The conserved Asn49 of maize glutathione S-transferase I modulates substrate binding, catalysis and intersubunit communication.

The functional and structural role of the conserved Asn49 of theta class maize glutathione S-transferase was investigated by site-directed mutagenesis. Asn49 is located in the type I beta turn formed by residues 49-52, and is involved in extensive hydrogen-bonding interactions between alpha helix 2 and the rest of the N-terminal domain. The substitution of Asn49 with Ala induces positive cooperativity for 1-chloro-2,4-dinitrobenzene (CDNB) binding as reflected by a Hill coefficient of 1.9 (S(0.5)CDNB = 0.43 mm). The positive cooperativity is also confirmed by following the isothermic binding of 1-hydroxyl-2,4-dinitrobenzene (HDNB) by UV-difference spectroscopy. In addition, the mutated enzyme exhibits: (a) an increase in the Km(GSH) value of about 6.5-fold, and decrease in kcat value of about fourfold; (b) viscosity-independent kinetic parameters; (c) lower thermostability, and (d) increased susceptibility to proteolytic attack by trypsin, when compared to the wild-type enzyme. It is concluded that Asn49 affects the rate-limiting step of the catalytic reaction, and contributes significantly to the structural and binding characteristics of both the glutathione binding site (G-site) and the electrophile substrate binding site (H-site) by affecting the structural integrity of a type I beta turn (comprising residues 49-52) and probably the flexibility of the highly mobile short 310 helical segment of alpha helix 2 (residues 35-46). These structural perturbations are probably transmitted, via Phe51 and Phe65, to alpha helix H3" of the adjacent subunit which contains key residues that interact with the electrophile substrate and contribute to the monomer-monomer contact region. This may accounts for the positive cooperativity observed.     

Analysis of sequence determinants of F1Fo-ATP synthase in the N-terminal region of alpha subunit for binding of delta subunit

The stator in F(1)F(o)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(o) and to the alpha(3)beta(3) hexagon of F(1). Previous work has shown that N-terminal residues of alpha subunit are involved in binding delta. A synthetic peptide consisting of the first 22 residues of alpha (alphaN1-22) binds specifically to isolated wild-type delta subunit with 1:1 stoichiometry and high affinity, accounting for a major portion of the binding energy between delta and F(1). Residues alpha6-18 are predicted by secondary structure algorithms and helical wheels to be alpha-helical and amphipathic, and a potential helix capping box occurs at residues alpha3-8. We introduced truncations, deletions, and mutations into alphaN1-22 peptide and examined their effects on binding to the delta subunit. The deletions and mutations were introduced also into the N-terminal region of the uncA (alpha subunit) gene to determine effects on cell growth in vivo and membrane ATP synthase activity in vitro. Effects seen in the peptides were well correlated with those seen in the uncA gene. The results show that, with the possible exception of residues close to the initial Met, all of the alphaN1-22 sequence is required for binding of delta to alpha. Within this sequence, an amphipathic helix seems important. Hydrophobic residues on the predicted nonpolar surface are important for delta binding, namely alphaIle-8, alphaLeu-11, alphaIle-12, alphaIle-16, and alphaPhe-19. Several or all of these residues probably make direct interaction with helices 1 and 5 of delta. The potential capping box sequence per se appeared less important. Impairment of alpha/delta binding brings about functional impairment due to reduced level of assembly of ATP synthase in cells.     

Structure and tropomyosin binding properties of the N-terminal capping domain of tropomodulin 1

Two families of actin regulatory proteins are the tropomodulins and tropomyosins. Tropomodulin binds to tropomyosin (TM) and to the pointed end of actin filaments and "caps" the pointed end (i.e., inhibits its polymerization and depolymerization). Tropomodulin 1 has two distinct actin-capping regions: a folded C-terminal domain (residues 160-359), which does not bind to TM, and a conserved, N-terminal region, within residues 1-92 that binds TM and requires TM for capping activity. NMR and circular dichroism were used to determine the structure of a peptide containing residues 1-92 of tropomodulin (Tmod1(1-92)) and to define its TM binding site. Tmod1(1-92) is mainly disordered with only one helical region, residues 24-35. This helix forms part of the TM binding domain, residues 1-35, which become more ordered upon binding a peptide containing the N-terminus of an alpha-TM. Mutation of L27 to E or G in the Tmod helix reduces TM affinity. Residues 49-92 are required for capping but do not bind TM. Of these, residues 67-75 have the sequence of an amphipathic helix, but are not helical. Residues 55-62 and 76-92 display negative 1H-15N heteronuclear Overhauser enhancements showing they are flexible. The conformational dynamics of these residues may be important for actin capping activity.     

