Dominant-negative mutants of human MxA protein: domains in the carboxy-terminal moiety are important for oligomerization and antiviral activity.

Human MxA protein is an interferon-induced 76-kDa GTPase that exhibits antiviral activity against several RNA viruses. Wild-type MxA accumulates in the cytoplasm of cells. TMxA, a modified form of wild-type MxA carrying a foreign nuclear localization signal, accumulates in the cell nucleus. Here we show that MxA protein is translocated into the nucleus together with TMxA when both proteins are expressed simultaneously in the same cell, demonstrating that MxA molecules form tight complexes in living cells. To define domains important for MxA-MxA interaction and antiviral function in vivo, we expressed mutant forms of MxA together with wild-type MxA or TMxA in appropriate cells and analyzed subcellular localization and interfering effects. An MxA deletion mutant, MxA(359-572), formed heterooligomers with TMxA and was translocated to the nucleus, indicating that the region between amino acid positions 359 and 572 contains an interaction domain which is critical for oligomerization of MxA proteins. Mutant T103A with threonine at position 103 replaced by alanine had lost both GTPase and antiviral activities. T103A exhibited a dominant-interfering effect on the antiviral activity of wild-type MxA rendering MxA-expressing cells susceptible to infection with influenza A virus, Thogoto virus, and vesicular stomatitis virus. To determine which sequences are critical for the dominant-negative effect of T103A, we expressed truncated forms of T103A together with wild-type protein. A C-terminal deletion mutant lacking the last 90 amino acids had lost interfering capacity, indicating that an intact C terminus was required. Surprisingly, a truncated version of MxA representing only the C-terminal half of the molecule exerted also a dominant-negative effect on wild-type function, demonstrating that sequences in the C-terminal moiety of MxA are necessary and sufficient for interference. However, all MxA mutants formed hetero-oligomers with TMxA and were translocated to the nucleus, indicating that physical interaction alone is not sufficient for disturbing wild-type function. We propose that dominant-negative mutants directly influence wild-type activity within hetero-oligomers or else compete with wild-type MxA for a cellular or viral target.     

Evidence for phosphorylation requirement for human bilirubin UDP-glucuronosyltransferase (UGT1A1) activity

Our discovery of rapid down-regulation of human bilirubin UDP-glucuronosyltransferase (UGT) in colon cell lines that was transient and irreversible following curcumin- and calphostin-C-treatment, respectively, suggested phosphorylation event(s) were involved in activity. Likewise, bilirubin-UGT1A1 expressed in COS-1 cells was inhibited by curcumin and calphostin-C. Because calphostin-C is a highly specific protein kinase C (PKC) inhibitor, we examined and found 4 to 5 predicted PKC phosphorylation sites in 11 UGTs examined. UGT1A1 incorporated [33P]orthophosphate, which was inhibited by calphostin-C. Also triple mutant, T75A/T112A/S435G-UGT1A1, at predicted PKC sites failed to incorporate [33P]orthophosphate. Individual or double mutants exhibited dominant-negative, additive, or no effect, while the triple mutant retained 10-15% activity towards bilirubin and two xenobiotics. Compared to wild-type, S435G and T112A/S435G shifted pH-optimum for eugenol, but not for bilirubin or anthraflavic acid, toward alkaline and acid conditions, respectively. This represents the first evidence that a UGT isozyme requires phosphorylation for activity.     

Investigating the structure of human RNase H1 by site-directed mutagenesis

In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis. The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and contains the amino acid residues of the putative catalytic site and basic substrate-binding domain of the E. coli RNase enzyme. The amino terminus of human RNase H1 contains a structure consistent with a double-strand RNA (dsRNA) binding motif that is separated from the conserved E. coli RNase H1 region by a 62-amino acid sequence. These studies showed that although the conserved amino acid residues of the putative catalytic site and basic substrate-binding domain are required for RNase H activity, deletion of either the catalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate. Deletion of the region between the dsRNA-binding domain and the conserved E. coli RNase H1 domain resulted in a significant loss in the RNase H activity. Furthermore, the binding affinity of this deletion mutant for the heteroduplex substrate was approximately 2-fold tighter than the wild-type enzyme suggesting that this central 62-amino acid region does not contribute to the binding affinity of the enzyme for the substrate. The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates approximately 2-fold faster than the rate observed for wild-type enzyme. Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heteroduplex substrate indicates that the deletion of this region resulted in a 5-fold loss in binding affinity. Finally, comparison of the cleavage patterns exhibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the observed strong positional preference for cleavage exhibited by human RNase H1.     

Structure and function of poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.

