Population shift of binding pocket size and dynamic correlation analysis shed new light on the anticooperative mechanism of PII protein

P-II protein is one of the largest families of signal transduction proteins in archaea, bacteria, and plants, controlling key processes of nitrogen assimilation. An intriguing characteristic for many P-II proteins is that the three ligand binding sites exhibit anticooperative allosteric regulation. In this work, P-II protein from Synechococcus elongatus, a model for cyanobacteria and plant P-II proteins, is utilized to reveal the anticooperative mechanism upon binding of 2-oxoglutarate (2-OG). To this end, a method is proposed to define the binding pocket size by identifying residues that contribute greatly to the binding of 2-OG. It is found that the anticooperativity is realized through population shift of the binding pocket size in an asymmetric manner. Furthermore, a new algorithm based on the dynamic correlation analysis is developed and utilized to discover residues that mediate the anticooperative process with high probability. It is surprising to find that the T-loop, which is believed to be responsible for mediating the binding of P-II with its target proteins, also takes part in the intersubunit signal transduction process. Experimental results of P-II variants further confirmed the influence of T-loop on the anticooperative regulation, especially on binding of the third 2-OG. These discoveries extend our understanding of the P-II T-loop from being essential in versatile binding of target protein to signal-mediating in the anticooperative allosteric regulation. Proteins 2014; 82:1048-1059. (c) 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.