Clara cell 10 kDa protein (CC10): comparison of structure and function to uteroglobin

The cellular localization, functional activities and structures of rat and human Clara cell 10 kDa proteins (CC10) are compared to rabbit uteroglobin. CC10 is present exclusively in the non-ciliated cells of the surface epithelium of the pulmonary airways, whereas uteroglobin is reported to be present in the lung and reproductive organs. There is about 55% identity between the amino acid sequences of rat CC10 and either rabbit uteroglobin or human CC10. The latter two have 61% identity. Using the known structure of uteroglobin as the model, correlations between the structure and function for this group of proteins are made. Substitution of the residues for the rat and human CC10 into the structure of uteroglobin suggests that these proteins may be members of a structurally homologous family. Some of the functional differences may be due to distortion of the hydrophobic pocket in the dimeric protein and a surface hypervariability located on one contiguous helix and beta turn. Rat CC10 and rabbit uteroglobin both, nearly equally, inhibit papain and bind progesterone. Human CC10 does not inhibit papain and has markedly lower progesterone binding (4.6% of rabbit uteroglobin). Antiinflammatory activity of synthetic peptides corresponding to a homologous sequence region of uteroglobin and the two Clara cell proteins was tested. The region chosen has sequence similarity to lipocortin I. The peptides not only failed to inhibit carrageenan-induced foot pad swelling but exacerbated it. All three proteins inhibit pancreatic phospholipase A2. The phospholipase A2 inhibitory effect of CC10 may be important in regulating the inflammatory responses in the lung.     

Inhibition of pancreatic phospholipase A2 activity by uteroglobin and antiflammin peptides: possible mechanism of action

We investigated the possible mechanism of inhibition of porcine pancreatic phospholipase A2 in vitro by rabbit uteroglobin and by the antiflammin peptides. We optimized the conditions of phospholipase A2 assay using a deoxycholate-phosphatidylcholine mixed micellar substrate and established the activity of these inhibitors under optimized conditions. The results of fluorescence studies and crosslinking experiments indicate that the inhibitors interact with the enzyme in solution and affect the increase in intrinsic fluorescence of phospholipase A2 observed upon interaction with a mixed micellar substrate. In addition, we identified a sequence similarity between the antiflammin peptides, the putative active region of uteroglobin and a region in pancreatic phospholipase A2. This region of phospholipase A2 has been previously identified as being involved in the regulation of dimerization of this enzyme, and is conserved in the pancreatic-type enzymes. Taken together, these observations suggest that uteroglobin and antiflammins interact with porcine pancreatic phospholipase A2 and this may, at least in part, explain the enzyme inhibitory effect of these molecules observed in vitro. One possible mechanism of this effect may be an interference with the dimerization process of phospholipase A2 which is associated with interfacial activation.     

Amino acid sequence analysis of the annexin super-gene family of proteins.

The annexins are a widespread family of calcium-dependent membrane-binding proteins. No common function has been identified for the family and, until recently, no crystallographic data existed for an annexin. In this paper we draw together 22 available annexin sequences consisting of 88 similar repeat units, and apply the techniques of multiple sequence alignment, pattern matching, secondary structure prediction and conservation analysis to the characterisation of the molecules. The analysis clearly shows that the repeats cluster into four distinct families and that greatest variation occurs within the repeat 3 units. Multiple alignment of the 88 repeats shows amino acids with conserved physicochemical properties at 22 positions, with only Gly at position 23 being absolutely conserved in all repeats. Secondary structure prediction techniques identify five conserved helices in each repeat unit and patterns of conserved hydrophobic amino acids are consistent with one face of a helix packing against the protein core in predicted helices a, c, d, e. Helix b is generally hydrophobic in all repeats, but contains a striking pattern of repeat-specific residue conservation at position 31, with Arg in repeats 4 and Glu in repeats 2, but unconserved amino acids in repeats 1 and 3. This suggests repeats 2 and 4 may interact via a buried saltbridge. The loop between predicted helices a and b of repeat 3 shows features distinct from the equivalent loop in repeats 1, 2 and 4, suggesting an important structural and/or functional role for this region. No compelling evidence emerges from this study for uteroglobin and the annexins sharing similar tertiary structures, or for uteroglobin representing a derivative of a primordial one-repeat structure that underwent duplication to give the present day annexins. The analyses performed in this paper are re-evaluated in the Appendix, in the light of the recently published X-ray structure for human annexin V. The structure confirms most of the predictions and shows the power of techniques for the determination of tertiary structural information from the amino acid sequences of an aligned protein family.     

