Characterization of wild-type and mutant forms of human tryptophan hydroxylase 2

Tryptophan hydroxylase (TPH) catalyses the rate-limiting step in the biosynthesis of serotonin. In vertebrates, the homologous genes tph1 and tph2 encode two different enzymes with distinct patterns of expression, enzyme kinetics and regulation. Variants of TPH2 have recently reported to be associated with reduced serotonin production and behavioural alterations in man and mice. We have produced the human forms of these enzymes in Esherichia coli and in human embryonic kidney cell lines (HEK293) and examined the effects of mutations on their heterologous expression levels, solubility, thermal stability, secondary structure, and catalytic properties. Pure human TPH2 P449R (corresponds to mouse P447R) had comparable catalytic activity (V(max)) and solubility relative to the wild type, but had decreased thermal stability; whereas human TPH2 R441H had decreased activity, solubility and stability. Thus, we consider the variations in kinetic values between wild-type and TPH2 mutants to be of secondary importance to their effects on protein stability and solubility. These findings provide potential molecular explanations for disorders related to the central serotonergic system, such as depression or suicidal behaviour.     

Functional domains of human tryptophan hydroxylase 2 (hTPH2)

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NDelta150 and NDelta150/CDelta24) using isopropyl beta-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH(2)-terminal regulatory domain. The solubility of hTPH2, NDelta150, and NDelta150/CDelta24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t((1/2)) at 37 degrees C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NDelta150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH(2)-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH(2)-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V(max) values. These data identify the NH(2)-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.     

Large hepatitis delta antigen in packaging and replication inhibition: role of the carboxyl-terminal 19 amino acids and amino-terminal sequences.

Hepatitis delta virus (HDV) encodes two proteins, the small delta antigen (SHDAg) and large delta antigen (LHDAg). The latter is identical to the former except for the presence of additional 19 amino acids at the C terminus. While SHDAg is required for HDV replication, LHDAg inhibits replication and, together with hepatitis B surface antigen (HBsAg), is required for the assembly of HDV. The last 19 C-terminal amino acids of LHDAg are essential for HDV assembly. Most of LHDAg (amino acids 19 to 146 and 163 to 195) had been shown to be dispensable for packaging with HBsAg. To discern whether the last 19 C-terminal amino acids solely constitute the signal for packaging with HBsAg, we constructed two LHDAg deletion mutants and tested their abilities to be packaged with HBsAg in cotransfection experiments. We found that deletion of amino acids 2 to 21 and 142 to 165 did not affect LHDAg packaging. This result suggested that only the last 19 C-terminal amino acids of LHDAg are required for packaging. We further constructed two plasmids which expressed c-H-ras with or without additional 19 C-terminal amino acids identical to those in LHDAg. Only c-H-ras with additional 19 amino acids could be cosecreted with HBsAg in the cotransfection experiment. This result confirmed that the C-terminal 19 amino acids are the packaging signal for HBsAg. We also tested the trans activation activity and trans-dominant inhibitory activity of the deletion mutants of SHDAg and LHDAg, respectively. In contrast to deletion of amino acids 142 to 165, deletion of amino acids 2 to 21 impaired the trans-dominant inhibitory activity of LHDAg. Deletion of amino acids 2 to 21 and 142 to 165 did not affect the trans activation activity of SHDAg. This result suggested that a functional domain which is important for the trans-dominant inhibitory activity of LHDAg exists in the amino terminus of HDAg.     

Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system. In vitro intragenic complementation: the roles of Arg126 in phosphoryl transfer and the C-terminal domain in dimerization