Relationships between heme incorporation, tetramer formation, and catalysis of a heme-regulated phosphodiesterase from Escherichia coli: a study of deletion and site-directed mutants

The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (DeltaN147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg2+ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe2+ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme.     

Studies of the erythrocyte spectrin tetramerization region

Human erythrocyte spectrin dimers associate at the N-terminal region of alpha spectrin (alpha N) and the C-terminal region of beta-spectrin (beta C) to form tetramers. We have prepared model peptides to study the tetramerization region. Based on phasing information obtained from enzyme digests, we prepared spectrin fragments consisting of the first 156 amino-acid residues and the first 368 amino-acid residues of alpha-spectrin (Sp alpha 1-156 and Sp alpha 1-368, respectively), and found that both peptides associate with a beta-spectrin model peptide, with an affinity similar to that found in alpha beta dimer tetramerization. Spin label EPR studies show that the region consisting of residues 21-46 in alpha-spectrin is helical even in the absence of its beta-partner. Multi-dimensional nuclear magnetic resonance studies of samples with and without a spin label attached to residue 154 show that Sp alpha 1-156 consists of four helices, with the first helix unassociated with the remaining three helices, which bundle to form a triple helical coiled coil bundle. A comparison of the structures of erythrocyte spectrin with other published structures of Drosophila and chicken brain spectrin is discussed. Circular dichroism studies show that the lone helix in Sp alpha-156 associates with helices in the beta peptide to form a coiled coil bundle. Based on NMR and CD results, we suggest that the helices in Sp alpha 1-156 exhibit a looser (frayed) conformation, and that the helices convert to a tighter conformation upon association with its beta-partner. This suggestion does not rule out possible conversion of a non-structured conformation to a structured conformation in various parts of the molecule upon association. Spectrin mutations at residues 28 and 45 of alpha-spectrin have been found in patients with hereditary elliptocytosis. NMR studies were also carried out on Sp alpha 1-156R28S, Sp alpha 1-156R45S and Sp alpha 1-156R45T. A comparison of the structures of Sp alpha 1-156 and Sp alpha 1-156R28S, Sp alpha 1-156R45S and Sp alpha 1-156R45T is discussed.     

Heme binding by the N-terminal fragment 1-44 of human growth hormone

Fragment 1-44 of human growth hormone (hGH), prepared in vitro by limited proteolysis of the hormone with pepsin at low pH, encompasses in full the N-terminal helix of this four-helix bundle protein [Spolaore, B., Polverino de Laureto, P., Zambonin, M., and Fontana, A. (2004) Biochemistry 40, 9460-9468]. Here, we report the new and interesting observation that fragment 1-44 can bind heme. The binding property is specific for the N-terminal helix of hGH, since heme binding does not occur with fragment 45-191 or the entire protein. The spectral characteristics of Fe-protoporphyrin IX are those of a low-spin, hexacoordinated iron ligated by two imidazole rings of His residues or His and Met residues. Far-UV circular dichroism (CD) measurements revealed that fragment 1-44 acquires a helical secondary structure upon heme binding. Heme appears to be bound to the fragment in a stereospecific way, since an induced dichroic signal is observed in the Soret region of the CD spectrum. The heme-fragment complex occurs in a 1:1 molar ratio, as determined by spectrophotometric titration, as well as by electrospray-ionization mass spectrometric analysis of the complex. The fragment alone is much more susceptible to tryptic digestion than the heme complex, implying a more folded and rigid structure of this last species. It is proposed that the molecular features of fragment 1-44 determining its heme-binding property reside in the amphipathic character of the helix adopted by the fragment, as well as in the presence in its polypeptide chain of His18, His21, and Met14. These residues can act as specific ligands for the heme-iron, as observed with cytochromes.     

