CHAPTER 6 PLASMODIUM YOELII ACETYL-COA CARBOXYLASE
6.2 Results …
6.2.5 Homology modelling of the biotinoyl domain of P. yoelii
generated by Proscan™ analysis in Figure 6.4. The phospho-serine residue is highly conserved and flanked by conserved sequences, with a 100% conserved lysine and tryptophan at positions –4 and +4 respectively (the phospho-serine residue is at position 0). The serine residue at position +5 in H. sapiens 1 and M. musculus is most favoured with a score of 99.7%.
The only modification site that appeared to be conserved on the BCCP domain is the biotin attachment motif. The N-myristoylation sites predicted to be conserved by the Proscan™ tool does not precede the N-terminal methionine, and therefore ingnored. N-glycosylation sites identified by the Proscan™ analysis were not conserved and were not included in the results.
A threonine phosphorylation site at N2 was identified by the programme on the carboxyl transferase domain, which appeared to be highly conserved. Except that the phospho- threonine residue is not at a conserved position across the sequences examined. The phospho- threonine residue is flanked by a 100% conserved lysine at position – 4 and + 4 (the phospho- serine residue is at position 0), in Plasmodium. A phospho-serine protein kinase site that was only conserved in Plasmodium was identified by NetPhos 2.0™ algorithm. This site has a consensus sequence KTAQSIEDF (phospho-serine in bold), with a 100% conserved lysine and phenylalanine residues at – 4 and + 4 positions, respectively.
6.2.5 Homology modelling of the biotinoyl domain of P. yoelii acetyl-CoA carboxylase
H.sapiens1. ---NKVIEKVLIANNGIAAVKCMRSIRRWSYEMFRNERAIRFV 154 M.musculus1. ---NKVIEKVLIANNGIAAVKCMRSIRRWSYEMFRNERAIRFV 153 H.sapiens2. ---DRVIEKVLIANNGIAAVKCMRSIRRWAYEMFRNERAIRFV 296 M.musculus2. ---NRVIEKVLIANNGIAAVKCMRSIRRWAYEMFRNERAIRFV 286 P.yoelii. YINYINERRYPYINYLKMKNEKIIKKLLIANNGMAALKCILSLKEWLFKTFNDENLIQII 456 P.falciparum. YVNYINERRYGYFDLLEKKNEKIIRKLLIANNGMAALKCILSLKDWLFKKFYDENLIKII 505 P.knowlesi. YTKYIEERRYPYFNFVKNKNGKIIKKLLIANNGMAAMKCILSIKEWLFKSFSEENLIKII 445 P.vivax. YTTYIEERRYPYFNFAKEKNGKIIKKLLIANNGMAAMKCILSIKEWLFKTFSEENLIKII 485 : *..:******:**:* : *:: * :: * *. * ::
H.sapiens1. SGMIAGESSLAYNEIITISLVTCRAIGIGAYLVRLGQRTIQVENSHLILTGAGALNKVLG 1859 M.musculus1. SGMIAGESSLAYDEVITISLVTCRAIGIGAYLVRLGQRTIQVENSHLILTGAGALNKVLG 1858 H.sapiens2. SGMIAGESSLAYEEIVTISLVTCRAIGIGAYLVRLGQRVIQVENSHIILTGASALNKVLG 1970 M.musculus2. SGMIAGEASLAYEKTVTISMVTCRALGIGAYLVRLGQRVIQVENSHIILTGAGALNKVLG 1960 P.yoelii. SGLIAGETSKAYDEIFTLSYVTGRSVGIGAYLVRLGKRTIQKKGSSLLLTGFNALNKILG 2453 P.falciparum. SGLIAGETSKAYEEIFTLSYVTGRSVGIGAYLVRLGKRTIQKKGSSLLLTGFNALNKILG 2771 P.knowlesi. SGLIAGETSKAYDEIFTLSYVTGRSVGIGAYLVRLGKRTIQKKGSSLLLTGFNALNKILG 2438 P.vivax. SGLIAGETSKAYDEIFTLSYVTGRSVGIGAYLVRLGKRTIQKKGSSLLLTGFNALNKILG 2577 :* *** * **:: .*:: ** *::***:*:.*** * ** .. ::*** :***:**
