Hemochromatosis: Sequence based predictions
Contents
Secondary Structure
In this task, the secondary structure of proteins is predicted using the programs reProf and PsiPred. The results are then compared to the DSSP[1] secondary structure assignments of corresponding crystal structures from the PDB.
<figtable id="sequences">
Uniprot | PDB | |||||
---|---|---|---|---|---|---|
UID | Name | Length | ID | Resolution [A] | Chain | Length |
Q30201 | Hereditary hemochromatosis protein | 348 | A16Z | 2.60 | A | 275 |
P10775 | Ribonuclease inhibitor | 456 | 2BNH | 2.30 | A | 457 |
Q9X0E6 | Divalent-cation tolerance protein CutA | 101 | 1VHF | 1.54 | A | 113 |
Q08209 | CAM-PRP catalytic subunit | 521 | 1M63 | 2.80 | A | 372 |
</figtable>
For this purpose, the crystal structures listed in <xr id="sequences"/> were selected for comparison. The first priority for selection was to get the protein in its native state and not bound to another molecule. The other criteria were the quality of the structure and the alignment to the protein sequence (having one continuous segment).
In the next step, the output of the prediction programs and the DSSP assignments have to be made comparable. DSSP assigns 8 different classes of secondary structure, whereas reProf and PsiPred only predict helix (H), sheet (E) and loop (L or C). The exact mappings can be found in the lab journal.
To assess the quality of the prediction, the class specific accuracy, coverage and F1-measure were used along with the Q3 and SOV3 <ref>Zemla. A, et al.;A Modified Definition of Sov, a Segment-Based Measure for Protein Secondary Structure Prediction Assessment; PROTEINS 34:220–223 (1999)</ref> measures. The Q3 is defined as follows:
Reprof takes as input either sequences or PSSMs. Therefore, PSSMs were generated by querying the HFE (Q30201) sequence against the big_80, SwissProt and PDB databases. The tool of choice for this was PsiBlast with standard parameters (2 iterations and e-value cutoff of 0.002).
<figtable id="prediction quality">
Prediction methods | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Reprof + Sequence | Reprof + Big80 | Reprof + SwissProt | Reprof + PDB | Psipred | |||||||||||
Q3 | 0.76 | 0.81 | 0.86 | 0.84 | 0.84 | ||||||||||
SOV3 | 0.66 | 0.75 | 0.84 | 0.84 | 0.73 | ||||||||||
Acc | Cov | F1 | Acc | Cov | F1 | Acc | Cov | F1 | Acc | Cov | F1 | Acc | Cov | F1 | |
H | 0.63 | 0.33 | 0.44 | 0.74 | 0.48 | 0.59 | 0.84 | 0.65 | 0.74 | 0.85 | 0.52 | 0.64 | 0.98 | 0.77 | 0.86 |
C | 0.57 | 0.69 | 0.63 | 0.63 | 0.75 | 0.68 | 0.68 | 0.81 | 0.74 | 0.64 | 0.83 | 0.72 | 0.61 | 0.95 | 0.74 |
E | 0.79 | 0.63 | 0.70 | 0.81 | 0.84 | 0.83 | 0.89 | 0.86 | 0.87 | 0.88 | 0.85 | 0.86 | 0.94 | 0.55 | 0.69 |
</figtable>
The deciding criteria for the Reprof method choice were the Q3, SOV3 and F1-measures. For the Q3 and the SOV3, the SwissProt PSSM combination performed best and the F1-measures were also reasonable (see <xr id="prediction quality"/>). Therefore, this method was chosen for all future predictions.
The DSSP assignments together with the ReProf and PsiPred predictions for each of the four proteins can be found here.
