Mapping functional domains of colicin M

Colicin M (Cma) lyses Escherichia coli cells by inhibiting murein biosynthesis through hydrolysis of the phosphate ester between C(55)-polyisoprenol and N-acetylmuramyl (MurNAc)-pentapeptide-GlcNAc in the periplasm. To identify Cma functional domains, we isolated 54 point mutants and small deletion mutants and examined their cytotoxicity levels. Activity and uptake mutants were distinguished by osmotic shock, which transfers Cma into the periplasm independent of the specific FhuA receptor and the Ton system. Deletion of the hydrophobic helix α1, which extends from the compact Cma structure, abolished interference with the antibiotic albomycin, which is transported across the outer membrane by the same system as Cma, thereby identifying α1 as the Cma site that binds to FhuA. Deletion of the C-terminal Lys-Arg strongly reduced Cma translocation across the outer membrane after binding to FhuA. Conversion of Asp226 to Glu, Asn, or Ala inactivated Cma. Asp226 is exposed at the Cma surface and is surrounded by Asp225, Asp229, His235, Tyr228, and Arg236; replacement of each with alanine inactivated Cma. We propose that Asp226 directly participates in phosphate ester hydrolysis and that the surrounding residues contribute to the active site. These residues are strongly conserved in Cma-like proteins of other species. Replacement of other conserved residues with alanine inactivated Cma; these mutations probably altered the Cma structure, as particularly apparent for mutants in the unique open β-barrel of Cma, which were isolated in lower yields. Our results identify regions in Cma responsible for uptake and activity and support the concept of a three-domain arrangement of Cma.     

Mapping the functional domains of elongation factor-2 kinase

A new class of eukaryotic protein kinases that are not homologous to members of the serine/threonine/tyrosine protein kinase superfamily was recently identified [Futey, L. M., et al. (1995) J. Biol. Chem. 270, 523-529; Ryazanov, A. G., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4884-4889]. This class includes eukaryotic elongation factor-2 kinase, Dictyostelium myosin heavy chain kinases A, B, and C, and several mammalian putative protein kinases that are not yet fully characterized [Ryazanov, A. G., et al. (1999) Curr. Biol. 9, R43-R45]. eEF-2 kinase is a ubiquitous protein kinase that phosphorylates and inactivates eukaryotic translational elongation factor-2, and thus can modulate the rate of polypeptide chain elongation during translation. eEF-2 was the only known substrate for eEF-2 kinase. We demonstrate here that eEF-2 kinase can efficiently phosphorylate a 16-amino acid peptide, MH-1, corresponding to the myosin heavy chain kinase A phosphorylation site in Dictyostelium myosin heavy chains. This enabled us to develop a rapid assay for eEF-2 kinase activity. To localize the functional domains of eEF-2 kinase, we expressed human eEF-2 kinase in Escherichia coli as a GST-tagged fusion protein, and then performed systematic in vitro deletion mutagenesis. We analyzed eEF-2 kinase deletion mutants for the ability to autophosphorylate, and to phosphorylate eEF-2 as well as a peptide substrate, MH-1. Mutants with deletions between amino acids 51 and 335 were unable to autophosphorylate, and were also unable to phosphorylate eEF-2 and MH-1. Mutants with deletions between amino acids 521 and 725 were unable to phosphorylate eEF-2, but were still able to autophosphorylate and to phosphorylate MH-1. The kinases with deletions between amino acids 2 and 50 and 336 and 520 were able to catalyze all three reactions. In addition, the C-terminal domain expressed alone (amino acids 336-725) binds eEF-2 in a coprecipitation assay. These results suggest that eEF-2 kinase consists of two domains connected by a linker region. The amino-terminal domain contains the catalytic domain, while the carboxyl-terminal domain contains the eEF-2 targeting domain. The calmodulin-binding region is located between amino acids 51 and 96. The amino acid sequence of the carboxyl-terminal domain of eEF-2 kinase displays similarity to several proteins, all of which contain repeats of a 36-amino acid motif that we named "motif 36".     

