Visualizing an Ultra‐Weak Protein–Protein Interaction in Phosphorylation Signaling

Abstract

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 K(D)≈25 mM interaction between two bacterial enzymes. Using paramagnetic NMR spectroscopy and with the introduction of a novel Gd(III)-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.

Transient protein–protein interactions visualized by solution NMR

“…When the exchange rate k ex between free and bound states (the sum of association and dissociation rates) is comparable to or slower than the theoretical CSP for an interfacial residue, the exchange time-scale falls to intermediate or slow exchange regimen, and the NMR peak for this residue would barely move during the titration. The CSPs can also be hardly observable, when the binding is ultraweak with K D values in the mM range [13,14] or when the complex is extremely large, i.e. several hundred kilodaltons to megadaltons [14][15][16].…”

“…The CSPs can also be hardly observable, when the binding is ultraweak with K D values in the mM range [13,14] or when the complex is extremely large, i.e. several hundred kilodaltons to megadaltons [14][15][16]. Methods have been developed to extract the chemical shift values for the otherwise invisible states.…”

“…2A). An ultraweak complex can be very short-lived, and therefore it is also called a fleeting complex [14]. As a result, intermolecular NOEs may not efficiently build up during the lifetime of the complex.…”

“…The PRE effect for a nucleus is inverse sixth power related to the distance between the protein nucleus and the paramagnetic center. Among the different paramagnetic probes, lanthanide ion Gd 3+ can elicit the strongest PRE effect [14,40], and can enhance the relaxation of protons up to 40 Å away from the paramagnetic center. When the χ tensor of unpaired electron in the paramagnetic center is anisotropic, such as a lanthanide ion other than Gd 3+ , introduction of the paramagnetic probe not only causes PRE, but also PCS and RDC to the protein nucleus.…”

“…As the percentage bound is low and the resulting complex is short-lived, identification of intermolecular NOE is out of the question. Tang and colleagues discovered a binary phosphorylation complex between enzymes EIN and EIIA-glucose with an estimated K D value of 25 mM [14]. Using the PRE technique with a Gd 3+ paramagnetic probe conjugated at three different sites at the periphery of the interface, the authors were able to model the structure of this fleeting complex that account for phosphoryl transfer reaction between the two proteins.…”