Canavan Task 2 - Sequence alignments
Contents
Protocol
Further information can be found in the protocol.
GO Term Enrichment
In the following we are performing different sequence searches with the protein Aspartoacylase (UniProt ID: P45381). In order to validate the found hits, we are looking for common GO classifications of the hits with the query sequence.
For our protein Aspartoacylase there are 17 annotated GO terms (using EMBLs QuickGO):
<figtable id="aspa_go_terms">
| GO ID | GO Name | |
| Cellular Compartment | GO:0005634 | nucleus (3X) |
| GO:0005634 | cytoplasm (4X) | |
| Biological Process | GO:0006533 | aspartate catabolic process |
| GO:0008152 | metabolic process | |
| GO:0022010 | central nervous system myelination | |
| GO:0048714 | positive regulation of oligodendrocyte differentiation | |
| Molecular Function | GO:0046872 | metal ion binding |
| GO:0004046 | aminoacylase activity | |
| GO:0016787 | hydrolase activity | |
| GO:0016788 | hydrolase activity, acting on ester bonds | |
| GO:0016811 | hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides | |
| GO:0019807 | aspartoacylase activity |
</figtable>
Pairwise Sequence Search
BLASTP
|
The blast search with default parameters yielded 196 results when using the default E-Value cutoff of 10. Being more restrictive and considering only hits with E-Values less than 0.002 we get 104 hits. The best alignment is with an uncharacterized Protein from Rattus norvegicus and has an E-Value of e-155. Most of the resulting proteins are Aspartoacylases of other species. There also are a lot of uncharacterized proteins, just as our best hit. Most of the results with EValue > e-15 are Succinylglutamate Desuccinylases, which are in the same protein family (Desuccinylase / Aspartoacylase family) and catalyze a reaction similar to Aspartoacylase. |
<figtable id="aspa_blastp">
</figtable> |
PSIBLAST
We run PSIBlast with four different parameter combinations:
- 2 iterations (j=2), default E-Value cutoff for inclusion of sequences into profile (h=0.002)
- 2 iterations (j=2), strict E-Value cutoff for inclusion of sequences into profile (h=10e-10)
- 10 iterations (j=10), default E-Value cutoff for inclusion of sequences into profile (h=0.002)
- 10 iterations (j=10), strict E-Value cutoff for inclusion of sequences into profile (h=10e-10)
Again we considered only hits with an E-Value up to 0.002 as significant. The Psiblast run with default parameters (2 iterations, EValue 0.002) results in an amount of 597 hits. The most significant hit again is the uncharacterized protein of Rattus Norvegicus with an E-Value of e-142. When restricting the search to include only sequences with an E-Value of up to 10e-10, we still get 502 hits. The best hit still is the Rattus Norvegicus protein.
In contrast to the simple Blast search, the PsiBlast runs with two iterations find more distant related proteins. This can be seen in the great amount of Succinylglutamate desuccinylasen that are found (though with higher E-Values). They belong, as already mentioned, to the same Pfam family as Aspartoacylase.
Increasing the number of iterations obviously results in many more hits. When using a less restrictive E-Value more than 3000 hits are found against 1500 when using a more restrictive E-Value. Interestingly, the best hits (Rattus Norvegicus again) are less significant (~e-70) than for the PsiBlast search with only 2 iterations (~e-140). The majority of found proteins now are Succinylglutamate Desuccinylases, even among the most significant hits (first Succinylglutamate Desuccinylase is ranked 15th). Only among the first 15 significant results orthologs of Aspartoacylase can be found. Additionally, even further relatives are found, like Zinc Carboxypeptidases, Carboxypeptidases, Endopeptidases, etc.
<figtable id="aspa_psiblast">
| Parameters | it2, def E-Value (h=2e-3) | it2, E-Value h=10e-10 | it10 def E-Value (h=2e-3) | it10 E-Value h=10e-10 |
| time | ~2m30 | ~2m30 | ~25m | ~22m |
| best E-Value | 1e-142 | 1e-145 | 2e-46 | e-68 |
| results (EVal: 2e-3) | 597 | 502 | 3211 | 1515 |
| hits with GO terms | 586 | 496 | 3152 | 1461 |
</figtable>
HHBLITS
We used the same parameters for the HHblits search as we used for PsiBlast. This means:
- 2 iterations (n=2), E-Value cutoff for inclusion in result alignment (e=0.002)
- 2 iterations (n=2), strict E-Value cutoff for inclusion in result alignment (e=10e-10)
- 8 iterations (n=8), E-Value cutoff for inclusion in result alignment (e=0.002)
- 8 iterations (n=8), strict E-Value cutoff for inclusion in result alignment (e=10e-10)
commentmixed results with Aspartoacylases and Succivery varying results: Aspartoacylasen, Succinylasen, Zinc Proteins
<figtable id="aspa_hhblits">
| Parameters | it 2 | it 8 |
| time | 2m50 | ~6m |
| results | 274 | 500 |
| best E-Value | 2e-110 | 2.9e-68 |
| worst E-Value | 0.0011 | 9.5e-09 |
</figtable>
Validation and Comparison
Along with the expactations one can find more hits with Psi-Blast than with a simple Blast search.
