Peptide Recognition Site Research Articles

New Perspectives on Reverse Translation: Brief History and Updates

Since the 1950s, reverse translation has been an enigmatic part of Crick’s central dogma of molecular biology. It might be described as the possibility to back-translate information from proteins to nucleic acids (or codons). A few studies have attempted to theorize and/or conduct in vitro experiments to test the likelihood of reverse translation, with ideas often involving the creation of peptide recognition sites that bridge the peptide and the codon. However, due to many constraints including an asymmetrical informational transfer, the stability of protein-peptide bonds, the structural non-uniformity of protein R-groups, and the informational loss in post-translational protein modifications, this concept requires follow-up studies. On the other hand, current bioinformatic tools that rely on computational programs and biological databases represent a growing branch of biology. Bioinformatics-based reverse translationcan utilize codon usage tables to predict codons from their peptide counterparts. In addition, the development of machine learning tools may allow for the exploration of biological reverse translation in vitro. Thus, while in vivo reverse translation appears to be nearly impossible (due to biological complexity), related biological and bioinformatics studies might be useful to understand better thecentral dogma’s informational transfer and to develop more complex biological machinery.

The prediction of a pathogenesis-related secretome of Puccinia helianthi through high-throughput transcriptome analysis

BackgroundMany plant pathogen secretory proteins are known to be elicitors or pathogenic factors,which play an important role in the host-pathogen interaction process. Bioinformatics approaches make possible the large scale prediction and analysis of secretory proteins from the Puccinia helianthi transcriptome. The internet-based software SignalP v4.1, TargetP v1.01, Big-PI predictor, TMHMM v2.0 and ProtComp v9.0 were utilized to predict the signal peptides and the signal peptide-dependent secreted proteins among the 35,286 ORFs of the P. helianthi transcriptome.Results908 ORFs (accounting for 2.6% of the total proteins) were identified as putative secretory proteins containing signal peptides. The length of the majority of proteins ranged from 51 to 300 amino acids (aa), while the signal peptides were from 18 to 20 aa long. Signal peptidase I (SpI) cleavage sites were found in 463 of these putative secretory signal peptides. 55 proteins contained the lipoprotein signal peptide recognition site of signal peptidase II (SpII). Out of 908 secretory proteins, 581 (63.8%) have functions related to signal recognition and transduction, metabolism, transport and catabolism. Additionally, 143 putative secretory proteins were categorized into 27 functional groups based on Gene Ontology terms, including 14 groups in biological process, seven in cellular component, and six in molecular function. Gene ontology analysis of the secretory proteins revealed an enrichment of hydrolase activity. Pathway associations were established for 82 (9.0%) secretory proteins. A number of cell wall degrading enzymes and three homologous proteins specific to Phytophthora sojae effectors were also identified, which may be involved in the pathogenicity of the sunflower rust pathogen.ConclusionsThis investigation proposes a new approach for identifying elicitors and pathogenic factors. The eventual identification and characterization of 908 extracellularly secreted proteins will advance our understanding of the molecular mechanisms of interactions between sunflower and rust pathogen and will enhance our ability to intervene in disease states.

Structural Basis of the Recognition of the SAMP Motif of Adenomatous Polyposis Coli by the Src-Homology 3 Domain

Elucidation of the basis of interactions between biological molecules is essential for the understanding of living systems. Src-homology 3 (SH3) domains play critical roles in interaction networks of proteins by recognizing a proline-rich sequence motif, PxxP. There are, however, several SH3 domains that specifically bind to polypeptide chains without the conventional recognition sequence. The SH3 domain of DDEF1 associates with the SAMP motifs of the adenomatous polyposis coli (APC) tumor suppressor. The SAMP motifs are indispensable for the normal function of APC in tumor suppression. Here we present the structural basis of the interaction between the DDEF1-SH3 domain and the APC-SAMP motifs. We determined the solution structures of the DDEF1-SH3 domain both in a free state and in a complex with APC-SAMP. As the affinity of the interaction was not sufficiently high for the determination of the complex structure in solution by conventional methods, we utilized a fusion protein of the DDEF1-SH3 domain and APC-SAMP. The structures revealed that the SAMP motif adopts a class II polyproline type II helix even though it does not contain the PxxP motif and that a characteristically large hydrophobic pocket of the SH3 domain confers high selectivity to the interaction. Furthermore, investigation into the backbone dynamics of the free and bound systems by NMR spin relaxation experiments demonstrated that the DDEF1-SH3 domain exhibits high flexibility at the peptide recognition site in the absence of the ligand and that most residues of the APC-SAMP motif display extensive local motions even in the stable complex.

