Structure ERp18

Methods

Study of the ERp18 protein (ID = 1sen-A) began by obtaining its file from the Protein Data Bank (PDB) and visualising the 3D structure of the protein using PyMOL. It was known from study about its function that ERp18 contains a CXXC motif[1], characteristic of all thiol-disulfide oxidoreductases - this motif was identified as residues ⁶⁶CGAC⁶⁹ of the amino acid sequence of mature ERp18. These catalytic cysteine residues were found to be localised to the surface of the protein and thus were predicted to form the catalytic site. Next, conserved sequences identified by a ClustalX multiple alignment from evolution study were highlighted on the protein. PDB ProteinWorkshop 3.4 was used to map conformation types and hydrophobicity of ERp18. Following this, searches were made with PDBsum, SCOP, and Prosite for more information about the folding and domain-annotation of the protein. A search with DALI revealed a list of structurally related proteins, from which a number of comparisons were made. Secondary structures of 1sen-A and other proteins identified via the DALI search were provided by PDBsum. After completing these fundamental protein structure searches, published papers were consulted and their findings on ERp18 were compared and integrated with our findings.

Results

Figure 1: PyMOL image displaying the catalytic cysteine residues (66,69) of ERp18. These residues fit the CXXC motif ubiquitous of all thiol–disulfide oxidoreductases.

Figure 2: PyMOL image (front view) displaying conserved surface residues, which surround the location of the cysteine residues. These conserved surface residues are hypothesised to form the active site of ERp18. Conserved regions on this front view are shown in pink, green, yellow, and dark blue. These regions correspond to continuous segments of highly conserved amino acids.

Pockets around the predicted catalytic site were identified as potential binding sites by Q-SiteFinder and CASTp, being in the top hits for these ligand binding site searches.

Figure 3: PyMOL image (rear view) displaying another area of conservation (shown as purple and turqoise regions). This area is not believed to have any catalytic activity; nevertheless, it is likely to have an important function - perhaps as a targeting signal or as an area for association with another protein, even another ERp18 protein to form a homodimer.

Figure 4: PyMOL image displaying the conserved sections of a ribbon diagram of ERp18. Notice the conservation of the three core beta sheets - probably vital for maintaining stability and correct folding of the protein, as well as correct catalytic activity.

SCOP Summary

Protein: Thioredoxin-like protein p19, TLP19 from Human (Homo sapiens) [TaxId: 9606] Lineage:

  1. Root: scop
  2. Class: Alpha and beta proteins (a/b) [51349]
     Mainly parallel beta sheets (beta-alpha-beta units)
  3. Fold: Thioredoxin fold [52832]
     core: 3 layers, a/b/a; mixed beta-sheet of 4 strands, order 4312; strand 3 is antiparallel to the rest
  4. Superfamily: Thioredoxin-like [52833]
  5. Family: Thioltransferase [52834]
  6. Protein: Thioredoxin-like protein p19, TLP19 [110604]
  7. Species: Human (Homo sapiens) [TaxId: 9606] [110605]

Figure ?: Image of ERp18 identifying each conformation type. Turn = Light Blue, Coil = Red, Helix = Green, Strand = Dark Blue

Figure ?: Hydrophobicity mapping of ERp18. Most hydrophobic = red, least hydrophobic = blue. Image produced using PDB ProteinWorkshop 3.4.