Poly(3-hydroxybutyrate) (PHB) depolymerase from Alcaligenes faecalis T1 is composed of three domains: the catalytic (C) domain, the fibronectin type III-like (F) domain, and the substrate-binding (S) domain. We constructed domain deletion, inversion, chimera, and extra-F-domain mutants and examined their enzyme activity and PHB-binding ability. In addition, we performed substitution of 214Asp and 273His with glycine and aspartate, respectively, to examine their participation in a catalytic triad together with 139Ser. The mutant with both the F and S domains deleted and the trypsin-digested enzyme showed no PHB-hydrolyzing activity and less PHB-binding ability than that of the wild-type enzyme but retained D-(-)-3-hydroxybutyrate trimer-hydrolyzing activity at a level similar to that of the wild-type enzyme. The mutant with the F domain deleted and the mutant which had the order of the F and S domains inverted retained PHB-binding ability and trimer-hydrolyzing activity at levels similar to those of the wild-type enzyme but lost PHB-hydrolyzing activity. The chimera mutant, in which the F domain was substituted with a Thr-rich domain of PHB depolymerase A from Pseudomonas lemoignei, and the extra-F-domain mutant, with an additional F domain, retained trimer- and PHB-hydrolyzing activities and PHB-binding ability at levels similar to those of the wild-type enzyme. Two mutants (D214G and H273D) showed no enzymatic activity toward trimer and PHB, and they were not labeled with [3H]diisopropylfluorophosphate.     

Biophysical analysis of the endoplasmic reticulum-resident chaperone/heat shock protein gp96/GRP94 and its complex with peptide antigen

Animals vaccinated with heat shock protein (HSP)--peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP--peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96--peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques. Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates. Size distributions ranged from dimers to octamers and higher. Circular dichroism and intrinsic Trp fluorescence suggested that the gp96 dimerization domain deletion mutant protein was more compact than the wild-type gp96. A fluorescent peptide antigen was synthesized, and the peptide-binding properties of wild-type and the dimerization domain deletion mutant gp96 were studied. Fluorescence lifetime and anisotropy decay showed that the bound antigenic peptide was located in a hydrophobic pocket, with considerable free space for the rotation of the probe. Deletion of the dimerization domain affected the peptide-binding microenvironment, although peptide-binding affinity was reduced by only a small extent. Peptide--gp96 complexes were extremely stable, persisting for many days in the cold. The extraordinary stability of peptide--gp96 complexes and the plasticity of the peptide-binding pocket support the proposed relay of diverse peptides to MHC and/or other molecules via molecular recognition.     

Lateral dimerization of the E-cadherin extracellular domain is necessary but not sufficient for adhesive activity

Cadherins are transmembrane glycoproteins involved in Ca(2+)-dependent cell-cell adhesion. Using L cells coexpressing E-cadherin constructs with different epitope tags, we examined the lateral dimerization of E-cadherin and its adhesive activity by co-immunoprecipitation and aggregation assays, respectively. Although the transmembrane domain is required for dimerization, tail-less constructs possessing the transmembrane domain of either N-cadherin or CD45 show dimerization and are active in aggregation assays. Two mutant constructs having either of two amino acid substitutions, W2A or substitutions that disrupt the recognition sequence for endoproteolytic enzymes involved in removal of the precursor segment, cannot form dimers and are inactive in aggregation. These monomeric proteins, like their wild-type dimerizing counterparts, retain their Ca(2+)-dependent resistance to trypsin digestion, suggesting that dimerization per se does not induce a large conformational change. Two other constructs, having either an amino acid substitution, D134A, or a C-terminal deletion of 70 amino acid residues, retain the ability to associate laterally but are inactive in aggregation assays. Staurosporine treatment of cells expressing the latter construct increases aggregation but does not increase the extent of lateral dimerization. Thus, lateral dimerization is necessary, but not sufficient for adhesive activity.     

Rescue of HIV-1 receptor function through cooperation between different forms of the CCR5 chemokine receptor

