Structural insights on binding mechanism of CAD complexes (CPSase, ATCase and DHOase)

Abstract

Pyrimidine biosynthetic pathway enzymes constitute an important target for the development of antitumor drugs. To understand the role of binding mechanisms underlying the inborn errors of pyrimidine biosynthetic pathway, structure and function of enzymes have been analyzed. Pyrimidine biosynthetic pathway is initiated by CAD enzymes that harbor the first three enzymatic activities facilitated by Carbamoyl Phosphate Synthetase (CPSase), Aspartate Transcarbamoylase (ATCase) and Dihydroorotase (DHOase). While being an attractive therapeutic target, the lack of data driven us to study the CPSase (CarA and CarB) and its mode of binding to ATCase and DHOase which are the major limitation for its structural optimization. Understanding the binding mode of CPSase, ATCase and DHOase could help to identify the potential interface hotspot residues that favor the mechanism behind it. The mechanistic insight into the CAD complexes were achieved through Molecular modeling, Protein-Protein docking, Alanine scanning and Molecular dynamics (MD) Studies. The hotspot residues present in the CarB region of carboxy phosphate and carbamoyl phosphate synthetic domains are responsible for the assembly of CAD (CPSase-ATCase-DHOase) complexes. Overall analysis suggests that the identified hotspot residues were confirmed by alanine scanning and important for the regulation of pyrimidine biosynthesis. MD simulations analysis provided the prolonged stability of the interacting complexes. The present study reveals the novel hotspot residues such as Glu134, Glu147, Glu154, Asp266, Lys269, Glu274, Asp333, Trp459, Asp526, Asp528, Glu533, Glu544, Glu546, Glu800, Val855, Asp877, Tyr884 and Gln919 which could be targeted for structure-based inhibitor design to potentiate the CAD mediated regulation of aggressive tumors.

Communicated by Ramaswamy H. Sarma

Keywords:

• CAD
• alanine scanning mutagenesis
• pyrimidine biosynthesis
• protein-protein interactions

Disclosure statement

The authors declare that they have no competing interests.

Additional information

Funding

The authors are grateful to the computational facilities provided by DBT, New Delhi, India [BT/PR4524/BID/7/388/2012], DBT [BT/PR15407/BRB/10/923/2011 dated 05.07.2012], University Grants Commission, New Delhi, India [F.14-13/2013 (Inno/ASIST)], DST [Grant number SR/SO/BB-0079/2012 dated 09.07.2013], DST- Fund for Improvement of S&T Infrastructure in Universities & Higher Educational Institutions (FIST) [SR/FST/LSI-667/2016)(C)], DST-Promotion of University Research and Scientific Excellence (PURSE) [SR/PURSE Phase 2/38 (G)], RUSA-Phase 2.0.[F.24-51/2014-U, Policy (TNMulti-Gen), Dept. of Edn. Govt. of India, dt: 09.10.2018] and Department of Bioinformatics, Alagappa University, Karaikudi.