Mapping the functional domains of nucleolar protein B23

Protein B23 is a multifunctional nucleolar protein whose cellular location and characteristics strongly suggest that it is a ribosome assembly factor. The protein has nucleic acid binding, ribonuclease, and molecular chaperone activities. To determine the contributions of unique polypeptide segments enriched in certain classes of amino acid residues to the respective activities, several constructs that produced N- and C-terminal deletion mutant proteins were prepared. The C-terminal quarter of the protein was shown to be necessary and sufficient for nucleic acid binding. Basic and aromatic segments at the N- and C-terminal ends, respectively, of the nucleic acid binding region were required for activity. The molecular chaperone activity was contained in the N-terminal half of the molecule, with important contributions from both nonpolar and acidic regions. The chaperone activity also correlated with the ability of the protein to form oligomers. The central portion of the molecule was required for ribonuclease activity and possibly contains the catalytic site; this region overlapped with the chaperone-containing segment of the molecule. The C-terminal, nucleic acid-binding region enhanced the ribonuclease activity but was not essential for it. These data suggest that the three activities reside in mainly separate but partially overlapping segments of the polypeptide chain.     

Mapping the functional domains of human transcobalamin using monoclonal antibodies

Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.     

Mapping the functional domains of the Golgi stacking factor GRASP65

The Golgi reassembly stacking protein (GRASP) family has been implicated in the stacking of Golgi cisternae and the regulation of Golgi disassembly/reassembly during mitosis in mammalian cells. GRASP65 is a dimer that can directly link adjacent surfaces through trans-oligomerization in a mitotically regulated manner. Here we show that the N-terminal GRASP domain (amino acids 1-201) is both necessary and sufficient for dimerization and trans-oligomerization but is not mitotically regulated. The C-terminal serine/proline-rich domain (amino acids 202-446) cannot dimerize nor can it link adjacent surfaces. It does, however, confer mitotic regulation on the GRASP domain through multiple sites phosphorylated by the mitotic kinases, cdc2/B1, and the polo-like kinase. Transient expression corroborated these results by showing that the GRASP domain alone inhibited mitotic fragmentation of the Golgi apparatus.     

Mapping two functional domains of clathrin light chains with monoclonal antibodies

The two forms of clathrin light chains (LCA and LCB) or clathrin-associated proteins (CAP1 and CAP2) have presented an immunochemical paradox. Biochemically similar, both possess two known functional parameters: binding the clathrin heavy chain and mediating the action of an uncoating ATPase. All previously reported anti-CAP mAbs, however, react specifically with only CAP1 (Brodsky, F. M., 1985, J. Cell Biol., 101:2047-2054; Kirchhausen, T., S. C. Harrison, P. Parham, and F. M. Brodsky, 1983, Proc. Natl. Acad. Sci. USA, 80:2481-2485). Four new anti-CAP mAbs are reported here: two, C-7H12 and C-6C1, react with both forms; two others, C-10B2 and C-4E5, react only with the lower form. Sandwich ELISAs indicated that C-10B2, C-4E5, C-6C1, and C-7H12 react with distinct epitopes. Monoclonal antibodies C-10B2 and C-4E5 immunoprecipitate clathrin-coated vesicles (CCVs) and react with CAP2 epitopes accessible to chymotrypsin on the vesicle. These mAbs inhibit phosphorylation of CAP2 by endogenous CCV casein kinase II. In contrast, C-6C1 and C-7H12 react with epitopes that are relatively insensitive to chymotrypsin. CAP peptide fragments containing these epitopes remain bound to reassembled cages or CCVs after digestion. Immunoprecipitation and ELISAs demonstrate that C-7H12 and C-6C1 react with unbound CAPs but not with CAPs bound to triskelions or CCVs. The data indicate that the CAPs consist of at least two discernible structural domains: a nonconserved, accessible domain that is relevant to the phosphorylation of CAP2 and a conserved, inaccessible domain that mediates the binding of CAPs to CCVs.     

Mapping of the functional domains in the amino-terminal region of calponin.

Three chymotryptic fragments accounting for almost the entire amino acid sequence of gizzard calponin (Takahashi, K., and Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288) were isolated and characterized. They encompass the segments of residues 7-144 (NH2-terminal 13-kDa peptide), 7-182 (NH2-terminal 22-kDa peptide), and 183-292 (COOH-terminal 13-kDa peptide). They arise from the sequential hydrolysis of the peptide bonds at Tyr182-Gly183 and Tyr144-Ala145 which were protected by the binding of F-actin to calponin. Only the NH2-terminal 13- and 22-kDa fragments were retained by immobilized Ca(2+)-calmodulin, but only the larger 22 kDa entity cosedimented with F-actin and inhibited, in the absence of Ca(2+)-calmodulin, the skeletal actomyosin subfragment-1 ATPase activity as the intact calponin. Since the latter peptide differs from the NH2-terminal 13-kDa fragment by a COOH-terminal 38-residue extension, this difference segment appears to contain the actin-binding domain of calponin. Zero-length cross-linked complexes of F-actin and either calponin or its 22-kDa peptide were produced. The total CNBr digest of the F-actin-calponin conjugate was fractionated over immobilized calmodulin. The EGTA-eluted pair of cross-linked actin-calponin peptides was composed of the COOH-terminal actin segment of residues 326-355 joined to the NH2-terminal calponin region of residues 52-168 which seems to contain the major determinants for F-actin and Ca(2+)-calmodulin binding.     

