and from full-length mutant huntingtin but not from full-length wild-type huntingtin. One prominent N-terminal product was blocked by Z-VAD-FMK. In summary, the formation of inclusions in clonal striatal cells corresponds to that seen in the HD brain and is separable from events that regulate cell death. N-terminal cleavage may be linked to mutant huntingtin's role in cell death.     

Functional analysis of human mitochondrial receptor Tom20 for protein import into mitochondria

The mitochondrial import receptor translocase of the outer membrane of mitochondria (Tom20) consists of five segments, an N-terminal membrane-anchor segment, a linker segment rich in charged amino acids, a tetratricopeptide repeat motif, a glutamine-rich segment, and a C-terminal segment. To assess the role of each segment, four C-terminally truncated mutants of the human receptor (hTom20) were constructed, and the effect of their overexpression in COS-7 cells was analyzed. Expression of a mutant lacking the tetratricopeptide repeat motif inhibited preornithine transcarbamylase (pOTC) import to the same extent as the wild-type receptor. Thus, overexpression of the membrane-anchor and the linker segments is sufficient for the inhibition of import. Expression of either the wild-type receptor or a mutant lacking the C-terminal end of 20 amino acid residues stimulated import of pOTC-green fluorescent protein (GFP), a fusion protein in which the presequene of pOTC was fused to green fluorescent protein. On the other hand, expression of mutants lacking either the glutamine-rich segment or larger deletions inhibited pOTC-GFP import. In vitro import of pOTC was inhibited by the wild-type hTom20 and the mutant lacking the C-terminal end, but much less strongly by the mutant lacking the glutamine-rich segment. On the other hand, import of pOTC-GFP was little affected by any of the forms of hTom20. In binding assays, pOTC binding to hTom20 was only moderately decreased by the deletion of the glutamine-rich segment, whereas pOTC-GFP binding was completely lost by this deletion. Binding of pOTCN-GFP a construct that contains an additional 58 N-terminal residues of mature OTC, resembled that of pOTC. All of these results indicate that the region 106-125 containing the glutamine-rich segment of hTom20 is essential for binding and import stimulation in vivo of pOTC-GFP and for inhibition of in vitro import of pOTC. The results also indicate that this region is important for mitochondrial aggregation. The different behaviors of pOTC and the pOTC-GFP chimera toward hTom20 mutants is explicable on the basis of the conformation of the precursor proteins.     

A controlled trial of the effects of pattern of alcohol intake on serum lipid levels in regular drinkers

Four minor protein components were detected in whey from Romagnola cows' milk by polyacrylamide gel isoelectric focusing and two dimensional gel electrophoresis. Individual protein spots were transferred by electroblotting on to a polyvinylidene difluoride membrane and isolated by cutting out the relevant area. After in situ trypsinolysis, a portion of the digest was analysed directly by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass profile allowed us to establish a correlation between beta-lactoglobulins A and B and the four minor whey protein components. They were identified as C-terminally truncated beta-lactoglobulin A and B variants with missing N-terminal peptides, beyond residues in the range 100-123 and 136-147 respectively. Two of the minor components were related to beta-lactoglobulin A and two to beta-lactoglobulin B.     

Molecular properties and activity of a carboxyl-terminal truncated form of xylanase 3 from Aeromonas caviae W-61

