Competing allosteric mechanisms modulate substrate binding in a dimeric enzyme

Abstract

Allostery has been studied for many decades yet it remains challenging to determine experimentally how it occurs at a molecular level. We have developed an approach combining isothermal titration calorimetry, circular dichroism, and nuclear magnetic resonance spectroscopy to quantify allostery in terms of protein thermodynamics, structure, and dynamics. This strategy was applied to study the interaction between aminoglycoside N-(6′)-acetyltransferase-Ii and one of its substrates, acetyl coenzyme A. It was found that homotropic allostery between the two active sites of the homo-dimeric enzyme is modulated by opposing mechanisms. One follows a classical KNF paradigm while the other follows a recently-proposed mechanism in which partial unfolding of the subunits is coupled to ligand binding. Competition between folding, binding, and conformational changes represents a new way to govern energetic communication between binding sites. Allostery is a key feature of biological systems in which covalent modification or ligand binding at one site influences the activity at distant sites in a macromolecule or macromolecular assembly. Allosteric regulation plays a central role in metabolism and cell signaling, and has been identified as a source of new drug targets 1-5. Thus detailed descriptions of allostery have far reaching implications. A number of systems have been explained in terms of mechanistic models, yet allostery takes a variety of forms and many details of this phenomenon remain unresolved 6-9. Homotropic allostery (or homotropic cooperativity) involves interactions between a macromolecular system and two or more identical ligands. Cooperativity implies that binding depends on the ligation state, i.e. the first ligand is bound with a different affinity than the second, etc. This occurs frequently in oligomeric proteins and many theoretical models have focused on such systems. For Correspondence should be addressed to A.M.

Thermodynamic Dissection of the Intrinsically Disordered N-terminal Domain of Human Glucocorticoid Receptor

“…Truncation of the R region results in a corresponding increase in the stability of the remaining ID NTD and an increase in its transcriptional activity. These results are suggestive of a general paradigm for allosteric control whereby the folding of disordered protein regions can control and be controlled by the folding of other ID regions (53,54). This strategy provides proteins with a general mechanism that can be utilized within all disordered segments, perhaps answering why ID sequences are found in such abundance in transcription factors and other cell signaling proteins (2).…”

Structural Dynamics, Intrinsic Disorder, and Allostery in Nuclear Receptors as Transcription Factors

“…This more structured NTD/AF1 shows increased affinity for important known CoFs. It is increasingly clear that ID is important for mediating allosteric control, as demonstrated theoretically and experimentally (91,92,100). By examining how the response of the protein ensemble to ligand can drive allosteric coupling, we have shown that the presence of one or more ID regions in a protein can modulate allosteric coupling between domains.…”

Experimental free energy surfaces reveal the mechanisms of maintenance of protein solubility

“…The internal motions associated with proteins are not only important for protein function (38)(39)(40)(41) but also because they act as crucial determinants of the ability of a protein to avoid aggregation (18,42). The present study has addressed this point in detail by showing the manner by which the native free energy landscape limits the accessibility of aggregation-prone regions of conformational space, thereby allowing the protein to maintain a soluble and functional state in solution.…”