COASY method
Expression Data
The expression of CoAsy in mouse tissues was determined using SymAtlas (Genomics Institute of Novartis Research Foundation, 2007). SymAtlas was also used to determine expression in mice of the other enzymes in the CoA synthesis pathway, including pantothenate kinase (Pank2), phosphopantothenoylcysteine synthase (6330579B17Rik) and phosphopantothenolycysteine decarboxylase (8430432M10Rik).
Domain Identification
Lalign was used to determine which residues of the full length CoAsy protein were present on chain A (Pearson, 2007).
NCBI Entrez Protein (NCBI, 2002) was used to predict domains of full-length CoAsy, and PFAM was used to predict domains of CoAsy chain A (Sanger Institute, 2005). Results from PFAM for Chain A were compared with those obtained by entering the 2f6rA PDB file into Profunc (Laskoski et. al., 2005).
Structural Conservation of Structurally Related Proteins
A DALI (Holm & Sander, 1993) search was performed on the Mus musculus Coenzyme A Synthase sequence to gather structurally related proteins (Dali output file). Fourteen proteins with a structure relation Z score greater than nine (Table 2) had their PDB entries collected and entered into VMD (Humphrey, Dalke, & Schulten, 1996). Using the STAMP structural alignment tool the molecules were aligned (Russell & Barton, 1992) and the resulting alignment was then coloured by Q per residue (structural Identity) using the MultiSeq (Roberts, Eargle, Wright, & Luthey-Schulten, 2006) tool for VMD (Figure 12). The proteins were then hidden such that only the 2F6R PDB molecule was visible, and the start and end zones of structurally related areas were then labeled. The resulting model can be seen in Figure 11.
Table 2
PDB/chain identifiers and structural alignment statistics for DALI (Holm & Sander, 1993) search
NR. STRID1 STRID2 Z RMSD LALI LSEQ2 %IDE REVERS PERMUT NFRAG TOPO PROTEIN 1: 3024-A 2f6r-A 40.4 0.0 230 230 100 0 0 1 S TRANSFERASE bifunctional coenzyme a synthase fragment 2: 3024-A 1jjv-A 21.1 2.1 187 194 22 0 0 5 S TRANSFERASE dephospho-coa kinase (dephosphocoenzyme a 3: 3024-A 1tev-A 12.7 2.3 154 194 14 0 0 11 S TRANSFERASE ump-cmp kinase (cytidylate kinase, deoxycy 4: 3024-A 1y63-A 11.9 2.6 143 168 15 0 0 10 S UNKNOWN FUNCTION lmaj004144aaa pr 5: 3024-A 1zin 11.1 2.9 147 217 18 0 0 11 S PHOSPHOTRANSFERASE adenylate kinase (adk)(bacillus) 6: 3024-A 1xrj-A 10.9 3.3 146 211 14 0 0 12 S TRANSFERASE uridine-cytidine kinase 2 (uck 2, uridine) 7: 3024-A 5tmp-A 10.7 3.2 154 210 9 0 0 13 S TRANSFERASE thymidylate kinase (escherichia coli) A. 8: 3024-A 1shk-A 10.3 3.0 140 158 12 0 0 9 S TRANSFERASE shikimate kinase biological_unit(erwinia) 9: 3024-A 1nks-A 10.1 3.6 149 194 14 0 0 12 S KINASE adenylate kinase (atp:ampphosphotransferase) 10: 3024-A 2jaq-A 10.0 2.8 108 189 17 0 0 10 S TRANSFERASE deoxyguanosine kinase (deoxyadenosine kina 11: 3024-A 1knq-A 9.6 3.0 141 171 15 0 0 13 S TRANSFERASE gluconate kinase (thermoresistant glucono) 12: 3024-A 1gky 9.6 2.3 111 186 13 0 0 11 S TRANSFERASE Guanylate kinase complex with guanosine 13: 3024-A 1jag-A 9.5 2.9 152 229 10 0 0 15 S 14: 3024-A 1qhs-A 9.4 3.3 139 178 14 0 0 14 S TRANSFERASE chloramphenicol phosphotransferase (cpt) 15: 3024-A 1via-A 9.1 3.0 132 159 15 0 0 13 S TRANSFERASE shikimate kinase(campylobacter jejuni) b. 16: 3024-A 1bif 9.0 3.2 133 432 9 0 0 12 S BIFUNCTIONAL ENZYME 6-phosphofructo-2-kinase fructose- 17: 3024-A 1dek-A 8.9 2.6 110 241 14 0 0 10 S PHOSPHOTRANSFERASE deoxynucleoside monophosphate kinas 18: 3024-A 3tmk-A 8.8 2.8 143 216 6 0 0 15 S KINASE thymidylate kinase biological_unit 19: 3024-A 1cke-A 8.5 