Proteins interact with each other to fulfill their functions. The importance of weak protein-protein interactions has been increasingly recognized. However, owing to technical difficulties, ultra-weak interactions remain to be characterized. Phosphorylation can take place via a KD≈25 mM interaction between two bacterial enzymes. Using paramagnetic NMR spectroscopy and with the introduction of a novel GdIII-based probe, we determined the structure of the resulting complex to atomic resolution. The structure accounts for the mechanism of phosphoryl transfer between the two enzymes and demonstrates the physical basis for their ultra-weak interaction. Further, molecular dynamics (MD) simulations suggest that the complex has a lifetime in the micro- to millisecond regimen. Hence such interaction is termed a fleeting interaction. From mathematical modeling, we propose that an ultra-weak fleeting interaction enables rapid flux of phosphoryl signal, providing a high effective protein concentration. Phosphorylation signaling takes place between two bacterial enzymes EI and EIIAGlc, which have a binding affinity of only 25 mM (see picture). The structure of the ultra-weak fleeting complex was determined to atomic resolution by a novel paramagnetic NMR technique, and it shows that electrostatic repulsion largely accounts for the low affinity between the two proteins.
Bibliographical noteFunding Information:
We thank the Chinese Ministry of Science and Technology (grant number 2013CB910200) and the National Natural Sciences Foundation of China (grant numbers 31225007 and 31170728) for support. C.T. was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute. The coordinates for the EIN-EIIAGlc complex at 2% occupancy together with experimental restraints are deposited in the PDB with the accession code 2MP0.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
- NMR spectroscopy
- paramagnetic relaxation enhancement
- protein-protein interactions
- signal transduction