Huang_2002_Biopolymers_63_247

Many examples exist of charged amino acids that play a role in attracting or holding a charged ligand toward or inside an oppositely charged binding pocket of the protein For example the enzymes superoxide dismutase triose-phosphate isomerase and acetylcholinesterase can steer ligands toward their oppositely charged binding pockets or gorges Interestingly in our Brownian dynamics simulations of a phosphate-binding protein we discovered that negatively charged phosphate HPO(2-)(4 could make its way into the negatively charged binding pocket In fact the phosphate-binding protein exhibits counterintuitive kinetics of association That is one would expect that the rate of association would increase on increases to the ionic strength since the interaction between the ligand with a charge of 2 and the electronegative binding pocket would be repulsive and greater screening should reduce this repulsion and increase the rate of association However the opposite is seen-i.e the rate of association decreases on increases in the ionic strength We used Brownian dynamics techniques to compute the diffusion limited association rate constants between the negatively charged phosphate ligand and several open forms of PBP wild-type and several mutants based on an x-ray structure of open-form PBP mutant T141D With the appropriate choices of reaction criteria and molecular parameters the ligand was able to diffuse into the binding pocket A number of residues influence binding of the ligand within the pocket via hydrogen bonds or salt bridges Arg135 partially neutralizes the charges on the HPO(2-)(4 ligand in the binding pocket allowing it to enter It is also found that the positive electrostatic patches above and below the binding entrance of PBP contribute the major attractive forces that direct the ligand toward the surface of the protein near the binding site