Crystal structure of colicin E3 immunity protein: an inhibitor of a ribosome-inactivating RNase

BACKGROUND: Colicins are antibiotic-like proteins of Escherichia coli that kill related strains. Colicin E3 acts as an RNase that specifically cleaves 16S rRNA, thereby inactivating the ribosomes in the infected cell. The producing organism is protected against colicin E3 by a specific inhibitor, the immunity protein Im3, which forms a tight 1:1 complex with colicin E3 and renders it inactive. Crystallographic studies on colicin E3 and Im3 have been undertaken to unravel the structural basis for the ribonucleolytic activity and its inhibition. RESULTS: The crystal structure of Im3 has been determined to a resolution of 1.8 A. The structure consists of a four-standard antiparallel beta sheet flanked by three alpha helices on one side of the sheet. Thr7, Phe9, Phe16 and Phe74 form a hydrophobic cluster on the surface of the protein in the vicinity of Cys47. This cluster is part of a putative binding pocket which also includes nine polar residues. CONCLUSIONS: The putative binding pocket of Im3 is the probable site of interaction with colicin E3. The six acidic residues in the pocket may interact with some of the numerous basic residues of colicin E3. The involvement of hydrophobic moieties in the binding is consistent with the observation that the tight complex can only be dissociated by denaturation. The structure of Im3 resembles those of certain nucleic acid binding proteins, in particular domain II of topoisomerase I and RNA-binding proteins that contain the ribonucleoprotein (RNP) sequence motif. This observation suggests that Im3 has a nucleic acid binding function in addition to binding colicin E3.     

Conformation of uteroglobin fragments.

The conformation of three fragments of uteroglobin in aqueous solution and in the presence of SDS micelles is described. Two of these fragments correspond to helix II and helix III of uteroglobin, the crystal structure of which is made of four helices. The third peptide comprises helices II and III, with the connecting beta-turn. While helix II does not interact strongly with the micelles, helix III adopts a rather clear alpha-helix in this system. The elongation of helix III with the addition of helix II at the N-terminus somewhat stabilizes the ordered structure. It is possible that the beta-turn found in the crystal is also present in solution.     

High level bacterial expression of uteroglobin, a dimeric eukaryotic protein with two interchain disulfide bridges, in its natural quaternary structure

Bacterial expression of eukaryotic proteins is a tool of ever-increasing importance in biochemistry and molecular biology. However, the majority of the recombinant eukaryotic proteins that have been expressed in bacteria are produced as fusion proteins and not in their native conformation. In particular, correct formation of quaternary structures by recombinant proteins in bacterial hosts has been reported very rarely. To our knowledge, correct intracellular formation of multimeric structures containing more than one interchain disulfide bridge has not been reported so far. We have constructed three plasmids which are able to direct expression of recombinant rabbit uteroglobin, a homodimeric protein with two interchain disulfide bridges, in Escherichia coli. Among these, the plasmid pLE103-1, in which the expression of recombinant uteroglobin is controlled by a bacteriophage T7 late promoter, is by far the most efficient. With pLE103-1, recombinant uteroglobin production reached about 10% of total bacterial soluble proteins. This protein accumulated in bacterial cells in dimeric form, as it is naturally found in the rabbit uterus. Recombinant uteroglobin was purified to near-homogeneity and its NH2-terminal amino acid sequence was confirmed to be identical to that of its natural counterpart, except for 2 Ala residues the codons for which were added during the plasmid construction. This protein was found to be as active a phospholipase A2 inhibitor as natural uteroglobin on a molar basis. To our knowledge, this is the first report of high level bacterial expression of a full length eukaryotic homodimeric protein with two interchain disulfide bridges in its natural, biologically active form. The plasmid pLE103-1 may be useful to explore structure-function relationships of rabbit uteroglobin. In addition, this plasmid may be useful in obtaining high level bacterial expression of other eukaryotic proteins with quaternary structure, as well as for other general applications requiring efficient bacterial expression of cDNAs.     

Colicin E2 release: lysis,leakage or secretion? Possible role of a phospholipase.

Results presented here and by others indicate that the release of colicins from producing cells can be uncoupled from the decline in culture turbidity which usually occurs within 2-3 h after the induction of colicin synthesis. This excludes lysis as a necessary event in colicin release. Conversely, the failure to dissociate colicin release from the normally simultaneous release of a specific subset of soluble proteins argues against the idea of a specific colicin secretion system sensu-stricto. Rather, colicin release appears to be a consequence of semi-specific leakage resulting from an alteration of the permeability properties of the cell envelope. This alteration is caused by the 'lysis protein' known to be encoded by most multiple copy number Col plasmids. The finding that the expression of the lysis gene of plasmid ColE2 renders the cells exquisitely sensitive to lysozyme demonstrates that the permeability of the outer membrane must indeed be altered. Evidence is presented that this alteration could be due at least in part to the activation of the detergent-resistant phospholipase A (pldA product). Lysophosphatidylethanolamine, a product of the action of phospholipase on phosphatidylethanolamine, is a membrane perturbant which could alter the permeability properties of the envelope and allow some proteins such as colicin to leak out of the cell.     