Aeromonas caviae W-61 produces five species of xylanases, xylanases 1, 2, 3, 4, and 5 [Nguyen, V.D. et al., Biosci. Biotechnol. Biochem., 56, 1708-1712 (1993) and Appl. Environ. Microbiol., 57, 445-449 (1991)]. While preserving a purified xylanase 3 preparation from A. caviae in solution at 4 degrees C, the xylanase 3 was found to be proteolyzed to give a truncated form with a smaller molecular mass than that of the intact one. It appears likely that the truncated form of xylanase 3 was produced in this particular purification experiment by the action of a contaminating protease. We isolated the truncated form of xylanase 3 (Xyn3tr), of which the C-terminal 102-residue segment is missing. By the chemical analysis of the N- and C-terminal amino acid residues of Xyn3tr and the DNA sequencing analysis of the xylanase 3 gene (xyn3), the N-terminal 398th proline residue of xylanase 3 was found to be the C-terminus of Xyn3tr. Xyn3tr had the activity to form xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) as main final products from oat spelt xylan. In contrast, intact xylanase 3 released X6 and higher xylo-oligosaccharides as main products. Xylanase 3 hydrolysed X4 through X6. However, Xyn3tr had no activity towards X4 and X5. The recombinant Xyn3tr and recombinant xylanase 3 (XYN3) were purified homogeneously from the periplasmic space of E. coli harboring the plasmids pXYN3 and pXYN3tr, which include xyn3 and xyn3tr genes, respectively, and their enzymatic activities were measured. The cleavage patterns of oat spelt and xylo-oligosaccharides by XYN3tr were identical with that by intact Xyn3tr. Thus, we conclude that the C-terminal region comprising a 102-residue segment in xylanase 3 is involved in governing the molecular size of xylo-oligosaccharides cleaved from beta-1,4-xylan by the enzyme and in the hydrolytic activity towards X4 and X5.     

Sensitivity of aerosol bolus behavior to methacholine-induced bronchoconstriction

Thanatin is the first inducible insect peptide that has been found to have, at physiological concentrations, a broad range of activity against bacteria and fungi. Thanatin contains 21 amino acids including two cysteine residues that form a disulfide bridge. Two-dimensional (2D) 1H-NMR spectroscopy and molecular modelling have been used to determine its three-dimensional (3D) structure in water. Thanatin adopts a well-defined anti-parallel beta-sheet structure from residue 8 to the C-terminus, including the disulfide bridge. In spite of the presence of two proline residues, there is a large degree of structural variability in the N-terminal segment. The structure of thanatin is quite different from the known structures of other insect defence peptides, such as antibacterial defensin and antifungal drosomycin. It has more similarities with the structures of various peptides from different origins, such as brevinins, protegrins and tachyplesins, which have a two-stranded beta-sheet stabilized by one or two disulfide bridges. Combined with activity test experiments on several truncated isoforms of thanatin, carried out by Fehlbaum et al. [Fehlbaum, P., Bulet, P., Chernysh, S., Briand, J. P., Roussel, J. P., Letellier, L., Hétru, C. & Hoffmann, J. (1996) Proc. Natl Acad. Sci. USA 93, 1221-1225], our structural study evidences the importance of the beta-sheet structure and also suggests that anti-Gram-negative activity involves a site formed by the Arg20 side-chain embedded in a hydrophobic cluster.     

Cutting at the right place--the importance of selective limited proteolysis in the activation of proproteinase E

Proteinase E is a proteolytic enzyme which belongs to a distinct subfamily of chymotrypsin-like serine endopeptidases. Its proform from the bovine pancreatic system has been structurally analyzed by X-ray crystallography for the intact native form, with a 11-residue N-terminal activation peptide, in a ternary complex with chymotrypsinogen C and procarboxypeptidase A [Gomis-Rüth, F. X., Gómez, M., Bode, W., Huber, R. & Avilés, F. X. (1995) The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C, EMBO J. 14, 4387-4394]. Also for a N-terminally truncated form, lacking the first 13 residues and called subunit III, a crystal structure is available [Pignol, D., Gaboriaud, C., Michon, T., Kerfelec, B., Chapus, C. & Fontecilla-Camps, J. C. (1994) Crystal structure of bovine procarboxypeptidase A-S6 subunit III, a highly structured truncated zymogen E, EMBO J. 8, 1763-1771]. Both structures are well defined by electron density, except for the first 7 residues of subunit III. However, both structures present large deviations of up to 2 nm in several regions, indicating that they correspond to two quite distinct states of low free energy, influenced by very few contacts made via the N-terminal segment. As no structure of an active proteinase E is known so far, pancreatic porcine elastase has been chosen as a model for this enzyme and an activation mechanism for this distinct serine endopeptidase subfamily is proposed.     