Interaction of the human immunodeficiency virus (HIV-1) envelope glycoproteins with the CCR5 chemokine receptor, a G-protein-coupled receptor, triggers a membrane fusion process and virus entry. Cooperation for HIV-1 receptor activity was observed when two forms of CCR5 were coexpressed, either the wild-type (WT) receptor and a defective mutant with deletion of the amino-terminal (NT) extracellular domain or the latter deltaNT mutant and a human-mouse CCR5 chimera bearing the NT domain from human CCR5. Cooperation was most efficient when the two forms of CCR5 were in a 1:1 ratio. It was not observed between the CCR5 deltaNT mutant and a chimeric receptor (5444) in which the NT domain of CCR5 was in the context of another G-protein-coupled receptor, the HIV-1 receptor CXCR4. These results suggested that physical association between two forms of CCR5 was required for their cooperation. Coimmunoprecipitation experiments in transfected cell lysates indeed showed that the deltaNT CCR5 mutant formed oligomeric complexes with the WT CCR5 or the HMMM chimera but not with the CXCR4-derived chimera 5444. These observations suggest that the formation of CCR5 oligomers is a constitutive process independent from activation by chemokine ligands. The interaction of HIV-1 with independent subunits of CCR5 oligomers could favor the local recruitment of fusiogenic proteins and the formation of a fusion pore.     

Dimerization of nitric oxide-sensitive guanylyl cyclase requires the alpha 1 N terminus

The enzyme nitric oxide-sensitive guanylyl cyclase is an obligate heterodimer consisting of an alpha and a beta subunit. Whereas the C-terminal parts of the subunits have been shown to be sufficient for catalysis, regulation was assigned to the N termini. The central domains have been postulated to be responsible for the formation of alphabeta heterodimers. Here, we have analyzed dimerization by precipitation of various N- and C-terminally truncated alpha(1) mutants with beta(1) wild type or deletion mutants thereof after coexpression in the baculovirus/Sf9 system. In contrast to the current hypothesis, our analysis revealed that an N-terminal region of the alpha(1) subunit (amino acids 61-128) is mandatory for quantitative dimerization. The central domain (amino acids 367-462) contributes but is not sufficient to mediate robust alphabeta interaction. Wild type-like binding of the identified minimum dimerization region of alpha(1) (amino acids 61-462) requires the N-terminal and central region of beta(1) (amino acids 1-385). Furthermore, we observed an unequal stability of the alpha(1) and beta(1) subunit. Whereas beta(1) forms heme containing homodimers and is stable, alpha(1) appears to be prone to misfolding and degradation when heterodimerization is impaired by deletion of important sequences.     

Activation mechanism of solubilized epidermal growth factor receptor tyrosine kinase.

Dimerization of epidermal growth factor receptor (EGFR) leads to the activation of its tyrosine kinase. To elucidate whether dimerization is responsible for activation of the intracellular tyrosine kinase domain or just plays a role in the stabilization of the active form, the activated status of wild-type EGFR moiety in the heterodimer with kinase activity-deficient mutant receptors was investigated. The kinase activity of the wild-type EGFR was partially activated by EGF in the heterodimer with intracellular domain deletion (sEGFR) or ATP binding-deficient mutant (K721A) EGFRs, while the wild-type EGFR in the heterodimer of wild-type and phosphate transfer activity-deficient mutant receptor D813N could be fully activated. After treatment with EGF, the ATP binding affinity and the V(max) of the wild-type EGFR increased. In the presence of sEGFR, a similar increase in the affinity for ATP was observed, but V(max) did not change. A two-step activation mechanism for EGFR was proposed: upon binding of EGF, the affinity for ATP increased and then, as a result of interaction between the neighboring tyrosine kinase domain, V(max) increased.     

Identification of a tetrameric assembly domain in the C terminus of heat-activated TRPV1 channels

Transient receptor potential (TRP) channels as cellular sensors are thought to function as tetramers. Yet, the molecular determinants governing channel multimerization remain largely elusive. Here we report the identification of a segment comprising 21 amino acids (residues 752-772 of mouse TRPV1) after the known TRP-like domain in the channel C terminus that functions as a tetrameric assembly domain (TAD). Purified recombinant C-terminal proteins of TRPV1-4, but not the N terminus, mediated the protein-protein interaction in an in vitro pulldown assay. Western blot analysis combined with electrophysiology and calcium imaging demonstrated that TAD exerted a robust dominant-negative effect on wild-type TRPV1. When fused with the membrane-tethered peptide Gap43, the TAD blocked the formation of stable homomultimers. Calcium imaging and current recordings showed that deletion of the TAD in a poreless TRPV1 mutant subunit suppressed its dominant-negative phenotype, confirming the involvement of the TAD in assembly of functional channels. Our findings suggest that the C-terminal TAD in TRPV1 channels functions as a domain that is conserved among TRPV1-4 and mediates a direct subunit-subunit interaction for tetrameric assembly.     

