Structure of a transiently phosphorylated switch in bacterial signal transduction

Abstract

Receiver domains are the dominant molecular switches in bacterial signalling. Although several structures of non-phosphorylated receiver domains have been reported, a detailed structural understanding of the activation arising from phosphorylation has been impeded by the very short half-lives of the aspartylphosphate linkages. Here we present the first structure of a receiver domain in its active state, the phosphorylated receiver domain of the bacterial enhancer-binding protein NtrC (nitrogen regulatory protein C). Nuclear magnetic resonance spectra were taken during steady-state autophosphorylation/dephosphorylation, and three-dimensional spectra from multiple samples were combined. Phosphorylation induces a large conformational change involving a displacement of beta-strands 4 and 5 and alpha-helices 3 and 4 away from the active site, a register shift and an axial rotation in helix 4. This creates an exposed hydrophobic surface that is likely to transmit the signal to the transcriptional activation domain.

Protein dynamics and function from solution state NMR spectroscopy

“…Shown is a structural superimposition of phosphorylated NtrC (orange) and nonphosphorylated NtrC (blue) indicating the structural difference between active and inactive states, respectively. This figure was adapted from reference Kern et al (1999). Tollinger et al 2001) and 1 H N (Ishima & Torchia, 2003) nuclei at two static magnetic field strengths.…”

Energetics-based Protein Profiling on a Proteomic Scale: Identification of Proteins Resistant to Proteolysis

“…This energy landscape is encoded within the amino acid sequence, and underlies biological properties such as catalysis, signal transduction, and protein turnover. [4][5][6][7] Thus, the entire energy landscape is subject to the same types of evolutionary pressures as is the native structure.…”

A network of molecular switches controls the activation of the two-component response regulator NtrC

“…We propose that atomistic simulations can help identify these key structural elements, and demonstrate this by examining the activation mechanism of nitrogen regulatory protein C (NtrC). This protein belongs to the family of two component regulatory systems ubiquitous in bacteria 4 , in which a phosphate is transferred from a sensor kinase (NtrB) to a response regulator (NtrC) 5 . This transfer activates the protein by triggering a structural rearrangement that exposes a hydrophobic surface and allows the protein to form oligomers essential for ATP hydrolysis 6 .…”

“…The active and inactive states are structurally very similar, with a root-mean-square deviation (RMSD) of only 2.9 Å (ref. 5). The inactive state is more flexible than the active state and could be described as a collection of states that rapidly interconvert 7 .…”

“…6). Recent nuclear magnetic resonance (NMR) studies have shown that both active and inactive states are populated in solution without phosphorylated D54, which demonstrates that phosphorylation is not required for the conformation change to occur 5 . Phosphorylation stabilizes the active state, which results in population inversion from a 2-10% active state population to 99% (ref.…”