Mechanism and Energetics of L-Arginine Binding to Arginine Repressor Protein in E. Coli

Saurabh Kumar Pandey1, David Řeha1,2, Milan Melichercik1,3, Jannette Carey4, Rüdiger Ettrich1,2, Babak Minofar1,2

1Center for Nanobiology and Structural Biology, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic

2Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Branišovská 1760, 37005 České Budějovice, Czech Republic

   3Department of Nuclear Physics and Biophysics, Comenius University, Mlynská dolina F1, 842 48 Bratislava, Slovak Republic

 4Chemistry Department, Princeton University, Princeton, New Jersey 08544-1009, USA

 

Arginine repressor protein provides feedback regulation of arginine metabolism upon activation by the negatively cooperative binding of L-arginine. Understanding this phenomenon requires the detailed analysis of each binding event and its effect on global motion of the complex.         

Umbrella sampling technique was used to calculate binding energy (potential of mean force) of L-arginines to the ArgRC. Unbinding of L-Arg from ArgR was performed using steered dynamics. Potential of mean force (PMF) was calculated using weighted histogram analysis method in GROMACS. Differently ligated states were prepared either by deleting (from holo-ArgR crystal structure) or adding (to apo-ArgR crystal structure), using YASARA tool.

PMF for holo-5 state was ~12 kcal/mol, while in corresponding apo+1 state it was ~7 kcal/mol. PMF for holo-4 state and apo+2 state were ~4 kcal/mol and ~15 kcal/mol respectively.

The PMF of +1 and -5 states have similar values while that of -4  and +2 states are very different. The huge difference in the PMF between -4 and +2 states could be due to the differently occupied binding pockets in these two systems. Few more repetitions of +2 and -4 states are undergoing, once completed these will hopefully allow us to compare the binding affinity of differently liganded states of ArgRC and their effect on global motion of protein.