Dynamic coordination of two-metal-ions orchestrates λ-exonuclease catalysis

Abstract

Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity. Here, we use λ-exonuclease as a model enzyme with two Mg²⁺ binding sites and probe activity at various concentrations of magnesium by single-molecule-FRET. We find that while Mg_A²⁺ and Mg_B²⁺ have similar binding constants, the dissociation rate of Mg_A²⁺ is two order of magnitude lower than that of Mg_B²⁺ due to a kinetic-barrier-difference. At physiological Mg²⁺ concentration, the Mg_B²⁺ ion near the 5'-terminal side of the scissile phosphate dissociates each-round of degradation, facilitating a series of DNA cleavages via fast product-release concomitant with enzyme-translocation. At a low magnesium concentration, occasional dissociation and slow re-coordination of Mg_A²⁺ result in pauses during processive degradation. Our study highlights the importance of metal-ion-coordination dynamics in correlation with the enzymatic reaction-steps, and offers insights into the origin of dynamic heterogeneity in enzymatic catalysis.

Fig. 1

Real-time measurements of processive degradation by λ-exonuclease. a The crystal structure of λ-exonuclease (PDB entry 1AVQ). b Experimental layout, depicting the DNA, protein binding to DNA, and processive degradation. c Schematic showing how the degradation time is measured using FRET signal. d Single-molecule FRET histogram obtained as in b (top: dsDNA only; middle: before the degradation in the absence of Mg²⁺; and bottom: after the degradation in the presence of Mg²⁺)

Fig. 2

Mg²⁺-dependent degradation of DNA by λ-exonuclease. a Representative traces showing that degradation reaction slows down at lower Mg²⁺ concentrations (0 mM, 0.03 mM, 0.08 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.5 mM, 1 mM, 3 mM, 6 mM, and 9 mM Mg²⁺: top to bottom) at the fixed 16 nM λ-exonuclease (trimer concentration unless otherwise stated). b The degradation rate (velocity) versus Mg²⁺ concentrations following a Hill fit (red line) with a maximum velocity of 17.2 nt s⁻¹, a Km value of 0.885 mM of Mg²⁺, and n = 1.6. Inset, blowing up at lower Mg²⁺ concentrations showing fitting to a sigmoidal kinetics (red) versus a Michaelis Menten kinetics (blue). Error bars denote the standard error of the mean (SEM). The velocity at [Mg^2 + ] = 15 mM is highlighted in red. c Time-dependent fractional growth at various Mg²⁺ concentrations. d The proportion of the pause population as a function of the Mg²⁺ concentration. e Distribution of pause times with varying concentrations of Mg²⁺

Fig. 3

Inhibition of λ-exonuclease activity by Ca²⁺ ions. a Single-molecule FRET histograms with various concentrations of Ca²⁺ at the fixed concentration of 3 mM Mg²⁺. b Ca²⁺-substitution-induced pause behavior fitted with an automated step-finding algorithm. c Pause histograms obtained from various Ca²⁺ concentrations at the fixed concentration of 3 mM Mg²⁺. d Three degradation patterns with pauses and backtracking when inhibited by Ca²⁺. e The proportion of pause and backtracking as a function of Ca²⁺ concentration. f Increase in the degradation time with increasing concentration of Ca²⁺