Enzyme I mutants of the Salmonella typhimurium phosphoenolpyruvate:sugar phosphotransferase system (PTS), which show in vitro intragenic complementation, have been identified as Arg126Cys (strain SB1690 ptsI34), Gly356Ser (strain SB1681 ptsI16), and Arg375Cys (strain SB1476 ptsI17). The mutation Arg126Cys is in the N-terminal HPr-binding domain, and complements Gly356Ser and Arg375Cys enzyme I mutations located in the C-terminal phosphoenolpyruvate(PEP)-binding domain. Complementation results in the formation of unstable heterodimers. None of the mutations alters the K(m) for HPr, which is phosphorylated by enzyme I. Arg126 is a conserved residue; the Arg126Cys mutation gives a V(max) of 0.04% wild-type, establishing a role in phosphoryl transfer. The Gly356Ser and Arg375Cys mutations reduce enzyme I V(max) to 4 and 2%, respectively, and for both, the PEP K(m) is increased from 0.1 to 3 mM. It is concluded that this activity was from the monomer, rather than the dimer normally found in assays of wild-type. In the presence of Arg126Cys enzyme, V(max) for Gly356Ser and Arg375Cys enzymes I increased 6- and 2-fold, respectively; the K(m) for PEP decreased to <10 microM, but the K(m) became dependent upon the stability of the heterodimer in the assay. Gly356 is conserved in enzyme I and pyruvate phosphate dikinase, which is a homologue of enzyme I, and this residue is part of a conserved sequence in the subunit interaction site. Gly356Ser mutation impairs enzyme I dimerization. The mutation Arg375Cys also impairs dimerization, but the equivalent residue in pyruvate phosphate dikinase is not associated with the subunit interaction site. A 37 000 Da, C-terminal domain of enzyme I has been expressed and purified; it dimerizes and complements Gly356Ser and Arg375Cys enzymes I proving that the association/dissociation properties of enzyme I are a function of the C-terminal domain.     

Different domains of the mitogen-activated protein kinases ERK3 and ERK2 direct subcellular localization and upstream specificity in vivo.

Extracellular signal-regulated kinase 3 (ERK3) is a member of the mitogen-activated protein (MAP) kinase family. ERK3 is most similar in its kinase catalytic domain to ERK2, yet it displays many unique properties. Among these, unlike ERK2, which translocates to the nucleus following activation, ERK3 is constitutively localized to the nucleus, despite the lack of a defined nuclear localization sequence. We created two chimeras between ERK2 and the catalytic domain of ERK3 (ERK3DeltaC), and some mutants of these chimeras, to examine the basis for the different behaviors of these two MAP kinase family members. We find the following: 1) the N-terminal folding domain of ERK3 functions in phosphoryl transfer reactions with the C-terminal folding domain of ERK2; 2) the C-terminal halves of ERK2 and ERK3DeltaC are primarily responsible for their subcellular localization in resting cells; and 3) the N-terminal folding domain of ERK2 is required for its activation in cells, its interaction with MEK1, and its accumulation in the nucleus.     

Mutations in the C-terminal domain of ALSV (Avian Leukemia and Sarcoma Viruses) integrase alter the concerted DNA integration process in vitro.

Integrase (IN) is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cell DNA. The C-terminal domain of IN is involved in DNA binding and enzyme multimerization. We previously performed single amino acid substitutions in the C-terminal domain of the avian leukemia and sarcoma viruses (ALSV) IN. Here, we modelled these IN mutants and analysed their ability to mediate concerted DNA integration (in an in vitro assay) as well as to form dimers (by size exclusion chromatography and protein-protein cross-linking). Mutations of residues located at the dimer interface (V239, L240, Y246, V257 and K266) have the greatest effects on the activity of the IN. Among them: (a) the L240A mutation resulted in a decrease of integration efficiency that was concomitant with a decrease of IN dimerization; (b) the V239A, V249A and K266A mutants preferentially mediated non-concerted DNA integration rather than concerted DNA integration although they were found as dimers. Other mutations (V260E and Y246W/DeltaC25) highlight the role of the C-terminal domain in the general folding of the enzyme and, hence, on its activity. This study points to the important role of residues at the IN C-terminal domain in the folding and dimerization of the enzyme as well as in the concerted DNA integration of viral DNA ends.     

