In vivo analysis of functional domains from villin and gelsolin

Transfected CV1 cells were used to compare the in vivo effects of various domains of villin and gelsolin. These two homologous actin modulating proteins both contain a duplicated severin-like sequence. Villin has in addition a carboxy-terminal domain, the headpiece, which accounts for its bundling activity. The effects of the villin-deleted mutants were compared with those of native villin. Our results show that essential domains of villin required to induce the growth of microvilli and F-actin redistribution are present in the first half of the core and in the headpiece. We also show that the second half of the villin core cannot be exchanged by its homolog in gelsolin. When expressed at high levels of CV1 cells, full length gelsolin completely disrupted stress fibers without change of the cell shape. Addition of the villin headpiece to gelsolin had no effect on the phenotype induced by gelsolin alone. Expression of the first half of gelsolin induced similar modifications as capping proteins and rapid cell mortality; this deleterious effect on the cell structure was also observed when the headpiece was linked to the first half of gelsolin. In cells expressing the second half of gelsolin, a dotted F-actin staining was often seen. Moreover elongated dorsal F-actin structures were observed when the headpiece was linked to the second gelsolin domain. These studies illustrate the patent in vivo severing activity of gelsolin as well as the distinct functional properties of villin core in contrast to gelsolin.     

Identification of a region in segment 1 of gelsolin critical for actin binding.

The actin severing and capping protein gelsolin contains three distinct actin binding sites. The smallest actin binding domain of approximately 15,000 Mr was originally obtained by limited proteolysis and it corresponds to the first of six repeating segments contained in the gelsolin sequence. We have expressed this domain (here termed segment 1 or N150 to define its amino acid length) in Escherichia coli, together with a series of smaller mutants truncated at either N- or C-terminal ends, in an attempt to localize residues critical of actin binding. Limited truncation of segment 1 by 11 residues at its N-terminal end has no observable effect on actin binding, but on removal of a further eight residues, actin binding is totally eliminated. Although this loss of actin binding may reflect ablation of critical residues, we cannot rule out the possibility that removal of these residues adversely affects the folding of the polypeptide chain during renaturation. Truncation at the C-terminus of segment 1 has a progressive effect on actin binding. Unlike intact segment 1, which shows no calcium sensitivity of actin binding within the resolution of our assays, a mutant with 19 residues deleted from its C-terminus shows unchanged affinity for actin in the presence of calcium, but approximately 100-fold weaker binding in its absence. Removal of an additional five residues from the C-terminus produces a mutant that binds actin only in calcium. Further limited truncation results in progressively weaker calcium dependent binding and all binding is eliminated when a total of 29 residues has been removed. Although none of the expressed proteins on their own binds calcium, 45Ca is trapped in the complexes, including the complex between actin and segment 1 itself. These results highlight a region close to the C-terminus of segment 1 that is essential for actin binding and demonstrate that calcium plays an important role in the high affinity actin binding by this domain of gelsolin.     

Identification of secreted and cytosolic gelsolin in Drosophila

We have cloned the gene for Drosophila gelsolin. Two mRNAs are produced from this gene by differential splicing. The protein encoded by the longer mRNA has a signal peptide and its electrophoretic mobility when translated in vitro in the presence of microsomes is higher than when it is translated without microsomes. The protein translated from the shorter mRNA does not show this difference. This indicates that Drosophila like vertebrates has two forms of gelsolin, one secreted, the other cytoplasmic. The mRNA for both is present ubiquitously in the early embryo. Later, the cytoplasmic form is expressed in parts of the gut. The RNA for the secreted form is expressed in the fat body, and the secreted protein is abundant in extracellular fluid (hemolymph). The cytoplasmic form of gelsolin co-localizes with F-actin in the cortex of the cells in the embryo and in larval epithelia. However, during cellularization of the blastoderm it is reduced at the base of the cleavage furrow, a structure similar to the contractile ring in dividing cells.     

Identification of complement regulatory domains in vaccinia virus complement control protein

Vaccinia virus encodes a homolog of the human complement regulators named vaccinia virus complement control protein (VCP). It is composed of four contiguous complement control protein (CCP) domains. Previously, VCP has been shown to bind to C3b and C4b and to inactivate the classical and alternative pathway C3 convertases by accelerating the decay of the classical pathway C3 convertase and (to a limited extent) the alternative pathway C3 convertase, as well as by supporting the factor I-mediated inactivation of C3b and C4b (the subunits of C3 convertases). In this study, we have mapped the CCP domains of VCP important for its cofactor activities, decay-accelerating activities, and binding to the target proteins by utilizing a series of deletion mutants. Our data indicate the following. (i) CCPs 1 to 3 are essential for cofactor activity for C3b and C4b; however, CCP 4 also contributes to the optimal activity. (ii) CCPs 1 to 2 are enough to mediate the classical pathway decay-accelerating activity but show very minimal activity, and all the four CCPs are necessary for its efficient activity. (iii) CCPs 2 to 4 mediate the alternative pathway decay-accelerating activity. (iv) CCPs 1 to 3 are required for binding to C3b and C4b, but the presence of CCP 4 enhances the affinity for both the target proteins. These results together demonstrate that the entire length of the protein is required for VCP's various functional activities and suggests why the four-domain structure of viral CCP is conserved in poxviruses.     