Identification of binding domains of the growth hormone-releasing hormone receptor by analysis of mutant and chimeric receptor proteins

The hypothalamic peptide GH-releasing hormone (GHRH) stimulates the release of GH from the pituitary through binding and activation of the GHRH receptor, which belongs to the family of G protein-coupled receptors. The objective of this study was to identify regions of the receptor critical for interaction with the ligand by expressing and analyzing truncated and chimeric epitope-tagged GHRH receptors. Two truncated receptors, GHRHdeltaN, in which part of the N-terminal domain between the putative signal sequence and the first transmembrane domain was deleted, and GHRHdeltaC, which was truncated downstream of the first intracellular loop, were generated. Both the receptors were deficient in ligand binding, indicating that neither the N-terminal extracellular domain (N terminus) nor the membrane-spanning domains with the associated extracellular loops (C terminus) are alone sufficient for interaction with GHRH. In subsequent studies, chimeric proteins between the receptors for GHRH and vasoactive intestinal peptide (VIP) or secretin were generated, using the predicted start of the first transmembrane domain as the junction for the exchange of the N terminus between receptors. The chimeras having the N terminus of the GHRH receptor and the C terminus of either the VIP or secretin receptor (GNVC and GNSC) did not bind GHRH or activate adenylate cyclase after GHRH treatment. The reciprocal chimeras having the N terminus of either the VIP or secretin receptors and the C terminus of the GHRH receptor (VNGC and SNGC) bound GHRH and stimulated cAMP accumulation after GHRH treatment. These results suggest that although the N-terminal extracellular domain is essential for ligand binding, the transmembrane domains and associated extracellular loop regions of the GHRH receptor provide critical information necessary for specific interaction with GHRH.     

Phosphorylation-induced distance change in a cardiac muscle troponin I mutant

Phosphorylation of two adjacent serine residues in the unique N-terminal extension of cardiac muscle troponin I (cTnI) is known to decrease the Ca2+-sensitivity of cardiac myofilaments. To probe the structural significance of the N-terminal extension, we have constructed two cTnI mutants each containing a single cysteine: (1) a full-length cTnI mutant (S5C/C81I/C98S) and (2) a truncated cTnI mutant (S9C/C50I/C67S) in which the N-terminal 32 amino acid residues were deleted. We determined the apparent binding constants for the complex formation between IAANS-labeled cardiac troponin C (cTnC) and the two cTnI mutants. The affinities of the cTnC for the truncated cTnI mutant were: (1) 1.5 x 10(6) M(-1) in EGTA, (2) 28.9 x 10(6) M(-1) in Mg2+, and (3) 87.5 x 10(6) M(-1) in Mg2+ + Ca2+. These binding constants were approximately 1.4-fold smaller than the corresponding values obtained with the full-length cTnI mutant, suggesting a very small contribution of the N-terminal extension to the binding of cTnI to cTnC. Cys-5 in the full-length cTnI mutant was labeled with IAANS, and the distribution of the separation between this site and Trp-192 was determined by analysis of the efficiency of fluorescence resonance energy transfer from Trp-192 to IAANS. The following mean distances were obtained with the unphosphorylated full-length mutant: 44.4 A (cTnI alone), 48.3 A (cTnI + cTnC), 46.3 A (cTnI + cTnC in Mg2+), and 51.6 A (cTnI + cTnC in Mg2+ + Ca2+). The corresponding values of the mean distance determined with the phosphorylated full-length cTnI mutant were 35.8, 36.6, 34.8, and 37.3 A. The phosphorylation of cTnI reduced the half-width of the distribution from 9.5 to 3.7 A. Similar but less pronounced decreases of the half-widths were also observed with the phosphorylated cTnI complexed with cTnC in different ionic conditions. Thus, phosphorylation of cTnI resulted in a decrease of 9-12 A in the mean distance between the sites located at the N- and C-terminal portion of cTnI. Our results indicate that phosphorylation elicits a change in the conformation of cTnI which underlies the basis of the phosphorylation-induced modulation of cTnI activity.     