F1F0-ATP synthase. Binding of delta subunit to a 22-residue peptide mimicking the N-terminal region of alpha subunit

The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to alpha(3)beta(3) hexagon of F(1). Proteolysis and cross-linking had suggested that N-terminal residues of alpha subunit are involved in binding delta. Here we demonstrate that a synthetic peptide consisting of the first 22 residues of alpha ("alpha N1-22") binds specifically to isolated wild-type delta subunit with high affinity (K(d) = 130 nm), accounting for a major portion of the binding energy when delta-depleted F(1) and isolated delta bind together (K(d) = 1.4 nm). Stoichiometry of binding of alpha N1-22 to delta at saturation was 1/1, showing that in intact F(1)F(0)-ATP synthase only one of the three alpha subunits is involved in delta binding. When alpha N1-22 was incubated with delta subunits containing mutations in helices 1 or 5 on the F(1)-binding face of delta, peptide binding was impaired as was binding of delta-depleted F(1). Residues alpha 6-18 are predicted to be helical, and a potential helix capping box occurs at residues alpha 3-8. Circular dichroism measurements showed that alpha N1-22 had significant helical content. Hypothetically a helical region of residues alpha N1-22 packs with helices 1 and 5 on the F(1)-binding face of delta, forming the alpha/delta interface.     

Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I

The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.     

Structural consequences of site-directed mutagenesis in flexible protein domains: NMR characterization of the L(55,56)S mutant of RhoGDI.

The guanine dissociation inhibitor RhoGDI consists of a folded C-terminal domain and a highly flexible N-terminal region, both of which are essential for biological activity, that is, inhibition of GDP dissociation from Rho GTPases, and regulation of their partitioning between membrane and cytosol. It was shown previously that the double mutation L55S/L56S in the flexible region of RhoGDI drastically decreases its affinity for Rac1. In the present work we study the effect of this double mutation on the conformational and dynamic properties of RhoGDI, and describe the weak interaction of the mutant with Rac1 using chemical shift mapping. We show that the helical content of the region 45-56 of RhoGDI is greatly reduced upon mutation, thus increasing the entropic penalty for the immobilization of the helix, and contributing to the loss of binding. In contrast to wild-type RhoGDI, no interaction with Rac1 could be identified for amino-acid residues of the flexible domain of the mutant RhoGDI and only very weak binding was observed for the folded domain of the mutant. The origins of the effect of the L55S/L56S mutation on the binding constant (decreased by at least three orders of magnitude relative to wild-type) are discussed with particular reference to the flexibility of this part of the protein.     

Structure of the Clade 1 catalase,CatF of Pseudomonas syringae,at 1.8 A resolution

Catalase CatF of Pseudomonas syringae has been identified phylogenetically as a clade 1 catalase, closely related to plant catalases, a group from which no structure has been determined. The structure of CatF has been refined at 1.8 A resolution by using X-ray synchrotron data collected from a crystal flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are, respectively, 18.3% and 24.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry. The crystallized enzyme is a homotetramer of subunits with 484 residues, some 26 residues shorter than predicted from the DNA sequence. Mass spectrometry analysis confirmed the absence of 26 N-terminal residues, possibly removed by a periplasmic transport system. The core structure of the CatF subunit was closely related to seven other catalases with root-mean-square deviations (RMSDs) of 368 core Calpha atoms of 0.99-1.30 A. The heme component of CatF is heme b in the same orientation that is found in Escherichia coli hydroperoxidase II, an orientation that is flipped 180 degrees with respect the orientation of the heme in bovine liver catalase. NADPH is not found in the structure of CatF because key residues required for nucleotide binding are missing; 2129 water molecules were refined into the model. Water occupancy in the main or perpendicular channel of CatF varied among the four subunits from two to five in the region between the heme and the conserved Asp150. A comparison of the water occupancy in this region with the same region in other catalases reveals significant differences among the catalases.     

