Crystal Structure of the Catalytic Domain of Drosophila β1,4-Galactosyltransferase-7

Boopathy Ramakrishnan,Pradman K Qasba

doi:10.1074/jbc.m109.099564

Abstract

The beta1,4-galactosyltransferase-7 (beta4Gal-T7) enzyme, one of seven members of the beta4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member beta4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of beta4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 A resolution. In the crystal structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of beta4Gal-T7 and beta4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH(2)OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the beta4Gal-T7 crystal structure shows that the aromatic side chain of Tyr(177) interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.

Highlights

The ␤1,4-galactosyltransferase-7 (␤4Gal-T7) enzyme, one of seven members of the ␤4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins
We report here the crystal structure of the catalytic domain of ␤4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 Aresolution
Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome

Summary

EXPERIMENTAL PROCEDURES

The Drosophila melanogaster ␤4Gal-T7 cDNA (AY094665) was purchased from Open Biosystems. The N-terminal fusion proteins P-Cd7⌬C and P1-Cd7⌬C, containing a 46- and a 33amino acid peptide (P and P1), respectively, from bovine ␤4Gal-T1 (residues 130 –175 and 143–175, respectively), were constructed by inserting the DNA fragments corresponding to the fusion peptides P and P1 at the N terminus of the Cd7⌬C protein between the restriction enzyme sites BamHI and NcoI (see Fig. 1A). Of the total 240 residues in the Cd7⌬C protein, 230 residues were automatically fitted into the solvent-flattened electron density maps generated by HKL2MAP by the web-based ARP/wARP program [29]. At this stage, the model was refined using the native data up to 1.81 Å resolution. The structure factors and coordinates have been deposited in the Protein Data Bank (code 3LW6)

RESULTS AND DISCUSSION

Ramachandran plotb Core region Allowed region Generously allowed region

Gal moiety in the crystals of the