Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle.

Gel-filtration experiments indicate that a peptide (P2) composed of the basic region of GCN4 fused to the leucine heptad repeats of Lac repressor forms tetrameric aggregates. Gel-shift experiments were performed to determine the orientation of the helices in the tetrameric P2 aggregate. Sandwich-complex formation of peptide P2 with two DNA fragments containing two symmetrical CRE binding sites (5'-ATGACGTCAT-3') at a distance of 21 bp suggests antiparallel aggregation of the Lac leucine heptad repeats. Thus, we conclude that the leucine heptad repeats of Lac repressor have the ability to form homomeric 4-helical bundles with an antiparallel arrangement of the helices. This topology enables the two DNA fragments in the sandwich complexes to be held together by two tetramers of peptide P2. Replacement of the uncharged amino acids of the helical g and e positions of peptide P2 by the corresponding charged residues of GCN4 (peptide P4) results in a dimeric and parallel aggregation of the leucine heptad repeats, and consequently abolishes the potential to form sandwich structures. Similarly, a hybrid Lac repressor in which the GCN4 leucine zipper replaces the natural Lac leucine heptad repeats forms dimers only. It regains the ability to form tetramers when the charged amino acids in helical positions g and e are replaced by uncharged alanines.     

Stability of a Lac repressor mediated "looped complex"

The quantitation of the stability of a protein-mediated "looped complex" of the Lac repressor and DNA containing two protein-binding sites whose centers of symmetry are separated by 11 helical turns (114 bp) was accomplished by footprint and gel mobility-shift titration techniques. Lac repressor binding to this DNA was only moderately cooperative; a cooperative free energy of -1.0 kcal/mol was calculated in a model-independent fashion from the individual-site loading energies obtained from the footprint titration studies. In order to partition the cooperative binding energy into components representing the dimer-tetramer association of Lac repressor and the cyclization probability of the intervening DNA, advantage was taken of the presence of experimental measures that were in proportion to the concentration of the looped complex present in solution. One measure was the DNase I hypersensitivity observed in footprint titrations in bands located between the two binding sites. The second measure resulted from the electrophoretic resolution in the gel mobility-shift titrations of the band representing the doubly liganded "tandem complex" from the band representing the singly liganded complexes, including the looped complex. Analysis of the footprint and mobility-shift titration data utilizing this additional information showed that approximately 65% of the molecules present in solution are looped complexes at pH 7.0, 100 mM KCl, and 20 degrees C when the binding sites on the DNA are saturated with protein. Reconciliation of the observed low binding cooperativity and the high proportion of looped complexes could only be obtained when the titration data were analyzed by a model in which Lac repressor tetramers dissociate into dimers in solution. The proportion of looped complexes present in solution is highly dependent on the dimer-tetramer association constant, delta Gtet. This result is consistent with the determination by high-pressure fluorescence techniques that Lac repressor tetramers dissociate with an association free energy comparable to their DNA-binding free energies [Royer, C. A., Chakerian, A. E., & Matthews, K. S. (1990) Biochemistry 29, 4959-4966]. However, when the value of delta Gtet of -10.6 kcal/mol (at 20 degrees C) reported by Royer et al. (1990) is assumed, the titration data demand that tetramers bind DNA with much greater affinity than dimers: a result inconsistent with the destabilization of tetramers by the operator observed in the dimer-tetramer dissociation studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oligomerization of Chicken Acetylcholinesterase Does Not Require Intersubunit Disulfide Bonds

Abstract: Acetylcholinesterase (AChE) is secreted from muscle and nerve cells and associates as multimers through intermolecular covalent and noncovalent bonds. The amino acid sequence of the C-terminus is thought to play an important role in these interactions. We generated mutants in the C-terminus of the catalytic T-subunit of chicken AChE to determine the importance of this region to oligomerization and to the amphipathic character of the protein. Wild-type recombinant chicken AChE secreted from human embryonic kidney 293 cells was assembled into dimers and tetramers exclusively. Mutants lacking the C-terminal Cys⁷⁶⁴, the only cysteine involved in interchain disulfide bonds, showed lower but significant levels of the secreted dimeric and tetrameric forms. A truncated mutant, lacking the C-terminal 39 amino acids, exhibited a severe decrease in content of the multimeric forms, yet small amounts of the dimer were detectable. The amphipathic character was dependent on the state of oligomerization. When analyzed by sucrose gradients, the sedimentation of tetramers was not affected by detergent, but monomers and dimers sedimented more slowly in the presence of detergent. Most of the recombinant wild-type enzyme, shown to be dimeric and tetrameric by sedimentation analysis, was monomeric when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that much of the secreted oligomeric AChE was not disulfide bonded. These data suggest that disulfide bonding of Cys⁷⁶⁴ is not required for the catalytic subunit of chicken AChE to form oligomers and that regions outside of the C-terminus contribute to the hydrophobic interactions that are important for stabilizing the oligomeric forms.     

