The phosphoenolpyruvate-dependent phosphotranferase system (PTS) is a multicomponent carbohydrate uptake system that is also involved in the regulation of metabolism, chemotaxis, and pathogenicity in bacteria. The PTS drives active transport of sugar by coupling the translocation of the ligand across the membrane with its concomitant covalent modification by phosphorylation to prevent efflux. The PTS has been the subject of extensive study for nearly half a century, but our understanding of the system has remained incomplete due to the lack of any structures for the integral membrane component EIIC responsible for the transport of the sugar across the inner membrane. The EIICs also confer specificity for a particular sugar to the PTS, and assist in the transfer of the phosphate from the cytoplasmic PTS protein EIIB to the sugar. We intend to address this gap in the mechanistic understanding of the PTS by combining structural and functional studies of ChbC, a member of the glucose EIIC superfamily that is specific for the uptake of N.N'- diacetylchitobiose. This sugar is produced by the breakdown of chitin, and as an important nutrient in the life cycle of pathogens such as Vibrio cholerae. To this end, we have solved the structure of a ChbC ortholog from Bacillus cereus (bcChbC), which has led us to propose hypotheses for how the transporter selectively binds sugar, translocates it across the membrane, and assists in coupling phosphorylation to transport. We will use this structure to understand the mechanism of EIIC function with three aims: (1) to determine the structural basis of bcChbC's substrate selectivity with binding and uptake assays, (2) to uncover the mechanism of phosphorylation by solving the structure of an bcChbC in complex with its partner EIIB, bcChbB, and (3) to reconstruct the conformational changes underlying the transport cycle by solving the structure of the outward- facing open state of bcChbC.

Public Health Relevance

N,N'-diactelychitobiose, a degradation product of chitin, is a vital food source for many bacteria, including the cholera-causing pathogen Vibrio cholerae, which survives on the chitin in the shells of its microcrustacean hosts during its water-bourne phase. Because the uptake system for N,N'-diactelychitobiose is unique to bacteria, it is a potential target for disrupting the lifecycle of this devastating pathogen. Our studies of the ChbC transporter will help shed light on the mechanism of the N,N'-diactelychitobiose uptake system.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098878-02
Application #
8317627
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2011-09-01
Project End
2013-01-31
Budget Start
2012-06-01
Budget End
2013-01-31
Support Year
2
Fiscal Year
2012
Total Cost
$232,000
Indirect Cost
$87,000
Name
Columbia University (N.Y.)
Department
Physiology
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
McCoy, Jason G; Levin, Elena J; Zhou, Ming (2015) Structural insight into the PTS sugar transporter EIIC. Biochim Biophys Acta 1850:577-85
Levin, Elena J; Zhou, Ming (2014) Recent progress on the structure and function of the TrkH/KtrB ion channel. Curr Opin Struct Biol 27:95-101
Huang, Hua; Levin, Elena J; Liu, Shian et al. (2014) Structure of a membrane-embedded prenyltransferase homologous to UBIAD1. PLoS Biol 12:e1001911
Zhou, Xiaoming; Levin, Elena J; Pan, Yaping et al. (2014) Structural basis of the alternating-access mechanism in a bile acid transporter. Nature 505:569-73
Cao, Yu; Pan, Yaping; Huang, Hua et al. (2013) Gating of the TrkH ion channel by its associated RCK protein TrkA. Nature 496:317-22
Pan, Yaping; Levin, Elena J; Quick, Matthias et al. (2013) Correction to Potentiation of the Kv1 family K(+) Channel by Cortisone Analogues. ACS Chem Biol 8:268
Cao, Yu; Jin, Xiangshu; Levin, Elena J et al. (2011) Crystal structure of a phosphorylation-coupled saccharide transporter. Nature 473:50-4