Identification of the topology and functional domains of PAQR10

PAQR10 (progestin and adipoQ receptor 10) is a Golgi-localized protein that is able to enhance the retention and activation of Ras proteins in the Golgi apparatus, subsequently leading to a sustained ERK (extracellular-signal-regulated kinase) signalling. However, little is known about the topology and functional domains of PAQR10. In the present study, we extensively dissected and analysed the structure of PAQR10. The topology analysis reveals that PAQR10 is an integral membrane protein with its N-terminus facing the cytosol. Multiple domains, including the membrane-proximal region at the N-terminus, the membrane-proximal region at the C-terminus and the three loops facing the cytosol, were found to be required for PAQR10 to reside in the Golgi apparatus, to stimulate ERK phosphorylation and to tether Ras to the Golgi apparatus. Furthermore, when PAQR10 was artificially forced to be expressed in the endoplasmic reticulum, it could neither mobilize Ras to the Golgi apparatus nor increase ERK phosphorylation. Finally, the PAQR10 mutants that lost Golgi localization failed to promote differentiation of PC12 cells. Collectively, the results of the present study indicate that Golgi localization is indispensable for PAQR10 to implement its regulatory functions in the Ras signalling cascade.     

In-frame deletions of BRCA1 may define critical functional domains

The identification of genomic rearrangements involving more than 0.5 kb of the BRCA1 gene has confirmed a more complex mutation spectrum than was initially appreciated. Genomic rearrangements in BRCA1 represent 15% of all mutations in a group of French and American breast and ovarian cancer families and 36% of all mutations in a group of Dutch families. The rearrangements described to date range in size from 510 bp to 23.8 kb, are found throughout the gene, and are most frequently attributable to homologous recombination. We describe the identification of rearrangements in two breast and ovarian cancer families that involve 3.4 and 11.5 kb of the BRCA1 gene and span multiple exons but maintain the reading frame. Both gene rearrangements appear to result from Alu-mediated homologous recombination and have been detected by using a combination of protein truncation analysis and Southern blot analysis. These rearrangements result in the loss of amino acids that lie at the carboxy-terminus of the protein and that have previously been shown to have functional significance. Because these rearrangements result in the deletion of exons but maintain the reading frame, they may provide insights into specific regions and amino acids that have functional significance for the BRCA1 protein.     

Identification of domains in IL-16 critical for biological activity.

IL-16 is a proinflammatory cytokine implicated in the pathogenesis of asthma and other conditions characterized by recruitment of CD4+ T cells to sites of disease. It is postulated that CD4 is an IL-16 receptor, although other receptors or coreceptors may exist. Among several known functions, IL-16 is a chemoattractant factor for CD4+ T cells and it inhibits MLR. We previously reported that an oligopeptide corresponding to the 16 C-terminal residues of human IL-16 inhibits chemoattractant activity. To identify functional domains with greater precision, shorter oligonucleotides containing native or mutated C-terminal IL-16 sequences were tested for IL-16 inhibition. Within the 16 C-terminal residues, the minimal peptide RRKS (corresponding to Arg106 to Ser109) was shown to mediate inhibition of IL-16 chemoattractant activity. Inhibition was lost when either arginine was substituted with alanine. Point mutations in IL-16 revealed that Arg107 is critical for chemoattractant activity, but MLR inhibition was unaffected by mutation of Arg107 or even deletion of the C-terminal tail through Arg106. Deletion of 12 or 22 N-terminal residues of IL-16 had no impact on chemoattractant activity, but MLR inhibition was reduced. Deletion of 16 C-terminal plus 12 N-terminal residues abolished both chemoattractant and MLR-inhibitory activity of IL-16. These data indicate that receptor interactions with IL-16 that activate T cell migration are not identical with those required for MLR inhibition, and suggest that both N-terminal and C-terminal domains in IL-16 participate in receptor binding or activation.     

