Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase

Abstract

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. Mutation of multiple conserved residues leads to increased substrate inhibition, suggesting a positive selection during evolution. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

Allostery can convert binding free energies into concerted domain motions in enzymes

“…By contrast, ATP induces the closing of the LID domain, and the subsequent arrival of AMP induces the closing of the NMP domain. These observations suggest the existence of an endosteric effect whereas: the initial binding of AMP inhibits the binding of ATP, or the initial binding of AMP forms a less active conformation, as previously suggested, 51 or the ATP-induced closing of the LID domain induces the formation of a higher-affinity binding site for AMP. In the M2-ternary-AMP:ATP:AK complex, the LID domain is more tightly closed (Figure 2I, right) than in the M2-binary-ATP:AK complex (koff = 118.9 ± 36.3 s -1 and 693.4 ± 29.6 s -1 , respectively, Figure 2I, left).…”

“…; when ATP and AMP or Ap5A are sampled, indicating that when both active sites are occupied the two domains cooperatively open and close. As previously suggested for the observed fast LID domain fluctuations, 51 these concerted motions might be instrumental to align the reactants in the proper conformation. A full kinetic scheme for the binding of ATP and AMP is shown in Figure 3.…”

“…30,33,50,56 The apo-enzyme showed one main current level that occasionally switched to a deeper state (M1, IRES M1 = 49.2 ± 0.1 %, kon M1 = 4.66 ± 0.72 s -1 and koff M1 = 55.2 ± 15.8 s -1 , Figure 2A, Figure S3). Previous work indicated that the enzyme might spontaneously close 33,36,41,45,51,53,[67][68][69][70] S2), most likely, representing all possible domain configurations that AK can adopt within the nanopore.…”

“…Structural studies have shown that the LID and NMP domains undergo major conformation changes [31][32][33] , closing over the active site to protect the ligands from hydrolysis. 34 Many studies have attempted to elucidate how dynamics and conformational changes in AK are associated with molecular recognition, catalysis and allostery 33,[35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] .…”

NMR and smFRET insights into fast protein motions and their relation to function

“…It was found that fast domain movements assist reorienting the substrates following incorrect initial binding to the enzyme, allowing them to reach an optimal orientation for catalysis. To shed further light on this question, mutants with different degrees of substrate inhibition by AMP were studied, both biochemically and using smFRET spectroscopy (112). It was shown that inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, pointing to an allosteric interaction between the domains of AK.…”