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. Fourteen proteins with a structure relation Z score greater than nine 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. 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 6.

1. 1. SUMMARY: PDB/chain identifiers and structural alignment statistics

 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    STRUCTURAL GENOMICS, UNKNOWN FUNCTION         lmaj004144aaa pr
  5: 3024-A 1zin   11.1  2.9  147   217   18      0      0    11 S    PHOSPHOTRANSFERASE         adenylate kinase (adk)         (bacillus s
  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:amp phosphotransferase) b
 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 mo
 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         (saccharomy
 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         (he

Sequence Conservation of Structurally related proteins

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 3.

MOTIF identification

MOTIF’s 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 2. 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 4.

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Abstract | Introduction | Results | Discussion | Conclusion | Method | References

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