Mapping functional domains of the guanylate cyclase regulator protein, GCAP-2

Guanylate cyclase regulator protein (GCAP)-2 is a Ca2+-binding protein that regulates photoreceptor outer segment membrane guanylate cyclase (RetGC) in a Ca2+-sensitive manner. GCAP-2 activates RetGC at free Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, and inhibits RetGC when free Ca2+ concentrations are above the 500 nM level, characteristic of dark-adapted photoreceptors. We have mapped functional domains in GCAP-2 by using deletion mutants and chimeric proteins in which parts of GCAP-2 were substituted with corresponding fragments of other closely related recoverin-like proteins that do not regulate RetGC. We find that in addition to the EF-hand Ca2+-binding centers there are three regions that contain GCAP-2-specific sequences essential for regulation of RetGC. 1) The region between Phe78 and Asp113 determines whether GCAP-2 activates outer segment RetGC in low or high Ca2+ concentrations. Substitution of this domain with the corresponding region from neurocalcin causes a paradoxical behavior of the chimeric proteins. They activate RetGC only at high and not at low Ca2+ concentrations. 2) The amino acid sequence of GCAP-2 between Lys29 and Phe48 that includes the EF-hand-related motif EF-1 is essential both for activation of RetGC at low Ca2+ and inhibition at high Ca2+ concentrations. Most of the remaining N-terminal region can be substituted with recoverin or neurocalcin sequences without loss of GCAP-2 function. 3) Region Val171-Asn189, adjacent to the C-terminal EF-4 contributes to activation of RetGC, but it is not essential for the ability of Ca2+-loaded GCAP-2 to inhibit RetGC. Other regions of the molecule can be substituted with the corresponding fragments from neurocalcin or recoverin, or even partially deleted without preventing GCAP-2 from regulating RetGC. Substitution of these three domains in GCAP-2 with corresponding neurocalcin sequences also affects activation of individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells.     

Mapping the functional domains of the eukaryotic elongation factor 1 beta gamma

The functional domains of the eukaryotic elongation factor (EF) 1 beta gamma have been delineated with the use of limited proteolysis, protein microsequencing, gel electrophoresis under non-denaturing conditions and antibodies against EF-1 beta and EF-1 gamma. By means of limited proteolysis, it was possible to obtain large fragments of EF-1 beta. In contrast to amino-terminal fragments, those derived from the carboxy-terminal part of EF-1 beta were still active in enhancing the guanine nucleotide exchange of GDP bound to EF-1 alpha. With the same technique of limited proteolysis, it was possible to isolate a trypsin-resistant core from EF-1 beta gamma containing polypeptide chain fragments derived from both subunits. A polyvalent antiserum against EF-1 beta and two monoclonal antibodies against EF-1 gamma were used to identify the protein fragments in this core. The monoclonal antibodies were shown to recognize different epitopes, one localized on the amino-terminal and another on the carboxy-terminal half of EF-1 gamma. The antiserum against EF-1 beta and one of the monoclonal antibodies (mAb 36E5), which recognized the amino-terminal half of EF-1 gamma, reacted with this trypsin-resistant core. We conclude that the amino-terminal halves of both EF-1 beta and EF-1 gamma are firmly attached to each other, and that the carboxy-terminal part of EF-1 beta interacts with EF-1 alpha.     

Mapping of MCP-1 functional domains by peptide analysis and site-directed mutagenesis

Monocyte chemoattractant protein-1 (MCP-1) is a member of the β chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP-1 protein which are important for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and chemotaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13–35 inhibits MCP-1 binding and chemotactic activity, while peptides representing the amino-terminus (amino acids 1–10), second loop (amino acids 37–51), and carboxy-terminus (amino acids 56–71) of MCP-1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the activity of this peptide to block MCP-1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins. While baculovirus produced wild type and R18A MCP-1 proteins are indistinguishable in their ability to induce THP-1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP-1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases.     