Identification of functional domains of Clostridium septicum alpha toxin

Alpha toxin (AT) is the major virulence factor of Clostridium septicum that is a proteolytically activated pore-forming toxin that belongs to the aerolysin-like family of toxins. AT is predicted to be a three-domain molecule on the basis of its functional and sequence similarity with aerolysin, for which the crystal structure has been determined. In this study, we have substituted the entire primary structure of AT with alanine or cysteine to identify those amino acids that comprise functional domains involved in receptor binding, oligomerization, and pore formation. These studies revealed that receptor binding is restricted to domain 1 of the AT structure, whereas domains 1 and 3 are involved in oligomerization. These studies also revealed the presence of a putative functional region of AT proximal to the receptor-binding domain but distal from the pore-forming domain that is proposed to regulate the insertion of the transmembrane beta-hairpin of the prepore oligomer.     

Capping and dynamic relation between domains 1 and 2 of gelsolin

Gelsolin is a protein that severs and caps actin filaments. The two activities are located in the N-terminal half of the gelsolin molecules. Severing and subsequent capping requires the binding of domains 2 and 3 (S2–3) to the side of the filaments to position the N-terminal domain 1 (S1) at the barbed end of actin (actin subdomains 1 and 3). The results provide a structural basis for the gelsolin capping mechanism. The effects of a synthetic peptide derived from the sequence of a binding site located in gelsolin S2 on actin properties have been studied. CD and IR spectra indicate that this peptide presented a secondary structure in solution which would be similar to that expected for the native full length gelsolin molecule. The binding of the synthetic peptide induces conformational changes in actin subdomain 1 and actin oligomerization. An increase in the polymerization rate was observed, which could be attributed to a nucleation kinetics effect. The combined effects of two gelsolin fragments, the synthetic peptide derived from an S2 sequence and the purified segment 1 (S1), were also investigated as a molecule model. The two fragments induced nucleation enhancement and inhibited actin depolymerization, two characteristic properties of capping. In conclusion, for the first time it is reported that the binding of a small synthetic fragment is sufficient to promote efficient capping by S1 at the barbed end of actin filaments. ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of functional domains of the Escherichia coli SeqA protein

The Escherichia coli SeqA protein, a negative regulator of chromosomal DNA replication, prevents the overinitiation of replication within one cell cycle by binding to hemimethylated G-mA-T-C sequences in the replication origin, oriC. In addition to the hemimethylated DNA-binding activity, the SeqA protein has a self-association activity, which is also considered to be essential for its regulatory function in replication initiation. To study the functional domains responsible for the DNA-binding and self-association activities, we performed a deletion analysis of the SeqA protein and found that the N-terminal (amino acid residues 1-59) and the C-terminal (amino acid residues 71-181) regions form structurally distinct domains. The N-terminal domain, which is not involved in DNA binding, has the self-association activity. In contrast, the C-terminal domain, which lacks the self-association activity, specifically binds to the hemimethylated G-mA-T-C sequence. Therefore, two essential SeqA activities, self-association and DNA-binding, are independently performed by the structurally distinct N-terminal and C-terminal domains, respectively.     

Identification of functional domains in herpes simplex virus 2 glycoprotein B

Glycoprotein B (gB) is one of four membrane proteins that are essential for the entry of herpes simplex viruses (HSV) into cells, and coexpression of the same combination of proteins in transfected cells results in cell fusion. The latter effect is reminiscent of the ability of virus infection to cause cell fusion, particularly since the degree of fusion is greatly increased by syncytial mutations in gB. Despite intensive efforts with the gB homologs of HSV and some other herpesviruses, information about functionally important regions in the 700-amino-acid ectodomain of this protein is very limited at present. This is largely due to the misfolding of the majority of the mutants examined. It was shown previously that the percentage of correctly folded mutants could be increased by targeting only predicted loop regions (i.e., not alpha-helix or beta-strand), and by using this approach new functional domains in HSV-2 gB have now been identified.     

Identification of regulatory domains in ADP-ribosyltransferase-1 that determine transferase and NAD glycohydrolase activities

Mono-ADP-ribosyltransferases (ART1-7) transfer ADP-ribose from NAD+ to proteins (transferase activity) or water (NAD glycohydrolase activity). The mature proteins contain two domains, an alpha-helical amino terminus and a beta-sheet-rich carboxyl terminus. A basic region in the carboxyl termini is encoded in a separate exon in ART1 and ART5. Structural motifs are conserved among ART molecules. Successive amino- or carboxyl-terminal truncations of ART1, an arginine-specific transferase, identified regions that regulated transferase and NAD glycohydrolase activities. In mouse ART1, amino acids 24-38 (ART-specific extension) were needed to inhibit both activities; amino acids 39-45 (common ART coil) were required for both. Successive truncations of the alpha-helical region reduced transferase and NAD glycohydrolase activities; however, truncation to residue 106 enhanced both. Removal of the carboxyl-terminal basic domain decreased transferase, but enhanced NAD glycohydrolase, activity. Thus, amino- and carboxyl-terminal regions of ART1 are required for transferase activity. The enhanced glycohydrolase activity of the shorter mutants indicates that sequences, which are not part of the NAD binding, core catalytic site, exert structural constraints, modulating substrate specificity and catalytic activity. These functional domains, defined by discrete exons or structural motifs, are found in ART1 and other ARTs, consistent with conservation of structure and function across the ART family.     

