Structure and activities of constrained analogues of human parathyroid hormone and parathyroid hormone-related peptide: implications for receptor-activating conformations of the hormones

Parathyroid hormone (PTH) has a helix-bend-helix structure in solution. Part of the C-terminal helix, residues 21-31, is amphiphilic and forms a critical receptor-binding region. Stabilization of this alpha-helix by lactam formation between residues spaced i, i + 4 on the polar face was previously reported to increase adenylyl cyclase-stimulating (AC) activity if between residues 22 and 26 but to diminish it if between residues 26 and 30 [Barbier et al. (1997) J. Med. Chem. 40, 1373-1380]. This work reports the effects of other cyclizations on the polar face, differing in ring size or position, on alpha-helix conformation, as measured by circular dichroism, and on AC-stimulating activity. All analogues cyclized between residues 22 and 26 had at least a 1. 5-fold increase in activity, suggesting an alpha-helical structure between about residues 21 and 26. Cyclization between residues 25 and 29 or residues 26 and 30 diminished activity by 20-30%, despite stabilizing alpha-helix, suggesting that residues 25-31 bind to the receptor in a helical, but not classical alpha-helical, conformation. Analogues cyclized between residues 13 and 17 had slightly increased activity. A bicyclic analogue, with lactams between residues 13 and 17 and residues 22 and 26, had about the same activity as that cyclized only between 22 and 26. Parathyroid hormone-related peptide (PTHrP) may bind in a manner similar to the common receptor, but hydrophobic moment calculations suggest that it must bind as a tighter helix in order to optimally present its hydrophobic residues to the receptor. Both PTHrP analogues cyclized between either residues 22 and 26 or residues 26 and 30 had more stable alpha-helices but reduced AC activities, consistent with this hypothesis.     

The role of tryptophan residues in the hemolytic activity of stonustoxin,a lethal factor from stonefish (Synanceja horrida) venom

Stonustoxin (SNTX) is a pore-forming cytolytic lethal factor, isolated from the venom of the stonefish Synanceja horrida, that has potent hemolytic activity. The role of tryptophan residues in the hemolytic activity of SNTX was investigated. Oxidation of tryptophan residues of SNTX with N-bromosuccinimide (NBS) resulted in loss of hemolytic activity. Binding of 8-anilino-1-naphthalenesulphonate (ANS) to SNTX resulted in occlusion of tryptophan residues that resulted in loss of hemolytic activity. Circular dichroism and fluorescence studies indicated that ANS binding resulted in a conformational change of SNTX, in particular, a relocation of surface tryptophan residues to the hydrophobic interior. NBS-modification resulted in oxidised surface tryptophan residues that did not relocate to the hydrophobic interior. These results suggest that native surface tryptophan residues play a pivotal role in the hemolytic activity of STNX, possibly by being an essential component of a hydrophobic surface necessary for pore-formation. This study is the first report on the essentiality of tryptophan residues in the activity of a lytic and lethal factor from a fish venom.     

Structure-function studies of human granulocyte-macrophage colony-stimulating factor. Identification of residues required for activity

Human granulocyte-macrophage colony stimulating factor (hGM CSF), a protein containing 127 amino acids, was chemically synthesized by using automated stepwise solid-phase methods. The unpurified synthetic hGM-CSF had the same range of actions on hemopoietic cells as the purified recombinant protein. The structural requirements for the activities of synthetic hGM-CSF were examined by the design and synthesis of fragments and analogs. The synthetic fragment, hGM-CSF (54-127), containing all four of the cysteine residues found in the intact protein, lacked detectable activity. Assays of fragments shortened at the N terminus showed that the residues 1-13 were not required for activity, but that the integrity of residues 14-25, particularly residues 16, 17, and 18, was critical for biologic activity. The 14-25 region is predicted to form the first alpha-helix in hGM-CSF. Synthetic peptides within the N-terminal 53 residue region lacked detectable activity. The synthetic analog hGM-CSF (1-121), which lacks the C-terminal 6 residues, had similar activity to hGM-CSF (1-127) indicating that residues 122-127 are not required for activity. An analog, [Ala88] hGM-CSF (14-96), which lacks the hydrophobic C-terminal region and 2 cysteine residues, had low but readily detectable activity suggesting that residues 14-96 are sufficient for detectable synthetic hGM-CSF activity, although the presence of residues 97-121 are required for full activity. No dissociation of the multiple biological activities of hGM-CSF was detected.     