Conformational dynamics and the energetics of protein--ligand interactions: role of interdomain loop in human cytochrome P450 reductase

A combination of mutagenesis, calorimetry, kinetics, and small-angle X-ray scattering (SAXS) has been used to study the mechanism of ligand binding energy propagation through human cytochrome P450 reductase (CPR). Remarkably, the energetics of 2',5'-ADP binding to R597 at the FAD-binding domain are affected by mutations taking place at an interdomain loop located 60 A away. Either deletion of a 7 amino acid long segment (T236-G237-E238-E239-S240-S241-I242) or its replacement by poly-proline repeats (5 and 10 residues) results in a significant increase in 2',5'-ADP enthalpy of binding (DeltaHB). This is accompanied by a decrease in the number of thermodynamic microstates available for the ligand-CPR complex. Moreover, the estimated heat capacity change (DeltaCp) for this interaction changes from -220 cal mol-1 K-1 in the wild-type enzyme to -580 cal mol-1 K-1 in the deletion mutant. Pre-steady-state kinetics measurements reveal a 50-fold decrease in the microscopic rate for interdomain (FAD --> FMN) electron transfer in the deletion mutant (kobs = 0.4 s-1). Multiple turnover cytochome c reduction assays indicate that these mutations impair the ability of the FMN-binding domain to shuttle electrons from the FAD-binding domain to the cytochrome partner. Binding of 2',5'-ADP to wild-type CPR triggers a large-scale structural rearrangement resulting in the complex having a more compact domain organization, and the maximum molecular dimension (Dmax) decreases from 110 A in ligand-free enzyme to 100 A in the ligand-bound CPR. The SAXS experiments also demonstrate that what is affected by the mutations is indeed the relative diffusional motion of the domains. Furthemore, ab initio shape reconstruction and homology modeling would suggest that-in the deletion mutant-hindering of domain motion occurs concomitantly with dimerization. The results presented here show that the energetics of this highly localized interaction (2',5'-ADP binding) have a global character, and are highly sensitive to functional structural dynamics involving distal domains. These findings support early theoretical studies which postulate a single protein molecule to be a real, independent thermodynamic ensemble.     

Human and Drosophila UDP-galactose transporters transport UDP-N-acetylgalactosamine in addition to UDP-galactose.

A putative Drosophila nucleotide sugar transporter was characterized and shown to be the Drosophila homologue of the human UDP-Gal transporter (hUGT). When the Drosophila melanogaster UDP-Gal transporter (DmUGT) was expressed in mammalian cells, the transporter protein was localized in the Golgi membranes and complemented the UDP-Gal transport deficiency of Lec8 cells but not the CMP-Sia transport deficiency of Lec2 cells. DmUGT and hUGT were expressed in Saccharomyces cerevisiae cells in functionally active forms. Using microsomal vesicles isolated from Saccharomyces cerevisiae expressing these transporters, we unexpectedly found that both hUGT and DmUGT could transport UDP-GalNAc as well as UDP-Gal. When amino-acid residues that are conserved among human, murine, fission yeast and Drosophila UGTs, but are distinct from corresponding ones conserved among CMP-Sia transporters (CSTs), were substituted by those found in CST, the mutant transporters were still active in transporting UDP-Gal. One of these mutants in which Asn47 was substituted by Ala showed aberrant intracellular distribution with concomitant destabilization of the protein product. However, this mutation was suppressed by an Ile51 to Thr second-site mutation. Both residues were localized within the first transmembrane helix, suggesting that the structure of the helix contributes to the stabilization and substrate recognition of the UGT molecule.     

Substrate recognition by nucleotide sugar transporters: further characterization of substrate recognition regions by analyses of UDP-galactose/CMP-sialic acid transporter chimeras and biochemical analysis of the substrate specificity of parental and chimeric transporters

Human UDP-Gal transporter 1 (hUGT1) and the human CMP-Sia transporter (hCST) are similar in structure, with amino acid sequences that are 43% identical, but they have quite distinct transport substrates. To define their substrate recognition regions, we constructed various chimeras between the two transporters and demonstrated that distinct submolecular regions of the transporter molecules are involved in the specific recognition of UDP-Gal and CMP-Sia (Aoki, K., Ishida, N., and Kawakita, M. (2001) J. Biol. Chem. 276, 21555-21561). In a further attempt to define the minimum submolecular regions required for the recognition of specific substrates, we found that substitution of helix 7 of hCST into the corresponding part of hUGT1 was necessary and sufficient for a chimera to show CST activity. Additional replacement of helix 2 or 3 of hUGT1 with the corresponding hCST sequence markedly increased the efficiency of CMP-Sia transport. For UGT activity, helices 1 and 8 of hUGT1 were necessary (but not sufficient), and helices 9 and 10 or helices 2, 3, and 7 derived from hUGT1 were also required to render the chimera competent for UDP-Gal transport. The in vitro analyses of a chimera with dual specificity indicated that it transported both UMP and CMP and mediated exchange reactions between these nucleotides and nucleotide sugars that are recognized specifically by either of the parental transporters.     