Evidence for functional homology in the F-actin binding domains of gelsolin and alpha-actinin: implications for the requirements of severing and capping

The F-actin binding domains of gelsolin and alpha-actinin compete for the same site on actin filaments with similar binding affinities. Both contain tandem repeats of approximately 125 amino acids, the first of which is shown to contain the actin-binding site. We have replaced the F-actin binding domain in the NH2-terminal half of gelsolin by that of alpha-actinin. The hybrid severs filaments almost as efficiently as does gelsolin or its NH2-terminal half, but unlike the latter, requires calcium ions. The hybrid binds two actin monomers and caps the barbed ends of filaments in the presence or absence of calcium. The cap produced by the hybrid binds with lower affinity than that of gelsolin and is not stable: It dissociates from filament ends with a half life of approximately 15 min. Although there is no extended sequence homology between these two different F-actin binding domains, our experiments show that they are functionally equivalent and provide new insights into the mechanism of microfilament severing.     

Two distinct domains of protein 4.1 critical for assembly of functional nuclei in vitro

Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly.     

Identification of two active functional domains of human adenylate kinase 5

A full length cDNA that partially corresponded to human adenylate kinase 5 (AK5) was identified and shown to encode for two separate domains. The full length protein could be divided in two distinct functional domains, a previously unidentified domain of 338 amino acids and a second domain of 198 amino acids that corresponded to the protein characterized as AK5, now called AK5p2. The first domain, AK5p1, phosphorylated AMP, CMP, dAMP and dCMP with ATP or GTP as phosphate donors similarly to AK5p2. Our data demonstrate that human AK5 has two separate functional domains and that both have enzymatic activity.     

Identification of functional domains in sarcoglycans essential for their interaction and plasma membrane targeting

Mutations in sarcoglycans have been reported to cause autosomal-recessive limb-girdle muscular dystrophies. In skeletal and cardiac muscle, sarcoglycans are assembled into a complex on the sarcolemma from four subunits (alpha, beta, gamma, delta). In this report, we present a detailed structural analysis of sarcoglycans using deletion study, limited proteolysis and co-immunoprecipitation. Our results indicate that the extracellular regions of sarcoglycans consist of distinctive functional domains connected by proteinase K-sensitive sites. The N-terminal half domains are required for sarcoglycan interaction. The C-terminal half domains of beta-, gamma- and delta-sarcoglycan consist of a cysteine-rich motif and a previously unrecognized conserved sequence, both of which are essential for plasma membrane localization. Using a heterologous expression system, we demonstrate that missense sarcoglycan mutations affect sarcoglycan complex assembly and/or localization to the cell surface. Our data suggest that the formation of a stable complex is necessary but not sufficient for plasma membrane targeting. Finally, we provide evidence that the beta/delta-sarcoglycan core can associate with the C-terminus of dystrophin. Our results therefore generate important information on the structure of the sarcoglycan complex and the molecular mechanisms underlying the effects of various sarcoglycan mutations in muscular dystrophies.     

Identification and characterization of two functional domains of the hemolysin translocator protein HlyD

Secretion of Escherichia coli hemolysin is mediated by a sec-independent pathway which requires the products of at least three genes, hlyB, hlyD and tolC. Two regions of HlyD were studied. The first region (region A), consisting of the 33-amino acid, C-terminal part of the HlyD protein, is predicted to form a potential helix-loop-helix structure. This sequence is conserved among HlyD analogues of similar transport systems of other bacterial species. Using site-directed mutagenesis, we showed that the amino acids Leu475, Glu477 and Arg478 of this region are essential for HlyD function. The last amino acid of HlyD, Arg478, is possibly involved in the release of the HlyA protein, since cells bearing a hlyD gene mutant at this position produce similar amounts of HlyA to the wild-type strain, but most of the protein remains cell-associated. Competition experiments between wild-type and mutant HlyD proteins indicate that region A interacts directly with a component of the secretion apparatus. The second region of HIyD (region B), located between amino acids Leul27 and Leu170, is highly homologous to the otherwise unrelated outer membrane protein TolC. Deletion of this region abolishes secretion of hemolysin. This sequence of HlyD also seems to interact with a component of the hemolysin secretion machinery since a hybrid HIyD protein carrying the corresponding TolC sequence, although inactive in the transport of HlyA, is able to displace wild-type HlyD from the secretion apparatus.