FHIT belongs to Histidine Triad (HIT) nucleotide-binding protein superfamily and is considered a tumor suppressor ⁽¹⁾. Genomic alterations and aberrant expression of FHIT have been correlated with many types of human cancers, including those of the lung ^(2–4), breast ^{(5, 6)}, cervix ⁽⁷⁾, colon ⁽⁸⁾, pancreas ⁽⁹⁾, prostate ⁽¹⁰⁾, stomach ^{(11, 12)}, head and neck ⁽¹³⁾.

Studies have shown the interaction of FHIT with MDM2 and the block of the interaction of MDM2 with p53, result in increased stability of p53 ⁽¹⁴⁾. Structurally, FHIT forms a dimer in solution (PDB code: 1FIT) and general structure of its protomer can be described as a common α + β type ⁽¹⁵⁾. An ordinary hydrophobic core is formed within the background of the dimer ⁽¹⁶⁾.

As prior studies demonstrate, MDM2 protein interacts with p53 directly ^{(17, 18)} and MDM2 can interact with FHIT (by immunoprecipitation) ⁽¹⁴⁾. Moreover, other studies confirm the interaction of p53 and FHIT ^{(14, 19)}. Thus, it is logical to consider that FHIT and p53 have binding sites on MDM2 and perhaps these proteins could influence each other in binding to MDM2.

As functional interacting domains of FHIT with MDM2 and/or p53 are not completely defined, therefore, by exploring the recognition site of interaction of FHIT-MDM2 with regard to p53 binding, one can evaluate the interaction and/or competition amid these proteins for therapeutical approaches. Furthermore, the research for functional domain of FHIT may reveal the protein domain responsible for tumor suppression. In addition, these studies will shed lights on the molecular mechanism of FHIT-MDM2-p53 complex.

In this study, we assessed FHIT constructs interaction with MDM2 and p53 optimized models in silico. Results can be useful for designing new inhibitors of this protein complex interaction which would result in tumor repression.

Three dimensional structures of proteins are essential for computational interaction research. As only parts of FHIT, MDM2 and p53 had been determined as three dimensional structures in protein databank, we performed modeling for their structures.

Docking is considered as an in silico method for investigating the best interaction between two molecules which can be used in the rational drug design ⁽²²⁾. HEX program actually accelerates the process compared with the classical FFT docking algorithms ⁽²³⁾. Earlier studies have proved that HEX method has no limitation for protein size ⁽²⁴⁾.

We tested docking of MDM2, p53 optimized parts as the receptor to compare the interaction tendencies of a special motif or a group of them within FHIT. The results of our previous docking study indicate that interaction of full FHIT with p53 (E-total: -568.66) and MDM2 (E-total: -459.53) is associated with lower total energy compared to the interaction of the complete MDM2 with p53 (E-total: -399.25). The abovementioned interaction occurred with higher total energy in comparison with the optimized p53 and MDM2 (E-total: -407.20). Moreover, subsequent to MDM2 and p53 optimization, it appeared that their relative tendency was augmented compared to their corresponding complete models.

Given the interaction of full FHIT with optimized models of p53 and MDM2, it is evident that FHIT truncates have higher affinity to interact with MDM2 optimized part than p53 optimized model. According to the interaction values, FHIT truncates interact with optimized MDM2 at lower E-total than optimized part of p53 and the total energies of docking interactions are directly related to shape energies of the mentioned interactions. Our results revealed that the tendency of β4-7, α1 segment of FHIT to p53 optimized model is more than other parts. Likewise, the β5-7, α1 structure of FHIT has more affinity to MDM2 optimized part than other forms. Thus, one can suggest the β5-7, α1 segment of FHIT as an interacting domain for both p53 and MDM2.

Having studied the above mentioned interactions, we found that FHIT remarkably has better affinity to bind MDM2 optimized part in the presence of p53 and MDM2 optimized models in most of the cases. Even though it can bind to p53 optimized model with low energy, when MDM2 optimized part is added to the model, the interaction with p53 optimized part is further attenuated (Table 4). Interestingly, complex of FHIT truncates interacting with MDM2 optimized part at lower total energy usually interacts with p53 optimized model at higher total energy. Thus, these findings indicate a sequence/conformation specificity of FHIT truncates for interacting with MDM2 or p53.

E-totals of interaction between MDM2 optimized model (for interaction with p53) and FHIT truncates reveal that α1 is important in these interactions. E-totals of interaction between p53 optimized model (for interaction with MDM2) and FHIT truncates show that β5-7 and α1 are important parts in these interactions.

Experimental reports using yeast two-hybrid ⁽²⁵⁾ and immunoprecipitation indicate that p53 at amino acids 1-41 ⁽²⁵⁾ or 1-52 ⁽²⁶⁾ interacts with MDM2. On MDM2, the interaction at amino acids 1-118 ⁽²⁵⁾ or 19-102 ⁽²⁶⁾ is the binding site to p53. Site-directed mutagenesis confirms the Leu14, Phe19, Leu22, and Trp23 of p53 are essential amino acids for interaction ⁽²⁷⁾. The co-crystal structure of MDM2-p53 complex shows that Phe19, Trp23, and Leu26 are three main interacting residues in p53 ⁽²⁸⁾.

MDM2 directly binds to p53 ⁽¹⁸⁾ and regulates p53 function and degradation ^(29–33). Moreover, a number of studies reported p53 and FHIT interaction ^{(14, 19)} and their possible association ⁽³⁴⁾. As shown in Table 3, in p53-MDM2 interaction, the significant part of p53 is amino acids 18-26, and the important part of MDM2 is amino acids 23-119 ⁽³⁵⁾. Based on our results, the interaction sites of FHIT with MDM2 and p53 have overlapping parts. The best interaction site for MDM2-FHIT is residues 34-106 containing β5-7, α1. Conversely, for FHIT-p53 interaction, amino acids 21-104 containing β4-7, α1 are involved. However, residues 34-104 are involved in both interactions (Table 2). Interestingly, when the interaction of FHIT with MDM2 optimized part is challenged with p53 optimized part, the interaction site is different from interaction with MDM2 optimized part alone.

Based upon these results, the interaction sites of FHIT with MDM2 and p53 are different with overlapping parts. FHIT binds to MDM2 with lower energy in the presence of p53 and the binding site shifts toward FHIT-MDM2 interaction. These data provide information involving competition of FHIT with p53 in binding to MDM2. Then, in the presence of FHIT, p53 is released from MDM2 and can increase apoptosis or cell cycle arrest. Figures 3 and 4 confirm the results indicated in the tables.

In conclusion, our findings provide valuable information to understand the molecular mechanism of FHIT-MDM2-p53 complex formation and the design of inhibitory compounds. The truncated parts of FHIT with higher absolute E-total energy interacting with MDM2 optimized part [parts (β5-7, α1) and/ or (α1)] can be effective in inhibiting degradation of p53 through altering MDM2 interaction with p53. Also, the truncated parts of FHIT with higher absolute E-total energy interacting with p53 optimized part [parts (β5-7, α1) and (β4-7, α1)] can be effective in inhibiting degradation of p53 through altering MDM2 interaction site with p53.