Altering the Tat-derived peptide bioactivity landscape by changing the arginine side chain length

Mutations of proteins with dual activities that lead to enhancement of one activity are frequently accompanied by attenuation of the other activity. However, this mutational negative trade-off phenomenon typically only involves the canonical 20 amino acids. To test the effect of non-canonical amino acids on the negative trade-off phenomenon, two bioactivities of HIV-1 Tat-derived peptides were monitored upon changing the Arg side chain length. In contrast to the expected mutational negative trade-off, shortening Arg by one methylene resulted in both higher TAR RNA binding specificity and higher cellular uptake. These results suggest that introducing previously unexploited building blocks, even if the difference is only one methylene, can alter the peptide bioactivity landscape leading to the enhancement of multiple bioactivities.     

Multivalency: Key Feature in Overcoming Drug Resistance with a Cleavable Cell-Penetrating Peptide-Doxorubicin Conjugate

Multivalency is often used in biological systems, to increase affinity and specificity through avidity. This inspired us to prepare a synthetic bioconjugate that mimics natural multivalent systems. It is composed of doxorubicin and two octaarginine cell-penetrating peptides, to strengthen the electrostatic interactions between the negatively charged glycosaminoglycans of the plasma membrane and the guanidinium groups of the arginine residues. The multivalent conjugate has improved cellular uptake and cytotoxicity, compared to a peptide-drug conjugate with only one polyarginine and as a result it can overcome drug resistance in Kelly-ADR cells. The synthetic approach and the multivalent structure reported here can be used further as model systems, to gain insight into the biological interaction of cell-penetrating peptides with artificial membranes or for the preparation of more complex multimers.     

Replacement of All Arginine Residues with Canavanine in MazF-bs mRNA Interferase Changes Its Specificity

Replacement of a specific amino acid residue in a protein with nonnatural analogues is highly challenging because of their cellular toxicity. We demonstrate for the first time the replacement of all arginine (Arg) residues in a protein with canavanine (Can), a toxic Arg analogue. All Arg residues in the 5-base specific (UACAU) mRNA interferase from Bacillus subtilis (MazF-bs(arg)) were replaced with Can by using the single-protein production system in Escherichia coli. The resulting MazF-bs(can) gained a 6-base recognition sequence, UACAUA, for RNA cleavage instead of the 5-base sequence, UACAU, for MazF-bs(arg). Mass spectrometry analysis confirmed that all Arg residues were replaced with Can. The present system offers a novel approach to create new functional proteins by replacing a specific amino acid in a protein with its analogues.     

Structure and function of RGD peptides derived from disintegrin proteins

The Arg-Gly-Asp (RGD) sequence serves as the primary recognition site in extracellular matrix proteins, and peptides containing this sequence can mimic the biological activities of matrix proteins. We have initiated structure-function studies of two RGD containing peptides, RGD-5(AGGDD) and cyclic RGD-6(CARGDDC). Assays have shown that cyclic RGD-peptides inhibit platelet aggregation more efficiently than linear ones. NMR data revealed that RGD-5 and RGD-6 have entirely different conformation. RGD-5 has a linear extended structure and RGD-6 has a stable loop conformation. In RGD-5 the guanidinium group of Arg2 and the carboxyl group of Asp4 lie in parallel, whereas the side-chains of Arg3 and Asp5 of RGD-6 are located in different planes, supporting the idea that the stability of the cyclic form derives from the packing of the side chain of the Arg and Asp residues. The structural features of these peptides could provide a basis for designing new drugs against diseases related to platelet aggregation and as cancer antagonists.     

Identification of secretin,vasoactive intestinal peptide and glucagon binding sites: from chimaeric receptors to point mutations

We have identified two basic residues that are important for the recognition of secretin and vasoactive intestinal peptide (VIP) by their respective receptors. These two peptides containing an Asp residue at position 3 interacted with an arginine residue in transmembrane helix 2 (TM2) of the receptor, and the lysine residue in extracellular loop 1 (ECL1) stabilized the active receptor conformation induced by the ligand. The glucagon receptor possesses a Lys instead of an Arg in TM2, and an Ile instead of Lys in ECL1; it markedly prefers a Gln side chain in position 3 of the ligand. Our results suggested that, in the wild-type receptor, the Ile side chain prevented access to the TM2 Lys side chain, but oriented the glucagon Gln(3) side chain to its proper binding site. In the double mutant, the ECL1 Lys allowed an interaction between negatively charged residues in position 3 of glucagon and the TM2 Arg, resulting in efficient receptor activation by [Asp(3)]glucagon as well as by glucagon.     

