*3.8. Binding Preference of Nucleotides for Amino Acids*

The binding preference of nucleotide with DOT residues has been calculated by counting the occurrence of nucleotides–amino acid interacting pairs under the distance of 3.5 Å.

#### *3.9. Interaction Energy between Amino Acids and Nucleotides at Binding Interface*

The interaction energy of amino acids with nucleotides is computed using van der Waals and coulombs potential using AMBER force field [50]. It is given by

$$\text{Energy} = \sum \left[ \left( \frac{A\_{ij}}{r\_{ij}^{-12}} - \frac{B\_{ij}}{r\_{ij}^{-6}} \right) + \frac{q\_i q\_j}{\varepsilon r\_{ij}} \right] \tag{5}$$

where, *Aij* = ε*ij*\* (*Rij*\*)<sup>12</sup> and *Bij* = 2 ε*ij*\* (*Rij*\*)6; *Rij*\*=(*Ri*\* + *Rj*\*); and ε*ij*\*=(ε*i*\* ε*j*\*)1/2; *R*\* and ε\* van der Waals radius and well depth, respectively, and these parameters are obtained from Gromiha et al. [51]; *qi* and *qj* is the charge on atom *i* and *j*, respectively and *Rij* is the distance separating atom *i* and *j*.

#### **4. Conclusions**

The analysis of DOT regions in protein–RNA complexes revealed that in each complex these regions are generally small in size. Electrostatic interactions are found to be important, with the involvement of positively charged residues (Arg, Lys and His) in DOT regions. Among nucleotide–amino acid pairs, guanine–Arg and uracil–Lys pairs are identified to be the most and the least preferred ones at the interface, respectively. Generally, nucleotides prefer to bind DOT regions than other regions of protein. Further, DOT regions are significantly more exposed to solvent than other residues of protein–RNA complexes. Specifically, hydrophobic residues have higher difference in RASA of DOT regions and complete proteins. DOT regions are preferred to form coils, turns, and bends than regular secondary structures such as helices and strands. On the RNA side, DOT residues prefer to bind unpaired A and U and paired regions of C and G. In pseudoknot condition, mostly C and G interact with DOT residues. The interaction energy calculations revealed the types of interactions and preferred amino acid-nucleotide pairs at the interface based on energy.

The frequencies and propensities obtained in the present study could be used for discriminating DOT binding residues from other residues. Further, the location of DOT binding residues based on solvent accessibility and secondary structure of protein and RNA along with energy calculations may help to understand the recognition mechanism.

We obtained the DOT regions by comparing 3D coordinates of the missing residues in protein–RNA complexes and their respective free proteins. This might be an under representation of DOT regions since the structures solved by crystallization often stabilize the residues and reduce the native disorder. Hence, the disordered residues having 3D coordinates in free proteins are not considered. The current study can further be refined with the availability of more numbers of protein–RNA complexes and the improvements in structure determination techniques. In addition, development of disorder specific databases for protein–nucleic acid complexes with large datasets could enhance the confidence level of the result reported in the present study.

#### **Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/6/1595/ s1.

**Author Contributions:** M.M.G. and S.A. conceived the project and designed experiments. A.S. constructed the dataset and performed the analysis. M.M.G, S.A. and A.S took part in discussions. A.S. drafted the manuscript. M.M.G and S.A. edited and refined the manuscript.

**Acknowledgments:** We thank the Department of Biotechnology, Indian Institute of Technology Madras for computational facilities. A.S. thank Ministry of Human Resource and Development (MHRD) for the fellowship. This project is partially supported by the Council of Scientific & Industrial Research (CSIR), Government of India to M.M.G. and S.A. (grant numbers: 37(1694)/17/EMR-II) and 37(1695)/17/EMRII respectively). S.A. would like to acknowledge a grant from University for potential of excellence (UpoE-II) #270 and support DST-PURSE.

**Conflicts of Interest:** The authors declare no conflict of interest.