Involvement of some amino acid residues in the enzymatic activity of solubilized cerebral fucosyltransferase

We have checked the effect of some chemical reagents specific for amino acid residues on the activity of a solubilized cerebral glycoprotein:fucosyltransferase. Diethylpyrocarbonate, 2,3-butanedione and tetranitromethane specific for histidyl, arginyl, and tyrosyl residues respectively, were strong inhibitors of the enzymatic activity This led us to conclude that these amino acid residues are "essential residues" in the cerebral fucosyltransferase activity.     

Chemical modification of liquefying alpha-amylase: role of tyrosine residues at its active center

Liquefying alpha-amylase from Bacillus amyloliquefaciens was inactivated by treatment with tetranitromethane and N-acetylimidazole. The loss of activity occurred with modification of five tyrosine residues. Preincubation of the enzyme with either the substrate or the competitive inhibitor at saturating levels provided complete protection against inactivation. However, the presence of substrate/inhibitor in the reaction mixture protected only two of the five modifiable tyrosine residues, suggesting the involvement of only two tyrosine residues at the active center. This was confirmed when hydroxylamine treatment of the acetylated enzyme fully restored the enzymatic activity. Both nitration and acetylation increased the apparent Km of the enzyme for soluble starch, which indicated that the tyrosine residues are involved in substrate binding. Reduction of nitrotyrosine residues to aminotyrosine residues failed to restore the enzymatic activity. So, the loss of activity on modification of tyrosine residues was ascribed to conformational perturbances and not simply to the changes in the ionic character of tyrosine residues.     

Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding.

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydrate-binding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.     

Site-directed mutagenesis reveals the importance of conserved charged residues for the transport activity of the PheP permease of Escherichia coli.

Site-directed mutagenesis has been used to identify a number of charged residues essential for the transport activity of the PheP protein. These residues are highly conserved in the cluster of amino acid transporters. However, some other conserved residues and a number of aromatic residues have been shown not to be essential for transport activity.     

A novel enhancing mechanism for hydrogen sulfide-producing activity of cystathionine beta-synthase

H2S is produced from cysteine by cystathionine beta-synthase (CBS) in the brain and functions as a neuromodulator. Although the production of H2S is regulated by Ca2+ and calmodulin in response to neuronal excitation, little is known about the molecular mechanism for the regulation in CBS activity. Here we show that four cysteine residues of CBS are involved in the regulation of its activity in the presence of Ca2+ and calmodulin. Sodium nitroprusside (SNP), a modifying agent for cysteine residues, enhances CBS activity, whereas N-ethylmaleimide, an alkylating agent for cysteine residues, completely abolished the effect of SNP. Site-directed mutagenesis of the 13 cysteine residues of CBS identified four cysteine residues that are involved in the regulation of CBS activity by SNP, and two of the four residues are involved in the regulation of the basal CBS activity. The enhancement of CBS activity by SNP is independent of nitric oxide production. In the presence of Staphylococcus aureus alpha-hemolysin, which permeabilizes the cell membrane, exogenously applied SNP enhances the activity of CBS in intact cells. The present study demonstrates a novel mechanism for the regulation of CBS activity and provides a possible therapeutic application of SNP for the diseases in which CBS activity is deficient.     

Homology modeling and mutational analysis of Ho endonuclease of yeast

Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast. Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI. Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain. The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain. We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization. We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region. C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential. Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity. Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.     

Requirements for peptidyl-prolyl isomerization activity: a comprehensive mutational analysis of the substrate-binding cavity of FK506-binding protein 12

Peptidyl-prolyl isomerase (PPIase) activity is exhibited by many proteins belonging to the PPIase family. However, the catalytic mechanism of this activity remains to be completely elucidated. Here, we selected human FK506-binding protein 12 (FKBP12) as the model PPIase and investigated the nature of amino acid residues essential for the activity. The crystal structures of several complexes of PPIase with short peptides revealed that the residues Asp37, Arg42, Phe46, Val55, Trp59, and Tyr82 in the substrate-binding cavity of FKBP12 appear to play key roles in the PPIase activity. Each of these six residues was substituted by 20 common amino acid residues. The activity of each mutant protein was measured using a peptide analog by the chymotrypsin digestion assay and then compared with wild-type FKBP12. It was found that site-specific interactions by the side chains of amino acid residues constituting the substrate-binding cavity were not essential for the PPIase activity, although the 37th, 55th, and 82nd amino acid residues significantly contributed to the activity. This suggests that the PPIase activity requires only the hydrophobic cavity that captures the Pro-containing peptide.     