CDNA cloning and in situ hybridization of a novel lysozyme in the Pacific oyster, Crassostrea gigas

A novel lysozyme cDNA from the Pacific oyster, Crassostrea gigas, was identified. This second lysozyme from the Pacific oyster was designated as CGL-2. The complete CGL-2 cDNA sequence comprises of 536 bp, and 429 bp of the open reading frame encodes 147 bp of amino acid residues. Estimated CGL-2 molecular characteristics (isoelectric point and numbers of peptide recognition sites) resembled those of cv-lysozyme 2, a digestive lysozyme of the eastern oyster, Crassostrea virginica. Moreover, CGL-2 is phylogenetically homologous to the cv-lysozyme 2, indicating that CGL-2 and cv-lysozyme 2 evolved from the same ancestor protein for adaptation to the digestive environment. In situ hybridization revealed that the CGL-2 gene is expressed in digestive cells. It is noteworthy that the other Pacific oyster lysozyme, CGL-1, was also transcribed in the same cells. Presence and expression of multiple lysozymes in the digestive diverticula suggest that CGL-1 and CGL-2 might play complementary roles in digestive organs.

Peptide Inhibitors of West Nile NS3 Protease: SAR Study of Tetrapeptide Aldehyde Inhibitors

A series of inhibitors related to the benzoyl-norleucine-lysine-arginine-arginine (Bz-nKRR) tetrapeptide aldehyde was synthesized. When evaluated against the West Nile virus (WNV) NS3 protease, the measured IC(50) ranges from approximately 1 to 200 microM. Concurrently, a modeling study using the recently published crystal structure of the West Nile NS3/NS2B protease complex (pdb code 2FP7) was conducted. We found that the crystal structure is relevant in explaining the observed SAR for this series of tetrapeptides, with the S1 and S2 pockets being the key peptide recognition sites. In general, a residue capable of both pi-stacking and hydrogen bonding is favored in the S1 pocket, while a positively charged residue is preferred in the S2 pocket. This study not only confirms the importance of the NS2B domain in substrate-based inhibitor binding of WNV, it also suggests that the crystal structure would provide useful guidance in the drug discovery process of related Flavivirus proteases, given the high degree of homology.

Functional Molecular Thin Films: Topological Templates for the Chemoselective Ligation of Antigenic Peptides to Self-Assembled Monolayers.

The combination of self-assembly and regioselective surface chemistry has made it possible to immobilize peptide recognition sites 1 on a template attached to a gold surface. Each of the seven individual reaction steps, including the final functional biomolecular recognition, was controlled in situ with surface-sensitive detection techniques. The presented strategy is of general importance for the formation of complex supramolecular structures with biologically interesting functionalities at the interfaces.

Integrins and matrix molecules in salivary gland cell adhesion, signaling, and gene expression.

Integrins play crucial roles in embryonic and adult cell adhesion, migration, morphogenesis, growth, and differentiation in many cell systems, including human salivary gland cells. Integrins function by binding through their extracellular domain to a specific peptide recognition site in a ligand, and then transmitting information to the cytoplasm by way of their cytoplasmic tails. By this transmembrane signaling process, integrins can mediate assembly of adhesion sites and organization of the actin-containing cytoskeleton by forming supermolecular complexes of cytoskeletal and signaling molecules. The specific steps in the assembly of these complexes as well as novel mechanisms for synergy between integrin and growth factor signaling pathways are still being determined. integrin-mediated interactions also have major effects on gene expression. For example, integrin-mediated adhesion to fibronectin by the HSG salivary gland cell line significantly alters the pattern of proteins synthesized and genes expressed. In fact, at least five transcription factors are activated, and over 30 genes (many of them novel) are found to be induced by such integrin-mediated interactions by salivary gland cells. The roles of integrins, in collaborative interactions with growth factors and signaling pathways, and in the induction of novel genes during salivary gland development, should provide fruitful areas of research for many years.

