i. Molecular Recognition of Nucleotides
Many groups have employed molecular recognition processes to design peptides, proteins, or small molecules which bind biologically relevant targets. Peter Dervan’s lab has designed a host of DNA binding small molecules. One example of his work is the design of hairpin polyamides which bind duplex DNA. The compounds are functionalized with a fluorophore, and a fluorescence increase is observed upon binding. This type of study has led to the development of sensors that could be used to detect certain DNA sequences such as
DNA mismatches.9 Schepartz and colleagues have designed mini-proteins that recognize DNA with high affinity and specificity through protein grafting. This method has afforded α- helices that are highly selective for their DNA targets.10 Using a different approach,
Robinson and colleagues have designed cyclic β-hairpin peptides which bind tightly to BIV TAR RNA and inhibit the Tat-TAR interaction of the bovine immunodeficiency virus.11a The group also inhibited the REV/RRE interaction.11b This type of binding achieved is significant because it is applicable to ongoing studies to stop HIV and other viruses.
Molecules such as Robinson’s peptidomimetic disrupt the Tat protein-TAR RNA interaction which inhibits the generation of full-length transcripts and decreases HIV replication.11
ii. Disrupting Protein-ssDNA Interactions
Robinson’s peptidomimetic is one of many molecules that disrupt key protein- nucleotide interactions to block debilitating processes from occurring. Inhibiting the Simian virus 40 function can be likened to disruption of the well-known Tat-TAR interaction. Simian virus 40 large tumor antigen (SV40 LTag) is crucial for DNA unwinding during the replication stage of the virus. This protein is a hexamer which interacts with DNA through a positively charged hexameric channel. Six β-hairpins are connected to each of these
subunits, and two Lys residues along with a histidine and a phenylalanine are positioned on
9 Rucker, V. C.; Foister, S.; Melander, C.; Dervan, P. B.
J. Am. Chem. Soc.2003, 125, 1195-
1202.
10 Yang, L.; Schepartz, A.
Biochemistry2005, 44, 7469-7478.
11 (a) Athanassiou, Z.; Patora, K.; Dias, R. L. A.; Moehle, K.; Robinson, J. A.; Varani, G. Biochemistry 2007, 46, 741-751. (b) Moehle, K.; Athanassiou, Z.; Patora, K.; Davidson, A.;
the tip of the hairpin.12 Their positions indicate that those residues are important for DNA recognition. A close examination of the interactions involved in DNA binding could lead to a similar peptide designed to inhibit viral replication.12 Using the knowledge of ssDNA binding protein characteristics, one could imagine several applications to the design of ssDNA binding ligands such as telomerase inhibition, inhibition of replication, and DNA damage detection.
iii. Mimicking the OB-fold Domain
Recognizing the importance of ssDNA and other nucleotides as target molecules, this laboratory has delved into the area of molecular recognition of ATP, ssDNA, and RNA.13 This research began with the synthesis of peptides designed to target ATP. A β-hairpin peptide was designed with a binding pocket to recognize the adenine or other nucleotide base.5 The binding pocket contains two aromatic Trp residues intended to form favorable stacking interactions with the base accompanied by two flanking Lys residues to form electrostatic interactions with the phosphates of the ATP. The peptide was named for its binding cleft, WKWK (Figure 1.4). Fluorescence binding studies were conducted to determine the binding affinity of the peptide for ATP. The dissociation constant for the interaction is about 170 µM. This is a good starting point for studying the interactions involved in DNA binding.
12Shen, J.; Gai, D.; Patrick, A.; Greenleaf, W. B.; Chen, X. S.
Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 11248-11253.
13(a) Stewart, A. L.; Waters, M. L. ChemBioChem 2009, 10,539-544. (b) Butterfield, S. M.;
Cooper, W. J.; Waters, M. L. J. Am. Chem. Soc.2005, 127, 24-25. (c) Butterfield, S. M.;
N H H N N H H N N H H N O O O O O H N H2N NH2 NH2 O O H N N H H N N H H N H2N O O O O NH2 O O HN O H H HN O NH NH3 NH3 H3N 1 2 3 4 5 6 7 8 9 10 11 12
Figure 1.4. WKWK peptide designed for molecular recognition of ATP and ssDNA.
A structural model of the interaction between WKWK and ATP was generated based on NMR structural studies. The model indicates that the peptide binds ATP as intended, with the tryptophans forming stacking interactions with the adenine and the lysines making
electrostatic interactions with the phosphate groups. This peptide mimics the recognition mode of OB-fold domains, and is a minimalist model system for understanding the driving forces for nucleotide binding by peptides and proteins.
Figure 1.5. Computational model of peptide WKWK interacting with ATP. The model is
based on NMR structural data with WKWK shown in yellow and Trp residues drawn. The adenine base is shown in green with the triphosphate group in red and white.