H.sapiens1. VPTKTPYDPRWMLAGRPHPTQKGQWLSGFFDYGSFSEIMQPWAQTVVVGRARLGGIPVGV 1985 M.musculus1. VPTKAPYDPRWMLAGRPHPTQKGQWLSGFFDYGSFSEIMQPWAQTVVVGRARLGGIPVGV 1984 H.sapiens2. LPSRAPYDPRWMLAGRPHPTLKGTWQSGFFDHGSFKEIMAPWAQTVVTGRARLGGIPVGV 2096 M.musculus2. TPTKAPYDPRWMLAGRPHPTLKGTWQSGFFDHGSFKEIMAPWAQTVVTGRARLGGIPVGV 2086 P.yoelii. NKINDVDMDNVNEADIIELLKGSDKKQGFLDKNSYFEYMNEWGKGIITGRGKLGSIPIGF 2669 P.falciparum. LHINDMDYDHIDDSNIIDLIKGTQEEQGFLDKNTYFEYMNEWGKGIITGRGKLGSIPVGF 3011 P.knowlesi. QRINDIDMDNIQNADVIELIKGTDTKQGFLDKNSYFEYMNEWGKGIITGRGKLGSIPVGI 2598 P.vivax. QRINDIDMDGLQNADIVELISGTDSKQGFLDKHSYFEYMNEWGKGILTGRGKLGSIPVGV 2737 . .. . *::* :: * : *.: ::.**.:**.:*:*.
Figure 6.4 Prosite™ prediction of protein kinase phosphorylation sites on enzyme domains of mammals and Plasmodium acetyl-CoA carboxylases. (A) Alignment produced by the ClustalW™
algorithm showing the predicted sites residues (highlighted) in the biotin carboxylase (BC) and the carboxyl transferase (CT) domains. (B). Cartoon structure of the multi-domain acetyl-CoA carboxylase illustrating the positions of the predicted phosphorylation sites. Double line indicates position fully or partially conserved across all organisms examined, broken line indicates position conserved only in Plasmodium.* indicates 100% conserved residue.
B
N P P P C
Biotin carboxylase Biotinoyl Carboxyl transferase
P P
A
BC
CT
P1
P3
P5 P2 P4
NetPhos Score (%)
H.sapiens1. 59.0 ---NKVIEKVLIANNGIAAVKCMRSIRRWSYEMFRNERAIRFV 154 M.musculus1. 59.0 ---NKVIEKVLIANNGIAAVKCMRSIRRWSYEMFRNERAIRFV 153 H.sapiens2. 53.9 ---DRVIEKVLIANNGIAAVKCMRSIRRWAYEMFRNERAIRFV 296 M.musculus2. 53.4 ---NRVIEKVLIANNGIAAVKCMRSIRRWAYEMFRNERAIRFV 286 P.yoelii. 79.4 YINYINERRYPYINYLKMKNEKIIKKLLIANNGMAALKCILSLKEWLFKTFNDENLIQII 456 P.falciparum. 95.3 YVNYINERRYGYFDLLEKKNEKIIRKLLIANNGMAALKCILSLKDWLFKKFYDENLIKII 505 P.knowlesi. 98.2 YTKYIEERRYPYFNFVKNKNGKIIKKLLIANNGMAAMKCILSIKEWLFKSFSEENLIKII 445 P.vivax. 98.2 YTTYIEERRYPYFNFAKEKNGKIIKKLLIANNGMAAMKCILSIKEWLFKTFSEENLIKII 485 : *..:******:**:* : *:: * :: * *. * ::
H.sapiens1. 91.4 VPTKTPYDPRWMLAGRPHPTQKGQWLSGFFDYGSFSEIMQPWAQTVVVGRARLGGIPVGV 1985 M.musculus1. 91.4 VPTKAPYDPRWMLAGRPHPTQKGQWLSGFFDYGSFSEIMQPWAQTVVVGRARLGGIPVGV 1984 H.sapiens2. 66.4 LPSRAPYDPRWMLAGRPHPTLKGTWQSGFFDHGSFKEIMAPWAQTVVTGRARLGGIPVGV 2096 M.musculus2. 66.4 TPTKAPYDPRWMLAGRPHPTLKGTWQSGFFDHGSFKEIMAPWAQTVVTGRARLGGIPVGV 2086 P.yoelii. 82.0 NKINDVDMDNVNEADIIELLKGSDKKQGFLDKNSYFEYMNEWGKGIITGRGKLGSIPIGF 2669 P.falciparum. 82.0 LHINDMDYDHIDDSNIIDLIKGTQEEQGFLDKNTYFEYMNEWGKGIITGRGKLGSIPVGF 3011 P.knowlesi. 82.0 QRINDIDMDNIQNADVIELIKGTDTKQGFLDKNSYFEYMNEWGKGIITGRGKLGSIPVGI 2598 P.vivax. 92.6 QRINDIDMDGLQNADIVELISGTDSKQGFLDKHSYFEYMNEWGKGILTGRGKLGSIPVGV 2737 . .. . *::* :: * : *.: ::.**.:**.:*:*.
