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Effects of mutation on key amino acid residues in the N-terminal of
p53 and its significance in binding with MDM2
Aditya Harisankar, Mohanraj Ramachandran, Vivek Anand Manivel and Roshan Vaid
Abstract
The tumor suppressor protein, p53 is absolutely necessary for the activation of various
downstream proteins involved in cell cycle regulation, metabolism and apoptosis. Binding of
MDM2 (Murine double minute 2) to the N-terminus of wild type p53 tags it for
ubiquitination and subsequent degradation. Over expression of MDM2 leads to excessive
degradation of p53 and hence the cell loses its ability to stop uncontrolled cell proliferation
leading to tumour development. Modifications in the N-terminus of p53 can prevent its
degradation and help initiate a cascade of signals, restoring its normal function. In this paper,
we have studied the importance of the key amino acids viz. Phe 19, Trp 23 and Leu 26, in the
N-terminal region responsible for binding interaction between p53 with the hydrophobic
pocket of MDM2. Using Swiss PDB Viewer we made in silico mutations in this region and
studied how these mutations affects binding. Using docking softwares like Hex dock we were
able to visualize the binding interactions before and after mutations.
1. Introduction:
The cell being the structural and functional unit of any organism is constantly
subjected to stress. This poses threat to its metabolic machinery and also the genetic
information viz. DNA. The molecule responsible to detect such DNA damage is the p53
tumor suppressor protein. Being on top of the response system, it functions as a transcription
factor and leads to expression of various genes that function in different cell cycle regulation
pathways and apoptosis. p53 is found in the cytoplasm in the free state and has a short half
life (ca. 20mins). Once DNA damage is detected, p53 is immediately transported in to the
nucleus and it activates the response system. MDM2 keeps p53 levels at bay in a normal cell.
Ironically, the p53 helps transcribe Mdm2 gene. The product, MDM2, an E3 Ubiquitin ligase,
binds and inhibits the activity of p53. It attaches a single ubiquitin tag onto p53 and marks it
for degradation by proteosomes. This type of downregulation is universal and happens in all
normal cells. In certain types of cancer, MDM2 is over expressed and leads to complete
suppression of p53 activity. In such a case the cell loses its ability to initiate apoptosis and
fail to show resistance against cancerous growth. Normally, p53 gets phosphorylated by the
enzymes Ataxia-telangiectasia mutated (ATM) and Chk1/Chk2 at different residues on the Nand C- terminal which gives it the ability to evade ubiquitin tagging by MDM2. Several
studies have aimed at disrupting the binding interactions between p53 and MDM2 as a
therapeutic approach for cancer.
1 2. Structure of p53:
Wild type p53 protein in its active form is a tetramer, each subunit having 393 amino
acid residues. Four structurally and functionally conserved domains viz. N-terminal domain,
proline rich region, central core DNA binding domain, and C- terminal domain have been
identified in each of the subunits. The N-terminal region (amino acid residues 1-42) is
responsible for interactions with transcription factors, binding to MDM2 and transactivation
activity [1]. Amino acid residues 17 – 29 in this region form an alpha-helix which binds to
the hydrophobic cleft in MDM2 [2]. The proline rich region (amino acid residues 61-94)
stabilizes p53 from degradation by MDM2 mediated pathway [3]. The central DNA binding
core domain (amino acid residues 102-292), most conserved among all the domains is
responsible for sequence specific binding to DNA and it is characterized by two copies of 10
bp consensus sequence 5’-PuPuPuC(A/T)-(T/A)GPyPyPy-3’[4]. The fold of this domain is
characterized by two anti parallel beta sheets forming a beta sandwich. One end of the beta
sandwich has three conserved secondary structure elements viz. a beta hairpin, an alpha helix
embedded with two Zn chelating atoms and another alpha helix that extends to the C-terminal
end of the protein. The other end of this beta sandwich has loops that connect beta strands [5].
Cancers developed due to non-functional p53 protein is mostly due to mutations in this
region, impairing its ability to bind DNA. The C- terminal region contains the tetramerization
domain (amino acid residues 324-355) and the strongly basic regulatory domain (amino acid
residues 363-393) [6]. It functions as a negative regulator by interacting with and blocking
the function of core DNA binding domain. This interaction is disrupted by posttranslational
modifications such as phosphorylation and acetylation at the C-terminal thus activating the
core DNA binding domain. Being flexible, the structure of the basic region is defined by the
‘induced fit model’, i.e. its structure changes depending on the ligand it interacts with. The
tetramerization domain is composed of four monomer subunits forming a dihedral symmetry,
each monomer composed of a beta sheet and an alpha helix forms a V like structure with the
help of tight turns stabilized by a conserved glycine residue (G334). Association of the four
monomers is governed by hydrophobic interactions between them [7].
Fig1: showing four different domains of p53.
2 3. p53 - MDM2 interaction
MDM2 mediated regulation of p53 is accomplished by the specific interaction of
these proteins. The binding pocket of MDM2 is composed of two beta sheets interspersed by
four helix bundles forming a β-α-β sandwich. Three dimensional structural studies of the p53MDM2 complex suggests that only three hydrophobic amino acid residues of p53 docks into
the hydrophobic cleft of Mdm2. These residues include Phe 19, Trp 23 and Leu 26 and they
hydrophobically interact with Leu 54, Met 62, Tyr 67 and Val 93 of MDM2 (shown in fig. 2).
