Effect of Single Residue Change on Conformational Dynamics of Proteins and Their Function: A Molecular Dynamics Study

Abstract

Proteins undergo conformational transitions and exhibit specific motion while performing their function. A single residue change (addition, deletion, or substitution) can sometimes influence the protein dynamics and disrupt its function. Molecular dynamics simulations can provide insights into the functional mechanism of proteins in scenarios where direct experimentation is not possible or feasible. Therefore, it is an excellent tool to study the effect of single residue change on the dynamics of proteins and, consequently, their function. This thesis investigates the following systems in which a single residue change significantly affects its function, using microsecond long all-atom molecular dynamics simulations in explicit solvent. First, how and why sickle haemoglobin aggregates to form fibrils in sickle cell anaemia. Sickle haemoglobin is produced by a mutation, leading to the substitution Glu6 → Val6 in the β chains of the haemoglobin tetramer. The aggregation of sickle haemoglobin occurs under a low partial pressure of oxygen, which deforms red blood cells causing sickle cell anaemia. Haemoglobin dynamics changes the relative orientation of its chains, changing the hydrogen bonding network at the interfaces with minimal change in the interface area or the average number of contacts at each interface. The system with sickle haemoglobin in the crystal arrangement has far more intermolecular contacts than normal haemoglobin in an identical arrangement. The dimer-dimer rotation was found to be a characteristic feature of the T and R states. Three potential wells were observed in most haemoglobin simulations in the free energy landscapes corresponding to the T state, R state, and an intermediate between them. The simulations together highlight the differences in normal and sickle haemoglobin behaviour and the basis of sickle haemoglobin aggregation and fibril formation. A new model for sickle haemoglobin fibrils present in sickle cells has been proposed to better agree to more experimental observations. In this model, the mutated residue β-Val6 interacts with a complementary pocket on the α chain instead of the putative pocket in the β chain. The inhibition of sickle haemoglobin aggregation due to S-glutathionylation has also been studied here. Glutathione bound to haemoglobin tetramer was observed in two dominant conformations, one extended and the other U-shaped. One of the conformers was more deeply entrenched in the intersubunit groove than the other. S-glutathionylation affected the flexibility of various segments and changed the tertiary and quaternary structure. These structural changes ultimately hinder the intermolecular contacts in sickle haemoglobin, inhibiting its aggregation. These insights may be useful in searching for therapeutics targetting sickle haemoglobin aggregation to treat sickle cell anaemia. Second, why does the fusion peptide from MHV-A59 containing central diproline promote cell–cell fusion while the fusion peptide from MHV-2 with one central proline is not? MHV-A59 and MHV-2 are two strains of the mouse hepatitis virus. MD simulations of the fusion domains showed that fusion peptides containing central diproline retain a stable helical conformation at this position instead of being destabilised in fusion peptides containing single proline. For the MD simulations in methanol, fusion peptides containing single and double proline both retain a stable helical conformation. The ability of the fusion apparatus to maintain a stable structure in both aqueous and hydrophobic environments allows the spike protein to promote fusion activity since a disordered structure would not be efficient in fusion. The study of trimeric fusion domain and the fusion peptide of the spike proteins from these viruses revealed the unique role of proline in the fusion peptide due to their atypical stereochemistry. Third, α1-antitrypsin is a metastable protein responsible for regulating the immune response, and a single amino acid substitution causes it to be non-functional and aggravate cases of lung disease. The mechanism behind the pathological behaviour of Z and M3 mutants of α1-antitrypsin has been studied here. In Z α1antitrypsin, glutamic acid is substituted by lysine near the reactive centre loop, and the electrostatic perturbation is per the substitution of a negatively charged residue by a positively charged one and localised in the vicinity. This mutation disrupts various hydrogen bonds stabilising the region where the reactive centre loop inserts during its conformational transition. In contrast, the mutation in M3 α1-antitrypsin changes the hydrogen-bonding network of the mutated residue with the loop between the strands s2B and s3B, consequently influencing the dynamics of the reactive centre loop through space. In conclusion, the location of the mutation and the stereochemical difference between the original and mutated residue is responsible for altering the structure and function of proteins. The extent of alteration is not discernible from sequence or structure alignment; a thorough molecular dynamics study is required to capture the differences and evaluate their importance. Similarly, insertion or deletion of residue at a critical position can have global effects not evident from simple sequence and structure alignment. Small local changes at critical positions manifest in complex global changes in protein structure and function. This work should provide a basis for future studies that wish to identify functionally important mutations that existing methods cannot interpret.