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Abstract Detail

Regulation of Metabolism

Jez, Joseph M. [1], Kumaran, Sangaralingam [2], Francois, Julie A. [3].

Regulatory Protein-Protein Interactions in Metabolism: The Case of Cysteine Biosynthesis.

Sulfur is an essential nutrient for plant growth and development. In plant sulfur assimilation, cysteine biosynthesis plays a central role in fixing inorganic sulfur from the environment. A key regulatory feature of cysteine biosynthesis is the association of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS) to form the cysteine synthase complex, which acts as a molecular sensor in the circuit that coordinates sulfate assimilation and modulates intracellular cysteine levels. Our goal is to understand the structural and energetic basis for formation of this macromolecular assembly and how protein-protein interactions regulate cysteine synthesis in plants. As a continuation of structural studies on OASS, we have determined the x-ray crystal structure of OASS bound with a peptide corresponding to the C-terminal ten residues of SAT (C10 peptide). An extensive hydrogen bond network, including interactions with key active site residues (Thr74, Ser75, and Gln147), locks the C10 peptide in the binding site. Binding of the peptide blocks access to the catalytic residues of OASS; this explains how OASS activity is down-regulated upon formation of the cysteine synthase complex. Comparison with the bacterial OASS suggests that structural plasticity in the active site allows for the binding of SAT C-termini with dissimilar sequences at structurally similar OASS active sites. In parallel, we have analyzed the thermodynamics of complex formation between OASS and the C10 peptide using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Our results suggest that the C-terminus of SAT provides the major contribution to total binding energy in the plant cysteine synthase complex. Interaction between OASS and the C10 peptide is tight (Kd=5-100 nM) over a range of temperatures (10-35 °C) and NaCl concentrations (0.02-1.3 M). OASS binding of the C10 peptide displays negative cooperativity at higher temperatures. ITC studies reveal compensating changes in enthalpy and entropy of binding that also depend on temperature. The heat capacity change and salt-dependence studies suggest that hydrophobic interactions drive formation of the OASSC10 peptide complex. The potential regulatory effect of temperature on the plant cysteine synthase complex is discussed. These results provide new insights into the molecular mechanism underlying formation of the cysteine synthase complex and provide a structural basis for the biochemical regulation of cysteine biosynthesis in plants.

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1 - Donald Danforth Plant Science Center, 975 N. Warson Rd., St. Louis, MO, 63132, USA
2 - Donald Danforth Plant Science Center
3 - Donald Danforth Plant Science Center & USDA-ARS Plant Genetics Researc

protein-protein interaction
x-ray crystallography.

Presentation Type: ASPB Minisymposium
Session: M27
Location: Continental A/Hilton
Date: Wednesday, July 11th, 2007
Time: 11:55 AM
Number: M27004
Abstract ID:726

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