Synthesis of modified peptides with C-terminal alpha-amino aldehydes]

Various synthetic approaches to modified peptides with the C-terminal aldehyde group, capable of inhibiting a number of proteolytic enzymes belonging to the classes of thiol, serine, and aspartyl proteases, are considered. Both chemical methods, including solid phase peptide synthesis now widely used, and biocatalytic synthetic methods for obtaining these substances are discussed in detail.     

Synthesis of Modified Peptides with C-Terminal α-Amino Aldehydes

Various synthetic approaches to modified peptides with the C-terminal aldehyde group, capable of inhibiting a number of proteolytic enzymes belonging to the classes of thiol, serine, and aspartyl proteases, are considered. Both chemical methods, including solid phase peptide synthesis now widely used, and biocatalytic synthetic methods for obtaining these substances are discussed in detail.     

C-terminal analogues of parathyroid hormone: effect of C-terminus function on helical structure, stability, and bioactivity

We have studied the effects of C-terminal group modifications (amide, methylamide, dimethylamide, aldehyde, and alcohol) on the conformation, adenylyl cyclase stimulation (AC), or binding of parathyroid hormone (hPTH) analogues, hPTH(1-28)NH(2) and hPTH(1-31)NH(2). hPTH(1-31)NH(2) has a C-terminal alpha-helix bounded by residues 17-29 [Chen, Z., et al. (2000) Biochemistry 39, 12766]. In both cases, relative to the natural analogue with a carboxyl C-terminus, the amide and methylamide had increased helix content whereas the dimethylamide forms had CD spectra more similar to the carboxyl one. Conformational effects were more pronounced with hPTH(1-28) than with hPTH(1-31), with increases in helix content of approximately 30% in contrast to 10%. Stabilization of the C-terminal helix of residues 1-28 seemed to correlate with an ability of the C-terminal function to H-bond appropriately. None of the analogues affected the AC stimulating activity significantly, but there was an up to 15-fold decrease in the level of apparent binding of the carboxyl hPTH(1-28) analogue compared to that of the methylamide and a 4-fold decrease in the level of binding of the aldehyde or dimethylamide. There was no significant change in binding activities for the 1-31 analogues. These observations are consistent with previous studies that imply the importance of a region of the hormone's C-terminal alpha-helix for tight binding to the receptor. They also show that modulation of helix stability does have an effect on the binding of the hormone, but only when the C-terminus is at the putative end of the helix. The similarity of AC stimulation even when binding changed 10-fold can be explained by assuming greater efficacy of the weaker binding PTH-receptor complexes in stimulating AC.     

Synthesis of peptide aldehydes via enzymatic acylation of amino aldehyde derivatives

Two ways for semi-enzymatic preparation of the peptide aldehydes are proposed: (1) enzymatic acylation of amino alcohols with acyl peptide esters and subsequent chemical oxidation of the resulting peptide alcohols with DMSO/acetic anhydride mixture or (2) enzymatic acylation of the preliminarily obtained by a chemical route amino aldehyde semicarbazones. Subtilisin 72, serine proteinase with a broad specificity, distributed over macroporous silica, was used as a catalyst in both cases. Due to the practical absence of water in the reaction mixtures the yields of the products in both enzymatic reactions were nearly quantitative. The second way seems to be more attractive because all chemical stages were carried out with amino acid derivatives, far less valuable compounds than peptide ones. A series of peptide aldehydes of general formula Z-Ala-Ala-Xaa-al (where Xaa-al=leucinal, phenylalaninal, alaninal, valinal) was obtained. The inhibition parameters for these compounds, in the hydrolysis reactions of corresponding chromogenic substrates for subtilisin and -chymotrypsin, were determined.     

