Measuring rates of ubiquitin chain formation as a functional readout of ligase activity

Abstract

Specificity within the pathways of ubiquitin conjugation are defined by protein-binding affinities among the components. Enzyme kinetics provides a facile high-resolution experimental approach for quantitating such protein-binding affinities and yields additional mechanistic insights into the transition state of the enzyme-catalyzed reaction. Most ubiquitin ligases form free polyubiquitin chains at a slow rate in the absence of their cognate target protein as a normal step in their overall catalytic cycle. Rates of polyubiquitin chain formation can, therefore, be used as a reporter function kinetically to characterize binding interactions within the ligation pathway. We describe experimental approaches for: (1) precisely quantitating functional E1 and E2 concentrations by their stoichiometric formation of (125)I-ubiquitin thiolester; (2) semiquantitative screens to define the cognate E2(s) for ubiquitin ligases based on their ability to support polyubiquitin chain formation; (3) initial rate studies to quantify K (m) and k (cat) as a measure of the ability of specific E2-ubiquitin thiolester substrates to support ligase-catalyzed polyubiquitin chain formation; and (4) an isopeptidase T-based technique for distinguishing between free and conjugated polyubiquitin chains formed in the functional assays. These kinetic methods provide mechanistic insights that are otherwise inaccessible by other experimental approaches and yield a precision in characterizing protein interactions that exceeds that of other techniques.

Fig. 1

Schematic mechanism of ubiquitin conjugation. Ubiquitin-activating enzyme (E1) couples hydrolysis of ATP to the formation of a ternary complex composed of a covalently bound ubiquitin thiolester and a tightly bound ubiquitin adenylate intermediate. The activating enzyme subsequently binds a ubiquitin-specifi c carrier protein (E2) and catalyzes the transfer of the former intermediate to the E2 to form a corresponding E2-ubiquitin thiolester. The ubiquitin-protein isopeptide ligase (E3) binds its cognate E2-ubiquitin thiolester among the total pool of such cellular intermediates and catalyzes a reaction that couples aminolytic cleavage of the E2-ubiquitin thiolester to formation of the new isopeptide bond on the target protein (P).

Fig. 2

Autoradiogram of stoichiometric quantitation of ubiquitin conjugation components. Incubations were performed as described in Subheading 3.1 for ¹²⁵I-ubiquitin in the absence (lane 1) or presence of the indicated components. Human Uba1 and recombinant UbcH7 E2 carrier protein were present at 50 nM. Recombinant GST–E6AP Hect domain fusion protein was present at 10 nM. Shown are the autoradiographic densities for the corresponding ¹²⁵I-ubiquitin thiolesters. Quantitation of these thiolester intermediates is achieved by excising the corresponding bands and quantitating associated ¹²⁵I by gamma counting.

Fig. 3

Autoradiogram of an E2 screen conducted in the presence of Trim25 ligase. Incubations were performed as described in Subheading 3.2 in 10-min incubations containing 100 nM Uba1 and 1 μM recombinant human GST-Trim25 (determined as total protein) in the absence (lane 2) or presence of 100 nM of the indicated recombinant E2 proteins. The concentrations of active E1 and E2 proteins were determined by the stoichiometric formation of ¹²⁵I-ubiquitin thiolester, as described in Subheading 3.1.

Fig. 4

Initial rate kinetic study of a GST-E6APF849Y point mutant. Incubations containing 100 nM Uba1, 30 nM GST-E6APF849Y, and the indicated concentrations of recombinant UbcH7 were incubated for 10 min at 37°C as outlined in Subheading 3.3. Conjugated ¹²⁵I-ubiquitin was quantitated by excising each lane above the 25-kDa relative molecular weight marker and used to calculate the initial velocity as described in the text. (a) Autoradiogram of the resulting SDS-PAGE resolution of the incubations. (b) Dependence of initial rate (v_o) on (UbcH7)_o (solid line represents the nonlinear regression fit for K_m = 52 ± 8 nM and k_cat = 1.4 ± 0.1 × 10^–3 s^–1). Inset – Double-reciprocal plot of the rate data.

Scheme 1

Michaelis–Menten scheme