Mapping functional domains of chloride intracellular channel (CLIC) proteins in vivo

Chloride intracellular channel (CLIC) proteins are small proteins distantly related to the omega family of glutathione S-transferases (GSTs). CLIC proteins are expressed in a wide variety of tissues in multicellular organisms and are targeted to specific cellular membranes. Members of this family are capable in vitro of changing conformation from a globular, soluble state to a membrane-inserted state in which they provide chloride conductance. The structural basis for in vivo CLIC protein function, however, is not well understood. We have mapped the functional domains of CLIC family members using an in vivo assay for membrane localization and function of CLIC proteins in the nematode Caenorhabditis elegans. A<70 amino acid N-terminal domain is a key determinant of membrane localization and function of invertebrate CLIC proteins. This domain, which we term the 'PTM' domain, named after an amphipathic putative transmembrane helix contained within it, directs distinct C. elegans CLIC homologs to distinct subcellular membranes. We find that within the PTM region, the cysteine residues required for GST-type activity are unnecessary for invertebrate CLIC function, but that specific residues within the proposed transmembrane helix are necessary for correct targeting and protein function. We find that among all tested invertebrate CLIC proteins, function appears to be completely conserved despite striking differences in the charged residues contained within the amphipathic helix. This indicates that these residues do not contribute to anion selectivity as previously suggested. We find that outside the PTM region, the remaining three-quarters of CLIC protein sequence is functionally equivalent not only among vertebrate and invertebrate CLIC proteins, but also among the more distantly related GST-omega and GST-sigma proteins. The PTM region thus provides both targeting information and CLIC functional specificity, possibly adapting GST-type proteins to function as ion channels.     

Mapping of functional domains within the Saccharomyces cerevisiae type 1 killer preprotoxin.

Strains of Saccharomyces cerevisiae harboring M1-dsRNA, the determinant of type 1 killer and immunity phenotypes, secrete a dimeric 19-kd toxin that kills sensitive yeast cells by the production of cation-permeable pores in the cytoplasmic membrane. The preprotoxin, an intracellular precursor to toxin, has the domain sequence delta-alpha-gamma-beta where alpha and beta are the 9.5-and 9.0-kd subunits of secreted toxin. Plasmids containing a partial cDNA copy of M1, in which alpha, gamma, and beta are fused to the PH05 promoter and signal peptide, have previously been shown to express phosphate-repressible toxin production and immunity. Here the construction of a complete DNA copy of the preprotoxin gene and its mutagenesis are described. Analysis of the expression of these mutants from the PH05 promoter elucidates the functions of the preprotoxin domains. delta acts as a leader peptide and efficiently mediates the secretion, glycosylation and maturation of killer toxin. Mutations within the beta subunit indicate it to be essential for binding of toxin to and killing of whole cells but unnecessary for the killing of spheroplasts. Mutations within the putative active site of alpha prevent killing of both cells and spheroplasts. The probable role of beta is therefore recognition and binding to the cell wall receptor whereas alpha is the active ionophore. Mutations within alpha causing loss of toxicity also cause loss of immunity, while the mutants described within gamma and beta retain partial or complete immunity. Expression of gamma without alpha or beta confers no phenotype. The immunity determinant may minimally consist of the alpha domain and the N-terminal portion of gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping the domains of CD134 as a functional receptor for feline immunodeficiency virus

The feline homologue of CD134 is the primary binding receptor for feline immunodeficiency virus (FIV), targeting the virus preferentially to activated CD4+ helper T cells. However, strains of FIV differ in utilization of CD134; the prototypic strain PPR requires a minimal determinant in the first cysteine-rich domain (CRD1) of feline CD134 to confer near-optimal receptor function, while strains such as GL8 require additional determinants in the CD134 CRD2. We map this determinant to a loop in CRD2 governing the interaction between the receptor and its ligand; the amino acid substitutions S78N-S79Y-K80E restored full viral receptor activity to the CDR2 of human CD134 in the context of feline CD134, with tyrosine-79 appearing to be the critical residue for restoration of receptor function.     

