Synthetic cross-links arrest the C-terminal region of the relaxin-like factor in an active conformation

All kinetic indicators suggest that the receptor-binding site of the relaxin-like factor (RLF) is located in the flexible C-terminal region of the B chain and is centered about the tryptophan in position B27. Conformational restraints of varying lengths were used to confine Trp (B27) to a narrow range of positions relative to the C terminus of the A chain. Total synthesis of variants involving residues proximate to Trp (B27) assured us that none had a role in receptor binding. Even arginine B26, next to the important tryptophan, can be replaced without deleterious effects. To fix the distance between the C-terminal end of the A chain and Trp (B27) at predetermined lengths, we synthesized RLF with covalent cross-links between a lysine, which was placed in position B26, and the alpha-carboxyl group at the C terminus of the A chain (A26). The affinity of the cross-linked ligands for the RLF receptor varied as a parabolic function of length whereby the range of 10.0-11.1 A provided the closest approach to the binding conformation. Wild-type transmembrane signaling activity, as determined by cAMP accumulation, was reached only with a glycine cross-linker.     

The C-terminal loop of the homing endonuclease I-CreI is essential for site recognition, DNA binding and cleavage

Meganucleases are sequence-specific endonucleases with large cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These enzymes open novel perspectives for genome engineering in a wide range of fields, including gene therapy. A new crystal structure of the I-CreI dimer without DNA has allowed the comparison with the DNA-bound protein. The C-terminal loop displays a different conformation, which suggests its implication in DNA binding. A site-directed mutagenesis study in this region demonstrates that whereas the C-terminal helix is negligible for DNA binding, the final C-terminal loop is essential in DNA binding and cleavage. We have identified two regions that comprise the Ser138–Lys139 and Lys142–Thr143 pairs whose double mutation affect DNA binding in vitro and abolish cleavage in vivo. However, the mutation of only one residue in these sites allows DNA binding in vitro and cleavage in vivo. These findings demonstrate that the C-terminal loop of I-CreI endonuclease plays a fundamental role in its catalytic mechanism and suggest this novel site as a region to take into account for engineering new endonucleases with tailored specificity.     

The conformation of the C-terminal region of actin: a site-specific photocrosslinking study using benzophenone-4-maleimide

The site-specific photocrosslinker, benzophenone-4-maleimide, was used to label G-actin specifically at Cys-374, the penultimate residue from the C terminus. The resultant BP-G-actin was polymerized to form BP-F-actin, and both forms of actin were irradiated to activate the benzophenone moiety. We found that for BP-F-actin both intersubunit and intrasubunit photocrosslinks were formed. For BP-G-actin only a small amount of an internally photocrosslinked species was formed. These findings suggest that in the F-actin polymer, the C-terminal peptide is localized in a region between neighboring subunits. In contrast, in the G-actin monomer, the C-terminal peptide is relatively distant from the surface of the molecule.     

Cloning and expression of rat histidase. Homology to two bacterial histidases and four phenylalanine ammonia-lyases

Histidase (histidine ammonia-lyase, EC 4.3.1.3) catalyzes the deamination of histidine to urocanic acid. Apart from phenylalanine ammonia-lyase, which is not expressed in animals, histidase is the only enzyme known to have a dehydroalanine residue in its active site. The amino site precursor and the mechanism of formation of dehydroalanine are not known. As an initial step to determining the precursor of dehydroalanine in histidase, we have isolated a functional cDNA clone for histidase from a rat liver cDNA library using an affinity-purified antiserum. The 2.2-kilobase cDNA has a 1,971-base pair open reading frame coding for a 657-amino acid polypeptide with a predicted molecular mass of 72,165 Da. The cDNA has a rare polyadenylation signal (AAUACA) that appears to inefficiently direct polyadenylation in transfected COS monkey kidney cells. Conversion of this sequence to the consensus polyadenylation signal (AAUAAA) resulted in increased levels of stable mRNA. COS cells transfected with a histidase expression vector produce active histidase. The formation of active histidase in cells that have no endogenous histidase activity suggests either that the requisite modifying enzyme is present in these cells or that the dehydroalanine residue forms by an autocatalytic mechanism. Rat histidase was found to have 41 and 43% amino acid identity to Pseudomonas putida and Bacillus subtilis histidases, respectively. Phenylalanine ammonia-lyases from parsley, kidney bean, and two yeast strains were also found to have approximately 20% amino acid identity to rat histidase. On the basis of the similarity of function of histidase and phenylalanine ammonia-lyase, dehydroalanine at the active sites, and the sequence conservation over a large evolutionary distance (mammals, bacteria, yeast, and plants), we propose that the genes for histidase and phenylalanine ammonia-lyase have diverged from a common ancestral gene, of which the most conserved regions are likely to be involved in catalysis or dehydroalanine formation.     

