Biochemical, Structural and Cellular Studies on IIGP1, a member of the p47 Family of GTPases

My thesis work focuses on the biochemical, structural and cellular characterisation of IIGP1, a member of the p47 family of GTPases. The p47 family of GTPases are induced transcriptionally from very low resting levels in mouse cells by interferons and are implicated in cell autonomous resistance to intracellular pathogens. A vast subset of genes are regulated by IFNs and the mechanistic details of only a few have been described. Therefore, to understand the function and features of this family of GTPases, an in depth study on IIGP1, a member of this family is investigated. Recombinant IIGP1 was expressed in (FROL and purified to homogeneity, and a detailed biochemical characterisation of IIGP1 was carried out. IIGP1 is a GTPase with low affinity for nucleotides (micromolar range) and a low GTPase activity. The GTPase activity is concentration dependent and functional interaction between IIGP1 molecules occur in a nucleotide dependent manner. IIGP1 shares micromolar nucleotide affinities, and oligomerisation-dependent hydrolytic activity with the 67 kDa GTPase hGBP1 (induced by type I and type II interferons), with the antiviral Mx proteins (type1 interferon induced) and with the paradigm of the self-activating large GTPases, the dynamins. Besides, IIGP1 differs by having a high affinity for GDP and low GTPase activity. The crystal structure of IIGP1 has a N- terminal helical domain followed by a typical G- domain fold and C- terminal helical regions. IIGP1 structure is a representative structure for the p47 family of GTPases. This conclusion arises firstly, from secondary structure analysis of other members of the family which are very similar to IIGP-1 and conform to the expectation derived from the crystal structure. Secondly, from the conservation of several pairs of distant residues in other p47 family members which interact to stabilise the IIGP-1 structure. This conservation could not be explained unless the structures of the other p47 GTPases are very similar to IIGP-1. Structural analysis of IIGP1 with respect to the sequence comparison with the members show several conserved regions, forming the core regions. However large deviations in the primary structure between the members, especially at the N- and C- termini explains their non- redundancy as indicated by their distinct subcellular locations, and resistance to specific pathogens. Cellular characterisation of IIGP1 was investigated in order to understand the features of IIGP1 in the cell. Previous studies on IIGP1 has shown the association of IIGP1 with the endoplasmic reticulum. Since the primary sequence does not indicate signal sequences, or ER retention or retreival signals, IIGP1 is rather peripherally associated with the ER. Cellular fractionation studies reveal differential distribution of the protein, present mostly in the membrane bound form and partly in the soluble form. Membrane attachment is dependent on myristoyl modification, although not solely. There is evidence for ionic interactions which could allow the association of IIGP1 to the membranes, and another pool of the protein is independent of the above two modifications for membrane attachment. This could be accomplished by the C- terminal CaaX like motif for IIGP1 (CLRN) which has not been tested so far. IIGP1 could be immunoprecipitated with D 165 serum, as well as with monoclonal antibodies. Co-precipitation of three putative proteins was acheived, but the identity of these molecules remains to be unknown. IIGP1 is a monomer in the nucleotide-free state in solution, and also in the presence of GDP, but cyrstallised as a dimer with or without GDP. In the presence of nucleotide triphosphates, IIGP1 forms higher oligomers. The importance of the dimer on the properties of IIGP1 was investigated by interfering with the dimer interfaces. The interface mutants have no defects in nucleotide binding but have abrogated cooperativity unlike the wild type, suggesting the functional importance of the dimer. However, the oligomerisation properties of the interface mutants has not been analysed. The data described here formulates a platform for further analysis on IIGP1 function and provides essential parameters to understand the molecular mechanism by which IIGP1 participates in this complex resistance programme. The characterisation of IIGP1 gives an understanding to the behaviour and properties of the protein LQ YLWUR and LQ YLYR.