Dali Summary

   No:  Chain   Z    rmsd lali nres  %id PDB  Description
  1:  1sen-A 31.6  0.0  134   134  100 PDB  MOLECULE: THIOREDOXIN-LIKE PROTEIN P19;                              
  2:  3f9u-A 13.0  2.2  114   145   12 PDB  MOLECULE: PUTATIVE EXPORTED CYTOCHROME C BIOGENESIS-                 
  3:  3f9u-B 12.3  2.2  114   148   12 PDB  MOLECULE: PUTATIVE EXPORTED CYTOCHROME C BIOGENESIS-                 
  4:  2fwe-A 11.7  2.4  107   122   21 PDB  MOLECULE: THIOL:DISULFIDE INTERCHANGE PROTEIN DSBD;                  
  5:  2fwf-A 11.6  2.4  107   123   21 PDB  MOLECULE: THIOL:DISULFIDE INTERCHANGE PROTEIN DSBD;                  
  6:  1se1-C 11.1  2.1  103   231   22 PDB  MOLECULE: THIOL:DISULFIDE INTERCHANGE PROTEIN DSBD;                  
  7:  1vrs-F 11.1  2.1  103   118   22 PDB  MOLECULE: THIOL:DISULFIDE INTERCHANGE PROTEIN DSBD;                  
  8:  1vrs-D 11.1  2.2  102   118   22 PDB  MOLECULE: THIOL:DISULFIDE INTERCHANGE PROTEIN DSBD;                  
  9:  1se1-A 11.1  2.2  102   239   22 PDB  MOLECULE: THIOL:DISULFIDE INTERCHANGE PROTEIN DSBD;                  
 10:  1ep7-B 10.9  2.4  102   112   13 PDB  MOLECULE: THIOREDOXIN CH1, H-TYPE;                                   
 11:  2fwg-A 10.9  2.7  106   122   22 PDB  MOLECULE: THIOL:DISULFIDE INTERCHANGE PROTEIN DSBD;                  
 12:  2fwh-A 10.9  2.2  102   117   23 PDB  MOLECULE: THIOL:DISULFIDE INTERCHANGE PROTEIN DSBD;                  
 13:  1uc7-A 10.8  2.2  104   124   22 PDB  MOLECULE: THIOL:DISULFIDE INTERCHANGE PROTEIN DSBD;                  
 14:  1ep8-B 10.8  2.5  103   112   13 PDB  MOLECULE: THIOREDOXIN CH1, H-TYPE;                                   
 15:  1ep8-A 10.7  2.5  103   112   13 PDB  MOLECULE: THIOREDOXIN CH1, H-TYPE;                                   
 16:  2ju5-A 10.6  2.2  105   144   17 PDB  MOLECULE: THIOREDOXIN DISULFIDE ISOMERASE;                           
 17:  1ep7-A 10.6  2.6  103   112   13 PDB  MOLECULE: THIOREDOXIN CH1, H-TYPE;                                   
 18:  1vrs-E 10.6  2.2  102   118   22 PDB  MOLECULE: THIOL:DISULFIDE INTERCHANGE PROTEIN DSBD;                  
 19:  1uc7-B 10.5  2.3  104   124   23 PDB  MOLECULE: THIOL:DISULFIDE INTERCHANGE PROTEIN DSBD;                  
 20:  3d22-A 10.4  2.8  108   129   13 PDB  MOLECULE: THIOREDOXIN H-TYPE;                                        
 21:  3d21-A 10.4  2.5  101   111   16 PDB  MOLECULE: THIOREDOXIN H-TYPE;                                        
 22:  3d21-B 10.3  2.5  101   111   16 PDB  MOLECULE: THIOREDOXIN H-TYPE;                                        
 23:  3fk8-A 10.2  2.6  106   131   17 PDB  MOLECULE: DISULPHIDE ISOMERASE;                                      
 24:  2vlv-B 10.2  2.3   99   113   22 PDB  MOLECULE: THIOREDOXIN H ISOFORM 2.;                                  
 25:  1se1-B 10.2  2.2  102   243   22 PDB  MOLECULE: THIOL:DISULFIDE INTERCHANGE PROTEIN DSBD;                  
 26:  2vlu-B 10.2  2.3  100   112   22 PDB  MOLECULE: THIOREDOXIN H ISOFORM 2.;                                  
 27:  2vm1-A 10.2  2.3   99   110   16 PDB  MOLECULE: THIOREDOXIN H ISOFORM 1.;                                  
 28:  2vlv-A 10.1  2.4  100   111   22 PDB  MOLECULE: THIOREDOXIN H ISOFORM 2.;                                  
 29:  2vm2-A 10.1  2.3   99   109   16 PDB  MOLECULE: THIOREDOXIN H ISOFORM 1.;                                  
 30:  2vlt-B 10.1  2.4  100   110   22 PDB  MOLECULE: THIOREDOXIN H ISOFORM 2.;                                  
 

Figure 5: The top thirty hits from a DALI search with 1sen-A. DALI lists proteins in order of their structural similarity. The protein names displayed are almost entirely thiol:disulfide interchange proteins and thioredoxins (thioredoxins being a related protein to the catalytic domain of thiol:disulfide interchange proteins), with the exception of 'putative exported cytochrome c biogenesis-related protein' for 2 and 3. However, hits 2 and 3 contain the CXXC motif and are therefore likely to be members of the thioredoxin family. Interestingly, the percent identification (%id) of these hits ranges between 12-23% - quite low considering the degree of structural homology as assigned by the rmsd value, for which structure similarity is considered significant if the value is less than 3.5.