The conformational flexibility of the carboxy terminal residues 105-114 is a key modulator of the catalytic activity and stability of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is a multifunctional protein and a major mediator of innate immunity. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo and the factors governing its oligomerization and stability remain poorly understood. The C-terminal region of MIF is highly conserved and participates in several intramolecular interactions that suggest a role in modulating the stability and biochemical activity of MIF. To determine the importance of these interactions, point mutations (A48P, L46A), insertions (P107) at the monomer-monomer interfaces, and C-terminal deletion (Delta 110-114NSTFA and Delta 105-114NVGWNNSTFA) variants were designed and their structural properties, thermodynamic stability, oligomerization state, catalytic activity and receptor binding were characterized using a battery of biophysical methods. The C-terminal deletion mutants DeltaC5 huMIF 1-109 and DeltaC10 huMIF 1-104 were enzymatically inactive and thermodynamically less stable than wild type MIF. Analytical ultracentrifugation studies demonstrate that both C-terminal mutants sediment as trimers and exhibit similar binding to CD74 as the wild type protein. Disrupting the conformation of the C-terminal region 105-114 and increasing its conformational flexibility through the insertion of a proline residue at position 107 was sufficient to reproduce the structural, biochemical and thermodynamic properties of the deletion mutants. P107 MIF forms an enzymatically inactive trimer and exhibits reduced thermodynamic stability relative to the wild type protein. To provide a rationale for the changes induced by these mutations at the molecular level, we also performed molecular dynamics simulations on these mutants in comparison to the wild type MIF. Together, our studies demonstrate that intersubunit interactions involving the C-terminal region 105-114, including a salt-bridge interaction between Arg73 of one monomer and the carboxy terminus of a neighboring monomer, play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties. Our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity.     

Bcl-XL protects BimEL-induced Bax conformational change and cytochrome C release independent of interacting with Bax or BimEL

The Bcl-2 homology (BH) 3-only pro-apoptotic Bcl-2 family protein Bim plays an essential role in the mitochondrial pathway of apoptosis through activation of the BH1-3 multidomain protein Bax or Bak. To further understand how the BH3-only protein activates Bax, we provide evidence here that BimEL induces Bax conformational change and apoptosis through a Bcl-XL-suppressible but heterodimerization-independent mechanism. Substitution of the conserved leucine residue in the BH3 domain of BimEL for alanine (M1) inhibits the interaction of BimEL with Bcl-XL but does not abolish the ability of BimEL to induce Bax conformational change and apoptosis. However, removal of the C-terminal hydrophobic region from the M1 mutant (M1DeltaC) abolishes its ability to activate Bax and to induce apoptosis, although deletion of the C-terminal domain (DeltaC) alone has little if any effect on the pro-apoptotic activity of BimEL. Subcellular fractionation experiments show that the Bim mutant M1DeltaC is localized in the cytosol, indicating that both the C-terminal hydrophobic region and the BH3 domain are required for the mitochondrial targeting and pro-apoptotic activity of BimEL. Moreover, the Bcl-XL mutant (mt1), which is unable to interact with Bax and BimEL, blocks Bax conformational change and cytochrome c release induced by BimEL in intact cells and isolated mitochondria. BimEL or Bak-BH3 peptide induces Bax conformational change in vitro only under the presence of mitochondria, and the outer mitochondrial membrane fraction is sufficient for induction of Bax conformational change. Interestingly, native Bax is attached loosely on the surface of isolated mitochondria, which undergoes conformational change and insertion into mitochondrial membrane upon stimulation by BimEL, Bak-BH3 peptide, or freeze/thaw damage. Taken together, these findings indicate that BimEL may activate Bax by damaging the mitochondrial membrane structure directly, in addition to its binding and antagonizing Bcl-2/Bcl-XL function.     

Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton-translocase; it contains a single type of polypeptide of approximately 81kDa. A line of evidence demonstrated that the carboxyl terminus of V-PPase is relatively conserved in various plant V-PPases and presumably locates in the vicinity of the catalytic site. In this study, we attempt to identify the roles of the C-terminus of V-PPase by generating a series of C-terminal deletion mutants over-expressed in Saccharomyces cerevisiae, and determining their enzymatic and proton translocating reactions. Our results showed that the deletion mutation at last 5 amino acids in the C-terminus (DeltaC5) induced a dramatic decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase; but the mutant lacking last 10 amino acids (DeltaC10) retained about 60-70% of the enzymatic activity of wild-type. Truncation of the C-terminus by more than 10 amino acids completely abolished the enzymatic activity and proton translocation of V-PPase. Furthermore, the DeltaC10 mutant displayed a shift in T(1/2) (pretreatment temperature at which half enzymatic activity is observed) but not the optimal pH for PP(i) hydrolytic activity. The deletion of the C-terminus substantially modified apparent K(+) binding constant, but exert no significant changes in the Na(+)-, F(-)-, and Ca(2+)-inhibition of the enzymatic activity of V-PPase. Taken together, we speculate that the C-terminus of V-PPase may play a crucial role in sustaining enzymatic activity and is likely involved in the K(+)-regulation of the enzyme in an indirect manner.     

Deletions in the acidic lipid-binding region of the plasma membrane Ca2+ pump. A mutant with high affinity for Ca2+ resembling the acidic lipid-activated enzyme

The C-terminal segment of the loop between transmembrane helices 2 and 3 (A(L) region) of the plasma membrane Ca(2+) pump (PMCA) is not conserved in other P-ATPases. Part of this region, just upstream from the third transmembrane domain, has been associated with activation of the PMCA by acidic lipids. cDNAs coding for mutants of the Ca(2+) pump isoform h4xb with deletions in the A(L) region were constructed, and the proteins were successfully expressed in either COS or Chinese hamster ovary cells. Mutants with deletions in the segment 296-349 had full Ca(2+) transport activity, but deletions involving the segment of amino acids 350-356 were inactive suggesting that these residues are required for a functional PMCA. In the absence of calmodulin the V(max) of mutant d296-349 was similar to that of the recombinant wild type pump, but its K(0.5) for Ca(2+) was about 5-fold lower. The addition of calmodulin increased the V(max) and the apparent Ca(2+) affinity of both the wild type and d296-349 enzymes indicating that the activating effects of calmodulin were not affected by the deletion. At low concentrations of Ca(2+) and in the presence of saturating amounts of calmodulin, the addition of phosphatidic acid increased about 2-fold the activity of the recombinant wild type pump. In contrast, under these conditions phosphatidic acid did not significantly change the activity of mutant d296-349. Taken together these results suggest that (a) deletion of residues 296-349 recreates a form of PMCA similar to that resulting from the binding of acidic lipids at the A(L) region; (b) the A(L) region acts as an acidic lipid-binding inhibitory domain capable of adjusting the Ca(2+) affinity of the PMCA to the lipid composition of the membrane; and (c) the function of the A(L) region is independent of the autoinhibition by the C-terminal calmodulin-binding region.     

Carboxyl-terminal hydrophilic tail of a NhaP type Na+/H+ antiporter from cyanobacteria is involved in the apparent affinity for Na+ and pH sensitivity

Little information is available on the C-terminal hydrophilic tails of prokaryotic Na(+)/H(+) antiporters. To address functional properties of the C-terminal tail, truncation mutants in this domain were constructed. Truncation of C-terminal amino acid residues of NhaP1 type antiporter from Synechocystis PCC6803 (SynNhaP1) did not change the V(max) values, but increased the K(m) values for Na(+) and Li(+) about 3 to 15-fold. Truncation of C-terminal tail of a halotolerant cyanobacterium Aphanothece halophytica (ApNhaP1) significantly decreased the V(max) although it did not alter the K(m) values for Na(+). The C-terminal part of SynNhaP1 was expressed in E. coli and purified as a 16kDa soluble protein. Addition of purified polypeptide to the membrane vesicles expressing the C-terminal truncated SynNhaP1 increased the exchange activities. Change of Glu519 and Glu521 to Lys in C-terminal tail altered the pH dependence of Na(+)/H(+) and Li(+)/H(+) exchange activities. These results indicate that the specific acidic amino acid residues at C-terminal domain play important roles for the K(m) and the pH dependence of the exchange activity.     

