Lecture 8 Protein Chemistry

Chemical Strategies Used in Studying ProteinProtein Interactions and DNA-Protein Interactions Examples

Targeting DNA-Protein Interactions

DNA-protein interaction are involved in processes related to gene regulation, DNA repair and DNA transcription. An example of studying the mechanism of DNA repair: Human 8-oxoguanine DNA glycosylase I (hOGG1)
8-oxoguanine (oxoG)

Cleaved by hOGG1

oxoG is misread to T (thymine) during replication of DNA. The resulting oxoG T mispair in another round of replication is mutated to T A. Only 50,000 molecules of hOGG1 protect the entire 6x10^9 base-pair nuclear genomic. Banerjee et al. Nature, 2005, 434, 612

Structural Studies have revealed that oxoG nucleoside is completely extruded from the DNA duplex, and inserted deeply into a lesion recognition pocket.

Catalytic site G0 C

Lesion recognition pocket G0 C

G0 C

G0 C

Banerjee et al. Nature, 2005, 434, 612

How the lesion recognition pocket can distinguish G from oxoG?

Is this single interaction enough to distinguish the oxoG from G? One H-B is equivalent of 1.5-3 kcal/mol.

Banerjee et al. Nature, 2005, 434, 612

Solution of Verdines group (Harvard) in addressing the issue of how hOGG1 is able to discriminate between oxoG and G

Strategy: Comparing the structure of hOGG1 bound to DNA presenting an oxoG lesion site to that of the enzyme bound to DNA presenting the normal base G, and use them as a basis for free energy difference calculations. The problem: the hOGG1:DNA(G) failed to form. Solution: Forcing the DNA(G) and the protein to form a stable complex by implanting a disulfide crosslink into the interface between the protein and DNA

Banerjee et al. Nature, 2005, 434, 612

Disulfide-Crosslinking Strategy

Banerjee et al. Nature, 2005, 434, 612

Disulfide-Crosslinking Strategy

Banerjee et al. Nature, 2005, 434, 612

X-Ray crystal structures

oxoG complex

G complex

Banerjee et al. Nature, 2005, 434, 612

The free energy calculation indicated that there is indeed a stabilization effect coming from the H-B formed between Gly(C=O) and the N7H, worth of 3.5 kcal/mol

Banerjee et al. Nature, 2005, 434, 612

Studying packing of transmembrane segments:

Disulfide-crosslinking technology (disulfide trapping)

Human multiple drug resistance P-glycoprotein

Loo et al. JBC, 2004, 279, 7692

Targeting Protein-Protein Interactions

Protein-protein interaction are a central regulatory feature to nearly all biological processes in a living organism. They have been a target either for development of novel chemotherapeutic treatments or for understanding protein function. Peptides have been an important strategy to target proteinprotein interaction, through mimicking these interactions.

Mimicking protein-protein interface through peptidomimetics

Peptide-based therapeutics are limited to: 1) Low bioavailability; 2) Inefficiency in crossing cell membranes (due to their size); 3) Poor metabolic stability in vivo. Peptidomimetics contain nonnatural amino acids. They mimic the structural and functional properties of their native parental peptides. They often are more stable in vivo, and an increased ability to penetrate cell membranes.

Peptides and peptidomimetics have been used quite often to target vital biological processes in cancer cells, as protein-protein interactions play an important role in the biology of cancer. For example, Apoptosis (programmed cell death) is important in preventing the cells to become carcinogenic, escape from this mechanism correlates with tumor aggressiveness and resistance to anticancer drug treatments.

Apoptotic pathways: Intrinsic pathways and the Extrinsic pathways

Bax, (cytoplasmic) and Bak (within mitochondria) are inducers of apoptosis. They are multipledomains and contain a domain known as BH3, an all helical domain. Bid is a proapoptotic protein that contain only BH3 domain that interacts with Bax and Bak causing their oligomerization.

DISC: death inducing signaling complex Caspase 3: executioner enzyme of apoptosis

An effective mechanism of inducing apoptosis is to cause oligomerization of the proapoptotic proteins such as Bak and Bax.

Strategy: design a peptidomimetic, well-folded ligand, that mimics interaction of BH3 with Bax and Bak.
Verdines Group: Used a technique called hydrocarbon stapling to generated a staple that stabilized the -helical structure of BH3 peptide
Non-natural AA BH3 Stabilized -helix

The staple increased the secondary structural feature of the ligands

Walensky LD., et al. Science, 2004

Use of protein scaffolds to design small-well folded proteins Protein Grafting

Golemi-Kotra D. JACS, 2004

Native Chemical Ligation

Has been used in synthesizing proteins Design of novel tertiary structures Glycosylation of proteins

Native Chemical Ligation

Mechanism of Native Chemical Ligation

Synthesis of complex tertiary structures

Synthetic strategy

Dolphin, GT, JACS, 2005, 128, 7287.

Synthesis of glycoproteins Glycosylation enhances the stability of the peptides and proteins in vivo. It enhances the blood-brain barrier, reduce the susceptibility to proteases. It is very difficult to incorporate glycosylation into peptides and protein produced in bacteria.

Tolbert TJ, et al. Biorg. Med. Chem. 2005, 13, 909.