H.sapiens1. VAVETRTVELSIPADPANLDSEAKIIQQAGQVWFPDSAFKTYQAIKDFNREGLPLMVFAN 2045 M.musculus1. VAVETRTVELSIPADPANLDSEAKIIQQAGQVWFPDSAFKTYQAIKDFNREGLPLMVFAN 2044 H.sapiens2. IAVETRTVEVAVPADPANLDSEAKIIQQAGQVWFPDSAYKTAQAIKDFNREKLPLMIFAN 2156 M.musculus2. IAVETRTVEVAVPADPANLDSEAKIIQQAGQVWFPDSAYKTAQVIRDFNKERLPLMIFAN 2146 P.yoelii. 99.1 IAVNKNLVTQTVPCDP-ALKTKAIKTTNAPCVFVPDNSYKTAQSIEDFNKENLPLFVFAN 2728 P.falciparum. 99.3 IAVNKNLVTQSIPCDP-ALKTKAQKLIQAPCVFFPDNSFKTAQSIEDFNKENLPLFIFAN 3070 P.knowlesi. 99.1 IAVNRNLVTQDVPCDP-ALKTKAVRSTQAPCVFFPDNSYKTAQSIEDFNKENLPLFVFAN 2657 P.vivax. 99.1 IAVNRNLVTQVTPCDP-ALKTKAVRSTHAPCVFFPDNSYKTAQSIEDFNKENLPLFVFAN 2796 :**: . * *.** *.:. :* *: **.: ** * : **.:* ***:::**
Figure 6.5 NetPhos 2.0™ prediction of protein kinase phosphorylation sites on enzyme domains of mammals and Plasmodium acetyl-CoA carboxylases. (A) Alignment produced by ClustalW™
algorithm showing phosphorylated residues (highlighted) in the biotin carboxylase (BC) and the carboxyl transferase (CT) domains. The residue between the +4 and –4 positions of the phosphorylated residues are underlined. The numbers in bold represents the probability score (%). (B).
Cartoon structure of the multi-domain acetyl-CoA carboxylase illustrating the positions of the predicted phosphorylation sites. Broken and double lines indicate a position 100% conserved in Plasmodium and across all organisms, respectively. Open and closed circles indicate a serine and a threonine protein kinase sites, respectively. * indicates 100% conserved residue.
B
N C
P P P
Biotin carboxylase Biotinoyl Carboxyl transferase
A
BC
CT N1
N2
N3
The Ramachandran analysis showed that 83.3% of non-glycine and non-proline residues in PyBCCP-model have conformational angles (φ andψ) in the most favoured regions of the Ramachandran plot (Figure 6.6). This is comparable to EcBCCP+ with 86.6%. 2.8% of the residues in PyBCCP-model fall in the disallowed region of the Ramachandran plot, while no residues in Ec-BCCP+ falls in the disallowed region.
Figure 6.6 Ramachandran analyses of the homology modelling of the biotinoyl proteins by ProCheck™. The Ramachandran plots were derived after energy minimization of (A) EcBCCP+ (B) PyBCCP-model. The different coloured regions indicate “most favoured” (red), “additionally allowed’
(yellow), “generously allowed” (light yellow) and “disallowed’ (white).
The PyBCCP-model is made of seven β-sheets, one turn and five loops, while EcBCCP+ has an extra β-sheet (eight β-sheet), six loops and one turn. The predicted secondary structure can be classified as a β-sheet because it lacked α-helix secondary structures (Figure 6.7).
