Temperature-dependent hydrogen deuterium exchange shows impact of analog binding on adenosine deaminase flexibility but not embedded thermal networks

Abstract

The analysis of hydrogen deuterium exchange by mass spectrometry as a function of temperature and mutation has emerged as a generic and efficient tool for the spatial resolution of protein networks that are proposed to function in the thermal activation of catalysis. In this work, we extend temperature-dependent hydrogen deuterium exchange from apo-enzyme structures to protein-ligand complexes. Using adenosine deaminase as a prototype, we compared the impacts of a substrate analog (1-deaza-adenosine) and a very tight-binding inhibitor/transition state analog (pentostatin) at single and multiple temperatures. At a single temperature, we observed different hydrogen deuterium exchange-mass spectrometry properties for the two ligands, as expected from their 10⁶-fold differences in strength of binding. By contrast, analogous patterns for temperature-dependent hydrogen deuterium exchange mass spectrometry emerge in the presence of both 1-deaza-adenosine and pentostatin, indicating similar impacts of either ligand on the enthalpic barriers for local protein unfolding. We extended temperature-dependent hydrogen deuterium exchange to a function-altering mutant of adenosine deaminase in the presence of pentostatin and revealed a protein thermal network that is highly similar to that previously reported for the apo-enzyme (Gao et al., 2020, JACS 142, 19936-19949). Finally, we discuss the differential impacts of pentostatin binding on overall protein flexibility versus site-specific thermal transfer pathways in the context of models for substrate-induced changes to a distributed protein conformational landscape that act in synergy with embedded protein thermal networks to achieve efficient catalysis.

Keywords: adenosine deaminase; conformational landscape; protein flexibility; temperature-dependent hydrogen deuterium exchange (TDHDX-MS); thermal activation.

Figure 1

Structure, catalytic mechanism, and ligands of mADA.A, the overall structure of mADA in complex with 1-deaza-adenosine (PDB:1ADD), with active site residues shown in (B) where zinc ion is colored magenta and the mutation position Phe61 for the study is shown in yellow stick. C, proposed catalytic mechanism of mADA in which the active site base His238 converts zinc-bound water to hydroxide; attack of hydroxide ion at C-6 of substrate, concomitant with proton transfer from Glu217 to N-1 of the purine ring, leads to the tetrahedral adduct (see details in reference 12); R group represents the ribose ring of substrate. D, chemical structures of the substrate, ground state analog (1-deaza-adenosine, DAA), and tight binding inhibitor/transition state (2′-deoxycoformycin, pentostatin) for mADA. DAA, 1-deaza-adenosine; mADA, murine adenosine deaminase.

Figure 2

Deuteron uptake comparisons between ligand free, ground state analog (ES), and tight binding inhibitor(ETS)bound statesfor WT mADA.A, HDX plots showing deuteron uptake differences between any two of the three states are presented. Red: ligand-free state; blue: ground state analog bound state; green: tight binding inhibitor bound state. B, dual-color mapping (blue and red, blue indicates less deuteron uptake and red indicates more deuteron uptake) was used to show HDX changes for ground state analog relative to ligand-free enzyme states (left) and for tight binding inhibitor bound relative to ligand-free enzyme states (right). HDX, hydrogen deuterium exchange; mADA, murine adenosine deaminase.

Figure 3

Interactions in regions of peptide 180-200 (from the structure of mADA in complex with DAA).A, Asp185 (in H-bonding distance to Thr187) initiates a series of hydrophobic interactions that connect, in turn, Leu59, Phe61, and Phe65; the latter is in direct contact with the bound analog. B, Asp181 and Leu182 form hydrogen bonds with the zinc ligand His214, which is in contact with the bound analog. DAA, 1-deaza-adenosine; mADA, murine adenosine deaminase.

Figure 4

Deuteron uptake comparisons between ligand free, ground state analog bound (ES), and tight binding inhibitor(ETS)bound statesto F61A mADA.A, HDX plots showing deuteron uptake differences between any two of the three states. Red: ligand-free state; blue: ground state analog bound state; green: tight binding inhibitor bound state. B, dual-color mapping (blue and red, blue indicates less deuteron uptake and red indicates more deuteron uptake) was used to show HDX changes for the DAA bound relative to free enzyme states (left) and tight binding inhibitor relative to free enzyme states (right). DAA, 1-deaza-adenosine; HDX, hydrogen deuterium exchange; mADA, murine adenosine deaminase.

Figure 5

Bar graph representations of the experimental activation energies Ea_HDXfor peptides from substrate free, DAA bound (ES), and pentostatin bound (ETS) WT mADA and mapping of ΔEa values on the structure of mADA.A, Ea_HDX values for each form of the protein. B, ΔEa (ES-apo). C, ΔEa (ETS-apo)_. We note that in four out of fifty one Arrhenius plots, WT-apo-(201–229), WT-DAA-(260–267), WT-DAA-(301–320), and WT-pentostatin-(301–320), the 40 °C data point was anomalously low, possibly indicating a transitioning above 30 °C from intermediate HDX to the very fast HDX regime that reduces the weighted average rate constant; in these limited instances, fitting was restricted to 10, 20, 25, and 30 °C. D, structural mapping of (left) ΔEa (ES-apo)_, (middle) ΔEa (ETS-apo)_, and (right) ΔEa (ETS-ES). Heat maps are used to show the direction and magnitude of flexibility changes for regions that have become either more flexible (red tones) or more rigid (blue tones). The active site faces toward the reader in the top three structures. DAA, 1-deaza-adenosine; HDX, hydrogen deuterium exchange; mADA, murine adenosine deaminase.

Figure 6

Active site comparison of mADA in the presence of various ligands. Comparison of active sites of HDPR (A) and pentostatin (B)-bound enzyme complexes. Distances (in Å) between the Zn²⁺ and its ligands are labeled in green dashed lines. Distances between the residues Phe58, Phe61, and Phe65 are labeled in marine dashed lines. Ribose moiety interactions in the HDPR complex (C), pentostatin complex (D), and DAA complex (E). DAA, 1-deaza-adenosine; HDPR, 6-hydroxyl-1,6-dihydropurine ribonucleoside. The zinc ion is labeled magenta and bound water molecules are indicated as red spheres.

Figure 7

Comparison of mADA thermal networks under different experimental conditions. Derived thermal networks for (A) ΔEa_HDX(F61A-WT) with apo-enzyme (27) and (B) ΔEa_HDX(F61A-WT) for enzyme complexed to pentostatin (this work). The measured Ea values for WT and F61A are on the left and the mapping of the regions affected onto the WT mADA structure (PDB 2ADA) on the right. In the displayed thermal networks, heat maps are used to show the direction and magnitude of flexibility changes for regions that have become either more flexible (red tones) or more rigid (blue tones). The active site faces toward the reader in the left-hand structures. mADA, murine adenosine deaminase.

Figure 8

Comparison of HDX results under different experimental conditions. Heat maps are used to show the direction and magnitude of flexibility changes for regions that have become either more flexible (red tones) or more rigid/protected (blue tones). A, protection of mADA from HDX upon binding of the tight binding inhibitor pentostatin at a single temperature; B, the impact of pentostatin binding on protein flexibility obtained from multiple temperature HDX experiments; and C, thermal network for catalysis deduced for mADA in the presence of pentostatin through a comparison of the functionally impaired mutation F61A to native enzyme. HDX, hydrogen deuterium exchange; mADA, murine adenosine deaminase.