Date of Completion

3-25-2014

Embargo Period

3-25-2014

Major Advisor

Andrei Alexandrescu

Associate Advisor

Debra Kendall

Associate Advisor

James Cole

Field of Study

Structural Biology and Biophysics

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Bacterial receiver domain proteins modulate the intracellular response to external stimuli in two-component systems. There is a new subclass of receiver domains associated with Histidine Tryptophan Glutamate kinases (HWE-kinases) that have divergent sequence properties. The enzyme Sma0114 is a receiver domain that is a substrate for an HWE-kinase. The structure and dynamics of the Sma0114 protein were explored using nuclear magnetic resonance (NMR). Differences between the NMR structures of the inactive and activated states occur in helix α1, the active site loop that connects strand β3 and helix α3, and inthe 455 (α4-β5-α5) face. In most receiver domains, the 455 face undergoes a structural rearrangement in the activated state to make it competent for binding downstream target molecules. Coupling between the 455 face and the active site phosphate is mediated through the rearrangement of a threonine and tyrosine residue via a mechanism called Y-T coupling. The NMR structure indicates that Sma0114 lacks Y-T coupling, and that communication between the active site and the 455 face is achieved through a conserved lysine residue that stabilizes the acyl phosphate in receiver domains. 15N-NMR relaxation experiments reveal a loss of entropy due to binding ligands at the active site with compensating entropic increases in the 455 face. The dynamic character of the 455 face in Sma0114, which in part results from replacement of helix α4 by a flexible loop, may facilitate induced-fit binding of target molecules.

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