CHAPTER 3 IN SILICO ANALYSIS OF SBP……………………………….. 40-72
3.3 RESULTS AND DISCUSSION
3.3.8 UgpABCE transporter
For the de novo biosynthesis of phospholipids, uptake of two essential metabolites viz. sn- glycerol-3-phosphate (G3P) and glycerophosphocholine (GPC) is accomplished mostly through UgpABCE transporters (Wuttge et al., 2012). Structurally, UgpABCE transporters are known to be similar to sugar ABC transporters and belong to the CUT1 family (Wuttge et al., 2012; Jiang et al., 2014). Owing to its high sequence and structural similarities with that of sugar ABC transporters, UgpABCE transporters, particularly its SBP component (referred to as UgpB), are often misannotated as sugar-binding proteins. In our previous work, we reported such a case where the UgpB protein (TtUgpB, ORF ID: TTHA0379) from T. thermophilus HB8 was misannotated as a sugar-binding protein (Chandravanshi et al., 2016). In UniProtKB database, other two ORFs TTHA1936 and TTHV034 from T.
thermophilus HB8 are annotated as UgpB proteins. Thus, here we report the possible reason(s) of the existence of multiple UgpB proteins in T. thermophilus HB8.
The proteins TTHA0379, TTHA1877, TTHA1936 and TTHV034 are functionally closer to UgpB rather than sugar-binding proteins
In UniProtKB database, ORFs TTHA0379 and TTHA1877 are annotated as “sugar ABC transporter, periplasmic sugar-binding protein” while TTHA1936 and TTHV034 as
“glycerol-3-phosphate ABC transporter, periplasmic glycerol-3-phosphate-binding protein” and “glycerol-3-phosphate ABC transporter substrate-binding protein”, respectively. However, a homology search of TTHA0379, TTHA1877, TTHA1936 and TTHV034 against UniProtKB database reveals their decent similarity (in the range of 22- 29%) with both the UgpB and sugar-binding proteins. Thus, to assign their exact function, an evolutionary analysis of these proteins and their homologs were performed. The result reveals their association with UgpB proteins rather than sugar-binding proteins and suggests a misannotation of the protein TTHA1877 (Figure 3.6A). To further substantiate the phylogenetic result, a multiple sequence alignment of these proteins with the UgpB proteins from T. thermophilus (TtUgpB), Mycobacterium tuberculosis (MtUgpB) and E.
coli (EcUgpB) was performed. The alignment confirms the conservation of the residues Tyr42, Glu66, Ser121, Trp169, Trp172, Gly284, Tyr323 and Arg374 (numbering according to EcUgpB) reported to coordinate G3P or GPC in the active site of UgpB
proteins (Figure 3.6B). Notably, the presence of either one or two tryptophan residues determines the binding of GPC or G3P, respectively (Jiang et al., 2014; Chandravanshi et al., 2016; Adhikari et al., 2017). As all these proteins TTHA0379, TTHA1877, TTHA1936 and TTHV034 possess only one tryptophan residue (Figure 3.6B), it can be concluded that they all might bind GPC rather than G3P.
To locate the GPC-binding site of the proteins TTHA1877, TTHA1936 and TTHV034, their tertiary structures were predicted using three different programs; all of which used the G3P-and GPC-binding proteins from E. coli (EcUgpB, PDB ID: 4AQ4) and M.
tuberculosis (MtUgpB, PDB ID: 4MFI), respectievely, as the default templates.