Effect of N-terminal truncation and solution conditions on chemokine dimer stability: nuclear magnetic resonance structural analysis of macrophage inflammatory protein 1 beta mutants

Chemokines (chemotactic cytokines) are a family of immune system proteins, several of which have been shown to block human immunodeficiency virus (HIV) infection in various cell types. While the solved structures of most chemokines reveal protein dimers, evidence has accumulated for the biological activity of individual chemokine monomers, and a debate has arisen regarding the biological role of the chemokine dimer. Concurrent with this debate, several N-terminal truncations and modifications in the CC subfamily of chemokines have been shown to have functional significance, in many cases antagonizing their respective receptors and in some cases retaining the ability to block HIV entry to the cell. As the dimer interface of CC chemokines is located at their N-terminus, a structural study of N-terminally truncated chemokines will address the effect that this type of mutation has on the dimer-monomer equilibrium. We have studied the structural consequences of N-terminal truncation in macrophage inflammatory protein 1 beta (MIP-1 beta), a CC chemokine that has been shown to block HIV infection. Examination of nuclear magnetic resonance (NMR) spectra of a series of N-terminally truncated MIP-1 beta variants reveals that these proteins possess a range of ability to dimerize. A mutant beginning at amino acid Asp6 [termed MIP(6)] has near wild-type dimer properties, while further truncation results in weakened dimer affinity. The mutant MIP(9) (beginning with amino acid Thr9) has been found to exist solely as a folded monomer. Relaxation measurements yield a rotational correlation time of 8.6 +/- 0.1 ns for wild-type MIP-1 beta and 4.5 +/- 0.1 ns for the MIP(9) mutant, consistent with a wild-type dimer and a fully monomeric MIP(9) variant. The presence of physiological salt concentration drastically changes the monomer-dimer equilibrium for both wild-type and most mutant proteins, heavily favoring the dimeric form of the protein. These results have implications for structure-function analysis of existing chemokine mutants as well as for the larger debate regarding the biological existence and activity of the chemokine dimer.     

Force-generating domain of myosin motor

To understand the underlying mechanism of force generation by myosin motor, it is crucial to know which part of the molecule is essential for the process. Recent structure determination of myosin motor domain at atomic resolution has revealed that the domain comprises two smaller domains, the "ATPase domain" consisting of only an N-terminal segment of the heavy chain and the "neck domain" consisting of a long alpha-helix of the heavy chain and two light chains. This atomic structure begs the question of whether both domains are required for force generation. To answer it, we genetically truncated the head to generate a recombinant fragment composed of the "ATPase domain" alone. The truncated head drove sliding movement of actin filaments and generated force in a novel in vitro assay system, which allows us to hold a specific site of the head on a glass surface. These results indicate that the compact ATPase domain functions as a force-generating machinery of the myosin motor.     

Water-soluble nicotinic acetylcholine receptor formed by alpha7 subunit extracellular domains

Water-soluble models of ligand-gated ion channels would be advantageous for structural studies. We investigated the suitability of three versions of the N-terminal extracellular domain (ECD) of the alpha7 subunit of the nicotinic acetylcholine receptor (AChR) family for this purpose by examining their ligand-binding and assembly properties. Two versions included the first transmembrane domain and were solubilized with detergent after expression in Xenopus oocytes. The third was truncated before the first transmembrane domain and was soluble without detergent. For all three, their equilibrium binding affinities for alpha-bungarotoxin, nicotine, and acetylcholine, combined with their velocity sedimentation profiles, were consistent with the formation of native-like AChRs. These characteristics imply that the alpha7 ECD can form a water-soluble AChR that is a model of the ECD of the full-length alpha7 AChR.     