1H NMR structural and functional characterisation of a cAMP-specific phosphodiesterase-4D5 (PDE4D5) N-terminal region peptide that disrupts PDE4D5 interaction with the signalling scaffold proteins, beta-arrestin and RACK1

The unique 88 amino acid N-terminal region of cAMP-specific phosphodiesterase-4D5 (PDE4D5) contains overlapping binding sites conferring interaction with the signaling scaffold proteins, betaarrestin and RACK1. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, encompasses the entire N-terminal RACK1 Interaction Domain (RAID1) together with a portion of the beta-arrestin binding site. (1)H NMR and CD analyses indicate that this region has propensity to form a helical structure. The leucine-rich hydrophobic grouping essential for RACK1 interaction forms a discrete hydrophobic ridge located along a single face of an amphipathic alpha-helix with Arg34 and Asn36, which also play important roles in RACK1 binding. The Asn22/Pro23/Trp24/Asn26 grouping, essential for RACK1 interaction, was located at the N-terminal head of the amphipathic helix that contained the hydrophobic ridge. RAID1 is thus provided by a distinct amphipathic helical structure. We suggest that the binding of PDE4D5 to the WD-repeat protein, RACK1, may occur in a manner akin to the helix-helix interaction shown for G(gamma) binding to the WD-repeat protein, G(beta). A more extensive section of the PDE4D5 N-terminal sequence (Thr11-Ala85) is involved in beta-arrestin binding. Several residues within the RAID1 helix contribute to this interaction however. We show here that these residues form a focused band around the centre of the RAID1 helix, generating a hydrophobic patch (from Leu29, Val30 and Leu33) flanked by polar/charged residues (Asn26, Glu27, Asp28, Arg34). The interaction with beta-arrestin exploits a greater circumference on the RAID1 helix, and involves two residues (Glu27, Asp28) that do not contribute to RACK1 binding. In contrast, the interaction of RACK1 with RAID1 is extended over a greater length of the helix and includes Leu37/Leu38, which do not contribute to beta-arrestin binding. A membrane-permeable, stearoylated Val12-Ser49 38-mer peptide disrupted the interaction of both beta-arrestin and RACK1 with endogenous PDE4D5 in HEKB2 cells, whilst a cognate peptide with a Glu27Ala substitution selectively failed to disrupt PDE4D5/RACK1 interaction. The stearoylated Val12-Ser49 38-mer peptide enhanced the isoprenaline-stimulated PKA phosphorylation of the beta(2)-adrenergic receptors (beta(2)AR) and its activation of ERK, whilst the Glu27Ala peptide was ineffective in both these regards.     

Analysis of parathyroid hormone (PTH)/secretin receptor chimeras differentiates the role of functional domains in the pth/ pth-related peptide (PTHrP) receptor on hormone binding and receptor activation.