Mapping of functional domains in the plasma membrane Ca2+ pump using trypsin proteolysis

The purified erythrocyte Ca2+ pump has been exposed to trypsin under conditions designed to enrich the fragments of molecular mass 90, 85, 81, and 76 kDa, respectively. In SDS-polyacrylamide gels, these fragments are accompanied by a product of molecular mass about 33 kDa. N- and C-terminal sequencing of the fragments blotted on PVDF membranes has located the four high molecular mass fragments and the 33-kDa fragment within the pump structure. The work has extended previous work on the organization of the calmodulin-interacting domain of the pump (Zurini et al., 1984; Benaim et al., 1984) and has tentatively placed the domain of the pump which interacts with acidic phospholipids between transmembrane helices 2 and 3.     

Mapping the landscape of the lymphocytic choriomeningitis virus stable signal peptide reveals novel functional domains

The stable signal peptide (SSP) of the lymphocytic choriomeningitis virus surface glycoprotein precursor has several unique characteristics. The SSP is unusually long, at 58 amino acids, and contains two hydrophobic domains, and its sequence is highly conserved among both Old and New World arenaviruses. To better understand the functions of the SSP, a panel of point and deletion mutants was created by in vitro mutagenesis to target the highly conserved elements within the SSP. We were also able to confirm critical residues required for separate SSP functions by trans-complementation. Using these approaches, it was possible to resolve functional domains of the SSP. In characterizing our SSP mutants, we discovered that the SSP is involved in several distinct functions within the viral life cycle, beyond translocation of the viral surface glycoprotein precursor into the endoplasmic reticulum lumen. The SSP is required for efficient glycoprotein expression, posttranslational maturation cleavage of GP1 and GP2 by SKI-1/S1P protease, glycoprotein transport to the cell surface plasma membrane, formation of infectious virus particles, and acid pH-dependent glycoprotein-mediated cell fusion.     

Mapping and characterization of the functional domains of the nucleolar protein RNA helicase II/Gu

RNA helicase II/Gu (RH-II/Gu) is a nucleolar RNA helicase of the DEAD-box superfamily. In this study, the functional domains of RH-II/Gu molecule were mapped by fusing the protein or its deletion mutants with a green fluorescence protein and subsequently transfecting or microinjecting the recombinant constructs into HeLa cells. In addition to the identification of a nuclear localization signal (NLS) in the N-terminus and a nucleolar targeting signal in the central helicase domain, a hidden NLS and a nucleolar targeting signal were found in the C-terminal arginine/glycine-rich domain. RH-II/Gu colocalized with fibrillarin, a component of the dense fibrillar region of the nucleolus. Overexpression of the entire RH-II/Gu protein or specific domains of the protein in HeLa cells did not interfere with the normal distribution of fibrillarin. However, when the helicase domain was truncated, the distribution pattern of fibrillarin was distorted. Microinjection of the wild-type RH-II/Gu cDNA into the nucleus of HeLa cells did not disrupt normal cell growth. However, when cells were injected with mutant DNA, only a small percentage of HeLa cells progressed through the cell cycle. Analysis of centrosomes in transfected cells demonstrated that most of the mutant-expressing cells were arrested early in the cell cycle. The results suggest that each of the structural domains of RH-II/Gu is necessary for cell growth and cell cycle progression.     

Mapping of functional domains in F plasmid partition proteins reveals a bipartite SopB-recognition domain in SopA

Active partition of the F plasmid to dividing daughter cells is assured by interactions between proteins SopA and SopB, and a centromere, sopC. A close homologue of the sop operon is present in the linear prophage N15 and, together with sopC-like sequences, it ensures stability of this replicon. We have exploited this sequence similarity to construct hybrid sop operons with the aim of locating specific interaction determinants within the SopA and SopB proteins that are needed for partition function and for autoregulation of sopAB expression. Centromere binding was found to be specified entirely by a central 25 residue region of SopB strongly predicted to form a helix-turn-helix structure. SopB protein also carries a species-specific SopA-interaction determinant within its N-terminal 45 amino acids, and, as shown by Escherichia coli two-hybrid analysis, a dimerization domain within its C-terminal 75 (F) or 97 (N15) residues. Promoter-operator binding specificity was located within an N-terminal 66 residue region of SopA, which is predicted to contain a helix-turn-helix motif. Two other regions of SopA protein, one next to the ATPase Walker A-box, the other C-terminal, specify interaction with SopB. Yeast two-hybrid analysis indicated that these regions contact SopB directly. Evidence for the involvement of the SopA N terminus in autoinhibition of SopA function was obtained, revealing a possible new aspect of the role of SopB in SopA activation.