A solvent effect on the side-chain conformation of phenylalanine derivatives and phenylalanine residuces in dipeptides

Three phenylalanine derivatives, Ac-Phe-NHMe, H-Phe-NHMe, and Ac-Phe-OH, were selected as models of Phe residues situated at the internal, the N-terminal, and the C-terminal positions of peptide chains, respctively. The side-chain conformations of the three compounds were analyzed from the vicnal coupling constants ³J_αβR and ³J_αβS, of their ¹H- nmr spectra measured in various organic sovlent. The two β-protons were unambiguously assined by use of sterospecifically β-monodeuterated phenylalanines. The pro-S β-proton was always situated at lower field than the pro-R one when they were observed separately. The results of a solvent effect on the conformation of the tree compounds demonstrated that the rotamer populations are remarkable sensitive of the three compounds demonstrated that the rotamer populations are remarkably sensitive to solvent polarity and that the tendencies of the solvent effects are quite different from each other. Ac-Phe-OH Showed a trend similar to that of Ac-Phe-OEt reported by early workers. The rotamer populations of other derivatives (Ac-Phe-NMe₂, Ac-Phe-NH₂, Ac-Phe-OBu^t, and Ac-Phe-OBzl) and of Phe residues in some N-acetyl dipeptde esters (Ac-Phe-Gly-OMe, Ac-Phe-Val-OMe, and Ac-Gly-Phe-OMe) were also examined in several sovent, and it was found that substituents of the Phe carboxyl group—amides or esters—determine the tendency of the solvent effect. These results are interesting in the side-chain conformations of Phe residues in peptides and proteins in an environment of low polarity can be disscussed on this experimental basis. Factors responsible for the solvent effect are discussed from (1) a structural comparison of the compunds with various carboxylic substituents, (2) an expriment with cyclohexylalanine derivatives, and (3) the measurement in mixed solvents wiht similar polarity.     

The role of the C-terminal region of rat brain natriuretic peptide in receptor selectivity.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have different C-terminal tail structures compared with the rather conservative ring structures which consist of 17 amino acid residues. To examine the different effects of the tail structures of ANP and BNP on their interaction with receptors, we synthesized several peptide analogs and measured their biological actions in three different assay systems. Deletion of the C-terminal tail from rat BNP did not effect the vasorelaxation activity against rat aorta, but it promoted cGMP production in cultured rat aortic smooth muscle cells (RASMC). Deletion of the C-terminal tail from rat ANP diminished both vasorelaxant and cGMP producing activities. In a binding competition assay with RASMC and [125I]rat ANP-(1-28), the competition activities of both ANP and BNP were greatly reduced by C-terminal deletion. In addition, we obtained agonists with novel receptor selectivity.     