Comparison of theoretical models with crystal structures of G3P- (EcUgpB) and GPC- binding (MtUgpB)proteins establishes that all three proteins TTHA1877, TTHA1936 and TTHV034 can bind GPC rather than G3P (Figure 3.6C). Since the GPC-bound structure of UgpB is unavailable in the literature, molecular docking was executed to obtain the substrate-binding mode to the UgpB protein; the protein TTHA0379, which is already annotated as GPC-binding protein, was taken as control. Results reveal that GPC binds to the active site of protein TTHA1877, TTHA1936 and TTHV034, wherein, the phosphate group of the GPC is held by the residues Tyr302, Tyr303 and Gly284, respectively (Table A.3). Similarly, the trimethylammonium moiety of GPC is held mostly through cation…π interactions by aromatic residues, which form a hydrophobic cage in the active site of these proteins (Figure A.10A-A.10C). Breathtakingly, the molecular docking experiment of GPC with the protein TTHA0379 suggests the binding of GPC in a secondary site, far from the putative active site. The possible reason for the non-binding of the GPC in the active site seems to be due to the hindrance created by the residue Gln274 of the protein TTHA0379 (Figure A.10D). This enforces to hypothesize that GPC may not be a physiological ligand for the protein TTHA0379. Thus, to understand the transport and metabolism of GPC, we looked into the genetic arrangement in the vicinity of all the four ORFs TTHA0379, TTHA1877, TTHA1936 and TTHV034. Astoundingly, the investigation reveals that unlike the E. coli UgpABCE transporter, T. thermophilus HB8 UgpABCE transporters lack genes for GPC metabolism in their locality. Moreover, the flanking genes in all four operons are different indicating distinct ligand for each transporter (Figure 3.6D). In
summary, all the four proteins are members of UgpB, however, their cognate ligands remain unknown.
Figure 3.6. Comparative assessment of four UgpB proteins with EcUgpB and MtUgpB. (A) Phylogenetic tree showing the evolutionary relationship of TTHA0379, TTHA1877, TTHA1936 and TTHV034 along with UgpBs and sugar-binding proteins. The phylogenetic tree is constructed using the protein sequences of TTHA0379, TTHA1877, TTHA1936, TTHV034, UgpBs from Salmonella choleraesuis (Q57IS0), S. typhimurium (Q7CPK0), S. paratyphi (Q5PJK8), Shigella dysenteriae serotype 1 (Q32AT3), E. coli (P0AG80), S. flexneri serotype 5b (Q0SZL9), Yersinia pestis bv. Antiqua (Q1CNC9), Y.
enterocolitica (A1JID7) and M. tuberculosis (A5U6I5) and sugar-binding proteins from Rhizobium radiobacter (P29822), Bacillus halodurans (Q9KEE7), B. subtilis (P94528, O06989 and O07009), Streptococcus mutans serotype c (Q00749), S. pneumoniae serotype 4 (P59213), Pyrococcus furiosus (P58300), E. coli O157:H7 (P0AEY0), Klebsiella aerogenes (P18815) and S. typhimurium (P19576). The UniProt ID of each protein is provided in parenthesis. In the phylogenetic tree, two separate clusters of UgpBs and sugar- binding proteins are shown in green and violet, respectively. The proteins TTHA0379, TTHA1877, TTHA1936 and TTHV034 are marked with red dots. (B) Multiple sequence alignment of TTHA1877, TTHA1936 and TTHV034 along with reference proteins EcUgpB (P0AG80), MtUgpB (A5U6I5) and TTHA0379 (Q5SLB4); the codes provided in
parenthesis are UniProt IDs. For the clarity of the figure, only a partial alignment has been shown here. The essential conserved residues of EcUgpB proteins are shaded in green and those similar to MtUgpB are enclosed in green box. (C) Comparison of the active-site pockets of TTHA1877, TTHA1936 and TTHV034 with EcUgpB, MtUgpB and TTHA0379. The active-site residues known to be involved in holding GPC are shown as ball-and-stick model while rest of the protein as cartoon. (D) A diagrammatic representation of the genetic cluster of EcUgpB, TTHA0379, TTHA1877, TTHA1936 and TTHV034 UgpABCE transporters. Each gene is represented by an arrow indicating its direction of transcription. The ORF numbers and its respective encoded protein names are furnished along each arrow. The ORFs encoding ABC transporters are shown in brown while ORFs for different proteins are filled with different color.