Structural model of the outer vestibule and selectivity filter of the Shaker voltage-gated K+ channel

A new generation of structural models were developed of the outer vestibule and ion-selective portion of the voltage-gated Shaker K+ channel. Some features of these models are similar to those that we have developed previously [Durrel S. R. and Guy H. R. (1992) Biophys. J. 62, 238-250; Guy H. R. (1990) In Monovalent Cations in Biological Systems (Pasternak C. A., Ed.), pp. 31-58, CRC Press, Boca Raton, FL; Guy H. R. and Durell S. R. (1994) In Molecular Evolution of Physiological processes (Fambrough D., Ed.), pp. 197-212, The Rockefeller University Press, NY; Guy H. R. and Durell S. R. (1995) In Ion Channels and Genetic Diseases (Dawson D., Ed.), pp. 1-16, The Rockefeller University Press, NY] and other features were modified to make the models more consistent with recent experimental findings. The first part of the P segment is postulated, as always, to form a short alpha helix that spans only the outer portion of the membrane. The helix is tilted so that its C-terminal is nearer the pore than its N-terminal. The latter part of the P segment, P2, is postulated to have a relatively elongated conformation that is positioned approximately parallel to the axis of the pore. Four of the P2 segments assemble to form an ion-selective region that has two narrow regions; one formed by the Y445 side-chains at the outer entrance of the pore and one formed by the backbone of the T442 residues near the innermost part of the P segments. The S6 segment is postulated to form two alpha helices. The first S6 helix packs next to the P segments in our models. The NMR structures of two scorpion toxins, charybdotoxin and agitoxin 2, have been docked into the models of the outer vestibules. The shape of the outer vestibule has been modeled so that specific toxin-channel residue-residue interactions correspond to those that have been identified experimentally.     

Enhanced tumor cell growth suppression by an N-terminal truncated retinoblastoma protein

The retinoblastoma (RB) gene encodes a nuclear phosphoprotein of 928 amino acids (pRB). Thus far, much effort in RB research has been focused on both the viral oncoprotein-binding domains and the C-terminal domain, whereas little is known about the N-terminal moiety of the protein. We report here that an N-terminal truncated RB protein of approximately 94 kDa (pRB94) exerts more potent cell growth suppression as compared to the full-length pRB protein in a diversity of tumor cell lines examined, including those having a normal endogenous RB gene. Tumor cells transfected with the pRB94-expressing plasmids displayed multiple morphological changes frequently associated with cellular senescence and/or apoptosis. They failed to enter S phase and rapidly died. The pRB94 expressed in recipient tumor cells had a longer half-life than the full-length pRB protein and tended to remain in an active un- or hypophosphorylated form. Since it has also been found that N-terminal truncated RB proteins often accumulated in growth-arrested and/or differentiated tumor cells, we suggest that N-terminal truncation of pRB may be one of the cellular mechanisms modulating the RB protein function in cell-cycle control.     

Lateral dimerization is required for the homophilic binding activity of C-cadherin

Regulation of cadherin-mediated adhesion can occur rapidly at the cell surface. To understand the mechanism underlying cadherin regulation, it is essential to elucidate the homophilic binding mechanism that underlies all cadherin-mediated functions. Therefore, we have investigated the structural and functional properties of the extracellular segment of Xenopus C-cadherin using a purified, recombinant protein (CEC 1-5). CEC 1-5 supported adhesion of CHO cells expressing C-cadherin. The extracellular segment was also capable of mediating aggregation of microspheres. Chemical cross-linking and gel filtration revealed that CEC 1-5 formed dimers in the presence as well as absence of calcium. Analysis of the functional activity of purified dimers and monomers demonstrated that dimers retained substantially greater homophilic binding activity than monomers. These results demonstrate that lateral dimerization is necessary for homophilic binding between cadherin extracellular segments and suggest multiple potential mechanisms for the regulation of cadherin activity. Since the extracellular segment alone possessed significant homophilic binding activity, the adhesive activity of the extracellular segment in a cellular context was analyzed. The adhesion of CHO cells expressing a truncated version of C-cadherin lacking the cytoplasmic tail was compared to cells expressing the wild-type C-cadherin using a laminar flow assay on substrates coated with CEC 1-5. CHO cells expressing the truncated C-cadherin were able to attach to CEC 1-5 and to resist detachment by low shear forces, demonstrating that tailless C-cadherin can mediate basic, weak adhesion of CHO cells. However, cells expressing the truncated C-cadherin did not exhibit the complete adhesive activity of cells expressing wild-type C-cadherin. Cells expressing wild-type C-cadherin remained attached to CEC 1-5 at high shear forces, while cells expressing the tailless C-cadherin did not adhere well at high shear forces. These results suggest that there may be two states of cadherin-mediated adhesion. The first, relatively weak state can be mediated through interactions between the extracellular segments alone. The second strong adhesive state is critically dependent on the cytoplasmic tail.     

