Design and crystal structure of bacteriophage T4 mini-fibritin NCCF

Fibritin is a fibrous protein that forms "whiskers" attached to the neck of bacteriophage T4. Whiskers interact with the long tail fibers regulating the assembly and infectivity of the virus. The fibritin trimer includes the N-terminal domain responsible for attachment to the phage particle and for the collar formation, the central domain forming a 500 A long segmented coiled-coil structure, and the C-terminal "foldon" domain. We have designed a "mini" fibritin with most of the coiled-coil domain deleted, and solved its crystal structure. The non-helical N-terminal part represents a new protein fold that tightly interacts with the coiled-coil segment forming a single domain, as revealed by calorimetry. The analysis of the crystal structure and earlier electron microscopy data on the collar-whisker complex suggests the necessity of other proteins to participate in the collar formation. Crystal structure determination of the N-terminal domain of fibritin is the first step towards elucidating the detailed structure and assembly mechanism of the collar-whisker complex.     

Very fast folding and association of a trimerization domain from bacteriophage T4 fibritin

The foldon domain constitutes the C-terminal 30 amino acid residues of the trimeric protein fibritin from bacteriophage T4. Its function is to promote folding and trimerization of fibritin. We investigated structure, stability and folding mechanism of the isolated foldon domain. The domain folds into the same trimeric beta-propeller structure as in fibritin and undergoes a two-state unfolding transition from folded trimer to unfolded monomers. The folding kinetics involve several consecutive reactions. Structure formation in the region of the single beta-hairpin of each monomer occurs on the submillisecond timescale. This reaction is followed by two consecutive association steps with rate constants of 1.9(+/-0.5)x10(6)M(-1)s(-1) and 5.4(+/-0.3)x10(6)M(-1)s(-1) at 0.58 M GdmCl, respectively. This is similar to the fastest reported bimolecular association reactions for folding of dimeric proteins. At low concentrations of protein, folding shows apparent third-order kinetics. At high concentrations of protein, the reaction becomes almost independent of protein concentrations with a half-time of about 3 ms, indicating that a first-order folding step from a partially folded trimer to the native protein (k=210 +/- 20 s(-1)) becomes rate-limiting. Our results suggest that all steps on the folding/trimerization pathway of the foldon domain are evolutionarily optimized for rapid and specific initiation of trimer formation during fibritin assembly. The results further show that beta-hairpins allow efficient and rapid protein-protein interactions during folding.     

The carboxy-terminal domain initiates trimerization of bacteriophage T4 fibritin.

Bacteriophage T4 fibritin is a triple-stranded, parallel, segmented alpha-helical coiled-coil protein. Earlier we showed that the C-terminal globular domain (foldon) of fibritin is essential for correct trimerization and folding of the protein. We constructed the chimerical fusion protein W31 in which the fibritin foldon sequence is followed by the small globular non-alpha-helical protein gp31 of the T4 phage. We showed that the foldon is capable of trimerization in the absence of the coiled-coil part of fibritin. A deletion mutant of fibritin (NB1) with completely deleted foldon is unable to fold and trimerize correctly. An excess of this mutant protein did not influence the refolding of fibritin in vitro, and the chimerical protein inhibited this process efficiently. Our conclusion is that the trimerization of the foldon is the initial step of fibritin refolding and is followed by the formation of the coiled-coil structure.     

A fluorescence assay for rapid detection of ligand binding affinity to HIV-1 gp41

The fusion-active conformation of the envelope protein gp41 of HIV-1 consists of an N-terminal trimeric alpha-helical coiled-coil domain and three anti-parallel C-terminal helices that fold down the grooves of the coiled-coil to form a six-helix bundle. Disruption of the six-helix bundle is considered to be a key component of an effective non-peptide fusion inhibitor. In the current study, a fluorescence resonance energy transfer (FRET) experiment for the detection of inhibitor binding to the gp41 N-peptide coiled-coil of HIV-1 was performed, utilizing peptide inhibitors derived from the gp41 C-terminal helical region. The FRET acceptor is a 31-residue N-peptide containing a known deep hydrophobic pocket, stabilized into a trimer by ferrous ion ligation. The FRET donor is a 16-18-residue fluorophore-labeled C-peptide, designed to test the specificity of the N-C interaction. Low microM dissociation constants were observed, correlated to the correct sequence and helical propensity of the C-peptides. Competitive inhibition was demonstrated using the assay, allowing for rank ordering of peptide inhibitors according to their affinity in the 1-20 microM range. The assay was conducted by measuring fluorescence intensity in 384-well plates. The rapid detection of inhibitor binding may permit identification of novel drug classes from a library.     

