A unique mechanism of protein oligomerization is domain swapping. It is a feature found in some proteins wherein a dimer or a higher oligomer is formed by the exchange of identical structural segments between protomers. Domain swapping is thought to have played a key role in the evolution of stable oligomeric proteins and in oligomerization of amyloid proteins. This thesis deals with studies to understand the significance of hinges involved in domain swapping for protein oligomerization and function. The stationary phase survival protein SurE from Salmonella typhimurium (StSurE) and Sesbania mosaic virus (SeMV) coat protein have been used as models for studies on domain swapping.
This thesis has been divided into eight chapters. Chapter 1 provides a brief introduction to domain swapping, while Chapters 2 to 6 describes the studies carried out on StSurE protein, Chapter 7 deals with studies on SeMV coat protein. The final Chapter 8 provides brief descriptions of various experimental techniques employed during these investigations.
Chapter 1 deals with a brief introduction to domain swapping in proteins. Examples where different domains are exchanged are cited. Then it describes physiological relevance of domain swapping in proteins and probable factors which promote swapping. Finally it also discusses the uncertainties that are inevitable in protein structure prediction and design.
Chapter 2 describes the structure of Salmonella typhimurium SurE (StSurE; Pappachan et al., 2008) determined at a higher resolution. The chapter also deals with the sequence and structure based comparison of StSurE with other known SurE homolog structures. A comparative analysis of the relative conservation of N- and C-terminal halves of SurE protomer and variations observed in the quaternary structures of SurE homologs are presented. Then a brief introduction is provided on function of StSurE. The conserved active site of StSurE that might be important for its phosphatase activity is described. A plausible mechanism for the phosphatase activity as proposed by Pappachan et al. (2008) is presented. Crystal structures of StSurE bound with AMP, pNPP and pNP that was determined with the view of better understanding the mechanism of enzyme function is presented. These
structures provide structural evidence for the mechanism proposed by Pappachan et al. (2008). Finally a substrate entry channel inferred from these structures is discussed.
SurE from Salmonella typhimurium (StSurE) was selected for studies on domain swapping as there is at least one homologous structure (Pyrobaculum aerophilum - PaSurE) in which swapping of the C-terminal helices appears to have been avoided without leading to the loss of oligomeric structure or function. It was of interest to examine if an unswapped dimer of StSurE resembling PaSurE dimer could be constructed by mutagenesis. To achieve this objective, a crucial hydrogen bond in the hinge involved in C-terminal helix swapping was abolished by mutagenesis. These mutants were constructed with the intention of increasing the flexibility of the hinge which might bring the C-terminal helices closer to the respective protomer as in PaSurE. Chapter 3 presents a comparative analysis of the hinges involved in C-terminal helix swapping in PaSurE and StSurE. Based on the comparison of structure and sequence, crucial residues important for C-terminal helix swapping in StSurE were identified as D230 and H234. The chapter describes the construction of mutants obtained by substituting D230 and H234 by alanine and their biophysical characterization. Finally it describes structural studies carried out on these mutants. The mutation H234A and D230A/H234A resulted in highly distorted dimers, although helix swapping was not avoided.
Comparative analysis of the X-ray crystal structures of native StSurE and mutants H234A and D230A/H234A reveal large structural changes in the mutants relative to the native structure. However the crystal structures do not provide information on the changes in dynamics of the protein resulting from these mutations. To gain better insights into the dynamics involved in the native and mutants H234A and D230A/H234A, MD simulations were carried on using GROMACS 4.0.7. Chapter 4 deals with a brief description of the theory of molecular dynamics, followed by results of simulation studies carried out on monomeric and dimeric forms of StSurE and dimeric forms of its mutants H234A and D230A/H234A. The conformational changes and dynamics of different swapped segments are discussed.
Crystal structures of H234A and D230A/H234A mutants reveal that they form highly distorted dimers with altered dimeric interfaces. Chapter 5 focuses on comparison of dimeric interfaces of the native StSurE and hinge mutants H234A and D230A/H234A. Based on the analysis, three sets of interactions were selected to investigate the importance of the interface formed by swapped segments in StSurE mutants H234A and D230A/H234A. One of the selected sites corresponds to a novel interaction involving tetramerization loop in the hinge mutants H234A and D230A/H234A resulting in a salt bridge between E112 – R179’ and E112’ – H180 (prime denotes residue from the other chain of the dimeric protein). This salt bridge seems to stabilize the distorted dimer. It is shown by structural studies that the loss of this salt bridge due to targeted mutation restores symmetry and dimeric organization of the mutants.
Loss of a crucial hydrogen bond in the hinge region involved in C-terminal helix swapping in SurE not only leads to large structural changes but also alters the conformation of a loop near the active site. It is of interest to understand functional consequences of these structural changes. StSurE is a phosphatase, and its activity could be conveniently monitored using the synthetic substrate para nitrophenyl phosphate (pNPP) at pH 7 and 25 ºC. Chapter 6 deals with the functional studies carried out with various StSurE mutants. The studies suggest that there is a drastic loss in phosphatase activity in hinge mutants D230A, H234A and D230A/H234A, while in the salt bridge mutants the function seems to have been restored. Few of these mutants also exhibit positive cooperativity, which could probably be due to altered dynamics of domains.
Sesbania mosaic virus (SeMV) is a plant virus, belonging to genus sobemovirus. SeMV is a T=3 icosahedral virus (532 symmetry) made up of 180 coat protein (CP) subunits enclosing a positive-sense RNA genome. The asymmetric unit of the icosahedral capsid is composed of chemically identical A, B and C subunits occupying quasi-equivalent environments. Residues 48 – 59 of the N-terminal arms of the C subunits interact at the nearby icosahedral three-fold axes through a network of hydrogen bonds to form a structure called the “β-annulus”. Residues 60 – 73 form the “βA-arm” that connects the N-terminal β-annulus to the rest of the protomer. Various studies on SeMV-CP suggest that different lengths of the N-terminal segments affect the assembly of virus. It might be possible to exploit this flexibility of the N-terminus in SeMV-CP to introduce swapping of this segment between two 2-fold related C subunits as is found in Rice yellow mottle virus (RYMV), another sobemovirus, with which SeMV shares significant sequence similarity. Chapter 7 focuses on attempts made to examine the mutational effects planned to introduce domain swapping. The strategy used for introducing swapping in SeMV-CP was based on the sequence of the βA-arm or the hinge involved in swapping of β-annulus in RYMV. TEM
images of the mutant virus like particles obtained suggest that they are heterogeneous. These mutants could not be crystallized, probably due to the heterogeneity. However, the assembly of the expressed proteins to virus like particles was profoundly influenced by the mutations.
Chapter 8 discusses various crystallographic, biophysical and biochemical techniques used during these investigations. Finally the thesis concludes with Conclusions and Future perspectives of the various studies reported in the thesis.
In summary, I have addressed the importance of amino acid residues and interactions of hinges involved in domain swapping for the quaternary structure and function of proteins.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.iisc.ernet.in:2005/3500 |
| Date | January 2014 |
| Creators | Yamuna Kalyani, M |
| Contributors | Murthy, M R N |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Relation | G26630 |
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