<figtable id="comparison">
Protein | Method | Q3 | SOV3 |
---|---|---|---|
Q08209 | ReProf | 0.83 | 0.78 |
Psipred | 0.87 | 0.79 | |
Q30201 | ReProf | 0.86 | 0.84 |
Psipred | 0.84 | 0.73 | |
P10775 | ReProf | 0.91 | 0.93 |
Psipred | 0.93 | 0.94 | |
Q9X0E6 | ReProf | 0.75 | 0.65 |
Psipred | 0.89 | 0.86 |
</figtable>
<xr id="comparison"/> shows a comparison of the ReProf and PsiPred prediction quality. The predictions were compared to the DSSP assignment of the corresponding pdb structures. While SOV3 and Q3 do not always agree which of the two methods performs better, they do so most of the time. Nevertheless, it was decided that SOV3 is to be trusted more than Q3, because it takes into account the per segment accuracy rather than just the per residue accuracy. Thus, PsiPred outperforms reprof in 3 out of 4 cases. It is also notable, that the SOV3 values range from 0.65 for Q9X0E6 (101 residues) to 0.94 for P10775 (456 residues). So the performance of the method depends on the length of the protein and the protein itself, but PsiPred performed best overall.
Disorder
We searched DisProt for the best matches to the four proteins, but there was only one direct match for Q08209. We used the PsiBlast and Smith Waterman search to find matches for the other three proteins. The first best matches are listed in <xr id="disprot id"/>.
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<figtable id="disprot id">
UID | DisProt ID | SeqID | E-value | Method |
---|---|---|---|---|
Q30201 | DP00670 | 0.29 | 2e-30 | psiblast |
P10775 | DP00554 | 0.4 | 7e-30 | psiblast |
Q9X0E6 | DP00564 | 0.25 | 0.36 | smith-waterman |
Q08209 | DP00092 | - | - | direct match |
</figtable>
IUPred is a disorder prediction server that predicts three different types:
- long disorder
- short disorder
- global (globular not disordered domains)
When predicting the long and short types of disorder, the output contains a likelihood of disorder for each residue and the globular domain prediction contains the start and end positions.
MetaDisorder (MD) is a predictor that is based on several prediction programs from predict protein. It combines those predictions into one value for each residue, which is the likelihood of being part of a disordered region.
<figure id=disorder pred1">
</figure>
<figure id="disorder pred2">
</figure>
All predictions are combined into plots in <xr id="disorder pred2"/>. A residue is predicted to be disordered if its likelihood is higher than 0.5, indicated by the gray horizontal line.
Unfortunately we could not find good matches in the DisProt database, apart from the direct match for Q08209. The matches for Q30201 and Q9X0E6 have a sequence identity below 30% and the e-value for Q9X0E6 is too high with 0.36. But the hit for P10775 with a sequence identity of 40% and an e-value of 7e-30 can possibly be used for an annotation transfer. Nevertheless, we included the annotations for the Q30201 and Q9X0E6 matches for the sake of completeness.
All predictions for the HFE protein Q30201 show nearly no disordered regions. The MD tendencies are always below 0.5 and there is only a short region after residue 250 where IUPred predicted long and short disordered regions. The DisProt annotation contains a small disordered region after residue 150, but this region can be neglected due to the low sequence identity to the corresponding DisProt protein. IUPred predicted, that the HFE protein consists of a single globular not disordered domain. In summary, the HFE protein is correctly predicted to be not disordered.
P10775 was also predicted to be not disordered by all methods. The region of the DisProt entry DP00465 that matched P10775 does not contain a disordered region, so the predictions are also right in this case.
The DisProt annotation of the entry DP00564 MetaDisorder likelihoods are higher, always above 0.3. But we trust DisProt the most, since all IUPred predictions, including the global, agree that Q9X0E6 is not disordered and DP00564 has an e-value of 0.36.
In contrast to the first three proteins, Q08209 is clearly disordered. The DisProt annotation of the protein contains several disordered regions near the C-terminus. IUPred's global prediction agrees that the C-terminus is disordered, and also the long, short and MetaDisorder predictions are above 0.5.
All N and C-termini of the proteins have short disorder tendencies with values above 0.5, but since the ends of all protein chains can be characterized as disordered to some extent, we would not count that as false predictions.