Thermostability of bacteriophage T4 fibritin and its deletion mutants]

The thermal stability of a series of recently obtained mutants of fibritin from bacteriophage T4 (a superhelical fibrous homotrimer with parallel-packed subunits each containing 486 amino acid residues) progressively truncated from the subunit N-end was studied during incubation at 40-90 degrees C in the presence of a surfactant (2% SDS). The mutant fibritins, G, B, C, and E, contained 443, 276, 231, and 120 amino acid residues, respectively. One more truncated mutant (fibritin S1, 108 amino acid residues) was obtained. The 2% SDS-PAGE showed that the migration mobilities of all these proteins corresponded to apparent molecular masses substantially greater than those of the preliminarily heated samples (3 min at 100 degrees C). The heating of the intact fibritin and the mutant G at 50-70 degrees C for 10 min resulted in the formation of a form with an apparent molecular mass higher than 200 kDa. This form probably represented a trimeric protein with a partly denatured N-terminal part. Fibritins B and C were more stable and were only partly denatured into monomers even at 70-90 degrees C. The short mutants E and S1 dissociated into monomers at temperatures from 45 to 50 degrees C. The denaturation of mutants B, C, E, and S1 proceeded in one stage without formation of any intermediate form. The stability of the trimeric molecules of native fibritin under PAGE denaturing conditions and the behavior of the intact protein during heating in the temperature range of 50-70 degrees C might be used for the identification of fibritin intermediate forms upon folding in vivo. The refolding capability was found for fibritin and its mutants denatured by heating at low temperatures in the presence of 2% SDS.     

Proteolytic degradation of ribonuclease A in the pretransition region of thermally and urea-induced unfolding.

The method of limited proteolysis has proven to be appropriate for the determination of unfolding rate constants (k(U)) of ribonuclease A in the transition region of thermal denaturation [Arnold, U. & Ulbrich-Hofmann, R. (1997) Biochemistry 36, 2166-2172]. The aim of the present paper was to extend this procedure to the pretransition region of thermally and urea-induced denaturation where spectroscopic methods do not allow direct measurement of k(U). The results show that the approach can be applied successfully to denaturing (free energy of unfolding Delta G < 10 kJ.mol(-1)) and to marginally native conditions (Delta G = 10-25 kJ.mol(-1)). Under moderately (Delta G = 25-30 kJ.mol(-1)) and strongly native conditions (Delta G > 30 kJ.mol(-1)), however, the determination of kU was not possible in this way as the proteolytic degradation of ribonuclease A by thermolysin or trypsin was no longer determined by global unfolding. Here, proteolysis proceeds via the native RNase A. In the presence of low concentrations of urea, the rate constants of proteolysis were, surprisingly, smaller than in the absence of urea. As the protease activity has been taken into account, this result points to a local stabilization of the RNase A molecule.     

Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41

The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36(Mut(e,g)) and N36(Mut(a,d)), were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546-581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36(Mut(e,g)) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil. N36(Mut(a,d)) contains nine substitutions designed to block formation of the trimeric coiled-coil but retains residues that interact with the C-terminal region of gp41. N36(Mut(a,d)) is monomeric, is largely random coil, does not interact with the C34 peptide derived from the C-terminal region of gp41 (residues 628-661), and does not inhibit fusion. The trimeric coiled-coil structure is therefore a prerequisite for interaction with the C-terminal region of gp41. N36(Mut(e,g)) forms a monodisperse, helical trimer in solution, does not interact with C34, and yet inhibits fusion about 50-fold more effectively than the parent N36 peptide (IC(50) approximately 308 nm versus approximately 16 microm). These results indicate that N36(Mut(e,g)) acts by disrupting the homotrimeric coiled-coil of N-terminal helices in the pre-hairpin intermediate to form heterotrimers. Thus N36(Mut(e,g)) represents a novel third class of gp41-targeted HIV fusion inhibitor. A quantitative model describing the interaction of N36(Mut(e,g)) with the pre-hairpin intermediate is presented.     