Expression and activity of chimeric molecules between human UDP-galactose transporter and CMP-sialic acid transporter.

Human UDP-galactose transporter (hUGT1) and CMP-sialic acid transporter (hCST) are related Golgi proteins with eight putative transmembrane helices predicted by computer analysis. We constructed chimeric molecules in which segments of various lengths from the C- or N-terminus of hUGT1 were replaced by corresponding portions of hCST. The chimeras were transiently expressed in UGT-deficient mutant Lec8 cells, and their UGT activity was assessed by the binding of GS-II lectin to the transfected cells. The replacement of either the N- or C-terminal cytoplasmic segment by that of hCST did not affect the expression or activity of hUGT1. A chimera in which the eighth helix and the C-terminal tail were replaced also retained the UGT activity, indicating that this helix is not involved in the determination of substrate specificity. In contrast, three types of chimeras, in which the first helix, the first and the second helices, and a segment from the seventh helix to the C-terminus were replaced, respectively, were expressed very infrequently in the transfected cells, and had no UGT activity. They are likely folded incorrectly and degraded by a quality-control system, since the amounts of their mRNAs were normal and the proteins were mainly localized in the ER. The first and the seventh helices are important for the stability of the transporter protein.     

The N-terminal domain of the reticulocyte-type 15-lipoxygenase is not essential for enzymatic activity but contains determinants for membrane binding

The rabbit reticulocyte-type 15-lipoxygenase is capable of oxygenating biomembranes and lipoproteins without the preceding action of ester lipid cleaving enzymes. This reaction requires an efficient membrane binding, and the N-terminal beta-barrel domain of the enzyme has been implicated in this process. To obtain detailed information on the structural requirements for membrane oxygenation, we expressed the rabbit wild-type 15-lipoxygenase, its beta-barrel deletion mutant (catalytic domain), and several lipoxygenase point mutations as His-tagged fusion proteins in Escherichia coli and tested their membrane binding characteristics. We found that: (i) the beta-barrel deletion mutant was catalytically active and its enzymatic properties (K(M), V(max), pH optimum, substrate specificity) were similar to those of the wild-type enzyme; (ii) when compared with the wild-type lipoxygenase, the membrane binding properties of the N-terminal truncation mutant were impaired but not abolished, suggesting a role of the catalytic domain in membrane binding; and (iii) Phe-70 and Leu-71 (constituents of the beta-barrel domain) but also Trp-181, which is located in the catalytic domain, were identified as sequence determinants for membrane binding. Mutation of these amino acids to more polar residues (F70H, L71K, W181E) impaired the membrane binding capacity of the recombinant enzyme. These data indicate that the C-terminal catalytic domain of the rabbit 15-lipoxygenase is enzymatically active and that the membrane binding properties of the enzyme are determined by a concerted action of the N-terminal beta-barrel and the C-terminal catalytic domain.     

Identification of key residues involved in the dimerization of the secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1

The "secretory" Na(+)-K(+)-2Cl(-) cotransporter, NKCC1, belongs to the SLC12 gene family of electroneutral cation-chloride cotransporters. A number of these proteins, including NKCC1 itself, exist as homodimers in the membrane, suggesting that this may be a common feature of the SLC12 family. We have previously demonstrated that replacing the C-terminus of NKCC1 with that of its close homologue NKCC2 produced a fully functional chimeric protein that formed homodimers but did not dimerize with NKCC1. Here we employ a novel co-immunoprecipitation assay to study the dimerization interaction of NKCC1 using additional NKCC1/NKCC2 C-terminal chimeras and point mutants. Our results indicate that the substitution of a number of regions of the C-terminus of NKCC1 with the corresponding sequence from NKCC2 results in weakened dimerization with wild-type NKCC1, demonstrating that various residues play a role in this interaction. Most interestingly, however, we find that the replacement of a single NKCC1 residue, G812, with cysteine, the corresponding amino acid in NKCC2, results in a point mutant that displays no significant dimerization with the wild-type protein. In addition to this effect on heterodimer formation, we also find that G812 mutants can nevertheless form homodimers but that this interaction can be weaker than that observed for wild-type NKCC1. We demonstrate that our results are consistent with at least one established mechanism of protein dimer formation, that of "domain swapping", as well as with a recently reported crystal structure of the C-terminus of a bacterial SLC12 homologue.