The role of the C-terminal region of pulchellin A-chain in the interaction with membrane model systems

Pulchellin is a Ribosome Inactivating Protein containing an A-chain (PAC), whose toxic activity requires crossing the endoplasmic reticulum (ER) membrane. In this paper, we investigate the interaction between recombinant PAC (rPAC) and Langmuir monolayers of dipalmitoyl phosphatidyl glycerol (DPPG), which served as membrane model. Three catalytically active, truncated PACs with increasing deletion of the C-terminal region, possessing 244, 239 and 236 residues (rPAC(244), rPAC(239) and rPAC(236)), were studied. rPAC had the strongest interaction with the DPPG monolayer, inducing a large expansion in its surface pressure-area isotherm. The affinity to DPPG decreased with increased deletion of the C-terminal region. When the C-terminal region was deleted completely (rPAC(236)), the interaction was recovered, probably because other hydrophobic regions were exposed to the membrane. Using Polarization Modulated-Infrared Reflection Absorption Spectroscopy (PM-IRRAS) we observed that at a bare air/water interface rPAC comprised mainly α-helix structures, the C-terminal region had unordered structures when interacting with DPPG. For rPAC(236) the α-helices were preserved even in the presence of DPPG. These results confirm the importance of the C-terminal region for PAC-ER membrane interaction. The partial unfolding only with preserved C-terminal appears a key step for the protein to reach the cytosol and develop its toxic activity.     

Maintenance and thermal stabilization of NADH dehydrogenase-2 conformation upon elimination of its C-terminal region

Development of an artificial enzyme with activity and structure comparable to that of natural enzymes is an important goal in biological chemistry. Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein, belonging to a group of enzymes with scarce structural information. By eliminating the C-terminal region of NDH-2, a water soluble version with significant enzymatic activity was previously obtained. Here, NDH-2 structural features were established, in comparison to those of the truncated version. Far-UV circular dichroism, Fourier transform infrared spectroscopy and limited proteolysis analysis showed that the overall structure of both proteins was similar at 30 °C. Experimental data agree with the predicted NDH-2 structure (PDB: 1OZK). The absence of C-terminal region stabilized in ∼5–10 °C the truncated protein conformation. However, truncation impaired enzymatic activity at low temperatures, probably due to the weak interaction of the mutant protein with FAD cofactor.     

The essential role of C-terminal residues in regulating the activity of hepatitis C virus RNA-dependent RNA polymerase

We have previously determined the crystal structure of a non-structural 5B (NS5B) protein, an RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). NS5B protein with the hydrophobic C-terminal 21 amino acid residues truncated, designated NS5B(570), shows a typical nucleotide polymerase structure resembling a right-hand shape. In the crystal structure, a C-terminal region between Leu545 and His562 occupies a putative RNA-binding cleft of this polymerase and seems to inhibit the polymerase activity. Varieties of recombinant NS5B proteins (NS5B(552), NS5B(544), NS5B(536) or NS5B(531), with C-terminal 39, 47, 55 or 60 amino acid residues truncated, respectively) were systematically constructed to elucidate effects of the region on the polymerase activity. NS5B(544), NS5B(536) and NS5B(531) showed markedly higher RdRp activities compared to the activities of NS5B(570) or NS5B(552). Furthermore, when the hydrophobic amino acid residues Leu547, Trp550 and Phe551 (LWF) in NS5B(570) and NS5B(552) were changed to alanine, their activities were higher than that of the original NS5B(570). The crystal structures of the various recombinant NS5B proteins were also determined. Structural comparison of the NS5B proteins indicates that the activation was caused by elimination of a unique hydrophobic interaction between the three C-terminal residues and a shallowly concave pocket consisting of thumb and palm domains.     

The role of the diphthamide-containing loop within eukaryotic elongation factor 2 in ADP-ribosylation by Pseudomonas aeruginosa exotoxin A

eEF2 (eukaryotic elongation factor 2) contains a post-translationally modified histidine residue, known as diphthamide, which is the specific ADP-ribosylation target of diphtheria toxin, cholix toxin and Pseudomonas aeruginosa exotoxin A. Site-directed mutagenesis was conducted on residues within the diphthamide-containing loop (Leu693-Gly703) of eEF2 by replacement with alanine. The purified yeast eEF2 mutant proteins were then investigated to determine the role of this loop region in ADP-ribose acceptor activity of elongation factor 2 as catalysed by exotoxin A. A number of single alanine substitutions in the diphthamide-containing loop caused a significant reduction in the eEF2 ADP-ribose acceptor activities, including two strictly conserved residues, His694 and Asp696. Analysis by MS revealed that all of these mutant proteins lacked the 2'-modification on the His699 residue and that eEF2 is acetylated at Lys509. Furthermore, it was revealed that the imidazole ring of Diph699 (diphthamide at position 699) still functions as an ADP-ribose acceptor (albeit poorly), even without the diphthamide modification on the His699. Therefore, this diphthamide-containing loop plays an important role in the ADP-ribosylation of eEF2 catalysed by toxin and also for modification of His699 by the endogenous diphthamide modification machinery.     

