Developing structure-based model of viral RdRp of SARS-CoV-2: Deciphering fly-casting phenomena

Angel, Mary C. T. (2023) Developing structure-based model of viral RdRp of SARS-CoV-2: Deciphering fly-casting phenomena. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS dissertation of Angel Mary C T (18MS147)) Thesis_18MS147.pdf - Submitted Version Restricted to Repository staff only Download (41MB)

Abstract

The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is a crucial enzyme that plays a key role in the replication and transcription of the virus. RdRp is a therapeutic target being the viral replication-transcription machinery, accommodating RNA to operate its required functions. Structural biophysical experiments have discovered its “apo” and ”bound” folded states. However, their microscopic folding mechanism elucidating functions is yet to be explored. In this thesis project, we have developed a coarse-grained structure-based model, integrating the theories and concepts of statistical mechanics of folding and the funneled energy landscape. We have specifically explored the folding mechanism of apo and bound RdRp proteins by employing coarse-grained simulations and by varying scaled temperature as a perturbative tool. The RdRP protein has a canonical cupped right-hand shape consisting of fingers and a thumb, two important functional domains. The fingers and thumb cooperate to accommodate an RNA to make the overall RdRP machine functional. We find a unique temperature window where the apo RdRp dynamically breathes between two distinct states corresponding to ”open” and ”closed” conformations. The closed conformation is attributed to the interactive finger-thumb association, while in the open state, these two domains are disconnected. Interestingly, we find that a finger (f1) domain specifically induces the folding of the thumb (t1) domain. This induced domain folding mechanism is analogous to the fly-casting phenomenon often found in flexible biomolecular associations. We have explained the induced domain folding mechanism using capillarity theory and statistical mechanics.: Chapter-I covers the description of our system of concern, and early biophysical experiments and other relevant works on RdRp were thoroughly reviewed to obtain various structural information. The drug repurposing studies targeting RdRp are also covered in this introductory chapter. The concept of the energy landscape of biopolymers and the detailed description of our coarse-grained structure-based model Hamiltonian are covered in Chapter II (Theory and Methods). In this chapter, we also explain how we have harnessed the folding energy landscape theory to tackle large-length scale systems and long-timescale bioprocesses. We explain the Langevin dynamics for the stochastic process (implicit conditions) and the statistical mechanics that underlie the coarse-grained simulations. The findings from our investigation into the breathing dynamics of “apo” protein at a unique temperature are presented in Chapter III (Results). We have analyzed and compared the fluctuation of different domains by measuring root mean square fluctuation (RMSF). We have also analyzed domain-domain correlation using covariance analysis and correlation matrix both for the apo and bound forms of the RdRp protein. The free energy calculations and contact evolution are also presented in this chapter. All the calculations point out an induced folding mechanism analogous to the fly-casting phenomenon of the thumb domain which is under the influence of the finger domain. In Chapter IV, we explain the induced folding mechanism of the thumb domain with an analytical model describing the fly-casting mechanism through the capillarity theory of protein folding. This model, with its basis in polymer physics, describes how a relatively ordered domain (fingers of RdRp) helps in folding a malleable/disordered domain (thumb domain) and to overcome the folding barrier.

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