Binding of bovine prion protein to heparin: a fluorescence polarization study

Glycosaminoglycans (GAGs) are believed to be associated with prion disease pathology and also with metabolism of the prion protein. Fluorescence polarization assay (FPA) of binding between bovine recombinant prion protein (brecPrP) and heparin labelled with AlexaFluor488 was used in model experiments to study glycosaminoglycan-prion protein interaction. Heparin binding to brecPrP was a rapid reversible event which occurred under defined conditions. The interaction of brecPrP with fluorophore-labelled heparin was inhibited by the presence of Cu(2+) ions and was sensitive to competition with heparin, heparan sulphate, and dextran. The dissociation constant of the heparin-brecPrP complex was 73.4+/-3.7 nM. Circular dichroism (CD) experiments indicated that the structure of brecPrP was less helical in the presence of heparin.     

Structural and Functional Analysis of Campylobacter jejuni PseG: A UDP-SUGAR HYDROLASE FROM THE PSEUDAMINIC ACID BIOSYNTHETIC PATHWAY*

Flagella of the bacteria Helicobacter pylori and Campylobacter jejuni are important virulence determinants, whose proper assembly and function are dependent upon glycosylation at multiple positions by sialic acid-like sugars, such as 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid (Pse)). The fourth enzymatic step in the pseudaminic acid pathway, the hydrolysis of UDP-2,4-diacetamido-2,4,6-trideoxy-β-l-altropyranose to generate 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose, is performed by the nucleotide sugar hydrolase PseG. To better understand the molecular basis of the PseG catalytic reaction, we have determined the crystal structures of C. jejuni PseG in apo-form and as a complex with its UDP product at 1.8 and 1.85 Å resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His¹⁷-coordinated water molecule as the putative nucleophile and suggests the UDP-sugar substrate adopts a twist-boat conformation upon binding to PseG, enhancing the exposure of the anomeric bond cleaved and favoring inversion at C-1. Furthermore, based on these structures a series of amino acid substitution derivatives were constructed, altering residues within the active site, and each was kinetically characterized to examine its contribution to PseG catalysis. In conjunction with structural comparisons, the almost complete inactivation of the PseG H17F and H17L derivatives suggests that His¹⁷ functions as an active site base, thereby activating the nucleophilic water molecule for attack of the anomeric C–O bond of the UDP-sugar. As the PseG structure reveals similarity to those of glycosyltransferase family-28 members, in particular that of Escherichia coli MurG, these findings may also be of relevance for the mechanistic understanding of this important enzyme family.The gastrointestinal pathogens Campylobacter jejuni and Helicobacter pylori have been shown to modify their flagellins with the sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid or pseudaminic acid (Pse),³ via O-linkage at up to 19 sites per flagellin monomer (1, 2). Not only is this sialic acid-like modification necessary for flagellar assembly and motility (1, 2), it has also been shown to be important for C. jejuni virulence (3). In addition to its role in autoagglutination of bacterial cells, Pse and related derivatives may also influence pathogenesis through bacterial adhesion, invasion, and immune evasion (4, 5), since sialic acids in humans have been shown to mediate a myriad of cell-cell and cell-molecule interactions (6). As flagellin glycosylation in these organisms is required for host colonization and ultimately virulence (3, 7, 8), these novel sugar biosynthetic pathways provide an excellent platform for therapeutic development.The reliance of H. pylori pathogenicity on Pse biosynthesis, in combination with the prevalence of H. pylori resistance to existing antibiotic treatments (9), prompted and led to the complete elucidation of the CMP-pseudaminic acid (CMP-Pse) biosynthetic pathway in both C. jejuni and H. pylori (10–15). The CMP-Pse biosynthetic pathway (Fig. 1) is similar to that of CMP-sialic acid, involving condensation of an N-acetylhexosamine intermediate with the three-carbon pyruvate molecule forming a nine-carbon sialic acid-like nonulosonate, although in contrast the CMP-Pse pathway consists of several more steps between the initial building block UDP-GlcNAc and the condensation reaction. PseG, a UDP-sugar hydrolase, produces the final 6-deoxy-N-acetylhexosamine intermediate in the CMP-Pse pathway by removing the