Antiparallel four-stranded coiled coil specified by a 3-3-1 hydrophobic heptad repeat

Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.     

A parallel coiled-coil tetramer with offset helices

Specific helix-helix interactions are fundamental in assembling the native state of proteins and in protein-protein interfaces. Coiled coils afford a unique model system for elucidating principles of molecular recognition between alpha helices. The coiled-coil fold is specified by a characteristic seven amino acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions. Nonpolar side chains spaced three and four residues apart are referred to as the 3-4 hydrophobic repeat. The presence of apolar amino acids at the e or g positions (corresponding to a 3-3-1 hydrophobic repeat) can provide new possibilities for close-packing of alpha-helices that includes examples such as the lac repressor tetramerization domain. Here we demonstrate that an unprecedented coiled-coil interface results from replacement of three charged residues at the e positions in the dimeric GCN4 leucine zipper by nonpolar valine side chains. Equilibrium circular dichroism and analytical ultracentrifugation studies indicate that the valine-containing mutant forms a discrete alpha-helical tetramer with a significantly higher stability than the parent leucine-zipper molecule. The 1.35 A resolution crystal structure of the tetramer reveals a parallel four-stranded coiled coil with a three-residue interhelical offset. The local packing geometry of the three hydrophobic positions in the tetramer conformation is completely different from that seen in classical tetrameric structures yet bears resemblance to that in three-stranded coiled coils. These studies demonstrate that distinct van der Waals interactions beyond the a and d side chains can generate a diverse set of helix-helix interfaces and three-dimensional supercoil structures.     

DNA-binding properties of a lac repressor mutant incapable of forming tetramers

The interaction of proteins bound to sites widely separated on the genome is a recurrent motif in both prokaryotic and eukaryotic regulatory systems. Lac repressor mediates the formation of "DNA loops" by the simultaneous interaction of a single protein tetramer with two DNA-binding sites. The DNA-binding properties of a Lac repressor mutant (LacIadi) deficient in the association of protein dimers to tetramers was investigated. The results of quantitative footprint and gel mobility-shift titrations suggest that the wild-type Lac repressor (LacI+) binds cooperatively to two operator sites separated by 11 helical turns on a linear DNA restriction fragment by the formation of a "looped complex." LacIadi binds to this two-site operator non-cooperatively and without formation of a looped complex. These results demonstrate that the dimer-tetramer association of LacI+ is directly responsible for its cooperative binding and its ability to mediate formation of a looped complex. The Iadi mutation disrupts the monomer-dimer as well as eliminating the dimer-tetramer association equilibria while the DNA binding affinity of LacIadi to a single site is unchanged relative to the wild-type protein. These results suggest that DNA binding and dimer-tetramer association are functionally unlinked. The similarity of the DNA-binding properties of LacIadi and Gal repressor, a protein believed to function by mediating the formation of a looped complex, are discussed.     

The roles of residues 5 and 9 of the recognition helix of Lac repressor in lac operator binding

We constructed expression libraries for Lac repressor mutants with amino acid exchanges in positions 1, 2, 5 and 9 of the recognition helix. We then analysed the interactions of residues 5 and 9 with operator variants bearing single or multiple symmetric base-pair exchanges in positions 3, 4 and 5 of the ideal fully symmetric lac operator. We isolated 37 independent Lac repressor mutants with five different amino acids in position 5 of the recognition helix that exhibit a strong preference for particular residues in position 2 and, to a lesser extent, in position 1 of the recognition helix. Our results suggest that residue 5 of the recognition helix (serine 21) contributes to the specific recognition of base-pair 4 of the lac operator. They further suggest that residue 9 of the recognition helix (asparagine 25) interacts non-specifically with a phosphate of the DNA backbone, possibly between base-pairs 2 and 3.     