In general, one can distinguish between two kinds of proteins, that frequently are identified by the sequence searches:
- Aspartoacylases
- Succinylglutamate Desuccinylases
BlastP
<figtable id="blastp_comp">
|
With a simple blast search we were able to identify the closest related sequences. The most significant hit(Rattus Norvegicus) has a sequence identity of 82%. In <xr id="blastp_comp"/> the distribution of the sequence identity of all hits with E-Value < 0.002 is depicted. As one can easily see, there are only few hits with high sequence identities and the majority of hits has sequence identities of about 30%.
For all 94 hits found with an E-Value cutoff of 10e-10, there are annotated GO terms. Furthermore all founds hits share the GO term "hydrolase activity, acting on ester bonds" and "metabolic process". Also, as one can see in <xr id="go_blastp_10e10" /> all hits share the most GO terms with Aspartacylase. Again, "Zinc binding" could also be associated with Aspartoacylase. Therefore, all GO terms that are found more than 5 times, are also associated with Aspartoacylase. The results are more accurate concerning shared GO terms with Aspartoacylase. This is what one would expect when restricting the EValue for finding the closer related proteins. |
<figure id="blastp_comp"> </figure> |
</figtable>
<figtable id="go_blastp">
| E-Value | default (10) | 10e-10 |
| GO terms |
<figure id="go_blastp_def"> </figure> |
<figure id="go_blastp_10e10"> </figure> |
</figtable>
Psi Blast
Increasing the amount of iterations performed in a PSI-Blast search, obviously increases the running time. One can see, that the best ranked hits of the runs with 10 iterations have lower E-Values than the best hits of the runs with less iterations. Yet, the result includes a larger amount of significant hits with higher E-Values. This means, increasing the iterations finds further distantly related sequences, which is the expected outcome. This outcome is also represented in the distribution of sequence identities. As one can see in <xr id="PSI_10e10_seqd"/>, running PSI-Blast with 10 iterations firstly results in more significant hits and secondly most hits have lower sequence identity compared to the run with two iterations.
When not restricting the E-Value Cutoff for the profile built-up, we found that hits with lower sequence identity (meaning more distantly related) are included in the final hit list. This goes along with our observation that more hits are classified as Succinylglutamate Desuccinylases than as Aspartoacylases when using the default cutoff at 0.002. Furthermore, when the more restrictive cutoff is used, simply less hits are being found (see <xr id="PSI_10it_seqid"/>).
The majority of the results from the runs with only two iterations, has moderate sequence identities with a broad distribution between 15% and 45%. In contrast, the results from the run with 10 iterations split up into two groups of hits which form cluster at about 10% and between 30% and 40% sequence identity.
These observations are also represented in the E_Value distribution. For runs with two iterations there are some results covering the range of E-Values between e-145 and e-60 and majority of hits with low E-Values between e-8 and the cutoff at 0.002. The runs with 10 iterations almost exclusively result in lower significant hits. For the run with the restricted cutoff, hits have E-Values ranging from e-60 to the cutoff at 0.002. There is a peak for hits with E-Values just about the cutoff Value of 0.002. The run with the default E-Value cutoff results in hits with E-Values ranging from e-30 to 0.002.
|
<figure id="PSI_10e10_seqd"> </figure> |
<figure id="PSI_10it_seqid"> </figure> |
<figure id="eval_distri_psiblast"> </figure> |
For the run with 2 iterations and the default cutoff value of 0.002, we got 915 hits. We considered 597 hits as significant (E-Value cutoff 2e-3). 586 of these significant proteins have GO terms annotated. As one can see in <xr id="psi_2it_def"/>, the four most often occuring GO terms are shared with Aspartoacylase. the fifth most often occuring GO term is "Zinc ion Binding", which is, as already mentioned, not annotated with Aspartoacylase, even though it does bind Zinc. Often occuring terms are on processes involving arginine, which might belong to the Succinylglutamate Desuccinylases.
A similar GO term statistic is obtained run with two iterations and an E-Value cutoff of 10e-10 (see <xr id="psi_2it_10e10"/>). Here we received 835 hits out of which we considered 502 proteins as significant. 496 proteins have GO terms annotated.
