Identifying Sequential Substrate Binding at the Single-Molecule Level by Enzyme Mechanical Stabilization

Abstract

Enzyme-substrate binding is a dynamic process intimately coupled to protein structural changes, which in turn changes the unfolding energy landscape. By the use of single molecule force spectroscopy (SMFS), we characterize the open-to-closed conformational transition experienced by the hyperthermophilic ADP-dependent glucokinase from Thermococcus litoralis triggered by the sequential binding of substrates. In the absence of substrates, the mechanical unfolding of TlGK shows an intermediate I, which is stabilized in the presence of Mg·ADP-, the first substrate to bind to the enzyme. However, in the presence of this substrate, an additional unfolding event is observed, intermediate-1*. Finally, in the presence of both substrates, the unfolding force of intermediates-1 and -1*, increases as a consequence of the domain closure. These results show that SMFS could be used as a powerful experimental tool to investigate binding mechanisms of different enzymes with more than one ligand, expanding the repertoire of protocols traditionally used in enzymology.

Dissecting the functional roles of the conserved NXXE and HXE motifs of the ADP‐dependent glucokinase fromThermococcus litoralis

“…The preincubation of TlGK with AMP, AlCl 3 and NaF such that the concentration of AMP-AlF 3 formed is $1 mM, followed by titration with increasing concentrations of glucose, led to a notorious increase in fluorescence intensity (Fig. 3A), in good agreement with previous assessment of glucose binding in the presence of MgAMP [10]. We fitted the change in fluorescence intensity of wild type TlGK to Eq.…”

“…In line with this kinetic mechanism, small angle X-ray scattering and structure solution of TlGK in the presence of substrates and analogs have shown that the large and small domains of TlGK experience a sequential open-to-closed conformational transition, where MgADP À binding elicits a semi-closed state and the subsequent binding of glucose leads to formation of the ternary complex, triggering total domain closure [7]. Moreover, this sequential binding of substrates in TlGK has been recently confirmed by singlemolecule force spectroscopy experiments [10].…”

“…The addition of glucose to Tl GK does not exert changes in the fluorescence intensity of any of its four Trp residues, unless a MgADP − analog is also present[10]. Therefore, assessment of glucose binding was carried out by measuring the change in fluorescence intensity upon titration of Tl GK in complex with the non‐hydrolysable transition state analog of MgADP − , AMP–AlF 3 .…”

Molecular Strategy for Blocking Isopeptide Bond Formation in Nascent Pilin Proteins

“…Specifically, we compare the unfolding mechanics of the modified pilus proteins with the intact Spy0128 and the mutant E258A, which lacks of isopeptide bond, as previously demonstrated (24). As shown in Figure 2B, our polyprotein constructs have two Spy0128 proteins flanked by titin I91 domains, used as fingerprint to discard spurious traces (24,35). When pulling intact Spy0128 constructs, only the three I91 domains unfold, which are identified as unfolding events with a contour length increment of 29 nm, as determined by fits to the worm-like chain model (36), and unfolding forces of ~200 pN (Figure 3A).…”

“…We characterized the mechanical properties of the intact Spy0128 domain, and mutated Glu258Ala, where the isopeptide bond is abolished (15). In our polyprotein construct, two Spy0128 domains are flanked by I27 titin domains, allowing to use the I27 unfolding pattern as a fingerprint to discard spurious traces (14,17). While in the intact Spy0128 polyproteins only the I27 domains unfold (Figure 3A), the Glu258Ala mutant is extensible, showing a contour length increment of 50 nm, and an unfolding force of ~300 pN (Figure 3B).…”