Effect of arginine 172 on the binding of apolipoprotein E to the low density lipoprotein receptor

The region of apolipoprotein E (apoE) that interacts directly with the low density lipoprotein (LDL) receptor lies in the vicinity of residues 136-150, where lysine and arginine residues are crucial for full binding activity. However, defective binding of carboxyl-terminal truncations of apoE3 has suggested that residues in the vicinity of 170-183 are also important. To characterize and define the role of this region in LDL receptor binding, we created either mutants of apoE in which this region was deleted or in which arginine residues within this region were sequentially changed to alanine. Deletion of residues 167-185 reduced binding activity (15% of apoE3), and elimination of arginines at positions 167, 172, 178, and 180 revealed that only position 172 affected binding activity (2% of apoE3). Substitution of lysine for Arg(172) reduced binding activity to 6%, indicating a specific requirement for arginine at this position. The higher binding activity of the Delta167-185 mutant relative to the Arg(172) mutant (15% versus 2%) is explained by the fact that arginine residues at positions 189 and 191 are shifted in the deletion mutant into positions equivalent to 170 and 172 in the intact protein. Mutation of these residues and modeling the region around these residues suggested that the influence of Arg(172) on receptor binding activity may be determined by its orientation at a lipid surface. Thus, the association of apoE with phospholipids allows Arg(172) to interact directly with the LDL receptor or with other residues in apoE to promote its receptor-active conformation.     

Effects of denaturation and amino acid modification on fluorescence spectrum and hemagglutinating activity of Hericium erinaceum Lectin

In a broad sense, lectins are proteins or glycoproteins ofnon-immune origin that bind specifically to carbohydrates[1]. But most lectins are usually multivalent, which meansthey have more than one carbohydrate-binding site in onemolecule, a property that enables them to agglutinate eryth-rocytes and other cells [2,3]. Some lectins exhibit blood-group specificity [4] and can be used in blood grouping;some agglutinate transformed cells better than the normalones [5]. Therefore, clinical research…     

Identification of amino acid residues of Ras protein that are essential for signal-transducing activity but not for enhancement of GTPase activity by GAP.

To determine the amino acid residues required for the signal-transducing activity of the human c-Ha-Ras protein, we introduced point mutations at residues 45-54 near the 'effector region' (residues 32-40). We transfected PC12 cells with these mutant genes and also micro-injected the mutant proteins, bound with an unhydrolyzable GTP analog, into PC12 cells. Both procedures showed that Val45----Glu and Gly48----Cys mutations impaired the ability of the Ras protein to induce morphological change of PC12 cells. These mutations did not affect the guanine nucleotide-binding activity or GTPase activity in the absence or presence of bovine GTPase-activating protein (GAP). Therefore, the Val45 and Gly48 residues should be included by definition in the effector region responsible for the signal transduction, while only a subset of the effector-region residues is required for enhancement of the GTPase activity by GAP.     

Functional consequences of tryptophan modification in human fibrinogen.

When human fibrinogen was modified with H2O2, inter- and intra-molecular cross-links of fibrinogen were formed, accompanied with oxidation of tryptophan, methionine and tyrosine residues. These cross-links may be closely associated with oxidation of tryptophan residues. The polymerization activity of fibrinogen with thrombin was decreased markedly by this modification. Modification of tryptophan residues in fibrinogen was also performed with 2-hydroxy-5-nitrobenzyl bromide. Modification of two out of a total 78 tryptophan residues in the molecule with the reagent led to the intensification (1.7 times) of the polymerization activity with thrombin and further modification of the next two residues led to complete loss of the polymerization activity. The first two tryptophan residues to be modified are in Fragment D, and the next two occur in Fragment E.     