The molecular basis for the pH-activation mechanism in the channel-forming bacterial colicin E1

The in vitro activity of the channel-forming bacteriocins such as colicin E1 in model membranes requires the specific activation of the protein by an acidic environment in the presence of a membrane potential. Acid activation of the C-terminal domain results in the formation of an insertion-competent intermediate with an enhanced ability to penetrate and perforate cell membranes. We report novel findings of this activation process through the design and study of mutant proteins involving the replacement of conserved Asp residues Asp-408, Asp-410, and Asp-423 within helices 5a and 4 in the colicin E1 channel domain that resulted in enhanced membrane binding, bilayer insertion rates, and ion channel activities at near neutral pH values. This activation process involves the destabilization of a critical salt bridge (Asp-410 and Lys-406) and H-bonds (Asp-408 and Ser-405 main chain; Asp-423 and Lys-420 main chain). The helix-to-coil transition of this motif was identified previously by time-resolved Trp fluorescence measurements (Merrill, A. R., Steer, B. A., Prentice, G. A., Weller, M. J., and Szabo, A. G. (1997) Biochemistry 36, 6874-6884), and here we use this approach to demonstrate that disruption of the helical structure of helices 4 and 5a results in a shift in this equilibrium to favor the coil state. Finally, we show that the essential components of the pH trigger motif are conserved among the channel-forming colicins and that it likely exists within other bacterial proteins and may even have evolved into more sophisticated devices in a number of microbial species.     

The binding of methylsulfonyl-polychloro-biphenyls to uteroglobin

The binding of methylsulfonyl-polychloro-biphenyls (methylsulfonyl-PCBs) to purified uteroglobin was studied by a dextran-coated charcoal assay using 4,4'-bis([ 3H]methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl [(3H-MeSO2)2TCB] as radiolabeled ligand. The specific binding of this ligand to uteroglobin was enhanced by the presence of dithiothreitol, and the optimal concentration of dithiothreitol for binding was 20 mM. The specific [(3H-MeSO2)2TCB] binding was inhibited by 4-methylsulfonyl-2,2',4',5,5'-pentachlorobiphenyl in a concentration-dependent manner. The molecular structures of methylsulfonyl-PCBs, and progesterone, were fitted into the X-ray crystallographic structure of uteroglobin using the molecular graphics program TOM. In these simulations the water-accessible surfaces of the ligands appeared quite similar, and fitted nicely in the internal water-accessible surface of uteroglobulin. Moreover, it appeared from the computer-supported ligand-binding studies that the sulfone oxygens of the studied methylsulfonyl-PCBs, as well as the carbonyl (C20) of progesterone, may form hydrogen bonds with the hydroxyl group of TYR 21 of uteroglobulin. These findings may explain why both steroids and methylsulfonyl-PCBs interact with the same protein, although these two types of ligands are structurally dissimilar.     

Functioning of the colicin A lysis protein is affected by Triton X-100, divalent cations and EDTA

The colicin A lysis protein, Cal, is synthesized at the same time as colicin A by Escherichia coli harbouring plasmid pColA after induction by mitomycin C. Its function in the induced bacteria involves the release of colicin A, quasi-lysis, the death of the producing cells and the activation of the outer membrane phospholipase A. We have found that these various functions are affected differently by treatment of the induced cells with Triton X-100, divalent cations or EDTA. Triton X-100 and EDTA caused increased quasi-lysis and a higher level of mortality of the producing cells, but while Triton X-100 enhanced the release of colicin A, EDTA reduced it. Divalent cations protected the cells against both killing and quasi-lysis without greatly affecting colicin release. The effects of these agents were similar for both wild-type and phospholipase A mutants and depended only on the presence of a functional cal gene.     