Transformation of a Fragment of β-Structural Bacteriophage T4 Adhesin to Stable α-Helical Trimer

Gene product 12 of bacteriophage T4, adhesin, serves to adhere the virus to host cells. Adhesin is a fibrous homotrimer, and a novel tertiary structure element, a -helix, is supposed to be a major structural feature of this protein. We have constructed two truncated gp12 mutants, 12N1 and 12N2, containing 221 and 135 N-terminal residues, respectively. When expressed in E. coli cells, these gp12 fragments formed labile -structural trimers. Another hybrid protein, 12FN, containing 179 N-terminal amino acid residues of gp12 fused to the C-terminal domain (31 amino acids) of T4 fibritin, was shown to have a trimeric proteolytically resistant a-helical structure. This structure is probably similar to that of fibritin, which has a triple -helical coiled-coil structure. Hence, we have demonstrated the possibility of global transformation of fibrous protein structure using fusion with a C-terminal domain that initiates trimerization.     

Foldon, the natural trimerization domain of T4 fibritin, dissociates into a monomeric A-state form containing a stable beta-hairpin: atomic details of trimer dissociation and local beta-hairpin stability from residual dipolar couplings

The C-terminal domain of T4 fibritin (foldon) is obligatory for the formation of the fibritin trimer structure and can be used as an artificial trimerization domain. Its native structure consists of a trimeric beta-hairpin propeller. At low pH, the foldon trimer disintegrates into a monomeric (A-state) form that has similar properties as that of an early intermediate of the trimer folding pathway. The formation of this A-state monomer from the trimer, its structure, thermodynamic stability, equilibrium association and folding dynamics have been characterized to atomic detail by modern high-resolution NMR techniques. The foldon A-state monomer forms a beta-hairpin with intact and stable H-bonds that is similar to the monomer in the foldon trimer, but lacks a defined structure in its N and C-terminal parts. Its thermodynamic stability in pure water is comparable to designed hairpins stabilized in alcohol/water mixtures. Details of the thermal unfolding of the foldon A-state have been characterized by chemical shifts and residual dipolar couplings (RDCs) detected in inert, mechanically stretched polyacrylamide gels. At the onset of the thermal transition, uniform relative changes in RDC values indicate a uniform decrease of local N-HN and Calpha-Halpha order parameters for the hairpin strand residues. In contrast, near-turn residues show particular thermal stability in RDC values and hence in local order parameters. This coincides with increased transition temperatures of the beta-turn residues observed by chemical shifts. At high temperatures, the RDCs converge to non-zero average values consistent with predictions from random chain polymer models. Residue-specific deviations above the unfolding transition reveal the persistence of residual order around proline residues, large hydrophobic residues and at the beta-turn.     

Filament-dependent and -independent localization modes of Drosophila non-muscle myosin II

Myosin II assembles into force-generating filaments that drive cytokinesis and the organization of the cell cortex. Regulation of myosin II activity can occur through modulation of filament assembly and by targeting to appropriate cellular sites. Here we show, using salt-dependent solubility and a novel fluorescence resonance energy transfer assay, that assembly of the Drosophila non-muscle myosin II heavy chain, zipper, is mediated by a 90-residue region (1849-1940) of the coiled-coil tail domain. This filament assembly domain, transiently expressed in Drosophila S2 cells, does not localize to the interphase cortex or the cytokinetic cleavage furrow, whereas a 500-residue region (1350-1865) that overlaps the NH(2) terminus of the assembly domain localizes to the interphase cortex but not the cytokinetic cleavage furrow. Targeting to these two sites appears to utilize distinct localization mechanisms as the assembly domain is required for cleavage furrow recruitment of a truncated coiled-coil tail region but not targeting to the interphase cortex. These results delineate the requirements for zipper filament assembly and indicate that the ability to form filaments is necessary for targeting to the cleavage furrow but not to the interphase cortex.     

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.     