Dimerisation mutants of Lac repressor. II. A single amino acid substitution, D278L, changes the specificity of dimerisation

Assembly of the lactose repressor tetramer involves two subunit interfaces, the C-terminal heptad repeats, and the monomer-monomer interface. Dimerisation between two monomers of Lac repressor of Escherichia coli lacking the two C-terminal heptad repeats occurs through the interactions between three alpha-helices of each monomer, which form a highly hydrophobic interface. Residues possibly involved in specific dimer formation are known from X-ray studies and from the phenotypes of more than 4000 single amino acid substitutions. During the examination of numerous mutants within the dimerisation interface of Lac repressor, we found that substitution of one amino acid, D278 to leucine, is sufficient to change the specificity of dimerisation. Analysis of this single substitution indicates that D278L mutant Lac repressor represses like wild-type. However, it no longer forms heterodimers with wild-type Lac repressor.     

Control of translational repression by protein-protein interactions.

The coat protein of the RNA bacteriophage MS2 is a translational repressor and interacts with a specific RNA stem-loop to inhibit translation of the viral replicase gene. As part of an effort to dissect genetically its RNA binding function, mutations were identified in the coat protein sequence that suppress mutational defects in the translational operator. Each of the mutants displayed a super-repressor phenotype, repressing translation from the wild-type and a variety of mutant operators better than did the wild-type coat protein. At least one mutant probably binds RNA more tightly than wild-type. The other mutants, however, were defective for assembly of virus-like particles, and self-associated predominantly as dimers. It is proposed that this assembly defect accounts for their super-repressor characteristics, since failure to assemble into virus-like particles elevates the effective concentration of repressor dimers. This hypothesis is supported by the observation that deletion of thirteen amino acids known to be important for assembly of dimers into capsids also resulted in the same assembly defect and in super-repressor activity. A second class of assembly defects is also described. Deletion of two amino acids from the C-terminus of coat protein resulted in failure to form capsids, most of the coat protein having the apparent molecular weight expected of trimers. This mutant (dl-8) was completely defective for repressor activity, probably because of an inability to form dimers. These results point out the inter-dependence of the structural and regulatory functions of coat protein.     

Characterization of a new four-chain coiled-coil: influence of chain length on stability.

Limited information is available on inherent stabilities of four-chain-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain alpha-helical chains 21, 28, and 35 amino acids in length are 3.1 x 10(-12), 6.7 x 10(-23), and 1.0 x 10(-38) M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 microM, 51 nM, and 280 fM, respectively. Finally, we discuss the rules governing coiled-coil formation in light of the work presented here.     

Characterization of the interaction between the plasma membrane H-ATPase of Arabidopsis thaliana and a novel interactor (PPI1)

Proton pump interactor, isoform 1 (PPI1) is a novel interactor of the C-terminus of Arabidopsis thaliana plasma membrane H(+)-ATPase (EC 3.6.3.6). We produced two fusion proteins consisting of, respectively, the first 88 amino acids or the entire protein deleted of the last 24 hydrophobic amino acids, and we show that the latter protein has a threefold higher affinity for the H(+)-ATPase. PPI1-induced stimulation of H(+)-ATPase activity dramatically decreased with the increase of pH above pH 6.8, but became largely pH-independent when the enzyme C-terminus was displaced by fusicoccin-induced binding of 14-3-3 proteins. The latter treatment did not affect PPI1 affinity for the H(+)-ATPase. These results indicate that PPI1 can bind the H(+)-ATPase independently of the C-terminus conformation, but is not able to suppress the C-terminus auto-inhibitory action.     

Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10.

The Tn10 derived Tet repressor contains an amino acid segment with high homology to the alpha-helix-turn-alpha-helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first alpha-helix without loss of activity. The last position of the first alpha-helix, the second position in the turn, and the fourth position in the second alpha-helix require mostly hydrophobic residues. The proposed C-terminus of the first alpha-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented.     