Membrane topology and predicted RNA-binding function of the 'early responsive to dehydration (ERD4)' plant protein

Functional annotation of uncharacterized genes is the main focus of computational methods in the post genomic era. These tools search for similarity between proteins on the premise that those sharing sequence or structural motifs usually perform related functions, and are thus particularly useful for membrane proteins. Early responsive to dehydration (ERD) genes are rapidly induced in response to dehydration stress in a variety of plant species. In the present work we characterized function of Brassica juncea ERD4 gene using computational approaches. The ERD4 protein of unknown function possesses ubiquitous DUF221 domain (residues 312-634) and is conserved in all plant species. We suggest that the protein is localized in chloroplast membrane with at least nine transmembrane helices. We detected a globular domain of 165 amino acid residues (183-347) in plant ERD4 proteins and expect this to be posited inside the chloroplast. The structural-functional annotation of the globular domain was arrived at using fold recognition methods, which suggested in its sequence presence of two tandem RNA-recognition motif (RRM) domains each folded into βαββαβ topology. The structure based sequence alignment with the known RNA-binding proteins revealed conservation of two non-canonical ribonucleoprotein sub-motifs in both the putative RNA-recognition domains of the ERD4 protein. The function of highly conserved ERD4 protein may thus be associated with its RNA-binding ability during the stress response. This is the first functional annotation of ERD4 family of proteins that can be useful in designing experiments to unravel crucial aspects of stress tolerance mechanism.     

Interchain cysteine bridges control entry of progesterone to the central cavity of the uteroglobin dimer.

The progesterone-binding protein uteroglobin has been expressed in Escherichia coli in an unfused, soluble form. Like mature uteroglobin from rabbit endometrium (UG), the E.coli produced uteroglobin (UG1) dimerizes in vitro, forms an antiparallel dimer with Cys3-Cys69' and Cys69-Cys3' disulfide bonds and binds progesterone under reducing conditions. In order to analyze the dimerization and the reduction dependence of progesterone binding in more detail, we separately replaced cysteine 3 and cysteine 69 by serines. Under reducing conditions, both uteroglobin variants (UG1-3Ser and UG1-69Ser) bind progesterone with the same affinity as the wild-type suggesting that both cysteine residues are not directly involved in progesterone binding. In contrast to the wild-type protein, both cysteine variants also bind progesterone with high affinity in the absence of reducing agents. In addition, UG1-3Ser and UG1-69Ser both form covalently linked homodimers. Thus, unnatural Cys69-69' and Cys3-3' disulfide bonds exist in UG1-3Ser and UG1-69Ser, respectively. These data together with computer models based on X-ray diffraction data strongly support the idea that progesterone reaches its binding site located in an internal hydrophobic cavity via a hydrophobic tunnel along helices 1 and 4. Under non-reducing conditions the tunnel is closed by two disulfide bridges (Cys3-Cys69' and Cys69-Cys3') that lie in the most flexible region of the dimer. Reduction or replacement of a cysteine residue enables conformational changes that open the channel allowing progesterone to enter.     

Immunity proteins to pore-forming colicins: structure-function relationships

Colicin A and B immunity proteins (Cai and Cbi, respectively) are homologous integral membrane proteins that interact within the core of the lipid bilayer with hydrophobic transmembrane helices of the corresponding colicin channel. By using various approaches (exchange of hydrophilic loops between Cai and Cbi, construction of Cbi/Cai hybrids, production of Cai as two fragments), we studied the structure-function relationships of Cai and Cbi. The results revealed unexpectedly high structural constraints for the function of these proteins. The periplasmic loops of Cai and Cbi did not carry the determinants for colicin recognition although most of these loops were required for Cai function; the cytoplasmic loop of Cai was found to be Involved in topology and function of Cai. The immunity function did not seem to be confined to a particular region of the immunity proteins.     

Delineation of the translocation of colicin E7 across the inner membrane of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The lysis protein of the colicinogenic operon is essential for colicin release and its main function is to activate the outer membrane phospholipase A (OMPLA) for the traverse of colicin across the cell envelope. However, little is known about the involvement of the lysis protein in the translocation of colicin across the inner membrane into the periplasm. The introduction of specific point mutations into the lipobox or sorting signal sequence of the lysE7 gene resulted in the production of various forms of lysis proteins. Our experimental results indicated that cells with wild-type mature LysE7 protein exhibited higher efficiency of colicin E7 translocation across the inner membrane into the periplasm than those with premature LysE7 protein. Moreover, the degree of permeability of the inner membrane induced by the mature LysE7 protein was significantly increased as compared to the unmodified LysE7 precursor. These results suggest that the efficiency of colicin movement into the periplasm is correlated with the increase in inner membrane permeability induced by the LysE7 protein. Thus, we propose that mature LysE7 protein has two critical roles: firstly mediating the translocation of colicin E7 across the inner membrane into the periplasm, and secondly activating the OMPLA to allow colicin release.     