nucleotide moiety from UDP-2,4-diacetamido-2,4,6-trideoxy-β-l-altropyranose or UDP-6-deoxy-AltdiNAc (Fig. 1). This sort of single enzymatic function is rare in nature, with the only other similar example being a GDP-mannose/GDP-glucose hydrolase (16), which belongs to the metal-dependent Nudix family of enzymes. In an elegant study, Liu and Tanner (11) demonstrated that PseG catalyzes nucleotide removal by a metal-independent C–O bond cleavage mechanism resulting in inversion of stereochemistry at C-1 of the product 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose or 6-deoxy-AltdiNAc, similar to the catalytic properties of some GT-B glycosyltransferases.Open in a separate windowFIGURE 1.Role of PseG within the CMP-pseudaminic acid biosynthetic pathway of C. jejuni and H. pylori. The biosynthetic step involving PseG is highlighted in blue. The enzymes and biosynthetic intermediates of the CMP-pseudaminic acid pathway are, in the following order, PseB (Cj1293/HP0840), NADP-dependent dehydratase/epimerase; PseC (Cj1294/HP0366), pyridoxal phosphate-dependent aminotransferase; PseH (Cj1313/HP0327), N-acetyltransferase; PseG (Cj1312/HP0326B), NDP-sugar hydrolase; PseI (Cj1317/HP0178), pseudaminic acid synthase; PseF (Cj1311/HP0326A), CMP-pseudaminic acid synthetase; and I, UDP-GlcNAc; II, UDP-2-acetamido-2,6-dideoxy-β-l-arabino-hexos-4-ulose; III, UDP-4-amino-4,6-dideoxy-β-l-AltNAc; IV, UDP-2,4-diacetamido-2,4,6-trideoxy-β-l-altropyranose; V, 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose; VI, pseudaminic acid; and VII, CMP-pseudaminic acid. Here, PEP refers to phosphoenolpyruvate. Pyranose rings are shown as their predominant chair conformation in solution as determined from nuclear Overhauser effects and J_H,H coupling constants (13).Together, glycosyltransferases and glycoside hydrolases compose the majority of enzymes in both eukaryotes and prokaryotes that manipulate glycosidic bonds. Glycosyltransferases of the Leloir classification use sugar-nucleotide derivatives as glycosyl donors resulting in transfer to acceptors such as a monosaccharide, oligosaccharide, or polysaccharide. It is therefore plausible that a “glycosyltransferase fold” in PseG has evolved to efficiently utilize water as an acceptor, instead of another carbohydrate, consequently behaving as a hydrolase (11). Based on structure, most glycosyltransferases fall into two groups, GT-A and GT-B, that exhibit different folds, respectively (17). For both families, depending on the particular enzyme, the outcome may result in either inversion or retention of stereochemistry for the donor anomeric carbon (see Fig. 2). In addition, GT-B family enzymes are metal-independent, lacking an important DXD motif present in most GT-A members. Based on the novelty of PseG and its role in H. pylori pathogenicity, we sought a greater structural and mechanistic understanding of this important enzyme.Open in a separate windowFIGURE 2.Functional comparison of enzymes belonging to the GT-B superfamily. A, UDP-sugar hydrolase PseG catalyzes the removal of UDP from UDP-2,4-diacetamido-2,4,6-trideoxy-β-l-Alt or UDP-6-deoxy-AltdiNAc. B, UDP-GlcNAc hydrolyzing 2-epimerase NeuC catalyzes the removal of UDP and the formation of ManNAc from UDP-GlcNAc. C, GlcNAc transferase MurG catalyzes the formation of undecaprenyl-phosphoryl-muramyl-pentapeptide-GlcNAc via formation of a glycosidic linkage between UDP-GlcNAc and undecaprenyl-phosphoryl-muramyl-pentapeptide. R represents the phosphoryl-undecaprenyl moiety, with the pentapeptide having the specific sequence l-Ala-d-γGlu-l-Lys-d-Ala-d-Ala. Both A and C activities result in an initial inversion of stereochemistry at C-1 for the donor substrate. In contrast, the activity for B results in an initial retention of C-1 stereochemistry. Enzymatically altered anomeric bonds are indicated in red.Here we report the crystal structure of PseG alone at 1.8 Å resolution and in complex with UDP, a product of the reaction, at 1.85 Å resolution. Although very few homologs have been identified based on sequence similarity alone, PseG bears the closest structural similarity to MurG, a GT-B family member (18). In addition, computational docking and molecular dynamics simulations were performed to gain insight into the binding mode of the PseG substrate UDP-6-deoxy-AltdiNAc. Based on the crystallographic and modeled structures, several potential active site residues were selected for mutagenesis and kinetic analyses to further characterize the PseG active site. The relevance of these findings to the structurally related MurG family of enzymes is discussed.     