Human 15-lipoxygenase gene promoter: analysis and identification of DNA binding sites for IL-13-induced regulatory factors in monocytes

Cystatins are protein inhibitors of papain and related cysteine proteinases. A series of continuous synthetic peptides corresponding to the entire sequence of rat salivary cystatin was used to localize the binding domains of the cystatin to papain. Several synthetic peptides, one from the aminoterminal sequence (peptide 1-24) and others from the carboxylterminal (peptides 66-79, 66-90, 79-90, 79-114), showed binding to papain, but none of the peptides showed inhibition of papain activity. Three recombinant rat salivary cystatin variants (N-terminal truncated protein lacking amino acid residues 1-9; variant 49-53, in which amino acid residues QVVAG of rat salivary cystatin had been replaced with amino acid residues LVL in mutant protein; and variant 65-78, in which amino acid residues 65-78 had been replaced with amino acids PG in mutant protein) were produced using the Escherichia coli expression system pGex-4T. To generate N-terminal truncated protein the desired coding region of the cystatin gene was amplified by polymerase chain reaction (PCR). To produce the variants 49-53 and 65-78, a PCR-based approach of gene splicing by overlap extension was used. Recombinant cystatin proteins were produced as insoluble inclusion bodies as fusion proteins with a glutathione S-transferase (GST) carrier. After solubilization with urea the GST carrier was cleaved from the fusion protein with thrombin and cystatin variants purified by fast liquid chromatography on a MonoQ column. The purified proteins reacted with antibodies to rat salivary cystatin. The N-terminal truncated and variant 49-53 exhibited very little inhibitory activity towards papain, whereas variant 65-78 exhibited papain-inhibitory activity similar to the full-length recombinant cystatin.     

Evidence for an RNA binding region in the Escherichia coli processing endoribonuclease RNase E

The processing endoribonuclease RNase E (Rne), which is encoded by the rne gene, is involved in the maturation process of messenger RNAs and a ribosomal RNA. A number of deletions were constructed in order to assess functional domains of the rne gene product. The expression of the deletion constructs using a T7 promoter/RNA polymerase overproduction system led to the synthesis of truncated Rne polypeptides. The smallest gene fragment in this collection that was able to complement a temperature sensitive rnets mutation and to restore the processing of 9 S RNA was a 2.3-kilobase pair fragment with a 1.9-kilobase pair N-terminal coding sequence that mediated synthesis of a 70.8-kDa polypeptide. Antibodies raised against a truncated 110-kDa polypeptide cross-reacted with the intact rne gene product and with all of the shorter C-terminal truncated polypeptides, indicating that the N-terminal part of the molecule contained strong antigenic determinants. Furthermore, by analyzing the Rne protein and the truncated polypeptides for their ability to bind substrate RNAs, we were able to demonstrate that the central part of the Rne molecule encodes an RNA binding region. Binding to substrate RNAs correlated with the endonucleolytic activity. RNAs that are not substrates for RNase E did not bind to the protein. The two mutated Rne polypeptides expressed from the cloned gene containing either the rne-3071 or ams1 mutation also had the ability to bind 9 S RNA, while their enzymatic function was completely abolished. The data presented here suggest that the endonucleolytic activity is encoded by the N-terminal part of the Rne protein molecule and that the central part of it possesses RNA binding activity.