PDBsum

PDB id: 1sen Name: Structural genomics, unknown function Title: Endoplasmic reticulum protein rp19 o95881

Structure: Thioredoxin-like protein p19. Chain: a. Synonym: endoplasmic reticulum protein erp19. Engineered: yes

Source: Homo sapiens. Human. Organism_taxid: 9606. Gene: tlp19. Expressed in: escherichia coli. Expression_system_taxid: 562. Other_details: the protein was cloned, expressed and purified by the secsg human protein production group (t.A. Dailey, m. Mayer) under the direction of h.A. Dailey.

UniProt: O95881 (TXD12_HUMAN) SAS Seq: 172 a.a. Struc: 135 a.a. Key: PfamB domain Secondary structure CATH domain

Enzyme class: E.C.1.8.4.2 [IntEnz] [ExPASy] [KEGG] [BRENDA]

Reaction: 2 glutathione + protein-disulfide = glutathione disulfide + protein- dithiol (see diagram below)

Resolution: 1.20Å

R-factor: 0.162

R-free: 0.183

Authors: Z.-J.Liu,L.Chen,W.Tempel,A.Shah,D.Lee,T.A.Dailey,M.R.Mayer, J.P.Rose,D.C.Richardson,J.S.Richardson,H.A.Dailey,B.-C.Wang Southeast Collaboratory For Structural Genomics (Secsg)

Key ref: z.-j.liu et al. Endoplasmic reticulum protein Rp19. To be Published, xsi:nil="true" />.

Date: 17-Feb-04

Release date: 13-Jul-04

Related entries: O95881 related db: targetdb

Figure 5: Secondary Structure of 1sen produced by PDBsum

Figure 6: Secondary Structure of 3f9u produced by PDBsum

Discussion

Localisation of the CXXC motif to a highly conserved surface region indicated that it forms the active site. This was supported by bioinformatics programs such as Q-SiteFinder and CASTp which predicted ligand binding sites at the hypothesised catalytic site. Other conserved areas such as the second surface region and the internal B-sheets are most likely indicative of their role in correct conformation and activity of the protein. The conformation type and hydrophobicity mapping of ERp18 provided by PDB ProteinWorkshop 3.4 supports the crystal structure conformation. The search results from SCOP, PDBsum, and Prosite identified ERp18 as a thioredoxin protein, which is consistent with DALI search. The literature identifies ERp18 as a homodimer, with a single protein having a molecular weight of 16.4 kDa[1]. Although the ⁶⁶CGAC⁶⁹ residues correspond to the CXXC motif of the thioredoxin superfamily, the ⁶⁶CGAC⁶⁹ residue sequence is unusual[2]. This, as well as other structurally conserved features, makes ERp18 a novel member of the thioredoxin superfamily[2]. This structural analysis supports the function of ERp18 as a thioredoxin and provides grounds for further elucidation of ERp18's function and evolutionary history.

References

1. Jeong, W., Lee, D., Park, S., & Rhee, S.G., (2008) "ERp16, an Endoplasmic Reticulum-resident Thiol-disulfide Oxidoreductase" The Journal of Biological Chemistry, Vol. 283, No. 37, pp. 25557–25566.

2. Liu, F., Rong, Y., Zeng, L., Zhang, X., & Han, Z.,(2003) "Isolation and characterization of a novel human thioredoxin-like gene hTLP19 encoding a secretory protein" Gene, Volume:315, pp. 71–78.

3. Alanen, H. I. et al. (2003) "Functional Characterization of ERp18, a New Endoplasmic Reticulum-located Thioredoxin Superfamily Member" The Journal of Biological Chemistry, Vol. 278, No. 31, Issue of August 1, pp. 28912–28920.