Discussion - 2qgnA
The crystal structure of tRNA isopentenyl transferase isolated from Bacillus halodurans was determined, allowing us to further analyse different aspects of this structure. This has enabled us to shed light on the structural composition and mechanism of action of this essential enzyme. Structural analysis of 2qgnA has resulted in the finding of several different aspects of tRNA-isopentenyl transferase. Beginning with the structure constructed from PyMOL(Figure 1)consisting of 322 amino acid, we have looked into the secondary structure(Figure 2), its surface properties covering positively charged regions together with negatively charged regions(Figure 3), and its domains as well as ligand binding sites and surface clefts, also conservation of residues across different species visually portrayed. Ligplots and protein-ligand interaction of tRNA isopentenyl were scrutinized, identifying hydrophobic and hydrophilic bonds between the protein and the ligand. Residues surrounding the ligand found to be crucial include Ala(13),Gly(15),Val(14),Lys(16) and Thr(17).
From CATH domain database, two main domains were found in 2qgn. The first domain ranges from residue 2-200 and residue 283-314 while the second domain boundaries included residues from 201-282. Topology of Domain 1 is known to be of Rossmann A-B-A fold, and is a representative of the homologous superfamily of P-loop containing nucleotide triphosphate hydrolases. Domain 2 is yet to be classified in CATH.
Binding sites and active sites of proteins and DNAs are often associated with structural pockets and cavities. From CASTp server, identification and measurements of surface accessible pockets as well as interior inaccessible cavities, were obtained. A total of 19 pockets were found in tRNA-isopentenyl transferase, with pocket 19 covering the largest area and volume. Liang et al. (1998) denotes the possibility that the largest pocket/cavity is the active site, despite a number of instructive exceptions. Shape and size parameters of protein pockets and cavities are thus are important for active site analysis.
Laskowski R.A postulated that cleft volumes in proteins relate to their molecular interactions and functions. It was found that in over 83% of proteins, ligands are usually bound in the largest cleft. This suggests that size is a functional requirement. Here, structural analysis of 2qgn resulted in the finding of sulfate ion as the main ligand in the structure, which coincidently is found in the surface cleft with the largest volume, corresponding to Laskowski's finding. However, based on the function of the enzyme, there is a possibility that the clefts allow ribosome to bind and interact with this enzyme during translation.
The CATH results were further supported by the DALI results, which show that adenylate kinase has structural similarities with tRNA-IPT. The former has a P-loop motif, formed between a β-strand and a α-helix, at its N-terminus. This implies that tRNA-IPT might have this motif as well. However, the pattern motif of the P-loop in the adenylate kinase is not found in tRNA-IPT. Moreover, guanylate kinase also has the P-loop but does not have the same motif pattern as that of adenylate kinase. Divergence of this motif has started then resulting in differences in the structures of both guanylate kinase and tRNA-IPT when they are superimposed (Figure 10 in Results). Thus the P-loop motif in tRNA-IPT is still to be identified but there is a high possibility that the P-motif is located near the ligand binding site.
As found from the structural analysis (PDB and Profunc), the ligand interacts with AVGKT at position 775-780 (Multiple Sequence Alignment). This is towards the end-terminus of the protein sequence and this sequence is between a β-strand and a α-helix (PDBsum). This supports the fact that the P-loop might be present in tRNA-IPT.
Cloning of the human tRNA isopentenyltransferase found a C2H2 Zn finger motif. From the article by Anna Glovko, this motif is always found in eukaryotic organisms although there are some exceptions for Arabidopsis thaliana, C.elegans and S.pombe. It is surprising to find that this motif is present as a single copy as this motif is usually interacting with more than one zinc finger. Moreover, the common role of this motif is in protein-RNA interation but this might not be the function in eukaryotes. The article suggested that the zinc finger motif may be involved in nuclear retention signal (La Casse 1995) and stability of enzyme conformation (Chong 1995).
Generally, the zinc finger motif have conserved glycine and tryptophan residues along the protein sequence together with cystein and histidine residues at extreme ends involved in coordination with zinc. The best conserved regions found to maintain the structural integrity of the protein in C2H2 zinc fingers are the conserved aliphatic and aromatic residues.
It is surprising that from LOCATE, the mouse enzyme is found in cytosol and mitochondria. According to our knowledge, tRNAs are involved in translation and this process occurs in the cytoplasm. Thus, the enzyme acting on them should be in the cytoplasm as well. However, the human enzyme is found in the nucleus. This may be because the human enzyme has a nuclear signal localization that is also found in yeast IPTs. This is not found in Bacillus halodurans as this signal peptide sequence is found in the additional residues in humans and yeast (Anna Glovko 2000). This may be the reason why human IPT is found in nucleus as well.
The DALI results obtained showed that there are several other enzymes that are structurally related to tRNA-IPT. They are isopentenyl transferase, guanylate kinase and adenylate kinase, starting from the most similar structure. According to biochemist view, similar structure proteins may contribute to similar functions as well. Thus, the functions of these enzymes are studied to further understand and support the existing function of our protein.
Using InterPro, the information on the functions of these enzymes is retrieved. Guanylate kinase catalyzes the ATP-dependent phosphorylation of GMP into GDP while adenylate kinase catalyse the Mg-dependent reversible conversion of ATP and AMP to two molecules of ADP, an essential reaction for many processes in living cells. Both of these enzymes may act on different molecules but the reactions that they catalyse involve phosphorylation.
The ability to phosphorylate is lost in isopentenyl transferases, which is also known as dimethylallyl transferase. This enzyme adds the isopentenyl group on adenine base situated at the 5'-terminal phosphate group. So far, tRNA-IPT only has the function of isopentenyl transferase and does not exhibit any phosphorylation function. However, there are still possibilities that tRNA-IPT has the ability to phosphorylate.
The presence of the P-loop motif found in humans, E.coli and S. cerevisiea (Anna Glovko 2000) might be involved in ATP/GTP binding. According to Saraste, this loop together with the zinc finger motif plays a role in ligand binding. Such functional motifs may be from the same superfamily initially but subsequently diverged away from each other, with some conserved functional regions.
Expression of tRNA-IPT is higher in several tissues but generally it is expressed in all cells. This is to ensure that efficient and correct translation takes place in each cell. This enzyme is particularly high in oocyte and adipose tissue. The former is constantly undergoing cell division and differentiation while the latter is often involved in energy production, energy storage, and hormone production. All of these processes require high levels of enzyme activity and the presence of different adaptor molecules and transcription factors.
Multiple sequence alignment(MSA) of 2qgn with other proteins, resulted in the outcome of several conserved regions. These regions include residues from (a) 750bp to 870 bps, (b) region from 880bp to 1000bp, (c) region from 1160bp to 1270bp and (d)region on the c-terminus. Combinatorial analysis of conserved regions with structural analysis unveiled that these conserved regions were located on regions surround the ligand. They may play a role in interaction with the ligand as well as form the active site for functional purposes. The conserved regions also annotated the presence of the P-loop motif that may well be conserved amongs other species as well.
Phylogenetic tree built incited the possiblity that this protein showed a high level of conservation from Bacteria to Eukaryotes. Despite constant evolution, presence of this protein conserved among a wide range of species implied the importance of this protein.
