Calculated pK_a Variations Expose Dynamic Allosteric Communication Networks

Abstract

Allosteric regulation of protein function, the process by which binding of an effector molecule provokes a functional response from a distal site, is critical for metabolic pathways. Yet, the way the allosteric signal is communicated remains elusive, especially in dynamic, entropically driven regulation mechanisms for which no major conformational changes are observed. To identify these dynamic allosteric communication networks, we have developed an approach that monitors the pKa variations of ionizable residues over the course of molecular dynamics simulations performed in the presence and absence of an allosteric regulator. As the pKa of ionizable residues depends on their environment, it represents a simple metric to monitor changes in several complex factors induced by binding an allosteric effector. These factors include Coulombic interactions, hydrogen bonding, and solvation, as well as backbone motions and side chain fluctuations. The predictions that can be made with this method concerning the roles of ionizable residues for allosteric communication can then be easily tested experimentally by changing the working pH of the protein or performing single point mutations. To demonstrate the method's validity, we have applied this approach to the subtle dynamic regulation mechanism observed for Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of aromatic biosynthesis. We were able to identify key communication pathways linking the allosteric binding site to the active site of the enzyme and to validate these findings experimentally by reestablishing the catalytic activity of allosterically inhibited enzyme via modulation of the working pH, without compromising the binding affinity of the allosteric regulator.

Experimentally based structural model of Yih1 provides insight into its function in controlling the key translational regulator Gcn2

“…This part of Ancient Domain is not affected, at least directly, by the interdomain interactions within Yih1. We can speculate, that if these motifs are involved in binding yet‐to‐be‐discovered binding partners, their effect on interdomain interactions within Yih1, is likely to be via entropically based mechanisms, as is increasingly found in allosteric regulation [51–53]. The most likely binding partner for this region is DNA or RNA, consistent with the recently discovered nuclease activity of IMPACT proteins [54,55].…”

A single amino acid substitution uncouples catalysis and allostery in an essential biosynthetic enzyme in Mycobacterium tuberculosis

“…The subfamilies differ in both quaternary structure and in the types of structural elements decorating the catalytic core, and these decorations deliver the binding sites for allosteric ligands. Type Ia DAH7PS, including those from Escherichia coli, [3][4][5] Saccharomyces cerevisiae, 6,7 and Neisseria meningitidis 8 contains an N-terminal extension and extended loops to the core barrel, which host the binding site for a single allosteric ligand ( Figure 1A). Whereas type Ib contains a group of DAH7PS which has a discrete allosteric domain covalently linked to the catalytic barrel, such as the those from Thermotoga maritima 9 or Geobacillus sp ( Figure 1B).…”

Interdomain Conformational Changes Provide Allosteric Regulation en Route to Chorismate

“…These two quite distinct allosteric solutions suggest that adopting this structural approach for the control DAH7PS represents a general mechanism, albeit that both of these allosteric control mechanisms require the same basic tetrameric structure for the DAH7PS modules to operate. Other DAH7PS enzymes have been shown to adopt allosteric regulation mechanisms based on changes in protein dynamics (13,14) rather than using a physical gating mechanism, which reinforces the versatility of both the protein fold and the approaches to allosteric control.…”

“…Whereas the type I␤ DAH7PS from Thermotoga maritima undergoes significant ACT domain movement associated with the binding of inhibitor Tyr (7), for other enzymes, such as the type II M. tuberculosis DAH7PS (13) and the type I␣ N. meningitidis DAH7PS (14), allosteric inhibition is conferred by more subtle structural changes and alteration in protein dynamics. The variation in allosteric machinery and mechanism for this enzyme is intriguing both from a mechanistic and evolutionary point of view.…”