Protein-DNA Interactions Worksheet
Use Kinemage file: protDNAinter.kin
These questions are designed to approximate writing the first draft of a manuscript describing the 3dimensional structure of Protein X, based on the structural information shown in the three kinemages in file
protDNAinterWrksht.kin.
Background/ Introduction
Protein X controls the operon for the synthesis of ligand Y in Bacillus loco. In the
absence of ligand Y, the protein is inactive, the operon is switched on and Y is produced.
As the concentration of Y increases, it binds to protein X and converts it to an active form
so that it can bind to the operator region and switch off the gene.
The structure of protein X was previously determined by X-ray crystallography in two
different crystal forms, and you have now “Structure 1” (Kinemage 1), has both the DNA
operon and ligand Y bound to Protein X. The operon is represented by a canonical 18
base-pair two fold symmetrical duplex DNA. Protein X functions as a dimer. Structure 2
is unliganded Protein X (“apo”); without the bound ligand Protein X is inactive. Structure
3 has ligand Y bound to Protein X in the absence of DNA. The superposition of all three
Protein X structures is shown in Kinemage 2. Kinemage 3 shows one subunit of Protein
X, with sidechains grouped and color-coded.
Methods X-ray crystallography
1) List key information about the refined crystal structure of the Protein X / DNA
complex that should be included in the paper (possibly in a tabular form) to enable the
reviewer and reader to assess the quality and validity of the structure. Define your terms.
Justify why the information is necessary. Take into account that the reviewers and readers
of the target journal are very skeptical people.
Information
1. Protein and
DNA Sequence
2. Resolution
3. R-Factor
Definition
Amino acid and nucleotide sequences of
Protein X and DNA, respectively.
Justification
Allows for sequence validation and
helps in understanding interactions.
Indicates the quality of the
The crystallographic resolution, measured in experimental data. Lower values
Ångströms (Å), indicating the level of detail are preferable, suggesting higher
in the structure.
accuracy.
The R-factor or R-free factor, which
A lower R-factor indicates a better
measures the agreement between observed fit between the model and
and calculated structure factors.
experimental data.
Information
Definition
Justification
Helps in visualizing the model's fit
Maps showing the electron density
to the experimental data. Should
4. Electron
distribution in the crystal, including 2Fo-Fc show clear density for atoms and
Density Maps
and Fo-Fc maps.
ligands.
Validation scores, such as Ramachandran
plot statistics (e.g., percentage of residues in Assesses the quality of the model
5. Model Quality favored/allowed regions), clash score, and
and its compliance with
Assessment
MolProbity scores.
stereochemical norms.
Demonstrates the presence and
Electron density maps for ligands or cocorrect placement of ligands in the
6. Ligand Density factors bound to Protein X or DNA.
complex.
7. Bond Lengths Bond lengths and angles for protein and
Ensures that atomic coordinates are
and Angles
DNA, including hydrogen bonds.
chemically plausible.
8. Solvent and
Information about water molecules, ions,
Provides insight into solvent
Ions
and their interactions in the crystal structure. networks and metal-binding sites.
9. B-Factors
(Temperature
B-factors for atoms, indicating their thermal High B-factors may indicate
Factors)
motion.
flexible or disordered regions.
Additional validation measures to
10. Structural
Validation reports from software tools like support the accuracy of the
Validation
MolProbity, PROCHECK, or others.
structure.
The PDB (Protein Data Bank) accession
Allows readers to access the
11. Deposition
code, deposition date, and any relevant
structure and associated data
Information
supplementary data.
online.
Justification:
1. Sequence information is crucial for verifying the identity of the molecules and
understanding potential interaction sites.
2. Resolution is a fundamental parameter reflecting the quality of the experimental
data and model refinement.
3. R-Factors are critical for assessing the goodness of fit between the model and
experimental data.
4. Electron density maps show the quality of the fit between the model and
experimental electron density.
5. Model quality assessments indicate the reliability of the atomic model.
6. Ligand density confirms the presence and positioning of ligands within the
complex.
7. Bond lengths and angles ensure that the atomic coordinates are physically realistic.
8. Solvent and ion information is vital for understanding the chemical environment
within the crystal.
9. B-factors provide insights into the flexibility and mobility of atoms.
10. Structural validation from independent software tools reinforces the validity of the
structure.
11. Deposition information allows readers and reviewers to access the complete data
for independent analysis and validation.
By providing these details, you enable reviewers and readers to thoroughly assess the
quality, accuracy, and validity of the Protein X / DNA complex structure, addressing the
skepticism of the target journal's audience.
2) In addition to the above list, suggest two figures to support the validity of the structure
and justify why each should be included.
Description of the protein structure
3) Using the 3-D information in Kinemage 1, describe the structure of Protein X, including the
following aspects:
•
Number of chains, number of amino acids (are any disordered?);
•
type, number, & location of secondary-structure elements. What is ligand
Y? How is ligand Y bound? (That is, describe the major protein-ligand interactions,
both polar and hydrophobic.
You may want to draw the H-bonds into the kinemage, remembering that the
N...O distance should be <3.5A.)
Protein-DNA binding interactions
4) Base your answers for this part on the 3-D information for Structure 1, in Kinemage 1.
(Turn side chains on or off as needed.)
What common structural motif is present in Protein X?
What residues (numbers) form the motif?
What residues (numbers) form the DNA recognition helix?
5) What structural features contribute to sequence-specific DNA binding of Protein X? Illustrate each point
with specific examples (using residue numbers, if appropriate) from Kinemage 1. Consider the role of water.
6) List five protein-DNA interactions observed in the Protein X complex with DNA
(Kinemage 1) that are independent of the specific sequence of the operon.
Conformational changes
7) Use the dynamic 3-D information in Kinemage 2 to describe Protein X’s conformational changes for apo
vs liganded vs DNA-bound forms, including the following aspects:
•
Describe the relative movements; how large is the motion? (measure some
differences); is there a definite hinge point?
•
how much of the change results from ligand binding and how much from DNA
binding?
•
can you propose a reason why the ligand might cause this type of movement?
•
could the apo form bind to DNA? why, or why not?
•
how much does the DNA conformation change on binding Protein X? (assuming
it started as B form DNA)
Regulation of DNA binding; future experiments
8) Building on your answer to question 7, hypothesize how ligand Y regulates the
binding of Protein X to DNA.