3.2 146 212 12 0 0 13 S TRANSFERASE cytidine monophosphate kinase (ck, mssa) 20: 3024-A 1ltq-A 8.2 2.5 115 286 20 0 0 11 S TRANSFERASE polynucleotide kinase (pnk, polynucleotide 21: 3024-A 1g3u-A 8.2 3.1 134 208 11 0 0 14 S TRANSFERASE thymidylate kinase (tmk)(mycobacterium) 22: 3024-A 1d6j-A 8.0 3.2 128 177 16 0 0 14 S TRANSFERASE adenosine-5'phosphosulfate kinase (aps kin 23: 3024-A 1yr6-A 7.9 3.2 142 248 11 0 0 14 S HYDROLASE atp(gtp)binding protein fragment (pab0955 ge 24: 3024-A 1esm-A 7.6 2.7 129 311 15 0 0 11 S TRANSFERASE pantothenate kinase (pank) (eschericha c) 25: 3024-A 1xjq-B 7.4 3.3 128 590 19 0 0 14 S TRANSFERASE bifunctional 3'-phosphoadenosine 5'- phosp 26: 3024-A 1t3l-A 7.3 5.2 150 306 14 0 0 17 S TRANSPORT PROTEIN dihydropyridine-sensitive l-type, ca 27: 3024-A 1qhi-A 7.0 3.5 149 300 11 0 0 13 S TRANSFERASE thymidine kinase (tk) biological_unit
Once the structural conservation had been performed the MultiSeq (Roberts, Eargle, Wright, & Luthey-Schulten, 2006) tool was again used to colour the molecules, this time using the sequence identity option. The model was relabeled with highly conserved residues and the results can be seen in Figure 10.
MOTIF identification
MOTIFs were identified using the PROSITE motif search service (Bairoch, Bucher, & Hofmann, 1997) on the Mus musculus residue sequence. The identified MOTIF patterns can be found in Table 1
Domain Surface Map and Structural Comparison of Surface Maps
Locations of domains on the surface of the Coenzyme A Synthase protein were collected from Mus musculus sequence entry (NCBI, 2007). The residue ranges for these were highlighted and recoloured on the 2F6R PDB entry using PyMol (Delano, 2007). The results can be seen in Figure 4. PyMol was also used to model the structural surface map comparisons of 2F6R and 1JJV PDB entries with colouring by secondary structure. ATP ligands were placed on the 2F6R PDB entries by aligning 1JJV PDB entry with 2F6R and then hiding all of 1JJV except the attached ATP atoms (Figure 5).
B-Factor Surface Map
The 2F6R PDB entry was modeled using PyMol with colouring set to b-factor. The results can be seen in Figure 7.
Electrostatic Surface Potential Map
The electrostatic potential map was calculated for the PDB 2F6R entry using APBS (Baker, Sept, Joseph, Holst, & McCammon, 2001) and displayed in PyMol (Delano, 2007). ACO and ATP ligands were added by aligning 1JJV and 2F6R PDB entries and then hiding all but the ligands themselves Figure 6.
Surface Binding Clefts
The 2F6R PDB entry was supplied to Jmol (SourceForge, 2007) through the PDSum website (EMBL EBI, 2005) with options set to view all clefts. The resulting model can be seen in Figure 13.
Hydrophobicity Model
The hydrophobicity mapped CoAsy (2F6R PDB) ribbon structure was modeled in MBT Protein Workshop (Moreland, Gramada, Buzko, Zhang, & Bourne, 2005) with hydrophobic set as its colouring for ribbon structures. The results can be viewed in Figure 8.
Phylogenetic Tree
NCBI’s BLAST search was performed using the human bifunctional phosphopantetheine adenylyl transferase/dephospho CoA kinase (gi:46048207) as the query sequence. The sequences were aligned with ClustalX and edited to reduce gapping in the alignment. A final multiple sequence alignment was performed on ClustalX with 53 sequences (Figure 14). Phylip programmes were used to produce a tree from this multiple sequence alignment(Figure 15). Editing of the tree included elimination of some species for clarity and the addition of bootstrap indicators on branch lengths.
Abstract | Introduction | Results | Discussion | Conclusion | Method | References
