Contributed by Ann E. McDermott, December 3, 2018 (sent for review June 28, 2018; reviewed by Vincent J. Hilser, Ayyalusamy Ramamoorthy, and Gianluigi Veglia)
Allostery is a common strategy used by nature to transmit information in proteins, including membrane signaling systems, wherein binding of one ligand affects avidity for binding another ligand in a distal site. Methods are needed for elucidation of functional groups that are critical for coupling between the binding sites, which we call allosteric participants. Identifying and characterizing allosteric participants is important for understanding the allosteric mechanism and crucial for developing new drugs. We develop an NMR method to detect allosteric participants that relies on their multistate nature, and we apply this method to a membrane protein, the potassium channel KcsA. We confirm the importance of specific amino acid side chains for allostery through analysis of binding affinities.
Allosteric couplings underlie many cellular signaling processes and provide an exciting avenue for development of new diagnostics and therapeutics. A general method for identifying important residues in allosteric mechanisms would be very useful, but remains elusive due to the complexity of long-range phenomena. Here, we introduce an NMR method to identify residues involved in allosteric coupling between two ligand-binding sites in a protein, which we call chemical shift detection of allostery participants (CAP). Networks of functional groups responding to each ligand are defined through correlated NMR perturbations. In this process, we also identify allostery participants, groups that respond to both binding events and likely play a role in the coupling between the binding sites. Such residues exhibit multiple functional states with distinct NMR chemical shifts, depending on binding status at both binding sites. Such a strategy was applied to the prototypical ion channel KcsA. We had previously shown that the potassium affinity at the extracellular selectivity filter is strongly dependent on proton binding at the intracellular pH sensor. Here, we analyzed proton and potassium binding networks and identified groups that depend on both proton and potassium binding (allostery participants). These groups are viewed as candidates for transmitting information between functional units. The vital role of one such identified amino acid was validated through site-specific mutagenesis, electrophysiology functional studies, and NMR-detected thermodynamic analysis of allosteric coupling. This strategy for identifying allostery participants is likely to have applications for many other systems.
Author contributions: Y.X. and A.E.M. designed research; Y.X. and D.Z. performed research; D.Z. contributed new reagents/analytic tools; Y.X., C.M.N., and A.E.M. analyzed data; and Y.X., D.Z., R.R., C.M.N., and A.E.M. wrote the paper.
Reviewers: V.J.H., Johns Hopkins University; A.R., University of Michigan; and G.V., University of Minnesota.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1811168116/-/DCSupplemental.
Published under the PNAS license.