Backbone-methylated analogues of the principle receptor binding region of human parathyroid hormone. Evidence for binding to both the N-terminal extracellular domain and extracellular loop region

We have used backbone N-methylations of parathyroid hormone (PTH) to study the role of these NH groups in the C-terminal amphiphilic alpha-helix of PTH (1-31) in binding to and activating the PTH receptor (P1R). The circular dichroism (CD) spectra indicated the structure of the C-terminal alpha-helix was locally disrupted around the methylation site. The CD spectra differences were explained by assuming a helix disruption for four residues on each side of the site of methylation and taking into account the known dependence of CD on the length of an alpha-helix. Binding and adenylyl cyclase-stimulating data showed that outside of the alpha-helix, methylation of residues Asp30 and Val31 had little effect on structure or activities. Within the alpha-helix, disruption of the structure was associated with increased loss of activity, but for specific residues Val21, Leu24, Arg25, and Leu28 there was a dramatic loss of activities, thus suggesting a more direct role of these NH groups in correct P1R binding and activation. Activity analyses with P1R-delNT, a mutant with its long N-terminal region deleted, gave a different pattern of effects and implicated Ser17, Trp23, and Lys26 as important for its PTH activation. These two groups of residues are located on opposite sides of the helix. These results are compatible with the C-terminal helix binding to both the N-terminal segment and also to the looped-out extracellular region. These data thus provide direct evidence for important roles of the C-terminal domain of PTH in determining high affinity binding and activation of the P1R receptor.     

Role of amino acid side chains in region 17-31 of parathyroid hormone (PTH) in binding to the PTH receptor

The principal receptor-binding domain (Ser(17)-Val(31)) of parathyroid hormone (PTH) is predicted to form an amphiphilic alpha-helix and to interact primarily with the N-terminal extracellular domain (N domain) of the PTH receptor (PTHR). We explored these hypotheses by introducing a variety of substitutions in region 17-31 of PTH-(1-31) and assessing, via competition assays, their effects on binding to the wild-type PTHR and to PTHR-delNt, which lacks most of the N domain. Substitutions at Arg(20) reduced affinity for the intact PTHR by 200-fold or more, but altered affinity for PTHR-delNt by 4-fold or less. Similar effects were observed for Glu substitutions at Trp(23), Leu(24), and Leu(28), which together form the hydrophobic face of the predicted amphiphilic alpha-helix. Glu substitutions at Arg(25), Lys(26), and Lys(27) (which forms the hydrophilic face of the helix) caused 4-10-fold reductions in affinity for both receptors. Thus, the side chains of Arg(20), together with those composing the hydrophobic face of the ligand's putative amphiphilic alpha-helix, contribute strongly to PTHR-binding affinity by interacting specifically with the N domain of the receptor. The side chains projecting from the opposite helical face contribute weakly to binding affinity by different mechanisms, possibly involving interactions with the extracellular loop/transmembrane domain region of the receptor. The data help define the roles that side chains in the binding domain of PTH play in the PTH-PTHR interaction process and provide new clues for understanding the overall topology of the bimolecular complex.