EcBCCP+ has the “protruding thumb” structure connecting β2 to β3; this is consistent with the experimentally resolved structure (Athappilly and Hendrickson, 1995) as well as previously determined computer models of the E. coli biotinoyl protein (Chapman-Smith et al., 2001;
Cronan, 2001; Reche et al., 1998; Reche and Perham, 1999; Streaker and Beckett, 2006). A similar structure is also present in PyBCCP-model. Figure 6.8 shows that the β-sheets in PyBCCP-model and EcBCCP+ are completely superimposable, except the extra β-sheet on the EcBCCP+.
A B
Figure 6.7 Predicted three dimensional molecular models of biotinoyl protein. (A) EcBCCP+and (B) PyBCCP-model. The structure is coloured according to secondary structure succession using the spdbv programme. Blue secondary structure is at the N-terminus and red secondary structure is at the C-terminus of the protein. The residues represent the conserved MKM biotinylation motifs situated at β4→ β5 turn.
The PyBCCP-model was superimposed (Figure 6.9) on EcBCCP+, and the root mean square deviation of the α-carbon was deduced to be 0.22 Å. Figure 6.9 shows that the two models are Figure 6.8 Superimposed image PyBCCP-model and EcBCCP+ β-sheets. The β-sheets with yellow (EcBCCP+) and blue (PyBCCP-model) coloured β-sheets are completely superimposed, while the red β-sheet is the EcBCCP+ extra sheet
N-terminal
C-terminal
A
N-terminal
C-terminal
B
β3
Protruding thumb
β1
β2
β4
β5
β6
β8
Protruding thumb
β1
β2
β4
β5
β6
β7
β3
β7
virtually super-imposable. Although the “protruding thumb” loop structure connecting β2 to β3 of the PyBCCP-model protrudes out side the molecular surface of the positive control 3D model, as shown (arrow) in Figure 6.9A.
Figure 6.9 Superimposed image of E. coli biotinoyl protein positive control model and P. yoelii biotinoyl domain model. (A) Ribbon structure of PyBCCP-model superimposed in the molecular surface map of EcBCCP+. (B) Ribbon structure of EcBCCP+ superimposed with the backbone strcurure of PyBCCP-model.
The E. coli biotin-binding motif is identical to the Plasmodium acetyl-CoA carboxylase biotin-binding motif with a consensus sequence, VEAMKM. The lysine residue is the biotinoyl lysine (Beckett et al., 1999; Blanchard et al., 1999a; Chapman-Smith et al., 1999;
Cronan, 2002). Studies have shown that the E. coli biotin protein ligase interacts with the glutamic acid residue (Glu115) within the consensus sequence, as well as Glu147 (Chapman- Smith et al., 1999; Chapman-Smith et al., 2001; Polyak et al., 2001; Solbiati et al., 2002;
Streaker and Beckett, 2006). In P. yoelii, Lys1152 is at the equivalent position of the E. coli Glu147. Figure 6.10 shows that the side chain of Glu147 sticks out of the surface of EcBCCP+
(Figure 6.10A), with a similar orientation to Lys1152 in the PyBCCP-model (Figure 6.10B).
However Glu115 as well its equivalent in the PyBCCP-model is buried inside the structure.
The consensus biotinylation motif MKM forms a knob-like structure on the molecular surface B
A
of both predicted biotinoyl 3D structures. This is consistent with previous studies on the orientation of the MKM motif with respect to the whole molecule (Cronan, 2001; Reche and Perham, 1999; Solbiati et al., 2002; Yao et al., 1999). The VEAMKM and E115/K1152 residues are super-imposable (Figure 6.11) with a root mean square deviation (between PyBCCP- model and EcBCCP+) of 0.03 Å and 0.31 Å of equivalent α-carbon atoms and equivalent side chains, respectively.
A
E147 B
E147
C
K1152 D
K1152
Figure 6.10 Position of the biotinylation motif with respect to the molecular surface of the biotinoyl protein models. (A and B) EcBCCP+ and (C and D) PyBCCP-model. The backbone and side chain structures of the amino-acid residues are coloured green. A and C are in a similar orientation. B and D are in a similar orientation. The selected residues are coloured as follows: Val and Ala (cyan), Glu and Lys1152 (red), Met (yellow), biotinyl-Lys (blue).
6.2.6 Homology modelling of mutant forms of the biotinoyl domain of P. yoelii acetyl-