Investigating Intra- and Inter-Molecular Coevolution of Intrinsically Disordered Protein, Prothymosin-alpha

Abstract

Prothymosin-α (ProTα) is a small, highly acidic protein found in the nuclei of virtually all mammalian tissues. It belongs to a class of proteins known for their lack of a rigid three-dimensional structure called intrinsically disordered proteins (IDPs). ProTα has been shown to play essential roles in cell robustness. As an example, ProTα is involved in apoptosis, or programmed cell death by inhibiting apoptosome formation via binding Apaf1. This research focus is on detecting coevolution of ProTα and between ProTα and Apaf1 (ProTα-Apaf1 or ProTα-Apaf1 complex). Coevolution refers to correlated changes between pairs of interacting species to maintain or refine functional interaction. Coevolution can be defined at the molecular level as correlated sequence changes that occur to maintain a structural or functional interaction. Studying coevolution of ProTα and ProTα-Apaf1 may provide useful information such as structural contacts and specific residues necessary for complex formation. In this study, a pipeline for performing molecular coevolution studies was established at East Carolina University (ECU). This pipeline was used to analyze myoglobin, ProTα, and ProTα-Apaf1. Myoglobin has been a target of previous coevolutionary studies and was chosen to test the robustness of the pipeline developed in this study. Most of the coevolving residues that were found in myoglobin match closely with those detected in other work. ProTα, which has never been studied by way of coevolution, displays several coevolving residues involved in long range interactions or functionally important regions. These methods were also applied to ProTα- Apaf1 complex. Previous experimental studies using 1H-15N heteronuclear single quantum coherence (HSQC) NMR have revealed residues on ProTα necessary for interaction with Apaf1 however the residues on Apaf1 necessary for interaction with ProTα have not been resolved. Several residues of ProTα were found to have coevolution with Apaf1. Docking studies were performed to simulate binding between ProTα and Apaf1 at the sites detected in this study (ProTα: Thr8, Thr107; Apaf1: Ser1056, Asp1096). Six orientations of ProTα and Apaf1 were run for 9 nanoseconds (ns) and in each simulation, the two proteins did not drift apart from one another. This suggests that the residues detected by coevolution in this study may play a role in the interaction between ProTα and Apaf1.