Scanning Alanine Mutagenesis and De-peptidization of a Candida albicans Myristoyl-CoA:ProteinN-Myristoyltransferase Octapeptide Substrate Reveals Three Elements Critical for Molecular Recognition

Candida albicans produces a single myristoyl-CoA:protein N-myristoyltransferase (Nmt) that is essential for its viability. An ADP-ribosylation factor (Arf) is included among the few cellular protein substrates of this enzyme. An octapeptide (GLYASKLS-NH2) derived from a N-terminal Arf sequence was used as the starting point to identify elements critical for recognition by the acyltransferases's peptide-binding site. In vitro kinetic studies, employing purified Nmt and a panel of peptides with single Ala substitutions at each position of GLYASKLS-NH2, established that its Gly1, Ser5, and Lys6 residues play predominant roles in binding. ALYASKLS-NH2 was found to be an inhibitor competitive for peptide (Ki = 15.3 +/- 6.4 microM) and noncompetitive for myristoyl-CoA (Ki = 31.2 +/- 0.7 microM). A survey of 26 derivatives of this inhibitor, representing (i) a complete alanine scan, (ii) progressive C-terminal truncations, and (iii) manipulation of the physical-chemical properties of its residues 1, 5, and 6, confirmed the important stereochemical requirements for the N-terminal amine, the beta-hydroxyl of Ser5, and the epsilon-amino group of Lys6. Remarkably, replacement of the the N-terminal tetrapeptide of ALYASKLS-NH2 with an 11-aminoundecanoyl group produced a competitive inhibitor, 11-aminoundecanoyl-SKLS-NH2, that was 38-fold more potent (Ki = 0.40 +/- 0.03 microM) than the starting octapeptide. Removing the primary amine (undecanoyl-SKLS-NH2), or replacing it with a methyl group (dodecanoyl-SKLS-NH2), resulted in 26- and 34-fold increases in IC50, confirming the important contribution of the amine to recognition. Removal of LeuSer from the C terminus (11-aminoundecanoyl-SK-NH2) yielded a competitive dipeptide inhibitor with a Ki (11.7 +/- 0.4 microM) equivalent to that of the starting octapeptide, ALYASKLS-NH2. Substitution of Ser with homoserine, cis-4-hydroxyproline, or tyrosine reduces potency by 3-70-fold, emphasizing the requirement for proper presentation of the hydroxyl group in the dipeptide inhibitor. Substituting D- for L-Lys decreases its inhibitory activity >100-fold, while deletion of the epsilon-amino group (Nle) or masking its charge (epsilon-N-acetyl-lysine) produces 4-7-fold attenuations. L-His, but not its D-isomer, can fully substitute for L-Lys, producing a competitive dipeptide inhibitor with similar potency (Ki = 11.9 +/- 1.0 microM). 11-Aminoundecanoyl-SK-NH2 and 11-aminoundecanoyl-SH-NH2 establish that a simple alkyl backbone can maintain an appropriate distance between three elements critical for recognition by the fungal enzyme's peptide-binding site: a simple omega-terminal amino group, a beta-hydroxyl, and an epsilon-amino group or an imidazole. These compounds contain one peptide bond and two chiral centers, suggesting that it may be feasible to incorporate these elements of recognition, or functionally equivalent mimics, into a fully de-peptidized Nmt inhibitor.

High affinity binding of the heat-stable protein kinase inhibitor to the catalytic subunit of cAMP-dependent protein kinase is selectively abolished by mutation of Arg133.

