Donor substrate binding and enzymatic mechanism of human core α1,6-fucosyltransferase (FUT8).

BACKGROUND

Fucosylation is essential for various biological processes including tumorigenesis, inflammation, cell-cell recognition and host-pathogen interactions. Biosynthesis of fucosylated glycans is accomplished by fucosyltransferases. The enzymatic product of core α1,6-fucosyltransferase (FUT8) plays a major role in a plethora of pathological conditions, e.g. in prognosis of hepatocellular carcinoma and in colon cancer. Detailed knowledge of the binding mode of its substrates is required for the design of molecules that can modulate the activity of the enzyme.

METHODS

We provide a detailed description of binding interactions of human FUT8 with its natural donor substrate GDP-fucose and related compounds. GDP-Fuc was placed in FUT8 by structural analogy to the structure of protein-O-fucosyltransferase (cePOFUT) co-crystallized with GDP-Fuc. The epitope of the donor substrate bound to FUT8 was determined by STD NMR. The in silico model is further supported by experimental data from SPR binding assays. The complex was optimized by molecular dynamics simulations.

RESULTS

Guanine is specifically recognized by His363 and Asp453. Furthermore, the pyrophosphate is tightly bound via numerous hydrogen bonds and contributes affinity to a major part. Arg365 was found to bind both the β-phosphate and the fucose moiety at the same time.

CONCLUSIONS

Discovery of a novel structural analogy between cePOFUT and FUT8 allows the placement of the donor substrate GDP-Fuc. The positioning was confirmed by various experimental and computational techniques.

GENERAL SIGNIFICANCE

The model illustrates details of the molecular basis of substrate recognition for a human fucosyltransferase for the first time and, thus, provides a basis for structure-based design of inhibitors.