The type 1 parathyroid hormore receptor (PTH1r) belongs to the class II family of G protein-coupled receptors. To delineate the sites in the PTH1r's N-terminal region, and the carboxy-core domain (transmembrane segments + extracellular loops) involved in PTH binding, we have evaluated the functional properties of 27 PTH1-secretin chimeras receptors stably expressed in HEK-293 cells. The wild type and chimeric receptors were analyzed for cell surface expression, binding for PTH and secretin, and functional responsiveness (cAMP induction) toward secretin and PTH. The expression levels of the chimeric receptors were comparable to that of the PTH1r (60-100%). The N-terminal region of PTH1r was divided into three segments that were replaced either singly or in various combinations with the homologous region of the secretin receptor (SECr). Substitution of the carboxy-terminal half (residues 105-186) of the N-terminal region of PTH1r for a SECr homologous segment did not reduced affinity for PTH but abolished signaling in response to PTH. This data indicate that receptor activation is dissociable from high affinity hormone binding in the PTH1r, and that the N-terminal region might play a critical role in the activation process. Further segment replacements in the N-termini focus on residues 105-186 and particularly residues 146-186 of PTH1r as providing critical segments for receptor activation. The data obtained suggest the existence of two distinct PTH binding sites in the PTH1r's N-terminal region: one site in the amino-terminal half (residues 1-62) (site 1) that participates in high-affinity PTH binding; and a second site of lower affinity constituted by amino acid residues scattered throughout the carboxy-terminal half (residues 105-186) (site 2). In the absence of PTH binding to site 1, higher concentrations of hormone are required to promote receptor activation. In addition, elimination of the interaction of PTH with site 2 results in a loss of signal transduction without loss of high-affinity PTH binding. Divers substitutions of the extracellular loops of the PTH1r highlight the differential role of the first- and third extracellular loop in the process of PTH1r activation after hormone binding. A chimera containing the entire extracellular domains of the PTH1r and the transmembrane + cytoplasmic domains of SECr had very low PTH binding affinity and did not signal in response to PTH. Further substitution of helix 5 of PTH1r in this chimera increased affinity for PTH that is close to the PTH affinity for the wild-type PTH1r but surprisingly, did not mediate signaling response. Additional substitutions of PTH1r's helices in various combinations emphasize the fundamental role of helix 3 and helix 6 on the activation process of the PTH1r. Overall, our studies demonstrated that several PTH1r domains contribute differentially to PTH binding affinity and signal transduction mechanism and highlight the role of the N-terminal domain and helix 3 and helix 6 on receptor activation.     

High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation.

The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.     

Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy

A critical point of interaction between F(1) and F(0) in the bacterial F(1)F(0)-ATP synthase is formed by the alpha and delta subunits. Previous work has shown that the N-terminal domain (residues 3-105) of the delta subunit forms a 6 alpha-helix bundle [Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201] and that the majority of the binding energy between delta and F(1) is provided by the interaction between the N-terminal 22 residues of the alpha- and N-terminal domain of the delta subunit [Weber, J., Muharemagic, A., Wilke-Mounts, S., and Senior, A. E. (2003) J. Biol. Chem. 278, 13623-13626]. We have now analyzed a 1:1 complex of the delta-subunit N-terminal domain and a peptide comprising the N-terminal 22 residues of the alpha subunit by heteronuclear protein NMR spectroscopy. A comparison of the chemical-shift values of delta-subunit residues with and without alpha N-terminal peptide bound indicates that the binding interface on the N-terminal domain of the delta subunit is formed by alpha helices I and V. NOE cross-peak patterns in 2D (12)C/(12)C-filtered NOESY spectra of the (13)C-labeled delta-subunit N-terminal domain in complex with unlabeled peptide verify that residues 8-18 in the alpha-subunit N-terminal peptide are folded as an alpha helix when bound to delta N-terminal domain. On the basis of intermolecular contacts observed in (12)C/(13)C-filtered NOESY experiments, we describe structural details of the interaction of the delta-subunit N-terminal domain with the alpha-subunit N-terminal alpha helix.     

Characterization of heme-regulated eIF2alpha kinase: roles of the N-terminal domain in the oligomeric state, heme binding, catalysis, and inhibition

Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed.     

Structure and activity of the N-terminal region of the eukaryotic cytolysin equinatoxin II

The actinoporins are a family of proteins from sea anemones that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being resistant to these toxins. Pore formation by the actinoporin equinatoxin II (EqTII) proceeds by membrane binding via a surface rich in aromatic residues, followed by translocation of the N-terminal region to the membrane and, finally, across the bilayer to form a functional pore. A key feature of this mechanism is the ability of the N-terminal region to form a stable, bilayer-spanning helix in the membrane, which in turn requires dissociation of the N-terminus from the bulk of the protein and significant extension of the N-terminal helix of native EqTII. In this study the structures of three peptides corresponding to residues 11-29, 11-32, and 1-32, respectively, of EqTII have been investigated by high-resolution nuclear magnetic resonance and Fourier transform infrared spectroscopy. The 32-residue peptide lacks ordered secondary structure in water, but residues 6-28 form a helix in dodecylphosphocholine micelles. Although this helix is long enough to span a bilayer membrane, this peptide and the shorter analogues display limited permeabilizing activity in large unilamellar vesicles and very weak hemolytic activity in human red blood cells. Thus, while the N-terminal region has the structural features required for this unusual mechanism of pore formation, the lack of activity of the isolated N-terminus shows that the bulk of the protein is essential for efficient pore formation by facilitating initial membrane binding, interacting with sphingomyelin, or stabilizing the oligomeric pore.     

