A Computational Investigation of Allostery and Binding Interactions in Proteins

Abstract

Protein allostery is a fundamental biological mechanism that plays a pivotal role in regulating various cellular processes. It allows proteins to change their shape and function in response to specific signals or ligands. In the context of nucleotide excision repair (NER), proteins involved in the repair process, such as the Rad4/XPC protein, undergo conformational changes when they bind to damaged DNA, which begin the lesion excision process by locating the lesion site, which is followed by the recruitment of other repair factors and facilitates the removal of damaged DNA segments. Essentially, protein allostery ensures that the repair machinery is only activated when and where it is needed, contributing to the precision and efficiency of DNA repair processes in cells. Molecular dynamics (MD) simulations enable the investigation of the conformational changes that facilitate complex, selective, and biologically significant processes involving proteins binding to a variety of substrates. Hence, the present work uses MD to study two processes that proteins participate in: (a) transcription modulation through mediator allostery and (b) locating UV-lesions present in DNA for their ultimate excision and repair. F-helices in CAP are able to ultimately recognise and bind to DNA for transcription when they undergo reorientation after cAMP, and a mediator has situated itself into the binding pockets of CAP. The present study uses a simulation-based approach to investigate the mechanism of cAMP-induced changes in the conformation and energetics of F-helices observed during the allosteric regulation of CAP by cAMP. The free energy profiles obtained by two-dimensional umbrella sampling of CAP and cAMPbound CAP provide a detailed picture of the elasticity modifications observed in the DNA-binding domain of CAP when cAMP is appropriately situated. Residue-wise interaction energy maps w.r.t. CAP residues under the different conformations of CAP, cAMP-bound CAP, and cAMP-bound DNAcomplexed CAP are created, which ultimately offer clues on the microscopic origin of the inter-subunit cooperativity and dimer stability of CAP. UV radiation-induced DNA damage has adverse effects on genome integrity and cellular function. The most prevalent DNA lesion is the cyclobutane pyrimidine dimer (CPD), implicated in a variety of genetic skin-related diseases and cancers in humans. Rad4/XPC is a damage-sensing protein that recognises and helps repair CPD lesions with high affinity. This binding efficiency of Rad4 depends on how efficiently the BHD2 and BHD3 domains have been associated with the CPD-containing lesion site of DNA. The present thesis investigates the mechanism, energetics, dynamics, and molecular basis for this Rad4-DNA association using CPD-containing perfectly matched DNA. This key molecular event that occurs in NER is studied using suitable reaction coordinates, and the resultant free energy surface when compared with the same of TTT/TTT mismatched DNA reveals that Rad4 has a higher tendency to stay in the associated conformation with CPD-containing DNA than TTT/TTT mismatched DNA, hence having a higher lesion-recognition efficiency on the former than the latter.