Expression of human cytochrome P450 46A1 in Escherichia coli: effects of N- and C-terminal modifications

Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic and substrate-binding properties of four truncated cytochromes P450 46A1 were investigated in the present study. All four lacked the N-terminal transmembrane region (residues 3-27), and, in addition, Delta 46A1H had a 4x His-tag fused to the C-terminus; H Delta 46A1 had the N-terminal 4x His-tag; H Delta 46A1 Delta had a 4x His-tag at the N-terminus and did not contain a proline-rich region at the C-terminus (residues 494-499); and Delta 46A1 Delta lacked the C-terminal proline-rich region. The truncated enzymes were expressed at 390-650 nmol/L culture levels, distributed at about a 1:1 ratio between the membrane fraction and the cytosol in low ionic strength buffer, and were predominantly monomers in detergent-free buffer. They had moderately decreased catalytic efficiencies for either cholesterol or 24S-hydroxycholesterol or both, whereas their substrate-binding constants were either unchanged or decreased 2-fold. The two forms, Delta 46A1 Delta and H Delta 46A1 Delta, both lacking the C-terminal proline-rich region seem to be good candidates for future crystallographic studies because they contain only 0.3-0.8% of high molecular weight aggregates and their catalytic efficiencies are decreased no more than 2.3-fold.     

Far3p domains involved in the interactions of Far proteins and pheromone-induced cell cycle arrest in budding yeast

Far3p (factor arrest), a protein that interacts with Far7-11p, is required for the pheromone-mediated cell cycle arrest in G1 phase. We used a combination of computational and experimental strategies to identify the Far3p self-association, to map the Far3p domains that interact with Far3p itself and with other Far proteins, and to reveal the importance of the two coiled-coil motifs of Far3p in the integrity and function of the Far complex. We show that Far3p self-associates through its central region and its C-terminal coiled-coil domain, that the amino acid 61-100 region of Far3p interacts with Far7p, and that the Far3p N-terminal coiled-coil domain interacts with Far9p and Far10p. Mutation of the N-terminal coiled coil disrupts the interactions of Far3p with Far9p and Far10p, and mutation of the C-terminal domain weakens the Far3p self-interaction. Although the N- and C-terminal coiled-coil mutants reserve some of the interactions with itself and some other Far proteins, both mutants are defective in the pheromone-mediated G1 arrest, indicating that both coiled-coil motifs of Far3p are essential for the integrity and the function of the Far complex.     

Very short peptides with stable folds: building on the interrelationship of Trp/Trp, Trp/cation, and Trp/backbone-amide interaction geometries

By combining a favorable turn sequence with a turn flanking Trp/Trp interaction and a C-terminal H-bonding interaction between a backbone amide and an i-2 Trp ring, a particularly stable (DeltaG(U) > 7 kJ/mol) truncated hairpin, Ac-WI-(D-Pro-D-Asn)-KWTG-NH(2), results. In this construct and others with a W-(4-residue turn)-W motif in severely truncated hairpins, the C-terminal Trp is the edge residue in a well-defined face-to-edge (FtE) aryl/aryl interaction. Longer hairpins and those with six-residue turns retain the reversed "edge-to-face" (EtF) Trp/Trp geometry first observed for the trpzip peptides. Mutational studies suggest that the W-(4-residue turn)-W interaction provides at least 3 kJ/mol of stabilization in excess of that due to the greater beta-propensity of Trp. The pi-cation, and Trp/Gly-H(N) interactions have been defined. The latter can give rise to >3 ppm upfield shifts for the Gly-H(N) in -WX(n)G- units both in turns (n = 2) and at the C-termini (n = 1) of hairpins. Terminal YTG units result in somewhat smaller shifts (extrapolated to 2 ppm for 100% folding). In peptides with both the EtF and FtE W/W interaction geometries, Trp to Tyr mutations indicate that Trp is the preferred "face" residue in aryl/aryl pairings, presumably because of its greater pi basicity.     

Functional domains present in the mycobacterial hemagglutinin, HBHA

Identification and characterization of mycobacterial adhesins and complementary host receptors required for colonization and dissemination of mycobacteria in host tissues are needed for a more complete understanding of the pathogenesis of diseases caused by these bacteria and for the development of effective vaccines. Previous investigations have demonstrated that a 28-kDa heparin-binding mycobacterial surface protein, HBHA, can agglutinate erythrocytes and promote mycobacterial aggregation in vitro. In this study, further molecular and biochemical analysis of HBHA demonstrates that it has three functional domains: a transmembrane domain of 18 amino acids residing near the N terminus, a large domain of 81 amino acids consistent with an alpha-helical coiled-coil region, and a Lys-Pro-Ala-rich C-terminal domain that mediates binding to proteoglycans. Using His-tagged recombinant HBHA proteins and nickel chromatography we demonstrate that HBHA polypeptides which contain the coiled-coil region form multimers. This tendency to oligomerize may be responsible for the induction of mycobacterial aggregation since a truncated N-terminal HBHA fragment containing the coiled-coil domain promotes mycobacterial aggregation. Conversely, a truncated C-terminal HBHA fragment which contains Lys-Pro-Ala-rich repeats binds to the proteoglycan decorin. These results indicate that HBHA contains at least three distinct domains which facilitate intercalation into surface membranes, promote bacterium-bacterium interactions, and mediate the attachment to sulfated glycoconjugates found in host tissues.     