The conformation of the tRNAPhe anticodon loop monitored by fluorescence

The intrinsic fluorescence of the Wye base was used to study the conformational change of the anticodon loop of yeast tRNAPhe brought about by the addition of magnesium. The fluorescence emission and excitation spectra show dramatic changes as magnesium is added to the solution. The rotational relaxation time changes from 6 nsec without added magnesium to 33 nsec with 10 mM magnesium at an ionic strength of 0.1 M. Stern-Volmer quenching by iodide or iodoethanol shows greater access of the base to the quencher with no added magnesium. A plausible interpretation of this data is that the base stack of the anticodon loop is altered by tilting or twisting the Wye base with respect to the adjacent bases and the base becomes parallel to its neighbors upon the addition of magnesium.     

The C-terminal region of TIM17 links the outer and inner mitochondrial membranes in Arabidopsis and is essential for protein import

The translocase of the inner membrane 17 (AtTIM17-2) protein from Arabidopsis has been shown to link the outer and inner mitochondrial membranes. This was demonstrated by several approaches: (i) In vitro organelle import assays indicated the imported AtTIM17-2 protein remained protease accessible in the outer membrane when inserted into the inner membrane. (ii) N-terminal and C-terminal tagging indicated that it was the C-terminal region that was located in the outer membrane. (iii) Antibodies raised to the C-terminal 100 amino acids recognize a 31-kDa protein from purified mitochondria, but cross-reactivity was abolished when mitochondria were protease-treated to remove outer membrane-exposed proteins. Antibodies to AtTIM17-2 inhibited import of proteins via the general import pathway into outer membrane-ruptured mitochondria, but did not inhibit protein import via the carrier import pathway. Together these results indicate that the C-terminal region of AtTIM17-2 is exposed on the outer surface of the outer membrane, and the C-terminal region is essential for protein import into mitochondria.     

The N-terminal region of an entomopoxvirus fusolin is essential for the enhancement of peroral infection, whereas the C-terminal region is eliminated in digestive juice

The spindles of Anomala cuprea entomopoxvirus (AncuEPV), which are composed of glycoprotein fusolin, are known to enhance the peroral infectivity of AncuEPV itself and of nucleopolyhedroviruses. This has been demonstrated to involve the disruption of intestinal peritrophic membrane (PM), composed of chitin matrix, glycosaminoglycans, and proteins. To identify essential and nonessential regions for this enhancement activity, AncuEPV fusolin and its deletion mutants were expressed in Sf21 cells using a baculovirus system, and their enhancement abilities were analyzed. The recombinant fusolin enhanced the peroral infectivity of Bombyx mori nucleopolyhedrovirus up to 320-fold and facilitated the infection of host insect with AncuEPV. Deletion mutagenesis revealed that the N-terminal region (amino acids 1 to 253), a possible chitin-binding domain, is essential for the enhancement of infection, whereas the C-terminal region is entirely dispensable. The glycosylation-defective mutants N191Q, whose Asn₁₉₁ is replaced with Gln, and ΔSIG, whose signal peptide is deleted, showed considerably reduced and abolished enhancing activities, respectively, indicating that the carbohydrate chain is important in the enhancing activity. Interestingly, the C-terminal dispensable region was digested by a serine protease(s) in insect digestive juice. Moreover, both the N-terminal conserved region and the carbohydrate chain were necessary not only for chitin binding but also for stability in digestive juice. A triple amino acid replacement mutant, IHE (Ile-His-Glu₁₆₁ to Ala-Ala-Ala), was stable in digestive juice and had chitin-binding ability but did not retain its enhancing activity. These results suggest that the enhancement of infectivity involves more than the tolerance to digestive juice and chitin-binding ability.     

