Protein Modeling/Human Genome

The 2015 topic for Protein Modeling deals with editing the human genome. The pre-build model is of the catalytic domain of the endonuclease protein fokI.

See the CBM webpage for more details.

Endonuclease FokI
The 2015 Pre-Build Model should represent amino acids 421-560 of chain A of the restriction endonuclease protein fokI based on the PDB file 2FOK.pdb.

Restriction endonucleases are proteins that can cut DNA at a specific point in a specific sequence, allowing genome editing. The specific protein FokI, the focus of this year's event, occurs naturally in bacteria as a defense mechanism against invading viruses. This protein, like other restriction enzymes, has two domains (functional parts): the cleavage domain (nuclease) and the DNA-binding domain, composed of zinc fingers. In designing genome-editing restriction enzymes, the nuclease of the FokI is typically removed from its natural DNA binding domains and attached to new binding domains, to create a new specialized restriction enzyme. The pre-build model focuses solely on the nuclease. This nuclease functions solely as a dimer, meaning it requires two copies (one attached to each strand of DNA) in order to successfully cleave the DNA.

Zinc-Finger Proteins
The 2015 regional and invitational onsite models will represent zinc-finger proteins. The invitational onsite model will be based on the PDB file 1psv.pdb and the regionals onsite model will be based on the file 1mey.pdb.

Zinc finger proteins are composed of a 2-stranded beta sheet and a single alpha helix. They are stabilized by a zinc ion coordinated by 4 cysteine and histidine amino acids. The zinc finger domain binds to the main groove of the DNA, while individual side chains interact with the DNA bases to "read" them. This allows chains of zinc finger domains to bind to very specific genomic sequences.

TALE Nuclease Proteins
The 2015 State onsite model will represent Transcription Activator Like Effector Nuclease Proteins (TALE) and will be based on the PDB file 3v6t.pdb.

CRISPR
The 2015 National onsite model will represent Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) proteins and will be based on the PDB file 4un3.pdb.

HIV Treatment
Although there are various examples of genetic editing, this year's event focuses on potential treatment for HIV infection through the use of therapeutic zinc-finger nucleases. An overview is presented here, but it is a good idea to visit the CBM website for more in-depth information.

HIV Receptor Proteins
HIV is a virus that affects T helper cells, a type of immune cell. In order to infect these cells, the envelope proteins of the virus bond to the host cell protein CD4. This alters the morphology of both proteins, allowing the complex to bind to CCR5, the host cell chemokine receptor. This allows the virus to penetrate the cell membrane and enter.

Genetic Treatment
Various treatments have focused on blocking the action of the virus once it is in the cell. However, patients with naturally suppressed CCR5 proteins due to a genetic 32 base pair deletion, or who have reduced levels due to heterozygous alleles, have T-cells that are resistant to the virus.

Therefore, introducing this 32 bp deletion into the patient's genetic material can create resistant T-cells. This can, in theory, be done with zinc-finger nucleases. This mutation works by preventing CCR5 proteins from being brought to the cell surface, and has no other known ill effects.

Zinc-Finger Nucleases
Zinc finger nucleases are sequence specific DNA binding proteins. They are composed of several zinc fingers, which each bind three bases, as well as a cleavage domain. As the nuclease can only function as a dimer, two nucleases are created, one with each half of the nuclease domain. One binds to each strand of DNA, and together they form a functional homodimer able to cut both strands of DNA. Then, repair enzymes in the cell attempt to fix the breach, introducing mutations as they do so.