Protein_O_fucosyltransferase

The presence of Protein O-fucosyltransferase 1 (PDB ID: 5KXH) from Mus musculus has been correlated to animal evolution. It binds EGF-like domains of the hEGF type and uses GDP-fucose as donor substrate. The protein also plays a crucial role in NOTCH signaling. POFUT1 fucosylates the epidermal growth factor (EGF)-like domains that are found on the surface of the cell and secreted glycoproteins. These include Notch and its ligands. This protein is very important for study as mutations in it have been linked to Dowling–Degos disease in H. sapiens. Furthermore, clinical approaches to using POFUT1 as diagnostic targets for cardiac diseases and even cancer have been proposed (1). POFUT1 has a molecular weight of 44,688 Da and an isoelectric point of 3.93 (4).

Using the single-wavelength anomalous dispersion (SAD) technique with selenomethionine-labeled protein, the X-ray crystal structure of POFUT1 was determined in complex with GDP or GSP-fucose. The results showed that in a groove between the N- and C- terminal domains of the GT-B fold lie the EGF-LDs. The EGF-LDs are positioned in an identical manner in all of the complexes which satisfied POFUT1’s requirement for folded EGF-LDs. POFUT1 is in contact with three regions of the EGF-LD: the C1-C2 loop, the C2-C3 loop, and the C5-C6 subdomain. The C1-C2 loop and the C5-C6 subdomain have contact with the flexible 133-143 segment of the POFUT1. Meanwhile, the C2-C3 loop contains the fucosylation motif. The seven residues of this motif make the closest contact with the protein. These residues are Cys45, Met46, Gly47, Arg48, His80, Tyr78, and Gly150.They make up the POFUT1 binding groove and interact with the canonical type I' beta turn (1).

Bioinformatics software, including PSI-BLAST and the Dali Server, were used in order to find structures with similar primary and tertiary structures to that of the query protein. PSI-BLAST utilizes the amino acid sequence of one or more subunits of the query protein in order to search for molecules with a similar primary structure to it. PSI-BLAST assigns an E value to each search result. This value is calculated by studying the total sequence homology and assigning gaps. An amino acid or a group of amino acids that are not in the query protein’s sequence but are present in the subject protein’s sequence is termed a gap. The total sequence homology between the search protein and the query protein decreases the E value, while gaps increase the E value. Subject entries with E values less than 0.05 are considered significantly similar in primary structure and were used to find comparison structures (3).

The Dali Server searches for proteins with a similar tertiary structure to that of the query protein. Dali Server uses a sum-of-pairs method which measures the similarity between the subject and query proteins by the comparison of intramolecular distances. The Server then assigns the subject protein a Z- score. Subject structures that have similar folds, and thus significantly similar structures, have a Z- score that is greater than 2. Therefore, the subject proteins with Z- scores above 2 were considered appropriate comparison structures. After conducting both PSI- BLAST and Dali Server searches, the subject protein with both an E-value of 3e-130, and a Z-score of 40.3 was discovered to be the Protein O-fucosyltransferase 2 mutant from Homo sapiens (PDB ID: 4AP6, POFUT2) and was used as the comparison structure (2). POFUT2 has a significantly larger structure than that of POFUT1. While POFUT1 only has two subunits, POFUT2 has four subunits. While both proteins contain GDP, only POFUT1 contains glycerol.

In conclusion, the study of this protein, POFUT1, is very important as it leads to insight on genetic mutations such as Dowling–Degos disease. Furthermore, it has been proposed that POFUT1 could be used as a diagnostic target for other diseases such as those of the heart and cancer (5).