Modulation of the oligomerization state of the bovine F1-ATPase inhibitor protein, IF1, by pH

Bovine IF(1), a basic protein of 84 amino acids, is involved in the regulation of the catalytic activity of the F(1) domain of ATP synthase. At pH 6.5, but not at basic pH values, it inhibits the ATP hydrolase activity of the enzyme. The oligomeric state of bovine IF(1) has been investigated at various pH values by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that the protein forms a tetramer at pH 8, and below pH 6.5, the protein is predominantly dimeric. By covalent cross-linking, it has been found that at pH 8.0 the fragment of IF(1) consisting of residues 44-84 forms a dimer, whereas the fragment from residues 32-84 is tetrameric. Therefore, some or all of the residues between positions 32 and 43 are necessary for tetramer formation and are involved in the pH-sensitive interconversion between dimer and tetramer. One important residue in the interconversion is histidine 49. Mutation of this residue to lysine abolishes the pH-dependent activation-inactivation, and the mutant protein is active and dimeric at all pH values investigated. It is likely from NMR studies that the inhibitor protein dimerizes by forming an antiparallel alpha-helical coiled-coil over its C-terminal region and that at high pH values, where the protein is tetrameric, the inhibitory regions are masked. The mutation of histidine 49 to lysine is predicted to abolish coiled-coil formation over residues 32-43 preventing interaction between two dimers, forcing the equilibrium toward the dimeric state, thereby freeing the N-terminal inhibitory regions and allowing them to interact with F(1).     

Crosslinking between alpha and beta subunits defines the orientation and spatial relationship of some of the transmembrane helices of the proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli

The proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta, organized as an alpha(2)beta(2) tetramer. The protein contains three recognizable domains, of which domain II is the transmembrane region of the molecule containing the pathway for proton translocation. Domain II is composed of four transmembrane helices at the carboxyl-terminus of the alpha subunit and either eight or nine transmembrane helices at the amino-terminal region of the beta subunit. We have introduced pairs of cysteine residues into a cysteine-free transhydrogenase by site-directed mutagenesis. Disulfide bond formation between some of these cysteine residues occurred spontaneously or on treatment with cupric 1, 10-phenanthrolinate. Analysis of crosslinked products confirmed that there are nine transmembrane helices in the domain II region of the beta subunit. The proximity to one another of several of the transmembrane helices was determined. Thus, helices 2 and 4 are close to helix 6 (nomenclature of Meuller and Rydstr?m, J. Biol. Chem. 274, 19072-19080, 1999), and helix 3 and the carboxyl-terminal eight residues of the alpha subunit are close to helix 7. In the alpha(2)beta(2) tetramer, helices 2 and 4 of one alpha subunit are close to the same pair of transmembrane helices of the other alpha subunit, and helix 6 of one beta subunit is close to helix 6 of the other beta subunit.     

The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis

The plasmid pRN1 encodes for a multifunctional replication protein with primase, DNA polymerase and helicase activity. The minimal region required for primase activity encompasses amino-acid residues 40–370. While the N-terminal part of that minimal region (residues 47–247) folds into the prim/pol domain and bears the active site, the structure and function of the C-terminal part (residues 248–370) is unknown. Here we show that the C-terminal part of the minimal region folds into a compact domain with six helices and is stabilized by a disulfide bond. Three helices superimpose well with the C-terminal domain of the primase of the bacterial broad host range plasmid RSF1010. Structure-based site-directed mutagenesis shows that the C-terminal helix of the helix bundle domain is required for primase activity although it is distant to the active site in the crystallized conformation. Furthermore, we identified mutants of the C-terminal domain, which are defective in template binding, dinucleotide formation and conformation change prior to DNA extension.