Transmembrane Helices
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</css> <figtable id="pdb structures">
Uniprot | PDB | |||||
---|---|---|---|---|---|---|
UID | Name | Length | ID | Resolution [A] | Chain | Length |
Q30201 | Hereditary hemochromatosis protein | 348 | A16Z | 2.60 | A | 275 |
P35462 | D(3) dopamine receptor | 400 | 3PBL | 2.89 | A | 400 |
Q9YDF8 | Voltage-gated potassium channel | 295 | 1ORQ | 3.20 | C | 223 |
1ORS | 1.90 | C | 132 | |||
P47863 | Aquaporin-4 | 323 | 2D57 | 3.20 | A | 301 |
</figtable>
HFE
The HFE protein is a single pass membrane protein and thus located at the membrane. Nevertheless, there are no entries in the OPM and PDBTM databases. A single pass membrane protein is a protein that has its N-terminus in the extracellular space and the C-terminus inside the mebrane. The N-terminal signal sequence is cleaved off after the translocation.
<figtable id="TM hfe">
HFE (Q30201) | |||
---|---|---|---|
Memsat | Polyphobius | OPM | PDBTM |
- | - |
</figtable>
Memsat predicts one transmembrane helix for the HFE protein and also a signal peptide consisting of the first 12 N-terminal residues (see <xr id="TM hfe"/>). The signal peptide is actually longer, it spans the first 22 residues (Uniprot), but the transmembrane helix from residue 306 to 329 is correctly predicted (307 to 330 in Uniprot).
Polyphobius prediction for HFE:
ID sp|Q30201|HFE_HUMAN FT SIGNAL 1 25 FT REGION 1 7 N-REGION. FT REGION 8 19 H-REGION. FT REGION 20 25 C-REGION. FT TOPO_DOM 26 304 NON CYTOPLASMIC. FT TRANSMEM 305 329 FT TOPO_DOM 330 348 CYTOPLASMIC.
Polyphobius is even more accurate in its prediction. The predicted signal sequence ranges from residue 1 to 25 and the transmembrane region from 305 to 329, which is only slightly shifted. The graphical prediction plot in <xr id="TM hfe"/> shows the transmembrane region in grey and the signal peptide in red. The cytoplasmic region is indicated by the green line and the non cytoplasmic region in blue.
Both prediction programs were able to predict the transmembrane helix topology of the HFE protein correct.
D(3) dopamine receptor
A D(3) dopamine receptor is a located in the cell membrane of neurons. It is a multi-pass membrane protein that spans the membrane more than once.
<figtable id="TM P35462">
D(3) dopamine receptor (P35462) | |||
---|---|---|---|
Memsat | Polyphobius | OPM | PDBTM |
</figtable>
The OPM and PDTM databases contain membrane assignments for crystal structures of known transmembrane proteins. Those reference assignments for 3PBL are shown in <xr id="TM P35462"/> In the OPM visualisation, the extracellular side of the membrane is marked with red and the cytoplasmic side with blue. The N-terminus of the protein is assigned to be extracellular. PDBTM colors the cytosolic part of the protein in red and the extracellular part in blue. Transmembrane helices are colored in yellow. Both databases have 7 transmembrane helices assigned to the structure of the D(3) dopamine receptor.
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</css> <figtable id="helices assignment P35462">
# TM helix | OPM | PDBTM |
---|---|---|
1 | 34-52 | 35-52 |
2 | 67-91 | 68-84 |
3 | 101-126 | 109-123 |
4 | 150-170 | 152-166 |
5 | 187-209 | 191-206 |
6 | 330-351 | 334-347 |
7 | 363-386 | 368-382 |
</figtable>
Memsat predicts a signal peptide from positions 1 to 29, however, the Uniprot entry does not contain a signal peptide. Besides, Memsat correclty locates the N-terminus of the sequence in the etracellular space and predicts 6 transmembrane helices in total. The positions of all transmambrane helices graetly overlap with those of the OPM and PDBTM assignments, only the 7th helix is not predicted.
The Polyphobius prediction is the following:
ID sp|P35462|DRD3_HUMAN FT TOPO_DOM 1 29 NON CYTOPLASMIC. FT TRANSMEM 30 55 FT TOPO_DOM 56 65 CYTOPLASMIC. FT TRANSMEM 66 88 FT TOPO_DOM 89 104 NON CYTOPLASMIC. FT TRANSMEM 105 126 FT TOPO_DOM 127 149 CYTOPLASMIC. FT TRANSMEM 150 170 FT TOPO_DOM 171 187 NON CYTOPLASMIC. FT TRANSMEM 188 212 FT TOPO_DOM 213 328 CYTOPLASMIC. FT TRANSMEM 329 352 FT TOPO_DOM 353 366 NON CYTOPLASMIC. FT TRANSMEM 367 386 FT TOPO_DOM 387 400 CYTOPLASMIC.
