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1. Design mutations in the gene encoding AcHE that will disrupt its binding to the
toxin. This way we will define the binding site. We need to target at least 3 residues
to achieve site-directed mutagenesis

'De-coding' Protein Data Bank Files

1ACJ
1. Method used: X-ray Diffraction EXPDTA
1. ATOM example
ATOM 1 N SER A 4 -12.503 89.084 35.130 1.00 66.28 N
1. B - Factor: this value represents the mobility of an atom. It positively correlates with
electronegativity of an atom.
Example: REMARK 3 GROUP 1 B-FACTOR (A** 2) : NULL ; NULL
1. Missing residues example (expected to be found mostly within the loops and the N-
and C- termini)
REMARK 465 MISSING RESIDUES
REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN
REMARK 465 IDENTIFIER; SSSEQ=SEQUENCE NUMBER; I=INSERTION CODE.)
REMARK 465
REMARK 465 M RES C SSSEQI
REMARK 465 ASP A 1
REMARK 465 ASP A 2
REMARK 465 HIS A 3
REMARK 465 HIS A 486
REMARK 465 SER A 487
REMARK 465 GLN A 488
REMARK 465 GLU A 489
REMARK 465 GLU A 536
REMARK 465 THR A 537

PyMol Model of 1ACJ (Secondary Protein Structure)

Basic PyMol Commands

1. PyMOL> hide all
1. PyMOL> show cartoon
2. PyMOL> show cartoon, 1ACJ
3. PyMOL> align 1B41, 1ACJ
4. PyMol> orient 1ACJ
Colour by B - Factor Values

The core of the protein has low B - Factor values with the highest B - factor values found in
the loops of the protein. That makes sense as they are found in the more mobile parts of the
protein that have the highest freedom of movement.
Aligned Structures
The PyMOL alignment algorithm creates a sequence alignment of the two proteins, and
then minimizes the root mean square distance (RMSD) between corresponding residues.
Active Site
The active site of the human acetylcholinesterase enzyme consists of a catalytic triad made
up of amino acids Ser203 (serine at position 203), Glu334 (glutamic acid at position 334),
and His447 (histidine at position 447)
PyMOL> select humantriad, 1B41 and resi 203+334+447
PyMOL> show sticks, humantriad
 Residues of the active site of human AcHE

Stick model of the active site

 Active site for Ray AcHE
PyMOL> select raytriad, 1ACJ and resi 200+440+327
PyMOL> show sticks, raytriad
Inhibitors
PyMOL> select tacrine, 1ACJ and resn THA
PyMOL> show sticks, tacrine
 The inhibitor lies close to the
active site. The snake produces a toxin, which is an inhibitor of acetylcholinesterase. This
toxin is a protein called fasciculin-II. Structure 1B41 is acetylcholinesterase in complex
with fasciculin-II
Calculate the distance of the alpha-carbon (CA) atom of Pro31 to the CA atom of the active
site residue His447

PyMOL> select toxinPro31, 1B41 and chain B and resi 31

PyMOL> show sticks, toxinPro31
To measure the distance between the CA atom of Pro31 and the CA atom of His447, we
label the atoms of Pro31 and His447
PyMOL> label 1B41 and i. 31, name
PyMOL> label 1B41 and i. 447, name
 Wizard ->
measurement -> select CA atoms of Pro31 and His447
The distance between Pro31 and His447 (active site) is 16.4 Å
Concluding remarks: Fasciculin-II blocks the entrance to the active site, preventing the
normal binding of acetylcholine.

Identifying Candidate Residues for Mutagenesis

Alter target resideues in order to prevent venom binding (mongoose and hedgehog are
resistant to snakes venom. The DNA sequence encoding aromatic residues in AcHE have
mutated to nonaromatic residues, and the DNA sequences encoding prolines have mutated
to leucines and histidines)
PyMOL> select tryptophans, resname TRP and chain A
PyMOL> show sticks, tryptophans
PyMOL> color yellow, tryptophans
Coloured Residues for TRP, TYR, PHE, PRO
Selecting and Mutating Residues
Wizard -> Mutagenesis
In species immune to venom the DNA sequence encoding aromatic residues in
acetylcholinesterase have mutated to nonaromatic residues, and the DNA sequence
encoding prolines have mutated to leucines and histidines. We can select a tyrosine,
tryptophan, or phenylalanine close to the toxin binding site to be mutated to a non-
aromatic residue like alanine or glycine. You could also select a proline close to the toxin
binding site to be mutated to a leucine or histidine.
Use Blast , a Basic Local Alignment Search Tool, to find more sequences for mutation.

Crystal Structures of MgrA

DNA-binding protein produced by Staphylococcus aureus (Transcriptional regulator)

Distance between the N and

the C termini
The quaternary structure of the protein is dimeric (this info can be found in the pdb file)
PyMOL> set_name sele, interface
PyMOL> show ribbon, (all &! interface)
PyMOL> hide cartoon, (all &! interface)