Strengthening the dimerisation interface of Lac repressor increases its thermostability by 40 deg. C

We increased drastically the heat stability of Lac repressor (LacR) of Escherichia coli. Wild-type tetrameric LacR denatures irreversibly at 53 degrees C. Improving hydrophobic packing at the dimerisation interface by a single substitution increases LacR heat-resistance by 40 deg. C without abolishing inducer binding at high and low temperatures. Tetrameric LacR mutants carrying substitutions of the positively charged amino acid Lys84 by each of the hydrophobic amino acids Leu, Ile and Met resist heating to temperatures up to 93 degrees C. We performed IPTG binding assays at 80 degrees C and found the mutant Lac repressors active and, thus, the core intact. Furthermore, the activity of LacR following heating is shown at room temperature by a gel retardation assay, which demonstrates normal oligomerisation state and function of the headpiece. The same mutations (K84L/I/M) in the dimer LacR331stop, carrying a stop codon in amino acid 331, increase thermostability of the dimer from 47 degrees C to 87 degrees C. LacRK84M represses beta-galactosidase activity in vivo as well as the wild-type and is sufficiently induced to allow growth on lactose. The results with both tetramer and dimer variants of LacR indicate mutual stabilisation of the tetramerisation region and the stable core.     

Secondary structure and oligomerization behavior of equilibrium unfolding intermediates of the lambda cro repressor

The thermal unfolding of the wild-type Cro repressor, its disulfide-bridged mutant Cro-V55C (with the Val-55 --> Cys single amino acid substitution), and a CNBr-fragment (13-66)2 of Cro-V55C was studied by Fourier transform infrared spectroscopy and dynamic light scattering. The combined approach reveals that thermal denaturation of Cro-WT and Cro-V55C proceeds in two steps through equilibrium unfolding intermediates. The first thermal transition of the Cro-V55C dimer involves the melting of the alpha-helices and the short beta-strand localized in the N-terminal part of the molecule. This event is accompanied by the formation of tetramers, and also impacts on the hydrogen-bonding interactions of the C-terminal beta-strands. The beta-sheet formed by the C-terminal parts of each polypeptide chain is the major structural feature of the intermediate state of Cro-V55C and unfolds during a second thermal transition, which is accompanied by the dissociation of the tetramers. Cutting of 12 amino acids in the N-terminal region is sufficient to prevent the formation of alpha-helical structure in the CNBr-fragment of Cro-V55C, and to induce tetramerization already at room temperature. The tetramers may persist over a broad temperature range, and start to dissociate only upon thermal unfolding of the beta-sheet structure formed by the C-terminal regions. The wild-type protein is a dimer at room temperature and at protein concentrations of 1.8-5.8 mg/mL. At lower concentrations, the dimers are stable until the onset of thermal unfolding, which is accompanied by the dissociation of the dimers into monomers. At higher protein concentrations, the unfolding is more complex and involves the formation of tetramers at intermediate temperatures. At these intermediate temperatures, the Cro-WT has lost all of its alpha-helical structure and also most of its native beta-sheet structure. Upon further temperature increase, a tendency for an intermolecular association of the beta-strands is observed, which may result in irreversible beta-aggregation at high protein concentrations.     

A disulfide bridge mediated by cysteine 574 is formed in the dimer of the 70-kDa heat shock protein

The 70-kDa heat shock protein (Hsp70) is predominantly present intracellularly as a monomer, but a small population is converted to dimers and oligomers under certain conditions. In the present study, we investigated the dimeric structure of human inducible Hsp70. As reported earlier, the C-terminal client-binding domain (amino acids 382-641) was required for the dimerization. A 40-amino acid deletion in the client-binding domain from either the N-terminus or C-terminus greatly enhanced the dimerization potential of Hsp70. Limited proteolysis indicated that the dimer formed through truncation from the C-terminus had a conformation similar to that of the non-truncated form. Truncation experiments demonstrated that the client-binding sub-domain (amino acids 382-520) with its adjacent region up to amino acid 541 was not sufficient for the dimerization but that the region up to amino acid 561 was sufficient. Interestingly, the dimer formed through truncation from the C-terminus acquired a homomeric disulfide bridge at Cys574.     

Assembly of Acanthamoeba myosin-II minifilaments. Definition of C-terminal residues required to form coiled-coils, dimers, and octamers

Acanthamoeba myosin-II forms bipolar octamers by three successive steps of dimerization of the C-terminal, coiled-coil tail. In this study, we generated N-terminal and C-terminal truncation constructs and point mutants of the Acanthamoeba myosin-II tail to delineate the structural requirements for assembly of bipolar mini-filaments. By the use of light-scattering, CD spectroscopy, analytical ultracentrifugation, and tryptophan fluorescence experiments, we determined that: (1) the C-terminal 14 heptad repeats plus most of the tailpiece (residues 1381-1509) are required to form antiparallel dimers of coiled-coils; (2) amino acid residues within heptads 23-32 (residues 1254-1325) are required to form tetramers; (3) the C-terminal 32 heptad repeats suffice to assemble octameric minifilaments; (4) A1378 is outside of the interaction interface; (5) the mutation L1475W inhibits dimerization; and (6) F1443 is involved in the dimerization interface but is exposed to the solvent. We propose that the tailpiece (residues 1483-1509) interacts with two heptads (13 and 14, residues 1381-1393), which are important for dimerization and coiled-coil formation. These results support a model in which hydrophobic as well as electrostatic interactions control the register between myosin-II coiled-coils and guide sequential steps of dimerization that generate stable, octameric mini-filaments.     