The two classes of physiological inhibitors of the catalytic subunit of cAMP-dependent protein kinase are the regulatory subunits and the heat-stable protein kinase inhibitors (PKIs), and both share a common mechanism of inhibition. Each has a similar inhibitor site that resembles a peptide substrate, and this occupies the P-3 to P+1 portion of the peptide recognition site. However, in addition to this consensus site, each inhibitor requires a peripheral binding site to achieve high affinity binding. Arg134 and Arg133 lie on the surface of the catalytic subunit with Arg133 coming close to the amphipathic helix of PKI(5-24) (Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N.-h., Taylor, S. S., and Sowadski, J. M. (1991) Science 253, 414-420). Replacement of Arg134 and Arg133 with Ala selectively abolishes the high affinity binding of PKI. Replacement of Arg133 alone is sufficient to give the same phenotype. In the presence of MgATP, the Kd,app, is increased from < 0.2 to 105 nM and, in the absence of ATP, the Kd is too large to be reliably measured. Based on the crystal structure, Arg133 hydrogen bonds to the P-7 backbone carbonyl of PKI(5-24). However, more importantly, it also contributes to the hydrophobicity of the P-11 binding site in the C.PKI(5-24) complex. We predict that it is the perturbation of this hydrophobic pocket that accounts for the effects of this mutation. In the absence of peptide, Arg133 may help to stabilize Glu230, a buried carboxylate that binds to the P-2 Arg in the crystal structure of C.PKI(5-24). Replacement of Arg133 and Arg134 with Ala has little effect on catalysis using a heptapeptide substrate and has no effect on the inhibition of the catalytic subunit by the regulatory subunit. The results thus demonstrate that these two inhibitor proteins that both bind to the catalytic subunit with a high affinity utilize different sites on the enzyme to achieve tight binding. The gamma isoform of the catalytic subunit is insensitive to inhibition by PKI and in this isoform Arg133 is replaced with Gln. We predict that this change accounts for the altered inhibitor properties of C gamma.

A three-dimensional model of the Cdc2 protein kinase: localization of cyclin- and Suc1-binding regions and phosphorylation sites.

The Cdc2 protein kinase requires cyclin binding for activity and also binds to a small protein, Suc1. Charged-to-alanine scanning mutagenesis of Cdc2 was used previously to localize cyclin A- and B- and Suc1-binding sites (B. Ducommun, P. Brambilla, and G. Draetta, Mol. Cell. Biol. 11:6177-6184, 1991). Those sites were mapped by building a Cdc2 model based on the crystallographic coordinates of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) (D. R. Knighton, J. Zheng, L. F. Ten Eyck, V. A. Ashford, N.-H. Xuong, S. S. Taylor, and J. M. Sowadski, Science 253:407-414, 1991). On the basis of this model, additional mutations were made and tested for cyclin A and Suc1 binding and for kinase activity. Mutations that interfere with cyclin A binding are localized primarily on the small lobe near its interface with the cleft and include an acidic patch on the B helix and R-50 in the highly conserved PSTAIRE sequence. Two residues in the large lobe, R-151 and T-161, influence cyclin binding, and both are at the surface of the cleft near its interface with the PSTAIRE motif. Cyclin-dependent phosphorylation of T-161 in Cdc2 is essential for activation, and the model provides insights into the importance of this site. T-161 is equivalent to T-197, a stable phosphorylation site in cAPK. On the basis of the model, cyclin binding very likely alters the surface surrounding T-161 to allow for T-161 phosphorylation. The two major ligands to T-197 in cAPK are conserved as R-127 and R-151 in Cdc2. The equivalent of the third ligand, H-87, is T-47 in the PSTAIRE sequence motif. Once phosphorylated, T-161 is predicted to play a major structural role in Cdc2, comparable to that of T-197 in cAPK, by assembling the active conformation required for peptide recognition. The inhibitory phosphorylation at Y-15 also comes close to the cleft interface and on the basis of this model would disrupt the cleft interface and the adjacent peptide recognition site rather than prevent ATP binding. In contrast to cyclin A, both lobes influence Suc1 binding; however, the Suc1-binding sites are far from the active site. Several mutants map to the surface in cAPK, which is masked in part by the N-terminal 40 residues that lie outside the conserved catalytic core. The other Suc1-binding site maps to the large lobe near a 25-residue insert and includes R-215.