Apart from these hydrophobic interactions, a hydrogen bond between Trp 23 and Leu 54 is
also involved in stabilization of this protein complex [8]. Tyr 100 is an important residue in
MDM2 and is found to impart structural stability to the ligand giving rise to stable
interactions. The orientation of Tyr100 controls the conformation of the hydrophobic cleft as
it moves in and out of the binding site. This is controlled by the N terminal part of MDM2
and to a lesser extent, conformation of p53 [9]. It has also been studied that apart from these
interactions, there is a stretch of amino acid residues which are necessary to signal MDM2
and coordinates p53 degradation. This has been identified in the region of 92-112. When this
region was switched with that of p73, a structurally similar protein, MDM2 bound and
degraded p73.
Fig 2: Showing key amino acid residues of p53 (green) interacting with MDM2 (red).
3 4. Experimental:
The structure of human p53 N-terminal peptide (wild type) complexed with MDM2
(1YCR) was obtained from RCSB-PDB. Using Swiss-PDB viewer, the molecules were
separated and saved as separate files. Two different groups of multi-point mutations were
done on the key hydrophobic amino acid residues in the N-terminal of p53 that interact with
MDM2. The wild type p53 N-terminal peptide was used as a reference. Table 1 shows the
mutations carried out in our study.
Amino Acid Residue
Wild Type p53
Mutant 1
Mutant 2
19
Phe (F)
Glu (E)
Asp (D)
23
Thr (T)
Tyr (Y)
Ser (S)
26
Leu (L)
Gly (G)
Gly (G)
Table 1: Showing the different mutation groups used in this study
Once the mutations were performed in silico, the sequence was fed into JPRED, a
secondary structure prediction server, to verify the helix forming ability of the mutated
peptide. The optimal rotamer conformations were applied to all the mutated amino acids.
Docking experiments were performed using the software Hex 4.2 [10]. The results obtained
in the form of E-values were used to validate the binding interactions.
5. Results and Discussions:
5.1 Mutations:
The multi-point mutations in the key amino acid residues were performed
successfully using Swiss-PDB viewer. The point mutations were chosen in such a
way that the helical region at the N-terminus of p53 was not disrupted. The
hydrophobic residues in the N-terminal of p53 peptide were substituted with
hydrophilic residues. This was done to check the significance of these hydrophobic
residues with respect to the binding ability of p53 to MDM2. It has been shown by
Kussie et. al., that the hydrophobic amino acids F19, W23 and L26 of p53 were
necessary to establish binding interactions with the hydrophobic cleft in MDM2.
Apart from this, hydrogen bonds were also visualized between the backbone atoms.
These could stabilize the complex along with the hydrophobic interactions. Other
amino acids such as Leu14 and Leu 22 were also reported to be important for
interactions but we choose to mutate only the 3 most important residues due to
reasons underlying stability and retention of helical structure.
4 5.2 Secondary Structure predictions:
The helix region is highly conserved as it is known to interact with many
transcription factors. JPRED results for the wild type and two mutated sequences are
shown in figure 3. The Jnet results show that both the mutated peptides form helices
in that region and it is backed by high Jnet Rel scores signifying high confidence level
of the predicted structures. Retention of the helical structure at the N-terminal of p53
is important to maintain binding interactions with other molecules such as p300 and
CBP which function as transcription factors.
Figure 3: Showing the JPRED results. a) Wild type N-terminal p53 sequence, b)
Mutant 1 N-terminal p53 sequence and c) Mutant 2 N-terminal p53 sequence.
5.3 Docking:
The energy minimized p53 N-terminal peptides were docked with the
hydrophobic cleft of MDM2 using Hex. For the wild type the E-total obtained after
docking was -511.43KJ/Mol while the E-total for the mutant 1 and mutant 2 were 413.80KJ/Mol and -393.70KJ/Mol respectively. It is evident from the E-values that
the wild type p53 is more stable and binds well with MDM2 compared with the two
multi-point mutated N-terminal p53 peptides. These results signify that the three
hydrophobic amino acid residues F19, W23 and L26 are very important for
interactions with MDM2.
5 6. Conclusions:
In this paper, we have studied the significance of the key amino acids in the Nterminus of p53 tumor suppressor protein. These hydrophobic residues are necessary for
interacting with the binding site in MDM2. The interaction is via conserved helix at Nterminal of p53 and beta sheets of MDM2. Replacing the amino acids F19, W23 and L26
with other hydrophilic amino acids do not disrupt the helix forming ability of this region, but
p53 loses its ability to hydrophobically interact with the cleft of MDM2 and this destabilizes
the binding interactions. So targeting the hydrophobic interactions between p53 and MDM2
makes it a potential cancer therapeutic.
Acknowledgements:
We sincerely thank all the teaching assistants who taught us Swiss-PDB viewer and
other bioinformatics tools. Special thanks to our mentor Henrik Keränen his encouraging
words and thoughtful criticism. We also thank the course coordinators Lars Liljas and Anna
Jansson for giving us an opportunity to do this project.
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