Binding sites in Escherichia coli dihydrofolate reductase communicate by modulating the conformational ensemble

Abstract

To explore how distal mutations affect binding sites and how binding sites in proteins communicate, an ensemble-based model of the native state was used to define the energetic connectivities between the different structural elements of Escherichia coli dihydrofolate reductase. Analysis of this model protein has allowed us to identify two important aspects of intramolecular communication. First, within a protein, pair-wise couplings exist that define the magnitude and extent to which mutational effects propagate from the point of origin. These pair-wise couplings can be identified from a quantity we define as the residue-specific connectivity. Second, in addition to the pair-wise energetic coupling between residues, there exists functional connectivity, which identifies energetic coupling between entire functional elements (i.e., binding sites) and the rest of the protein. Analysis of the energetic couplings provides access to the thermodynamic domain structure in dihydrofolate reductase as well as the susceptibility of the different regions of the protein to both small-scale (e.g., point mutations) and large-scale perturbations (e.g., binding ligand). The results point toward a view of allosterism and signal transduction wherein perturbations do not necessarily propagate through structure via a series of conformational distortions that extend from one active site to another. Instead, the observed behavior is a manifestation of the distribution of states in the ensemble and how the distribution is affected by the perturbation. M ost cellular processes, which are facilitated by proteins, are modulated by effectors. The basic features of such a mechanism of regulation are the presence of multiple binding sites for various ligands and communication between these binding sites, which often are situated many angstroms apart. An understanding of the ground rules of this regulatory mechanism requires a quantitative definition of the functional linkages between these binding sites. In 1964, Wyman introduced the thermodynamic concept of linked functions to establish a quantitative formulation for describing the mutual influence of binding sites on each other (1). Linkage theory is based on thermodynamic principles, is applicable to all biological systems, and exhibits quantitative predictive power. Although Wyman's theory provides the mathematical relationships, it does not address the mechanism through which different binding sites communicate. Thus, besides functional linkage, there are underlying structural-thermodynamic linkages that define the mechanism of site-site communication.Despite a significant body of literature showing that information is transmitted through biological systems via a series of interand intramolecular communication events (refs. 2-5 and references therein), a quantitative predictive theory of structural linkage analogous to the Wyman Linkage Theory is not available. Recently, however, a theoretical approach was established to treat the native state of a protein as an ensemble of conformationa...

Millisecond timescale fluctuations in dihydrofolate reductase are exquisitely sensitive to the bound ligands

“…This observation is consistent with theoretical simulations that reveal energetic and motional coupling between the active site and the adenosine binding site in E. coli DHFR (31)(32)(33)(34)(35). Interestingly, the coupling between these sites is lost in the binary and ternary product complexes E:THF and E:THF:NADP þ (Fig.…”

Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase

“…M.HhaI shares an AdoMet binding site architecture (Rossman fold) as found in the cofactor binding site of NADPH dependent enzymes (16). The NAD(P)H:flavin oxidoreductase, ferredoxinϪNADP ϩ reductase, E. coli dihydrofolate reductase, and R67 dihydrofolate reductase all have the capability to recognize ligands in multiple binding modes (31)(32)(33)(34) The dual energetic mode observed for the recognition of AdoMet and AdoHcy by M.HhaI, therefore, does not appear surprising. The dual mode recognition, therefore, implies plasticity in the recognition of bipartite molecules such as AdoMet and AdoHcy at concentrations high enough to saturate the cofactor binding site of M.HhaI.…”

Solution structure of a paradigm ArsR family zinc sensor in the DNA-bound state

“…In contrast, amide protons in the ␣5 helices in the allosteric ligand binding sites, for example, are comparatively less protected from solvent exchange relative to apo-CzrA (8) with k ex measurable for just one residue in this helix (A91) in the complex. These dynamics could strongly influence the shift in the native populations to the open or closed state (23), and suggest that DNA binding is effectively communicated to the allosteric sites via long-range dynamic disorder and native-state destabilization (24)(25)(26), likely as a consequence of a large structural change in this region of the molecule (Fig. 1).…”