Research News
Dr. Feng Shao's laboratory conducted structural studies elucidating the function of a novel heptosyltransferase family in bacterial pathogen.
On Oct. 13, 2014, Studies from Dr. Feng Shao's laboratory at National Institute of Biological Sciences (NIBS), Beijing, elucidate the structural and enzymatic mechanism for a novel heptosyltransferase family conserved in Gram-negative bacterial pathogens. The work entitled "A structural mechanism for bacterial autotransporter glycosylation by a dodecameric heptosyltransferase family" is published online in the journal eLife.
In a recent study from Dr. Shao's laboratory (Cell Host Microbe 16: 351-363, 2014), they identify a novel bacterial heptosyltransferase family (BAHT) that catalyzes hyper-glycosylation of AIDA-I and TibA autotransporters, which mediates bacterial adhesion onto host cells and efficient colonization in the mouse intestine. Interestingly, the BAHT family shows no sequence homology to known glycosyltransferases, and all BAHT proteins contain iron, forming a giant dodecamer with brownish color. The biochemical property of BAHT differs from that of all known glycosyltransferases, but its mechanism of action remains unknown.
In the current study aimed at revealing the structural mechanism of the BAHT family, researchers in the Shao laboratory first succeeded in crystallizing TibC, a BAHT family member from Enterotoxigenic E. coli (ETEC) catalyzing TibA heptosylation. The structure reveals a characteristic ring-shape dodecamer. The protomer features an N-terminal ß-barrel and a C-terminal catalytic domain with two unique structural insertions: a ß-hairpin thumb and a unique iron-finger motif. The iron-finger motif contributes to back-to-back dimerization; six dimers form the ring through ß-hairpin thumb-mediated hand-in-hand contact. The dodecamer has a cylinder shape with a large hollow channel. The catalytic centers of 12 protomers all face towards the inside of the hollow channel, suggesting that the autotransporter substrate likely gets glycosylated when passing through the channel.
Despite the lack of primary sequence homology, the catalytic domain of TibC protomer structurally more resembles the GT-B type of glycosyltransferase. The researcher further succeeded in crystallizing the TibC dodecamer in complex with its natural ligand ADP-D, D-heptose. Analyses of ADP-D, D-heptose-bound TibC reveals a sugar transfer mechanism and also the ligand stereoselectivity determinant for different BAHT-family members.
The researcher also obtained the TibC dodecamer (the K230A inactive mutant) in complex with TibA by recombinant co-expression in E. coli. The stable enzyme and substrate complex has a large size of about 750 kDa, and single particle cryo-electron microscopy analyses identify a TibC-TibA dodecamer/hexamer assembly with two enzyme molecules binding to one TibA substrate located in the central channel of the dodecamer. The complex structure also suggests that six autotransporter substrates are simultaneously modified by the dodecamer enzyme complex, highlighting a structural economy for high efficient catalysis.
The study for the first time determines the structures of a novel glycosyltransferase alone and in complex with its sugar ligand or glycosylation substrate. The study also reveals the substrate recognition and catalytic mechanism of the newly identified BAHT family. Together with Cell Host and Microbe publication, this study defines and characterizes the BAHT family from both genetics/biological function and biochemical/structural mechanism perspectives. These two studies not only broad our understanding of the glycosyltransferase mechanism but also may guide future development of new antibiotics targeting BAHT and bacterial glycosylation system.
Postdoc fellows Qing Yao and Qiuhe Lu from the Shao group are co-first authors of this paper; Dr. Xiaobo Wan from the Niu Huang laboratory and Feng Song from Dr. Ping Zhu's laboratory at Institute of Biophysics (CAS) have also made significant contributions; other contributors include PhD student Yue Xu (the Shao group), Professor Xiaoyun Liu from Peking University and his PhD student Mo Hu, Dr. Alla Zamyatina from the University of Natural Resources and Life Sciences (Austria) as well as Dr. Niu Huang. Dr. Ping Zhu and Dr. Feng Shao are co-corresponding authors. The research was supported by the 973 National High-Tech. Projects, the Beijing Municipal Government, China National Science Foundation, the Strategic Priority Research Program of the Chinese Academy of Sciences and Howard Hughes Medical Institute in the States, and carried out at National Institute of Biological Sciences, Beijing.