The location of the N-terminus is correct as non cytoplasmic and all 7 transmembrane helices are predicted at the right positions. The graphcal plot also shows that there is no signal peptide. Thus, Polyphobius was able to determine the toplogy of the D(3) dopamine receptor.
voltage-gated potassium channel
There are two different pdb structures that both match the Q9YDF8 sequence. The annotations in OMP and PDBTM differ a bit, thus we combined the annotations from both structures, see <xr id="helices assignment Q9YDF8"/>.
<figtable id="helices assignment Q9YDF8">
# TM helix | OPM | PDBTM | ||
---|---|---|---|---|
1ORS | 1ORQ | 1ORS | 1ORQ | |
1 | 37-58 | - | 39-62 | 33-64 |
2 | 67-90 | - | 67-87 | 69-92 |
3 | 98-109 | - | 100-119 | - |
4 | 112-121 | - | - | - |
5 | 129-160 | - | 130-154 | - |
6 | - | 165-184 | - | 163-183 |
7 | - | 195-207 | - | 196-212 (re-entrant helix) |
8 | - | 219-237 | - | 221-248 |
</figtable>
The OPM assignment contains eight transmambrane helices for both structures, but PDBTM six transmambrane helices and one loop that is actually a re-entrant helix in the structure. Both databases have the N-termini of the two pdb structures annotated as located in the cytoplasm.
<figtable id="TM Q9YDF8">
Voltage-gated potassium channel (Q9YDF8) | |||
---|---|---|---|
Memsat | Polyphobius | OPM (1ORQ) | PDBTM (1ORQ) |
</figtable>
Memsat predicts six transmembrane helices and one re-entrant helix (see <xr id="TM Q9YDF8"/>. It combines the third and fourth OPM helix into one, but all other helices can be counted as corret. If compared to the PDBTM assignment, all predicted helices are at the right positions, event the re-entrant helix. The location of the C-terminus in the cytoplasm is also correct, but not the N-terminus. It actually is located in the extracellular space and not the cytoplasm.
Polyphobius predicted the following topology:
ID sp|Q9YDF8|KVAP_AERPE FT TOPO_DOM 1 41 NON CYTOPLASMIC. FT TRANSMEM 42 60 FT TOPO_DOM 61 67 CYTOPLASMIC. FT TRANSMEM 68 88 FT TOPO_DOM 89 107 NON CYTOPLASMIC. FT TRANSMEM 108 129 FT TOPO_DOM 130 136 CYTOPLASMIC. FT TRANSMEM 137 157 FT TOPO_DOM 158 162 NON CYTOPLASMIC. FT TRANSMEM 163 184 FT TOPO_DOM 185 195 CYTOPLASMIC. FT TRANSMEM 196 213 FT TOPO_DOM 214 223 NON CYTOPLASMIC. FT TRANSMEM 224 244 FT TOPO_DOM 245 295 CYTOPLASMIC.
Seven transmembrane helices are predicted and the third and fourth OPM helix are also combined into one single helix (108 - 129), as in the Memsat output. Polyphobius does not predict a signal peptide and correcly locates the N and C-termini non-cytoplasm and cytoplasm, which is correct. So, the Polyphobius prediction does not match the PDBTM helices assignments as good as the Memsat prediction does, but the termini are located on the right sides.
Aquaporin-4
Aquaporin 4 is a potein that forms a channel through the cell membrane consisting of six transmambrane helices. The pore allows water molecules to poass the cell membrane quicker than by diffusion. <xr id="helices assignment P47863"/> lists the six transmambrane helices and two re-entrant helices assignments from OPM and PDBTM. Both the N and C-terminus are located in the extracellular space.
<figtable id="helices assignment P47863">
# TM helix | OPM | PDBTM |
---|---|---|
1 | 34-56 | 39-55 |
2 | 70-88 | 72-89 |
3 | 98-107 | 95-106 (re-entrant) |
4 | 112-136 | 116-133 |
5 | 156-178 | 158-177 |
6 | 189-203 | 188-205 |
7 | 214-223 | 209-222 (re-entrant) |
8 | 231-252 | 231-248 |
</figtable>
Both database annotations are very similar and contain the same number of helices, although OPM does not differentiate between transmambrane and re-entrant helices.
