PDB-UF: database of predicted enzymatic functions for unannotated protein structures from structural genomics
© von Grotthuss et al; licensee BioMed Central Ltd. 2006
Received: 06 August 2005
Accepted: 06 February 2006
Published: 06 February 2006
The number of protein structures from structural genomics centers dramatically increases in the Protein Data Bank (PDB). Many of these structures are functionally unannotated because they have no sequence similarity to proteins of known function. However, it is possible to successfully infer function using only structural similarity.
Here we present the PDB-UF database, a web-accessible collection of predictions of enzymatic properties using structure-function relationship. The assignments were conducted for three-dimensional protein structures of unknown function that come from structural genomics initiatives. We show that 4 hypothetical proteins (with PDB accession codes: 1VH0, 1NS5, 1O6D, and 1TO0), for which standard BLAST tools such as PSI-BLAST or RPS-BLAST failed to assign any function, are probably methyltransferase enzymes.
We suggest that the structure-based prediction of an EC number should be conducted having the different similarity score cutoff for different protein folds. Moreover, performing the annotation using two different algorithms can reduce the rate of false positive assignments. We believe, that the presented web-based repository will help to decrease the number of protein structures that have functions marked as "unknown" in the PDB file.
Over 30 structural genomics centers have been established worldwide with the common goal of large-scale, high-throughput structure determination using X-ray crystallography and NMR. One challenge is to predict the function of the proteins from their three-dimensional structures, primarily those which have no detectable sequence similarity to any protein of known function. Currently, the total size of the Protein Data Bank (PDB) is more than 32,000 entries, which contain over 29,000 different (63,000 redundant) protein chains. Many of the PDB chains have been mapped to Enzymatic Classification (EC) numbers via the Swiss-Prot database. The mapping information has been presented as a PDBSprotEC database , which is available on the Internet. SCOPEC  is another web-based repository which is similar to PDBSprotEC collection. The SCOPEC set contains a description of the protein catalytic domains with assigned enzyme function. Prediction of protein function has been conducted using sequence similarity in both web-accessible databases. There is no doubt the PDBSprotEC and SCOPEC databases are full of very useful EC number assignments. However, none of these services contains predictions for proteins that have no sequence similarity to known enzymes. Moreover, neither PDBSprotEC nor SCOPEC includes any data for recently deposited PDB structures. The "youngest" annotated in PDBSprotEC or SCOPEC protein was released by PDB in August 2004 or in February 2003, respectively. Therefore, we decided to use the structure-function relationship [7–9] for automatic assignment of the EC number to 499 protein structures that came from the structural genomics centers and whose function is marked as "unknown" in the PDB file. All assignments are combined into a web-accessible database, which will be updated as soon as the new structures from structural genomics projects are released. Because most of these PDB entries are still not published, we believe that our repository will help to reduce the number of proteins that have functions marked as "unknown" in the PDB file.
Construction and content
Two different strategies were applied to annotate the proteins with EC numbers: namely 3D-Hit and 3D-Fun. The first method simply scans using the 3D-Hit program  a sequentially non-redundant database of structures that are characterized by four cutoff values. Each value is defined by the highest, known score of structural similarity to any protein with different enzyme function at the corresponding or lower EC level. In the 3D-Hit strategy, the EC number of the protein with the strongest structural similarity is completely (or partially) assigned to the query, if the similarity score is greater than all (or any) of the cutoff values. As an example; let us consider a query protein which has the 3D-Hit score = 150 to the enzyme with the EC number 126.96.36.199 and the cutoff values = 100, 120, 180, 200, respectively. This structure will obtain an EC number assignment of 1.2.?.?.
All structural similarity scores are used for annotation in the 3D-Fun strategy. First, the query structure and all sequentially non-redundant proteins are hierarchically clustered (grouped) by structural similarity using complete-link algorithm[13, 14]. Next, the EC number is completely (or partially) assigned to each group in each clustering iteration, if all of the enzymes in the group have the same function at all (or any) of the EC levels; otherwise the EC number is assigned as unknown. As an example let us consider a cluster that contains 4 structures: the query protein and 3 enzymes with EC numbers 188.8.131.52, 184.108.40.206, and 220.127.116.11. This cluster will obtain an EC number assignment of 1.2.?.?. For the final prediction, the enzymatic function of the smallest cluster which contains the query structure is used. In the contrary to the 3D-Hit strategy, the 3D-Fun algorithm takes into account the enzymatic function of all structures that have greater values of similarity to the query than to all other proteins of the whole set.
We used both presented algorithms to infer the EC number for the 499 proteins from structural genomics that are currently available and have unknown functions. In order to avoid over-annotation due to partial EC numbers we used Green and Karp recommendation . If 3D-Hit and 3D-Fun methods were inconsistent in predicting enzyme function at any EC level it was indicated with a '?' symbol in its corresponding position (e.g. 2.3.4.?). If assignments were fully consistent, we indicated it with an 'n' in the fourth EC level (e.g. 2.3.4.n) which means that exact activity of this enzyme was predicted, but a sequence number has not been yet assigned by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB).
Utility and discussion
Structural genomics initiatives tend to target structures that are less typical of the PDB as a whole and so the cutoffs derived from the whole PDB may not be entirely applicable. Therefore, we analyzed 58 structures with predicted EC numbers, which were recently published and functionally annotated since this may give a truer indication of the accuracy. We found only one additional (except described above) incorrect prediction: 1VGY had been characterized as a succinyl diaminopimelate desuccinylase (3.5.1.?) while metallocarboxypeptidases function (3.4.17.n) was assigned. All such predictions will be manually corrected. However, as more structures are solved in the Protein Data Bank, the PDB-UF method will be more and more accurate and human intervention will not be required.
Example of PDB-UF record
The PDB-UF database is a collection of assigned EC numbers to protein structures of unknown function, which come from the structural genomics centers. Structure-based prediction of the EC number was conducted having different cutoff values for a different protein folds. In order to reduce the number of false positives the annotation was performed using the Meta-strategy. The web-based repository will be updated automatically when new protein structures are released.
We are indebted to Gert Vriend for his critical reading of the manuscript. MvG would like to thank the Foundation for Polish Science for the fellowship. The work was supported by 6FP GeneFun (LSHG-CT-2004-503567) and DataGenome (LSHB-CT-2003-503017) grants and by the Polish Ministry of Science and Information.
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