Relative spatial positions of tryptophan and cationic residues in helical membrane-active peptides determine their cytotoxicity

The cytotoxic activity of 10 analogs of the idealized amphipathic helical 21-mer peptide (KAAKKAA)3, where three of the Ala residues at different positions have been replaced with Trp residues, has been investigated. The peptide's cytotoxic activity was found to be markedly dependent upon the position of the Trp residues within the hydrophobic sector of an idealized α-helix. The peptides with Trp residues located opposite the cationic sector displayed no antitumor activity, whereas those peptides with two or three Trp residues located adjacent to the cationic sector exhibited high cytotoxic activity when tested against three different cancer cell lines. Dye release experiments revealed that in contrast to the peptides with Trp residues located opposite the cationic sector, the peptides with Trp residues located adjacent to the cationic sector induced a strong permeabilizing activity from liposomes composed of a mixture of zwitterionic phosphatidylcholine and negatively charged phosphatidylserine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS)) (2:1) but not from liposomes composed of zwitterionic phosphatidylcholine, POPC. Fluorescence blue shift and quenching experiments revealed that Trp residues inserted deeper into the hydrophobic environment of POPC/POPS liposomes for peptides with high cytotoxic activity. Through circular dichroism studies, a correlation between the cytotoxic activity and the α-helical propensity was established. Structural studies of one inactive and two active peptides in the presence of micelles using NMR spectroscopy showed that only the active peptides adopted highly coiled to helical structures when bound to a membrane surface.     

Iodination of ovotransferrin and its iron complex. Extent of involvement of tyrosine phenolic groups in the iron binding

Short-time iodination of metal-free ovotransferrin indicated that the tyrosine groups involved in the iron-binding activity are indistinguishable from other structural tyrosines. Modification of a minimum of 14 tyrosine residues per molecule of protein was required to achieve a complete loss of metal-binding activity. In contrast, a maximum modification of 10 tyrosine residues in iron-ovotransferrin complex could be produced with no loss of iron-binding activity. The difference in the extent of modification of tyrosines, therefore, indicated the involvement of four tyrosines in the binding of two atoms of iron. A minimal modification of histidine residues was also found, which was limited to one residue per molecule of both ovotransferrin and its iron complex. The possible participation of two tryptophan residues in the iron-binding activity is also suggested in the present study.     

Chemical modification of erythropoietin: an increase in in vitro activity by guanidination

Human recombinant erythropoietin (rHuEPO) was chemically modified with several group-specific reagents in order to study the role of each kind of amino-acid residue in its biological activity. Guanidination of the amino groups of the lysine residues yielded derivatives that showed higher activities in vitro than native rHuEPO, whereas amidination had no effect on the activity. By contrast, modification of the positive charges of the lysine residues to neutral or negative charges, such as in carbamylation, trinitrophenylation, acetylation or succinylation, caused a significant loss of rHuEPO activity. Chemical modification of other amino-acid residues, such as arginine and tyrosine residues or carboxyl groups, also led to loss of activity.     

Kinetic and site-directed mutagenesis studies of prevotella intermedia acid phosphatase

Site-directed mutagenesis was used to examine the roles of the conserved histidine, arginine and cysteine residues in acid phosphatase from Prevotella intermedia (PiACP). The replacement of histidine and arginine residues resulted in the elimination of the PiACP activity while the cysteine mutants retained activity. These results suggest that the histidine and arginine residues are essential for catalysis.     

粘质赛氏菌胞外蛋白酶的化学修饰

用九种化学修饰剂研究了粘质赛氏菌ＳｅｒｒａｔｉａＭａｒｃｅｓｃｅｎｓ４１００３（２）胞外蛋白酶分子中氨基酸侧链基团与酶催化活性的关系,结果表明组氨酸、丝氨酸、赖氨酸、精氨酸、谷氨酸及天冬氨酸等残基与酶活性无关;半胱氨酸残基与酶活性也无直接关系;而酪氨酸和色氨酸残基侧链的修饰引起酶活力大幅度下降,说明酪氨酸和色氨酸残基为酶活力必需．     

Characterization of residues in human IgE and IgG binding site by chemical modification of ovomucoid third domain.

Chemical modification of ovomucoid third domain (DIII) has been conducted to characterize the binding site residues that determine antigenecity and allergenecity of DIII. Nitration of Tyr, ethoxyformylation of His and succinylation of Lys residues led to a decrease of alpha-helix content of DIII. Modification of His, Tyr, Glu, Asp and Lys residues on DIII resulted in a reduction of human IgG binding activity, but little effect on IgE binding activity. These results suggest that hydrophilic residues appear to be more critical for human IgG binding site, whereas hydrophobic residues may be more important for IgG binding site.