Identification of caveolin-1-interacting sites in neuronal nitric-oxide synthase. Molecular mechanism for inhibition of NO formation

Caveolin is known to down-regulate both neuronal (nNOS) and endothelial nitric-oxide synthase (eNOS). In the present study, direct interactions of recombinant caveolin-1 with both the oxygenase and reductase domains of nNOS were demonstrated using in vitro binding assays. To elucidate the mechanism of nNOS regulation by caveolin, we examined the effects of a caveolin-1 scaffolding domain peptide (CaV1p1; residues (82-101)) on the catalytic activities of wild-type and mutant nNOSs. CaV1p1 inhibited NO formation activity and NADPH oxidation of wild-type nNOS in a dose-dependent manner with an IC(50) value of 1.8 microM. Mutations of Phe(584) and Trp(587) within a caveolin binding consensus motif of the oxygenase domain did not result in the loss of CaV1p1 inhibition, indicating that an alternate region of nNOS mediates inhibition by caveolin. The addition of CaV1p1 also inhibited more than 90% of the cytochrome c reductase activity in the isolated reductase domain with or without the calmodulin (CaM) binding site, whereas CaV1p1 inhibited ferricyanide reductase activity by only 50%. These results suggest that there are significant differences in the mechanism of inhibition by caveolin for nNOS as compared with those previously reported for eNOS. Further analysis of the interaction through the use of several reductase domain deletion mutants revealed that the FMN domain was essential for successful interaction between caveolin-1 and nNOS reductase. We also examined the effects of CaV1p1 on an autoinhibitory domain deletion mutant (Delta40) and a C-terminal truncation mutant (DeltaC33), both of which are able to form NO in the absence of CaM, unlike the wild-type enzyme. Interestingly, CaV1p1 inhibited CaM-dependent, but not CaM-independent, NO formation activities of both Delta40 and DeltaC33, suggesting that CaV1p1 inhibits interdomain electron transfer induced by CaM from the reductase domain to the oxygenase domain.     

Biochemical characteristics of functional domains using feline foamy virus integrase mutants

We constructed deletion mutants and seven point mutants by polymerase chain reaction to investigate the specificity of feline foamy virus integrase functional domains. Complementation reactions were performed for three enzymatic activities such as 3’-end processing, strand transfer, and disintegration. The complementation reactions with deletion mutants showed several activities for 3’-end processing and strand transfer. The conserved central domain and the combination of the N-terminal or C-terminal domains increased disintegration activity significantly. In the complementation reactions between deletion and point mutants, the combination between D107V and deletion mutants revealed 3’-end processing activities, but the combination with others did not have any activity, including strand transfer activities. Disintegration activity increased evenly, except the combination with glutamic acid 200. These results suggest that an intact central domain mediates enzymatic activities but fails to show these activities in the absence of the N-terminal or C-terminal domains. [BMB Reports 2013; 46(1):53-58]     

Structural and functional roles of deamidation and/or truncation of N- or C-termini in human alpha A-crystallin

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partially or fully recovered following either deletion in the alphaA-N123D mutant. The results of subunit exchange rates among alphaA mutants and WT-alphaB suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alphaA-crystallin.     

Mre11 nuclease and C-terminal tail-mediated DDR functions are required for initiating yeast telomere healing

Mre11 is a central factor in creating an optimal substrate for telomerase loading and elongation. We have used a G2/M synchronized telomere-healing assay as a tool to separate different functions of Mre11 that are not apparent in null alleles. An analysis of healing efficiencies of several mre11 alleles revealed that both nuclease and C-terminal mutations led to a loss of healing. Interestingly, trans-complementation of the 49 amino acid C-terminal deletion (DeltaC49) and the D16A mutant, deficient in nuclease activity and partially defective in MRX complex formation, restores healing. DeltaC49 provokes Rad53 phosphorylation after treatment with the radiomimetic agent MMS exclusively through the Tel1 pathway, suggesting that a Tel1-mediated function is initiated through the C-terminal tail.     

