Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 116, Issue 6, Pages 2078-2085Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1811168116
Keywords
solid-state NMR; allostery; membrane proteins; ion channel; ligand affinity
Categories
Funding
- NIH [P41 GM118302, R01 GM088724, R01 GM088352]
- Empire State Division of Science Technology and Innovation
- Office of Research Infrastructure Programs/NIH Facility Improvement Grant [CO6RR015495]
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available