Introduction
Capsular polysaccharides are of high molecular mass and low molecular weight cell-surface polysaccharides of Gram-positive and Gram-negative bacteria (Wang et al. 2000). They facilitate evading the immune system; therefore, the production and extraction of this layer is a prospective therapeutic target (rcsb.org). In the Gram-positive bacterium Streptococcus pneumoniae, strain TIGR4 the Cps4B is tyrosine protein phosphatase CpsB. The phosphatases have capability to dephosphorylate their analogous autokinases, instead of their similar functionality equivalence; they don’t demonstrate sequence homology. This capsule or CPS act as an interface border between a microbe and its environment that presents an expansive biological and clinical significance. CpsB belongs to the family of polymerase and histidinol phosphatase (PHP) which are metal-dependent enzymes (Gregor, et al. 2009).
Structure
The crystal structure of Cps4B capsule protein was first determined at a 2.799 Å X-ray resolutions. Cps4B is a Monomer protein with a sequence length of 247. It may contain one or more heterogenic compounds such as ligands (manganese and phosphate), ions, co-factors or modified amino acids (rcsb.org; ebi.ac.uk).
The secondary structure of Cps4B contains only one chain that is 45% helical (with 13 helices of 112 residues) and 15% beta-sheet (13 strands with 39 residues). The crystal structure analysis of Cps4B demonstrates that this protein is a member of the PHP-fold family (ebi.ac.uk). It contains two three-stranded parallel β-sheets and a ring of α-helices on the outer side of the structure. Between the β-sheets, metal ions are attached adjacently to one another. The distinct fold of Cps4B proved its relation with PHP family, which on applying SSM algorithm for the protein similarity search in protein structure database it showed similarity with only two proteins that were also phosphotriesterases (PHP) from E. coli and Sulfolobus solfataricus. Cps4B catalyzes reactions in phosphotriesterases (PHP) way through the hydrolysis of the phosphate ester bonds (figure 1) (Gregor, et al. 2009).
Cps4B contains three metal ions attached to its active site. Two axial ligands, Asp199 and Glu80 and three equatorial ligands, His5, His7 with water W1, synchronize metal M1. M2 is held by Glu80, Glu108, and His136 along with two water molecules, W1, and W2. Glu80 acts as a bidentate ligand for both metals, M1 and M2. The measured distance between first two metals is 3.5 Å, while M3 illustrates the distances of 4.7 and 5.6 Å to M1 and M2, respectively. M3 makes a tetragonal ligand sphere through attaching itself with His42, Asp14, His201 and water, W3 (figure 2). The loop βF/α6 of Cps4B that is located at 169-177 is involved in crystal contacts and protrudes into the contiguous solvent. It contains numerous hydrophobic amino acids that generate hydrophobic interactions with the surrounding substrate. Due to hydrophobicity these protein are non-polar in nature. The presence of aromatic amino acids, namely phenylalanine (F) and tyrosine (Y) in its protein give it an aromatic character. Thus, Cps4B is hydrophobic, aromatic and acidic protein (rcsb.org).
Cps4B has two ligands on its binding sites, phosphate and manganese cation (II+). Manganese is located at the active site of this protein and actively involved in its catalytic activities. Mn is required for the optimal activity in metal-binding phosphatase CpsB.
The Cps4B–PO43− complex from Steptococcus pneumoniae strain tigr4
There is no difference between the apo- and phosphate-bound structures of Cps4B. They both are nearly indistinguishable, with a rmsd of 0.14 Å. The phosphate is strongly attached to the active site of protein by a massive amount of interactions of three metal ions and catalytically active amino acid residues Arg139 and Arg206. In the phosphate bound complex, all three metal-bound solvent (Water) molecules are substituted by phosphate. Contrary of apo- structure Arg139 and Arg206 are better defined in terms of electron density due to their contact with the ligands. In apo structure, they were partially disordered (figure 3) (Gregor, et al. 2009). The aromatic residues at the side of the walls of the active site cavity show bulky relative movements (rcsb.org).
According to the study the phosphate ligands Arg139, and Arg206 are conserved and thus are probably significant for Cps4B-mediated catalysis. To verify this activity various mutants Arg139Ala and Arg206Ala (R139A, R206A) were prepared, and their catalytic activities were checked. Cps4BR139A illustrated only 5% of wild-type activity while Cps4BR206A showed around 40% remaining activity. The active-site metal ligands named His5, His7, His42 and His201 and residues connected with metal ligands (Asp3) are evidently significant for crystal strength as well as for the enzymatic function. Their role in catalysis is found to be indirect (Gregor, et al. 2009; Wang et al. 2000).
Moreover, some of the activity affecting residues are situated on the surface of the molecule at a distance from the active site of Cps4B and thus might not be implicated in catalysis. However at elevated temperatures (47 or 75 °C) a loss in activity is observed, due to the destabilization as after the effect of the mutations (Gregor, et al. 2009).
Significant Activities of Cps4B
Analysis of the genetic determinants of CPSs will be helpful in unravelling the CPS biosynthetic pathways. That can further help in exposing the prospect of the inhibitors design that would have the capability of hindering the expression of the virulence factor (Jiang, Wang and Reeves, 2001).
Several distinctive processes of CPS synthesis have been described such as Wzy-dependent pathway, where lipid-linked intermediates with repeat units of CPS are transported via the cytoplasmic membrane. They form substrates for the polymerization step, in the presence of oligosaccharide polymerase Wzy. For complete polymerization activity the involvement of a member of the polysaccharide co-polymerase family is required such as CPS in Streptococcus pneumoniae. Cps4B protein complex is believed to imitate Wzc, but it consumes different polypeptides for the cytoplasmic kinase (CpsD) and transmembrane/periplasmic kinase (CpsC). The phosphorylation state of CpsCD impacts both, the quantity of CPS created and the amount of CPS attached to the cell wall. Though many studies are evidenced that phosphorylation is not collectively essential for CPS assemblage, but likely to play a considerable role in numerous different but clinically significant bacterial systems (Gregor, et al. 2009).
Several molecular functions of Cps4B are catalytic activity, phosphoprotein phosphatase activity, hydrolase activity, protein tyrosine phosphatase activity and manganese ion binding. Cps4B is involved in numerous biological processes such as the dephosphorylation, polysaccharide biosynthetic process, peptidyl-tyrosine and capsule polysaccharide biosynthetic process (ebi.ac.uk; rcsb.org).
Works cited
Hagelueken, Gregor, et al. "Crystal Structures of Wzb of< i> Escherichia coli</i> and CpsB of< i> Streptococcus pneumoniae</i>, Representatives of Two Families of Tyrosine
Phosphatases that Regulate Capsule Assembly."Journal of molecular biology 392.3 (2009): 678-688.
Jiang, Sheng-Mei, Lei Wang, and Peter R. Reeves. "Molecular characterization of Streptococcus pneumoniae type 4, 6B, 8, and 18C capsular polysaccharide gene clusters." Infection and immunity 69.3 (2001): 1244-1255.
Wang, Shuishu, et al. "Crystal structures of a low-molecular weight protein tyrosine phosphatase from Saccharomyces cerevisiae and its complex with the substrate p-nitrophenyl phosphate." Biochemistry 39.8 (2000): 1903-1914.
