Protein toxins are determinants of bacterial virulence, vaccine targets, and tools for development of new therapeutic approaches. Diphtheria toxin is a protein that crosses cellular membranes in order to inactivate protein synthesis. The goal of this project is to determine the structure of diphtheria toxin in its membrane-inserted state, and the mechanism by which its catalytic A chain translocates across membranes. Fluorescence methods to analyze toxin structure were developed in the previous grant period. These methods: differentiate residues exposed to solution from those buried in the membrane; measure the depth of membrane-buried residues; differentiate residues facing the aqueous solution outside the membrane from those facing the internal aqueous solution; detect oligomerization; and reveal the size of toxin induced pores. Using these methods it was established that the T domain of the toxin, which is critical for translocation, can exist both in states in which helices 8 and 9 partially penetrate into membranes and another in which they penetrate fully. In the next grant period, the topography of membrane-inserted mutant toxins will be examined with single fluorescent residues introduced by site-directed mutagenesis and chemical labeling. The complete topography of the helices of will be determined in both conformations of membrane-inserted T domain. Next, the topography of membrane-inserted A chain and membrane-inserted A chain-T domain complex will be determined. Residues critical for formation of the fully inserted state will be identified using the fluorescence techniques to examine the effect of amino acid substitutions introduced by site-directed mutagenesis. In addition, the implications of our observation that membrane-associated T domain interacts specifically with proteins that partly unfold and form the so- called """"""""molten globule"""""""" state will be explored. Since the A chain also can partly unfold under physiological conditions, it is possible the T domain functions like a membrane-inserted chaperone of partly unfolded proteins. To test this idea, the nature of T domain interactions of with molten globule proteins will be compared to those with the A chain. Finally, the action of compounds we found to inhibit the pores induced by the toxin (and isolated T domain) in membranes will be examined, as well as whether they can block pores formed by other toxins. Thus, in addition to a better understanding of diphtheria toxin, these studies will yield new approaches for studying the membrane protein structure, insights into protein translocation across membranes, and clues to novel therapeutic agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM031986-16
Application #
2743753
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1983-04-01
Project End
2002-11-30
Budget Start
1998-12-01
Budget End
1999-11-30
Support Year
16
Fiscal Year
1999
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Wang, Jie; London, Erwin (2009) The membrane topography of the diphtheria toxin T domain linked to the a chain reveals a transient transmembrane hairpin and potential translocation mechanisms. Biochemistry 48:10446-56
Lai, Bing; Zhao, Gang; London, Erwin (2008) Behavior of the deeply inserted helices in diphtheria toxin T domain: helices 5, 8, and 9 interact strongly and promote pore formation, while helices 6/7 limit pore formation. Biochemistry 47:4565-74
Fujita, Kentaro; Krishnakumar, Shyam S; Franco, David et al. (2007) Membrane topography of the hydrophobic anchor sequence of poliovirus 3A and 3AB proteins and the functional effect of 3A/3AB membrane association upon RNA replication. Biochemistry 46:5185-99
Buchanan, Susan K; Lukacik, Petra; Grizot, Sylvestre et al. (2007) Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import. EMBO J 26:2594-604
White, Dawn; Musse, Abdiwahab A; Wang, Jie et al. (2006) Toward elucidating the membrane topology of helix two of the colicin E1 channel domain. J Biol Chem 281:32375-84
Wu, Zhengyan; Jakes, Karen S; Samelson-Jones, Ben S et al. (2006) Protein translocation by bacterial toxin channels: a comparison of diphtheria toxin and colicin Ia. Biophys J 91:3249-56
Wang, Jie; Rosconi, Michael P; London, Erwin (2006) Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1-TH3 as a hydrophilic tether. Biochemistry 45:8124-34
Zhao, Gang; London, Erwin (2006) An amino acid ""transmembrane tendency"" scale that approaches the theoretical limit to accuracy for prediction of transmembrane helices: relationship to biological hydrophobicity. Protein Sci 15:1987-2001
Musse, Abdiwahab A; Wang, Jie; Deleon, Gladys P et al. (2006) Scanning the membrane-bound conformation of helix 1 in the colicin E1 channel domain by site-directed fluorescence labeling. J Biol Chem 281:885-95
Zhao, Gang; London, Erwin (2005) Behavior of diphtheria toxin T domain containing substitutions that block normal membrane insertion at Pro345 and Leu307: control of deep membrane insertion and coupling between deep insertion of hydrophobic subdomains. Biochemistry 44:4488-98

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