Chemical shift imprint of intersubunit communication in a symmetric homodimer

Abstract

Allosteric communication is critical for protein function and cellular homeostasis, and it can be exploited as a strategy for drug design. However, unlike many protein-ligand interactions, the structural basis for the long-range communication that underlies allostery is not well understood. This lack of understanding is most evident in the case of classical allostery, in which a binding event in one protomer is sensed by a second symmetric protomer. A primary reason why study of interdomain signaling is challenging in oligomeric proteins is the difficulty in characterizing intermediate, singly bound species. Here, we use an NMR approach to isolate and characterize a singly ligated state ("lig 1 ") of a homodimeric enzyme that is otherwise obscured by rapid exchange with apo and saturated forms. Mixed labeled dimers were prepared that simultaneously permit full population of the lig 1 state and isotopic labeling of either protomer. Direct visualization of peaks from lig 1 yielded site-specific ligand-state multiplets that provide a convenient format for assessing mechanisms of intersubunit communication from a variety of NMR measurements. We demonstrate this approach on thymidylate synthase from Escherichia coli, a homodimeric enzyme known to be half-the-sites reactive. Resolving the dUMP 1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP 1 peaks are found beyond the limits set by apo and dUMP 2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers.A llosteric regulation in proteins is a ubiquitous mechanism for controlling cellular behavior and an attractive strategy for therapeutic development. Even though broadly recognized, longrange communication is not well understood mechanistically (1-4). Although there have been numerous strategies to reveal the structural and dynamic underpinnings of allostery, oddly these strategies have largely focused on complex oligomeric or, alternatively, on small monomeric allosteric proteins. A likely more straightforward approach is to study allosteric mechanisms using simple symmetric homodimeric proteins, which would allow for answering the basic and general question of how the occurrence of an event in one subunit is communicated to another subunit, as occurs in classical multisubunit allosteric proteins. Given the large number of homodimeric proteins involved in cellular regulation (such as growth factors, cytokines, kinases, G protein-coupled receptors, transcription factors, and metabolic proteins), insights into intersubunit communication should be widely beneficial (5).As a key step toward a broad understanding of allosteric mechanisms, it will be important to observe how binding a ligand in one subunit is communicated to a second subunit, even in the absence of conformational change. Although this communication is straightforwar...

Widespread Perturbation of Function, Structure, and Dynamics by a Conservative Single-Atom Substitution in Thymidylate Synthase

“…However, several lines of evidence show that the effects of substrate or cofactor binding are indeed communicated to the second subunit based on the following: 1) Changes in in enthalpy (and entropy) are not equivalent for the two binding events. 2) We observe quartets of resonances for a subset of amides at intermediate points in diligand NMR titrations (22, 62). Two of the resonances correspond to the free and doubly bound enzymes.…”

“…The other two are attributable to the free and bound subunits of the singly bound state, indicating that binding of the first diligand binding event is communicated to the second subunit. 3) Studies in which we used mixed dimers to isolate the singly bound dUMP state and measure the chemical shifts of all microstates also show crosstalk between binding and non-binding subunits (62). Thus the structure and/or dynamics of the second subunit are affected by binding the first dUMP, cofactor, or diligand molecules.…”

A Single Mutation Traps a Half-Sites Reactive Enzyme in Midstream, Explaining Asymmetry in Hydride Transfer

“…There is evidence from NMR spectroscopy that structural changes resulting from substrate binding propagate to and across the dimer interface in wild type EcTS as well 5, 26 . During titration of EcTS with the mechanism-based di-ligand inhibitor 5-fluoro-dUMP•mTHF, resonances not present in symmetric apo or doubly bound EcTS for several residues at or near the dimer interface could be assigned to a singly bound state 5 .…”

“…Similar but more exaggerated structural differences near the active site cysteine were observed in the asymmetric ternary complex of Pneumocystis carinii TS, which had CB3717 bound in only one active site, and the differences were proposed to be responsible for the half-the-sites reactivity of the enzyme 27 . NMR spectroscopy of EcTS heterodimers with inactivating mutations at one active site revealed that upon binding a single dUMP molecule, subtle structural changes occurred in these same interface residues 26 . This result suggests the residues may constitute a common pathway for communicating between active sites in EcTS, its C-terminal mutants, and other TS species.…”

Thermodynamic and NMR Assessment of Ligand Cooperativity and Intersubunit Communication in Symmetric Dimers: Application to Thymidylate Synthase

“…In principle, NMR titrations could also track dUMP binding, but we found that dUMP binds with kinetics that lead to chemical exchange mostly in the fast-intermediate timescale. As a result, peaks often became faint or disappeared in the midpoints of the dUMP titration, and quantitative interpretation was difficult or impossible (Falk et al, 2016 ). ITC yielded high-sensitivity data that could be fitted to multiple binding models (Figure 3 ).…”