Specific arginine mediated RNA recognition

In many biological systems substantial roles are played by interactions between amino acids and RNA. Among amino acids L-arginine seems to be particularly relevant, because the guanidinium group of arginine side chain can potentially form five hydrogen bonds with appropriately positioned acceptor groups of RNA. Extensive studies reveal that specific arginine recognition is achieved by many different RNAs over a broad range of binding affinities. Arginine is frequently found among amino acids in the nucleic acid-binding motifs in various proteins. For example, specific binding of the HIV-1 Tat protein to its RNA site (TAR) is mediated by a single arginine residue. Free arginine can be also bound by the guanosine site in the group I Tetrahymena ribosomal RNA intron catalytic centre, as well as by numerous RNA motifs, called arginine aptamers, which have been selected in vitro.     

Identification of electrostatic interactions that determine the phosphorylation site specificity of the cAMP-dependent protein kinase

"Charged-to alanine" scanning mutagenesis of the catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase (C1) identified three glutamate residues, E171, E214, and E274, that are involved in the recognition of a peptide substrate, kemptide (Leu1Arg2Arg3Ala4Ser5Leu6Gly7). These glutamate residues are conserved or conservatively substituted with asparate in the serine/threonine protein kinases that have a requirement for basic residues on the N-terminal side of their phosphorylation sites. Alanine replacement mutants in C1 were subjected to kinetic analysis using alanine-substituted peptides as substrates. The additivity or nonadditivity of the effects of the alanine substitutions on the catalytic efficiency (kcat/Km) was analyzed. This allowed the identification of electrostatic interactions between the three glutamate residues in the enzyme and the two arginine residues present in the peptide substrate. The data suggest that E171 interacts with Arg2 in the substrate and that E214 and E274 both interact with Arg3. This may be a general method for identifying simple intermolecular interactions involving proteins when there is no three-dimensional structure available of the complex of interacting species. The identification of these interactions provides the potential for rational protein engineering of enzymes with alternative specificities.     

Conformational features of bradykinin. A circular dichroism study of some peptide fragments and structural analogues of bradykinin.

The vasoactive hormone bradykinin, its N-and C-terminal fragments and some structural analogues were studied by Circular Dichroism. Conformational features of the peptide can be detected by comparative analysis of the various CD spectra recorded as a function of aqueous pH, solvent and temperature. It is shown that the two biologically essential arginine residues (Arg1 and Arg9) are important for the specific folded bradykinin conformation. Differences between bradykinin, its fragments and analogues become clearly established in conformational terms, and are discussed in relation to the biological activity of these peptides.     

Isolation and characterization of the cyanogen bromide fragments of lobster arginine kinase (homarus vulgaris).

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10 percent) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to differnt gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position: therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).     

Suppression of receptor interacting protein 140 repressive activity by protein arginine methylation

Receptor interacting protein 140 (RIP140), a ligand-dependent corepressor for nuclear receptors, can be modified by arginine methylation. Three methylated arginine residues, at Arg-240, Arg-650, and Arg-948, were identified by mass spectrometric analysis. Site-directed mutagenesis studies demonstrated the functionality of these arginine residues. The biological activity of RIP140 was suppressed by protein arginine methyltransferase 1 (PRMT1) due to RIP140 methylation, which reduced the recruitment of histone deacetylases to RIP140 and facilitated its nuclear export by enhancing interaction with exportin 1. A constitutive negative (Arg/Ala) mutant of RIP140 was resistant to the effect of PRMT1, and a constitutive positive (Arg/Phe) mutation mimicked the effect of arginine methylation. The biological activities of the wild type and the mutant proteins were examined in RIP140-null MEF cells. This study uncovered a novel means to inactivate, or suppress, RIP140, and demonstrated protein arginine methylation as a critical type of modification for corepressor.     