Genetic and biochemical studies of a mutant Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase, nmt72pLeu99–>Pro, that produces temperature-sensitive myristic acid auxotrophy.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme that transfers myristate from CoA to the amino-terminal glycine residue of at least 12 cellular proteins. Its reaction mechanism is Ordered Bi Bi with myristoyl-CoA binding occurring before binding of nascent polypeptides and release of CoA preceding release of the myristoylprotein product. nmt1-72 is a temperature-sensitive allele, identified by Stone et al. (Stone, D. E., Cole, G. M., Lopes, M. B., Goebl, M., and Reed, S. I. (1991) Genes & Dev. 5, 1969-1981) that causes arrest in the G1 phase of the cell cycle due to reduced acylation of Gpa1p. We have recovered this mutant allele and determined that it contains a single point mutation resulting in a Leu99 (CTA) to Pro (CCA) substitution. Addition of > or = 500 microM myristate but not palmitate to synthetic or rich media rescues the growth arrest caused by nmt1-72 at 37-39 degrees C, consistent with the observation that purified nmt72p has reduced affinity for myristoyl-CoA and that exogenous myristate but not palmitate increases cellular myristoyl-CoA pools. Metabolic labeling studies in S. cerevisiae and co-expression of nmt72p with several protein substrates of Nmt1p in Escherichia coli indicate that the Leu99-->Pro substitution causes a reduction in the acylation of some but not all protein substrates. Since formation of a myristoyl-CoA.Nmt1p complex appears to be required for synthesis/formation of a peptide binding site, these defects in acylation appear to arise either because Leu99 is a component of the enzyme's functionally distinguishable myristoyl-CoA and peptide recognition sites or because Pro99 alters the interaction between myristoyl-CoA and enzyme in a way that precludes formation of a normal peptide binding site. The reduction in affinity for myristoyl-CoA produced by Leu99-->Pro in nmt72p is less than that produced by the Gly451-->Asp mutation in nmt181p, which also produces temperature-sensitive myristic acid auxotrophy. Isogenic, haploid strains containing NMT1, nmt1-72, and nmt1-181 do not manifest any obvious differences in steady state levels of the acyltransferases during growth at permissive temperatures or in the biosynthesis of long chain saturated acyl-CoAs. The spectrum of cellular N-myristoylproteins whose level of acylation is affected by nmt1-72 and nmt1-181 is distinct.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a Thyroid Sulfotransferase Responsible for the 3-O-Sulfation of Terminal β-D-Galactosyl Residues in N-Linked Carbohydrate Units

Calf thyroid microsomes were found to contain an enzyme which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phospho[35S]sulfate (PAPS) to C-3 of terminal galactose residues in beta 1----4 linkage to GlcNAc. This sulfotransferase is believed to be involved in the biosynthesis of the recently described Gal(3-SO4) capping groups present in the N-linked oligosaccharides of thyroglobulin (Spiro, R.G., and Bhoyroo, V. D. (1988) J. Biol. Chem. 263, 14351-14358). Assays with various native and modified glycopeptides indicated that the enzyme acted optimally on complex-type carbohydrate units in which beta-linked Gal has been uncovered by desulfation or brought into a terminal position by removal of sialyl and/or alpha-galactosyl residues. With fetuin asialoglycopeptides as acceptors (Km = 0.1 mM) the transfer of sulfate from PAPS (Km = 6.3 microM) had a pH optimum of approximately 7.0, required Mn2+ ions (10-50 mM) and was markedly stimulated by Triton X-100 (0.1%) and ATP (2 mM). The same enzyme apparently sulfated free N-acetyllactosamine (LacNAc; Km = 0.69 mM) and its ethyl glycoside, indicating that it had no absolute requirement for a peptide recognition site. Studies with a number of disaccharides related to LacNAc provided information relating to the specifying role of the beta 1----4 galactosyl linkage and the configuration at C-2 of the sugar to which it is attached. Hydrazine-nitrous acid-NaBH4 treatment of the 35S-labeled products from sulfotransferase action on asialoglycopeptides as well as on the ethyl glycoside of LacNAc yielded the same disaccharide, Gal(3-SO4) beta 1----4 anhydromannitol, as is obtained from a similar treatment of thyroglobulin. Subcellular distribution studies indicated that the PAPS:galactose 3-O-sulfotransferase is located in the Golgi compartment which is consistent with the late occurrence of the requisite beta-galactosylation step. It is proposed that in certain tissues the ultimate nature of the capping groups attached to glycoproteins containing terminal Gal beta 1----4GlcNAc sequences could be the result of a competition between this 3-O-sulfotransferase and sialyl- and/or alpha-galactosyltransferases.