<figtable id="TM P47863">
Aquaporin-4 (P47863) | |||
---|---|---|---|
Memsat | Polyphobius | OPM | PDBTM |
</figtable>
Memsat wrongly predicts a signal peptide in the first 20 amino acids, but the six helices and even the re-entrant helices are predicted correct (see <xr id="TM P47863"/>. Polyphobius does predict all four transmambrane helices correct, but it does not predict re-entrant helices:
ID sp|P47863|AQP4_RAT FT TOPO_DOM 1 33 CYTOPLASMIC. FT TRANSMEM 34 58 FT TOPO_DOM 59 69 NON CYTOPLASMIC. FT TRANSMEM 70 91 FT TOPO_DOM 92 114 CYTOPLASMIC. FT TRANSMEM 115 136 FT TOPO_DOM 137 155 NON CYTOPLASMIC. FT TRANSMEM 156 177 FT TOPO_DOM 178 187 CYTOPLASMIC. FT TRANSMEM 188 208 FT TOPO_DOM 209 230 NON CYTOPLASMIC. FT TRANSMEM 231 252 FT TOPO_DOM 253 323 CYTOPLASMIC.
Both termini are also correctly predicted and Polyphobius not predict a signal peptide, in contrast to Memsat.
Summary
Memsat is a very good prediction tool, but it often predicts signal peptides although there are none. But apart from that and one wrong predicted N-terminus localisation, are the predictions very accurate and even re-entrant helices are found and reported. Polyphobius, on the other hand, always predicted the signal peptides and termini localsations right. However, it does not predict re-entrant helices.
Signal Peptides
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Protein Name | HFE_HUMAN | ALBU_HUMAN | LAMP1_HUMAN | AQP4_RAT | |
---|---|---|---|---|---|
UID | Q30201 | P02768 | P11279 | P47863 | |
SP id | 13435 | 22229 | 17551 | - | |
SP annotation | 1-22 | 1-18 | 1-18 | - | |
signalP prediction | 1-22 | 1-18 | 1-18 | no signal peptide | |
For all four proteins, the signalP predictions were completely correct, which is remarkable.
- ALBU_HUMAN, the human serum albumin, is the most abundant protein in human blood plasma. Its main responsibility is the regulation of the osmotic blood pressure and since it is a globular protein, it has no transmembrane helices.
- The lysosome-associated membrane glycoprotein 1 (LAMP1_HUMAN) is a single pass membrane protein that presents carbohydrate ligands to selectins. The selectins are a family transmembrane glycoproteins that are involved in the inflammatory process.
- Aquaporine 4 (AQP4_RAT) is an osmoreceptor that regulates body water balance in rats. It has no signal peptide and thus, the translocation signal is probably only recognizable in the folded structure of the protein, i.e. it is fragmented
across the sequence. Also, it is a membrane protein with 6 transmembrane alpha helices.
GO Terms
GoPet
GO id | Aspect | Confidence | GO term |
---|---|---|---|
GO:0004872 | F | 91% | receptor activity |
GO:0030106 | F | 88% | MHC class I receptor activity |
Although the prediction of GoPet is completely correct, it only recognises that the protein belongs to the MHC I receptor class. Any annotations hinting at the role of HFE in the iron metabolism are completely missing.
Unfortunately, ProtFun is currently not working and thus, results cannot be presented. For details please refer to the lab journal.
Pfam families The Pfam search for the 1A6Z_A structure of HFE revealed that it consists of two domains. The first domain is the MHC I binding domain that is responsible for the binding to ferritin. Most MHC I proteins are involved in the immune system and this specific domain and therefore need to be flexible. Therefore their sequence is not very well conserved as indicated by the low conservation values in the Pfam alignment. The second domain is the immunoglobulin C1-set domain, that corresponds to the constant domain of antibodies. This domain is better conserved than the MHC I domain, which reflects its function as a protein stabilizer. Beta-microglobulin, that is also part of a MHC I complex but not covalently bound to the protein, is also part of the Ig c1-set domain family.
References
<references />