Phospholipase-A-independent damage caused by the colicin A lysis protein during its assembly into the inner and outer membranes of Escherichia coli

The requirement for the activation of phospholipase A by the colicin A lysis protein (Cal) in the efficient release of colicin A by Escherichia coli cells containing colicin A plasmids was studied. In particular, we wished to determine if this activation is the primary effect of Cal or whether it reflects more generalized damage to the envelope caused by the presence of large quantities of this small acylated protein. E. coli tolQ cells, which were shown to be leaky for periplasmic proteins, were transduced to pldA and then transformed with the recombinant colicin A plasmid pKA. Both the pldA and pldA+ strains released large quantities of colicin A following induction, indicating that in these cells phospholipase A activation is not required for colicin release. This release was, however, still dependent on a functioning Cal protein. The assembly and processing of Cal in situ in the cell envelope was studied by combining pulse-chase labelling with isopycnic sucrose density gradient centrifugation of the cell membranes. Precursor Cal and lipid-modified precursor Cal were found in the inner membrane at early times of chase, and gave rise to mature Cal which accumulated in both the inner and outer membrane after further chase. The signal peptide was also visible on these gradients, and its distribution too was restricted to the inner membrane. Gradient centrifugation of envelopes of cells which were overproducing Cal resulted in very poor separation of the membranes. The results of these studies provide evidence that the colicin A lysis protein causes phospholipase A-independent alterations in the integrity of the E. coli envelope.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel peptides derived from a region of local homology between uteroglobin and lipocortin-1 inhibit platelet aggregation and secretion

The active site for uteroglobin inhibition of phospholipase A2 has been localized to a nonapeptide (P1) which is partially homologous to a nonapeptide (P2) in lipocortin, which also inhibits phospholipase A2. P1 and P2 share an identical tetrapeptide (P4) which is required for inhibition, although P4 alone does not inhibit this enzyme. We found the mechanism of inhibition of platelet aggregation and secretion by the nonapeptides and P4 varied depending on whether platelets were thrombin- or ADP-activated. All three peptides decrease thrombin esterolytic activity and thereby inhibit thrombin-induced platelet activation. P1 decreases ADP-induced aggregation and serotonin secretion by inhibiting phospholipase A2 whereas P4 decreases only aggregation by blocking fibrinogen binding to activated platelets. The P4 sequence in P1 may affect the interaction of P1 with platelets since the presence of P4 potentiates P1 inhibition of platelet activation.     

Conformation and molecular dynamics calculations on uteroglobin fragment 18-47

The conformational properties of fragment 18–47 of rabbit uteroglobin in aqueous solution containing SDS micelles were investigated by two-dimensional nmr spectroscopy and molecular dynamics calculations. The fragment comprises helices II and III and the β-turn connecting the two helices. The nmr results and nmr-restrained molecular dynamics calculations showed that in the isolated fragment the elements of secondary structure present in the intact protein are preserved only in part. Specifically, a well-defined α-helix was found in the sequence 33–44, corresponding to helix III of uteroglobin, while the regions of helix II and β-turn are characterized by high flexibility in the fragment. © 1994 John Wiley & Sons, Inc.     

Glucocorticoid and developmental regulation of uteroglobin synthesis in rabbit lung.

The synthesis of uteroglobin in rabbit lung was studied after the administration of glucocorticoids to intact adult animals as well as during the late stages of rabbit development. The synthesis of uteroglobin was compared with levels of translatable uteroglobin mRNA in the lung. Uteroglobin synthesis was determined both by incorporation of [25S]methionine into the protein by lung explants incubated in vitro and by radioimmunoassay measurements of uteroglobin concentration in lung. Lung poly(A)-containing mRNA, isolated by oligo(dT)--cellulose chromatography, was translated in cell-free systems and the activity of uteroglobin mRNA was determined after immunoprecipitation. Dexamethasone administration increased about 2-fold the synthesis of lung uteroglobin compared with the controls. The effect of cortisol was more moderate. Both glucocorticoids did not affect the degradation rate of lung uteroglobin, but produced increases in the translatable levels of uteroglobin mRNA parallel to those observed for uteroglobin synthesis. During the late stages of rabbit development, both the synthesis of lung uteroglobin and the translatable levels of its mRNA increase in parallel about 12-fold in a biphasic fashion. A first increase occurred between 2 days before and 2 days after birth. Starting at 5 days of age, there was a second increase in both parameters, which at 12 days of age reached values close to those observed in adult rabbits. Our results suggest that the rate of lung uteroglobin synthesis could be mainly determined by the translatable levels of its mRNA.