HIV-1 protease regulation: The role of the major homology region and adjacent C-terminal capsid sequences

The maturation of human immunodeficiency type-1 virions is accomplished through the proteolytic processing of Gag and GagPol precursor proteins by the viral protease (PR). Since virions must be assembled at the cell surface from uncleaved precursor molecules, intracellular activation of PR must be inhibited. We have previously developed a system where the intracellular activity of PR, associated with GagPol, was inhibited by the expression of Gagin trans. The disproportionate synthesis of Gag inhibits the activation of PR in the cytoplasm. Sequences capable of mediating this inhibition were localized to capsid. In this communication, the region of HIV-1 capsid capable of mediating inhibition was further defined and shown to require the major homology region of capsid within Gag.     

Importance of C-terminal flexible region of 4E-binding protein in binding with eukaryotic initiation factor 4E

Although the alpha-helical Y(X)4Lvarphi containing region of eIF4E-binding protein (4EBP) is the major binding region with eukaryotic initiation factor 4E (eIF4E), the roles of its N- and C-terminal regions in the binding are hardly known. To clarify the roles of these flexible regions in the interaction, the binding features of the sequentially N-, C-, or both-terminal-residue-deleted 4EBP2 mutants were investigated by surface plasmon resonance (SPR) analysis. It was shown that the C-terminal His74-Glu89 sequence has an auxiliary, but indispensable, function in stabilizing the binding to eIF4E. The possible interaction with eIF4E was estimated by molecular dynamics simulation. This is the first report on the importance of the C-terminal flexible region in the eIF4E-binding regulation of 4EBP.     

The role of phenylalanine in differentiating amphibian melanocytes

To see whether phenylalanine serves as a substrate in melanogenesis, hanging drop explants of neural crest from amphibian (Ambystoma maculatum and A. mexicanum) embryos were subjected on the seventh day in vitro to treatment with phenylalanine-³H and studied by means of light microscopic radioautography. All melanin-containing cells showed label. On the other hand, when puromycin, an inhibitor of protein synthesis, together with the labeled amino acid was administered to the cultures, no radioactivity was incorporated by pigmented cells. Comparable results were obtained when leucine was substituted for phenylalanine. In control experiments, puromycin and labeled tyrosine or 3,4-dihydroxyphenylalanine (DOPA), both known precursors for melanin synthesis, were administered to the neural crest cultures. In these experiments, puromycin had no effect on the incorporation of label by pigmented cells. Our data strongly indicate that in differentiating amphibian melanocytes with functional pigment-forming systems, phenylalanine is used in protein synthesis, but does not serve as a substrate for the tyrosine-tyrosinase system.In another series of experiments, explants of neuroepithelium (neural crest anlage) were grown from the time of explantation to the seventh day in vitro in the presence of phenyllactic acid, an analog of phenylalanine. Pigment cells developed normally.These results suggest that phenylalanine plays little or no role in pigment cell differentiation.     

染色质构象调控真核基因的表达

真核基因的表达受到各种顺式调控元件、反式作用因子、染色质DNA以及组蛋白表观遗传修饰等多因素、多层次的调控。染色质三维空间结构的变化在调控真核基因表达方面也发挥了至关重要的作用。染色质构象的变化一方面可以使增强子等调控元件与靶基因相互靠近,从而促进基因表达;同时也可能通过形成空间位阻结构阻碍调控元件作用于靶基因,抑制基因表达。虽然染色质结构变化调控真核基因表达的机制仍缺乏较为精确的分子模型,但在组蛋白修饰、核小体定位、染色体领域以及染色质间相互作用等表观遗传学研究中,已经发现有诸多证据支持染色质构象在真核基因表达调控中的重要地位。文章主要综述了染色质结构及其构象的变化等对真核基因表达调控的影响。