Fig. 4

Quantitative analysis of two-metal dynamics. a A kinetic model for the two-metal-ion dynamics consisting of three states: EMM (exonuclease:Mg_B²⁺:Mg_A²⁺ complex), EM (exonuclease:Mg_A²⁺ complex), and E (exonuclease only). In the model, Mg_B²⁺ dissociates upon DNA cleavage and translocation ($k_B^{off}$, red arrow, EMM to EM) whereas Mg_A²⁺ dissociates stochastically ($k_A^{\mathrm{off}}$, EM to E). The single cycle is completed upon rebinding of Mg_B²⁺ ($k_B^b$, EM to EMM). b The degradation time per nucleotide (τ₁) versus Mg²⁺ concentrations. The data are fitted to three models: (1) Michaelis Menten equation (orange line), (2) Hill equation (purple line, n = 1.6), and (3) Eq. 1 for the model shown in a (red line). Inset shows velocity ( = ${\tau }_1^{-1}$) versus Mg²⁺ concentrations data and their fits (solid lines). c Representative FRET time traces from simulations performed at three different choices of $k_A^{off}$: 0.01 s⁻¹ (top panel), 0.1 s⁻¹ (middle panel), and 1 s⁻¹ (bottom panel). [Mg²⁺] is set to 0.03 mM. d Degradation rate histograms at four different [Mg²⁺] conditions: 0.03 mM, 0.1 mM, 0.3 mM, and 3 mM. Gray bars represent experimental data, whereas solid lines are theoretical prediction calculated under three difference choices of $k_A^{off}$: 0.01 s⁻¹ (black), 0.1 s⁻¹ (red), and 1 s⁻¹ (orange). e Mean squared error (MSE) versus $k_A^{off}$. The errors are calculated by summing the squares of the difference between the experimental and theoretically predicted results

Fig. 5

FRET time trajectories and the free energy landscape during metal-ion dynamics. a Two experimental FRET time trajectories, showing Mg_B²⁺-dynamics-dependent slope change (purple) and a pause caused by the dissociation of two metal ions, Mg_A²⁺ and Mg_B²⁺ (pink). b A representative simulation trajectory, displaying three panels: a time-FRET trace (black line in the top panel), degradation position of exonuclease along DNA (the line in the middle panel), and its metal-ion state (green line in the bottom panel). The trajectory revealed that iteration between EM and EMM decreases the degradation slope in a Mg²⁺-dependent manner, and pauses occur due to the E state by dissociation of both metal ions. c Total fraction of single-molecule traces showing the pause state at three different Mg²⁺ states. d Free energy landscape along the reaction coordination of Mg²⁺ binding and dissociation. Processive degradation occurs during a repetitive cycle between the 1^st and 2^nd metal-ion binding states, whereas a pause occurs due to the dissociation of two metal ions. The energy barrier between the 2^nd and 1^st states is lower than the one between the 1^st and 2^nd states due to the different numbers of ligands surviving after the translocation (e.g., 1 versus 3 ligands with protein residues for Mg_B²⁺ and Mg_A²⁺, respectively). Transitions between ES** and EP* are indicated by two one-directional arrows to emphasize the transitions are non-reversible and not in equilibrium

Fig. 6

The mechanism of two-metal-ion dynamics. The active site of one subunit of the homotrimer is shown in green whereas two Mg²⁺ ions and nucleotides are represented by yellow circles and rectangles in various colors, respectively. Metal-ion coordination to the catalytic active site is highly dynamic so that two Mg²⁺ ions (Mg_A²⁺ and Mg_B²⁺) can coordinate with and dissociate from the surrounding ligands. Mg_A²⁺ remains stably bound to the active site, but Mg_B²⁺, which is close to the 5′ terminal side of the scissile phosphate, dissociates during every round of catalytic cleavage. As a result, the dissociation of Mg_B²⁺ facilitates product release and exonuclease translocation, promoting the overall processivity of exonuclease activity. More specifically, at high [Mg²⁺] (≈2 mM), λ-exonuclease degrades processively and its cleavage activity is mainly controlled by Mg_B²⁺ dynamics (green dashed square). The fast unbinding/rebinding dynamics of Mg_B²⁺ and strict requirement of two Mg²⁺ ions for the catalytic step yield a Mg²⁺-concentration-dependent exonuclease activity. Conversely at low [Mg²⁺] (≤0.3 mM), occasional unbinding of Mg_A²⁺ from the catalytic site and slow rebinding of Mg_A²⁺ stalls the exonuclease activity, giving rise to a long pause (red dashed square), thus elucidating the molecular origin of dynamic heterogeneity in exonuclease activity. The dynamic variation in the coordination states of two metal ions orchestrates the multistep process of exonuclease activity