Functional analysis of tail domains of Acanthamoeba myosin IC by characterization of truncation and deletion mutants

Acanthamoeba myosin IC has a single 129-kDa heavy chain and a single 17-kDa light chain. The heavy chain comprises a 75-kDa catalytic head domain with an ATP-sensitive F-actin-binding site, a 3-kDa neck domain, which binds a single 17-kDa light chain, and a 50-kDa tail domain, which binds F-actin in the presence or absence of ATP. The actin-activated MgATPase activity of myosin IC exhibits triphasic actin dependence, apparently as a consequence of the two actin-binding sites, and is regulated by phosphorylation of Ser-329 in the head. The 50-kDa tail consists of a basic domain, a glycine/proline/alanine-rich (GPA) domain, and a Src homology 3 (SH3) domain, often referred to as tail homology (TH)-1, -2, and -3 domains, respectively. The SH3 domain divides the TH-3 domain into GPA-1 and GPA-2. To define the functions of the tail domains more precisely, we determined the properties of expressed wild type and six mutant myosins, an SH3 deletion mutant and five mutants truncated at the C terminus of the SH3, GPA-2, TH-1, neck and head domains, respectively. We found that both the TH-1 and GPA-2 domains bind F-actin in the presence of ATP. Only the mutants that retained an actin-binding site in the tail exhibited triphasic actin-dependent MgATPase activity, in agreement with the F-actin-cross-linking model, but truncation reduced the MgATPase activity at both low and high actin concentrations. Deletion of the SH3 domain had no effect. Also, none of the tail domains, including the SH3 domain, affected either the K(m) or V(max) for the phosphorylation of Ser-329 by myosin I heavy chain kinase.     

Influence of ApoA-I structure on the ABCA1-mediated efflux of cellular lipids

The influence of apolipoprotein (apo) A-I structure on ABCA1-mediated efflux of cellular unesterified (free) cholesterol (FC) and phospholipid (PL) is not well understood. To address this issue, we used a series of apoA-I mutants to examine the contributions of various domains in the molecule to ABCA1-mediated FC and PL efflux from mouse J774 macrophages and human skin fibroblasts. Irrespective of the cell type, deletion or disruption of the C-terminal lipid-binding domain of apoA-I drastically reduced the FC and PL efflux ( approximately 90%), indicating that the C-terminal amphipathic alpha-helix is required for high affinity microsolubilization of FC and PL. Deletion in the N-terminal region of apoA-I also reduced the lipid efflux ( approximately 30%) and increased the K(m) about 2-fold compared with wild type apoA-I, whereas deletion of the central domain (Delta123-166) had no effect on either K(m) or V(max). These results indicate that ABCA1-mediated lipid efflux is relatively insensitive to the organization of the apoA-I N-terminal helix-bundle domain. Alterations in apoA-I structure caused parallel changes in its ability to bind to a PL bilayer and to induce efflux of FC and PL. Overall, these results are consistent with a two-step model for ABCA1-mediated lipid efflux. In the first step, apoA-I binds to ABCA1 and hydrophobic alpha-helices in the C-terminal domain of apoA-I insert into the region of the perturbed PL bilayer created by the PL transport activity of ABCA1, thereby allowing the second step of lipidation of apoA-I and formation of nascent high density lipoprotein particles to occur.     