Membrane adsorption and binding, cellular uptake and cytotoxicity of cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone: effects of hydrogen bonding and α-chirality in the β-peptoid residues

Cell-penetrating peptides (CPPs) provide a promising approach for enhancing intracellular delivery of therapeutic biomacromolecules by increasing transport through membrane barriers. Here, proteolytically stable cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone were studied to evaluate the effect of α-chirality in the β-peptoid residues and the presence of guanidinium groups in the α-amino acid residues on membrane interaction. The molecular properties of the peptidomimetics in solution (surface and intramolecular hydrogen bonding, aqueous diffusion rate and molecular size) were studied along with their adsorption to lipid bilayers, cellular uptake, and toxicity. The surface hydrogen bonding ability of the peptidomimetics reflected their adsorbed amounts onto lipid bilayers as well as with their cellular uptake, indicating the importance of hydrogen bonding for their membrane interaction and cellular uptake. Ellipsometry studies further demonstrated that the presence of chiral centers in the β-peptoid residues promotes a higher adsorption to anionic lipid bilayers, whereas circular dichroism results showed that α-chirality influences their overall mean residue ellipticity. The presence of guanidinium groups and α-chiral β-peptoid residues was also found to have a significant positive effect on uptake in living cells. Together, the findings provide an improved understanding on the behavior of cell-penetrating peptidomimetics in the presence of lipid bilayers and live cells.     

Structural basis of the highly efficient trapping of the HIV Tat protein by an RNA aptamer

An RNA aptamer containing two binding sites exhibits extremely high affinity to the HIV Tat protein. We have determined the structure of the aptamer complexed with two argininamide molecules. Two adjacent U:A:U base triples were formed, which widens the major groove to make space for the two argininamide molecules. The argininamide molecules bind to the G bases through hydrogen bonds. The binding is stabilized through stacking interactions. The structure of the aptamer complexed with a Tat-derived arginine-rich peptide was also characterized. It was suggested that the aptamer structure is similar for both complexes and that the aptamer interacts with two different arginine residues of the peptide simultaneously at the two binding sites, which could explain the high affinity to Tat.     

Identification of two novel arginine binding DNAs.

RNA tertiary structure is known to play critical roles in RNA-protein recognition and RNA function. To examine how DNA tertiary structure might relate to RNA structure, we performed in vitro selection experiments to identify single-stranded DNAs that specifically bind arginine, and compared the results with analogous experiments performed with RNA. In the case of RNA, a motif related to the arginine binding site in human immunodeficiency virus TAR RNA was commonly found, whereas in the case of DNA, two novel motifs and no TAR-like structures were found. One DNA motif, found in approximately 40% of the cloned sequences, forms of hairpin structure with a highly conserved 10 nucleotide loop, whereas the second motif is especially rich in G residues. Chemical interference and mutagenesis experiments identified nucleotides in both motifs that form specific arginine binding sites, and dimethylsulfate footprinting experiments identified single guanine residues in both that are protected from methylation in the presence of arginine, suggesting possible sites of arginine contact or conformational changes in the DNAs. Circular dichroism experiments indicated that both DNAs undergo conformational changes upon arginine binding and that the arginine guanidinium group alone is responsible for binding. A model for the G-rich motif is proposed in which mixed guanine and adenine quartets may form a novel DNA structure. Arginine binding DNAs and RNAs should provide useful model systems for studying nucleic acid tertiary structure.     

Crystal structure determinations of oxidized and reduced plastocyanin from the cyanobacterium Synechococcus sp. PCC 7942.

The crystal structures of oxidized and reduced plastocyanins from Synechococcus sp. PCC 7942 have been determined at 1.9 and 1.8 A resolution, respectively, at pH 5.0. The protein consists of only 91 amino acid residues, the smallest number known for a plastocyanin, and apparently lacks the mostly conserved acidic patch that is believed to be important for recognition with electron-transfer partners. The protein has two acidic residues, Glu42 and Glu85, around Tyr83, which is thought to be a possible conduit for electrons, but these are neutralized by Arg88 and Lys58. Residue Arg88 interacts with Tyr83 through a pi-pi interaction in which the guanidinium group of the former completely overlaps the aromatic ring of the tyrosine. Reduction of the protein at pH 5.0 causes a lengthening of one Cu-N(His) bond by 0.36 A, despite the small rms deviation of 0.08 A calculated for the backbone atoms. Moreover, significant conformational changes of Arg88 and Lys58, along with the movement of a water molecule adjacent to the OH group of Tyr83, were observed on reduction; the guanidinium group of Arg88 rotates by more than 11 degrees, and the water molecule moves by 0.42 A. The changes around the copper site and the alterations around Tyr83 may be linked to the reduction of the copper.