Requirement of the conserved, hydrophobic C-terminus region for the activation of heparanase

Heparanase is an endo-β-d-glucuronidase responsible for the cleavage of heparan sulfate, participating in extracellular matrix degradation and remodeling. Heparanase activity is well correlated with the potential for metastasis and angiogenesis in a large number of tumor-derived cell types, directly implicating the involvement of heparanase in tumor progression. Here, we provide the first evidence that the hydrophobic C-terminus region of heparanase has specific roles in intracellular trafficking, secretion, activation, and heparanase-mediated tumor cell migration. Furthermore, partial deletion of this hydrophobic C-terminus region, substitution within the hydrophobic C-terminus region to hydrophilic amino acids, and experiments of single amino acid mutations further point out the importance of the hydrophobic C-terminus region. Therefore, our findings suggest that the hydrophobic C-terminus region of heparanase is a determinant for its intracellular trafficking to the Golgi apparatus, followed by secretion, activation, and tumor cell migration.     

Kinetic characterization of adenylosuccinate synthetase from the thermophilic archaea Methanocaldococcus jannaschii

Adenylosuccinate synthetase (AdSS) catalyzes the Mg2+ dependent condensation of a molecule of IMP with aspartate to form adenylosuccinate, in a reaction driven by the hydrolysis of GTP to GDP. AdSS from the thermophilic archaea, Methanocaldococcus jannaschii (MjAdSS) is 345 amino acids long against an average length of 430-457 amino acids for most mesophilic AdSS. This short AdSS has two large deletions that map to the middle and C-terminus of the protein. This article discusses the detailed kinetic characterization of MjAdSS. Initial velocity and product inhibition studies, carried out at 70 degrees C, suggest a rapid equilibrium random AB steady-state ordered C kinetic mechanism for the MjAdSS catalyzed reaction. AdSS are known to exhibit monomer-dimer equilibrium with the dimer being implicated in catalysis. In contrast, our studies show that MjAdSS is an equilibrium mixture of dimers and tetramers with the tetramer being the catalytically active form. The tetramer dissociates into dimers with a minor increase in ionic strength of the buffer, while the dimer is extremely stable and does not dissociate even at 1.2 M NaCl. Phosphate, a product of the reaction, was found to be a potent inhibitor of MjAdSS showing biphasic inhibition of enzyme activity. The inhibition was competitive with IMP and noncompetitive with GTP. MjAdSS, like the mouse acidic isozyme, exhibits substrate inhibition, with IMP inhibiting enzyme activity at subsaturating GTP concentrations. Regulation of enzyme activity by the glycolytic intermediate, fructose 1,6 bisphosphate, was also observed with the inhibition being competitive with IMP and noncompetitive against GTP.     

Crystal structure of the lambda repressor C-terminal domain octamer.

The three-dimensional structure of the lambda repressor C-terminal domain (CTD) has been determined at atomic resolution. In the crystal, the CTD forms a 2-fold symmetric tetramer that mediates cooperative binding of two repressor dimers to pairs of operator sites. Based upon this structure, a model was proposed for the structure of an octameric repressor that forms both in the presence and absence of DNA. Here, we have determined the structure of the lambda repressor CTD in three new crystal forms, under a wide variety of conditions. All crystals have essentially the same tetramer, confirming the results of the earlier study. One crystal form has two tetramers bound to form an octamer, which has the same overall architecture as the previously proposed model. An unexpected feature of the octamer in the crystal structure is a unique interaction at the tetramer-tetramer interface, formed by residues Gln209, Tyr210 and Pro211, which contact symmetry-equivalent residues from other subunits of the octamer. Interestingly, these residues are also located at the dimer-dimer interface, where the specific interactions are different. The structures thus indicate specific amino acid residues that, at least in principle, when altered could result in repressors that form tetramers but not octamers.