Functional characterization of the placental fusogenic membrane protein syncytin

Syncytin is an envelope protein of the human endogenous retrovirus family W (HERV-W). Syncytin is specifically expressed in the human placenta and mediates trophoblast cell fusion into the multinucleated syncytiotrophoblast layer. It is a polypeptide of 538 amino acids and is predicted to be posttranslationally cleaved into a surface (SU) subunit and a transmembrane (TM) subunit. Functional characterization of syncytin protein can aid understanding of the molecular mechanism underlying syncytin-mediated cell fusion. In this report, we studied the structure-function relationship of syncytin in 293T and HeLa cells transiently expressing wild-type syncytin or syncytin mutants generated by linker scanning and deletion mutagenesis. Of the 22 linker-inserted mutants, mutants InS51, InV139, InE156, InS493, InA506, and InL529 were fusogenic, suggesting that regions around amino acids S51, V139, and E156 in the SU subunit and S493, A506, and L529 in the cytoplasmic domain (CTM) of syncytin are flexible in conformation. Of the 17 deletion mutants, nine mutants with deletions in the region from amino acids 479 to 538 were fusogenic. The deletion mutant DelI480, containing only the first four amino acid residues in the cytoplasmic domain, had enhanced fusogenic activity in comparison with the wild-type. In addition, two heptad repeat regions (HRA and B) were defined in the TM subunit of syncytin. A peptide inhibitor derived from the C-terminal heptad repeat region (HRB) was shown to potently inhibit syncytin-mediated cell fusion. Our results suggest that the cytoplasmic domain of syncytin is not essential for syncytin-mediated fusion but may play a regulatory role, and an intramolecular interaction between HRA and B is involved in the fusion process.     

Mouse tryptophan hydroxylase isoform 2 and the role of proline 447 in enzyme function

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin (5-HT). Once thought to be a single gene product, TPH is now known to exist in two isoforms. Isoform 1 (TPH1) is found in the pineal gland and gut, and isoform 2 (TPH2) is selectively expressed in brain. A single-nucleotide polymorphism in TPH2 results in a proline-to-arginine mutation at residue 447 and substantially lowers catalytic activity. In view of the importance of TPH in determining brain 5-HT function, we cloned TPH2 and produced the P447R mutant to assess the importance of this proline in enzyme function. Catalytically active TPH2 and the P447R mutant were expressed at the predicted subunit molecular mass of 56 kDa. The P447R mutant expressed less than 50% of the activity of TPH2. Mutation of this conserved proline in TPH1 (P403R) also resulted in an enzyme with significantly lower activity than the wild-type enzyme. The P447R mutant had a V(max) 50% lower than that of TPH2. The P447R mutation did not alter the oligomeric assembly of the protein, nor change its responsiveness to cysteine modification. The P447R mutation did not alter enzyme substrate specificity or stability, but conferred slightly enhanced sensitivity to inhibition by dopamine and diminished sensitivity to iron in catalysis. The conserved proline in TPH (residue 447 in TPH2 and 403 in TPH1) plays an important role in enzyme function by regulating V(max) of the catalytic reaction.     

Minimal catalytic domain of N-acetylglucosaminyltransferase V

UDP-GlcNAc: Manalpha1-6Manbeta-R beta1-6 N-acetylglucosaminyltransferase V (EC 2.4.1.155, GlcNAc-TV) is a Golgi enzyme that substitutes the trimannosyl core in the biosynthetic pathway for complex-type N-linked glycans. GlcNAc-TV activity is regulated by oncogenes frequently activated in cancer cells ( ras, src, and her2/neu ) and by activators of T lymphocytes. Overexpression of GlcNAc-TV in epithelial cells results in morphological transformation, while tumor cell mutants selected for loss of GlcNAc-TV products show diminished malignant potential in mice. In this report, we have expressed and characterized a series of N- and C-terminal deletions of GlcNAc-TV. Portions of GlcNAc-TV sequence were fused at the N-terminal domain to IgG-binding domains of staphylococcal Protein A and expressed in CHOP cells. The secreted fusion proteins were purified by IgG Sepharose affinity chromatography and assayed for enzyme activities. The peptide sequence S(213-740)of GlcNAc-TV was determined to be essential for the catalytic activity, the remaining amino acids comprising a 183 amino acid stem region, a 17 amino acid transmembrane domain and a 12 amino acid cytosolic moiety. Further deletion of 5 amino acids to produce peptide R(218-740)reduced enzyme activity by 20-fold. Similar K(m)and V(max)values for donor and acceptor were observed for peptide S(213-740), the minimal catalytic domain, and peptide Q(39-740), which also included the stem region. Truncation of five amino acids from the C-terminus also resulted in a 20-fold loss of catalytic activity. Secondary structure predictions suggest a high frequency of turns in the stem region, and more contiguous stretches of alpha-helix found in the catalytic domain.