Characterization of the roles of the 594-645 region in human endothelial nitric-oxide synthase in regulating calmodulin binding and electron transfer

It has been postulated that a segment (residues 594-645) inserted in the FMN subdomain of human endothelial nitric-oxide synthase (eNOS) plays a crucial role in controlling Ca(2+)-dependent CaM binding for eNOS activity. To investigate its functions, we expressed human eNOS in a baculovirus system with deletion of a 45-residue segment from this region (residues 594-606 and 614-645, designated as Delta45eNOS), and characterized the purified mutant enzyme. In contrast with wild-type eNOS, Delta45eNOS exhibited characteristics resembling inducible NOS (iNOS). It contained an endogenously bound CaM, which was essential in folding and stabilizing this mutant enzyme, and retained 60% of L-citrulline formation in 5 mM EGTA. We also produced four N-terminally truncated reductase domains with or without the 45-residue segment, and either including or excluding the CaM-binding sequence. Basal cytochrome c reductase activity of reductase domains without the 45-residue segment was up to 20 fold greater than that of corresponding insert-containing domains, and higher than CaM-stimulated activity of the wild-type enzyme. A series of mutants with smaller fragment deletion in this region such as Delta594-604, Delta605-612, Delta613-625, Delta626-634, Delta632-639, and Delta640-645 mutants were further characterized. The crude lysate of mutants Delta613-625 and Delta632-639 did not show activity in the presence of Ca(2+)/CaM, while other four mutants had activity comparable to that of WTeNOS. The purified Delta594-604 and Delta605-612 proteins had a 3-5-fold higher affinity for Ca(2+)/CaM, but their L-citrulline forming activity was still 80% dependent upon the addition of Ca(2+)/CaM. Both mutants exhibited a low level of the cytochrome c and ferricyanide reductase activities, which either did not respond to (Delta594-604) or slightly enhanced by (Delta605-612) the exogenous CaM. In contrast, activities of Delta626-634 and Delta640-645 like those of WTeNOS were largely Ca(2+)/CaM-dependent. Thus, our findings indicate that the N-terminal half of the 594-645 segment containing residues 594-612 plays a significant role in regulating Ca(2+)/CaM binding.     

Association of mammalian sterile twenty kinases, Mst1 and Mst2, with hSalvador via C-terminal coiled-coil domains, leads to its stabilization and phosphorylation

Genetic screens in Drosophila have revealed that the serine/threonine kinase Hippo (Hpo) and the scaffold protein Salvador participate in a pathway that controls cell proliferation and apoptosis. Hpo most closely resembles the pro-apoptotic mammalian sterile20 kinases 1 and 2 (Mst1 and 2), and Salvador (Sav) has a human orthologue hSav (also called hWW45). Here we show that Mst and hSav heterodimerize in an interaction requiring the conserved C-terminal coiled-coil domains of both proteins. hSav was also able to homodimerize, but this did not require its coiled-coil domain. Coexpression of Mst and hSav led to phosphorylation of hSav and also increased its abundance. In vitro phosphorylation experiments indicate that the phosphorylation of Sav by Mst is direct. The stabilizing effect of Mst was much greater on N-terminally truncated hSav mutants, as long as they retained the ability to bind Mst. Mst mutants that lacked the C-terminal coiled-coil domain and were unable to bind to hSav, also failed to stabilize or phosphorylate hSav, whereas catalytically inactive Mst mutants that retained the ability to bind to hSav were still able to increase its abundance, although they were no longer able to phosphorylate hSav. Together these results show that hSav can bind to, and be phosphorylated by, Mst, and that the stabilizing effect of Mst on hSav requires its interaction with hSav but is probably not due to phosphorylation of hSav by Mst.