For the PsiBlast run with 10 iterations and default cutoff, the GO term analysis looks quite different though (see <xr id="psi_10it_def"/>). This run resulted in 3211 hits out of which 3152 proteins had annotated GO terms. The GO term, that is found most often is "Zinc ion binding" which might as well be associated with Aspartoacylase. Other often occuring GO terms belong to other protein families like Succinylglutamate Desuccinylases or Carboxypeptidases, which we observed when checking the hit list.
Interestingly, when restricting the E-value to 10e-10, the most often occuring GO terms again are shared with Aspartoacylase (see <xr id="psi_10it_10e10"/>). This underlines the effect of finding more close relatives, that has already been seen in the higher sequence identity of found hits for the restricted run.
|
<figure id="psi_2it_def"> </figure> |
<figure id="psi_2it_10e10"> </figure> |
<figure id="psi_10it_def">  <xr nolink id="psi_10it_def"/> GO Term Enrichment for Hits of the PsiBlast run with 10 iterations and E-Value cutoff at 0.002. Most hits have different GO terms than Aspartoacylase, since they are mainly Succinylglutamate Desuccinylases. Yet, a second class of hits shares some GO terms with Aspartoacylase. </figure> |
<figure id="psi_10it_10e10"> </figure> |
HHBlits
Running HHBlits with 2 iterations yields a small amount of hits (270) with very low (2e-110) and very high (0.0011) E-Values. To increase the amount of hits, we repeated the HHBlits search with the maximum amount of 8 iterations which resulted in a broader output with more Hits with lower averaged E-Values (compare figure ??). Regarding the Sequence Identity distribution, running HHBlits with 8 iterations results in more distant related Hits (see Figure ??).
Overlap
As one can see in Figure ??, roughly 40 percent of the resulting hits are unique to each method. From our considerations, about 25 percent of the hits are significant hits, that could be further investigated (overlap of 50 percent).
Further Evaluation
We tried to further validate the sequence search hits via structural similarity. Unfortunately none of the resulting Hits was a PDB Hit. Furthermore we tried to map the sequence identifiers against the UniProtKB/Swiss-Prot PDB cross-references (http://www.uniprot.org/docs/pdbtosp.txt). Again, this mapping yielded no results, which is why we cannot include any structural information for our ongoing research. When inspecting the annotation for the sequence hits, we already found, that the majority of the hits codes for Aspartoacylases or respectively the highly related protein Succinylglutamate Desuccinylases. Since there already exists a crystal structure of the human Aspartoacylase, it is only reasonable that one will not find other structures for this class of proteins. Additionally, a huge amount of hits codes for not yet characterized proteins, which also will hardly be an interesting target for crystallization.
Multiple Sequence Alignments
For generating our dataset for the MSA we clustered all Hits into Sequence Identity groups:
- >90%: 1
- 60-89%: 59
- 40-59%: 197
- 20-39%: 1141
Since we only got one hit with an sequence Identity >90% we decided to group out hits as follows: three groups of sequences with eight members each:
- 60-99%
- 40-59%
- 20-39%
We chose those hits from the respective groups, that have been found by at least 4 methods (overlap of 50%).
id eVal identity # 60-99% sequence identity Q8BZC2 1.7e-25 90 E1BVP5 e-140 72 H2RVG4 e-141 63 G3VM93 e-105 72 F6ZFQ0 e-139 78 F8WFU8 e-145 86 Q28C61 e-132 68 H2M5L4 e-133 64 # 40-59% sequence identity G5BTW1 e-133 43 G6FRX8 e-103 39 F7NV91 e-112 39 G1Q6P7 e-120 42 H0WH68 e-135 44 F2PFG6 e-119 40 H2MX25 5e-81 40 Q1Z2X2 e-115 38 # 20-39% sequence identity Q2F9Q7 e-109 31 Q8YQC1 e-117 41 E1SMZ8 e-108 39 D7E1T3 e-110 36 A5GQV1 7e-92 33 E8LP14 e-107 31 F9TUZ3 e-106 30 A6VUE4 e-101 35
General Results
All in all the three Alignment methods yield comparable results. One can identify several conserved regions. Especially the two groups with sequence identities <60% show very similar MSAs.
There are three strongly conserved motivs located in the first half of the sequences:
- GGTHGNE
- DLNR
- DLHNT
For the second half of the sequence alignments there is no clear concensus about reserved motifs, but several residues are strongly conserved and may be of functional or structural importance.
In the alignment of the >60% group the first two motifs are not colored in the alignment. This is due to two very short sequences which produce gaps in the alignment and thus lower the consensus.
ClustalW
command: clustalw -align -infile=./db_over60.fa -outfile=./clustalw_msa_60.aln
TCoffee
Muscle
Concerning the wildtype human Aspartoacylase
The three identified motifs can also be foung in the wildtype protein (compare coloring of sequence on top of page). We colored the respective residues in the structure. They all are positioned in the same region of the protein and thus might implicate an important functional region.
