The dimeric structure of wild-type human glycosyltransferase B4GalT1

Abstract

Most glycosyltransferases, including B4GalT1 (EC 2.4.1.38), are known to assemble into enzyme homomers and functionally relevant heteromers in vivo. However, it remains unclear why and how these enzymes interact at the molecular/atomic level. Here, we solved the crystal structure of the wild-type human B4GalT1 homodimer. We also show that B4GalT1 exists in a dynamic equilibrium between monomer and dimer, since a purified monomer reappears as a mixture of both and as we obtained crystal forms of the monomer and dimer assemblies in the same crystallization conditions. These two crystal forms revealed the unliganded B4GalT1 in both the open and the closed conformation of the Trp loop and the lid regions, responsible for donor and acceptor substrate binding, respectively. The present structures also show the lid region in full in an open conformation, as well as a new conformation for the GlcNAc acceptor loop (residues 272–288). The physiological relevance of the homodimer in the crystal was validated by targeted mutagenesis studies coupled with FRET assays. These showed that changing key catalytic amino acids impaired homomer formation in vivo. The wild-type human B4GalT1 structure also explains why the variant proteins used for crystallization in earlier studies failed to reveal the homodimers described in this study.

Assembly of B4GALT1/ST6GAL1 heteromers in the Golgi membranes involves lateral interactions via highly charged surface domains

“…Recently, we presented the first human homodimeric crystal structure of B4GALT1 and showed that the interaction takes place at the surface surrounding the active site of the enzyme through concerted loop movements involving amino acids Met 340 and His 343 (13). To determine possible heterodimer interaction sites in the B4GALT1 catalytic domain, we first utilized molecular docking predictions using the ClusPro server (15) and obtained several potential residues that can mediate the interaction with ST6GAL1.…”

“…These events have been shown to be coordinated by formation of two disulfide bonds on two distinct surface areas within the catalytic domain of the enzyme (12). On the other hand, the first human B4GALT1 dimeric structure indicates that the homodimeric interaction takes place via the surface surrounding the active site, and two mutations there (M340H and H343A) prevented dimer formation, most likely by changing the affinity to the Mn 2ϩ binding and by altering the active site's loop dynamicity (13,14).…”

N-glycosylation of the human β1,4-galactosyltransferase 4 is crucial for its activity and Golgi localization

“…Overproduction of the mutant B4GalT4 version lacking a glycosylation site at Asn220 (MT2) showed significantly lower ability to correct the KO phenotype in A375 KO cell cultures. Although, to our knowledge, currently there are no available studies linking structural elements of B4GalT4 to its enzymatic activity, sequence comparison of B4GalTs shows that this glycosylation site of B4GalT4 is positioned closely to conserved regions found to be engaged in catalytic activity and acceptor recognition in B4GalT1 [12,32,33]. Interestingly, B4GalT4 is the only B4GalT to possess an N-glycosylation site near this region.…”

Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase is expendable

“…Moreover, presence of N-glycans impact protein folding and oligomerization, so eliminating N-glycans may change GT activity by altering its ability to oligomerize. Oligomerization may influence subcellular localization in the ER and/or Golgi, and so indirectly the activity of a GT [Harrus D., Khoder-Agha F. et al 2018;Nilsson T., Hoe M.H. et al 1994].…”