A dynamic allosteric pathway underlies Rad50 ABC ATPase function in DNA repair

Abstract

The Mre11-Rad50 protein complex is an initial responder to sites of DNA double strand breaks. Many studies have shown that ATP binding to Rad50 causes global changes to the Mre11-Rad50 structure, which are important for DNA repair functions. Here we used methyl-based NMR spectroscopy on a series of mutants to describe a dynamic allosteric pathway within Rad50. Mutations result in changes in the side chain methyl group chemical environment that are correlated with altered nanosecond timescale dynamics. We also observe striking relationships between the magnitude of chemical shift perturbations and Rad50 and Mre11 activities. Together, these data suggest an equilibrium between a ground state and an "active" dimerization competent state of Rad50 that has locally altered structure and dynamics and is poised for ATP-induced dimerization and eventual ATP hydrolysis. Thus, this sparsely populated intermediate is critical for Mre11-Rad50-directed DNA double strand break repair.

Figure 1

Domain architecture and NMR spectra of Rad50. (a) Cartoon representation of full length Rad50 (top) and Rad50^NBD (bottom). (b) Crystal structure of P. furiosus Mre11^HLH-Rad50^NBD (3QKS). Colors in (a) and (b) illustrate conserved domains important for Rad50 function. (c) 2D ¹³C,¹H methyl-TROSY HMQC spectrum of Mre11^HLH-Rad50^NBD collected at 14.1 T and 50 °C. The side chain methyl group assignments are given.

Figure 2

Mutations reveal an allosteric network in Rad50^NBD. (a) 2D ¹³C,¹H methyl-TROSY HMQC spectral overlay of WT and mutant Mre11^HLH-Rad50^NBD recorded at 14.1 T and 50 °C. Arrows highlight side chain methyl groups experiencing CSPs. (b) Structure of Mre11^HLH-Rad50^NBD emphasizing side chain methyl groups whose CSPs upon mutation cluster according to CHESCA as described in the Methods. Clusters are listed in Supplementary Table S1 and were determined via the dendrogram in Supplementary Fig. S4. Methyl groups not affected by the mutations are not shown.

Figure 3

Chemical shift perturbations and dynamics changes are correlated. (a) Representative η vs δ_Methyl scatter plots for Met808Cε (top) and Ile131Cδ1 (bottom). δ_Methyl values were determined according to equation (1), see Supporting Information. Pearson’s correlation coefficients (R_P) are given in the upper right corner. Insets show the build-up curves for the ratio of intensities arising from methyl group ¹H triple-quantum “forbidden” experiments (I_forbid/I_allow) vs. relaxation delay time for wildtype and mutant Mre11^HLH-ILVM labeled Rad50^NBD. These data were fit to equation (3) to determine the η rates, as described in the Methods, with errors determined from the covariance matrix of the fit. The coloring of the curves corresponds to the spectra of the mutants in Fig. 2a. (b) Structure of Mre11^HLH-Rad50^NBD showing side chain methyl groups with altered dynamics upon mutation. Red and orange spheres represent methyl groups that become more flexible upon mutation, whereas green and blue spheres represent methyl groups that become more rigid upon mutation. Red and green coloring denotes “Correlated” methyl groups with significant CSPs upon mutation (i.e., the range in δ_Methyl > 0.13 ppm) that also have a correlation for η vs δ_Methyl of |R_P| > 0.7. Orange and blue coloring denotes “Not Correlated” methyl groups with small CSPs upon mutation (i.e., the range in δ_Methyl < 0.13 ppm) but have a large difference in η rates between wildtype and the mutants (|η_WT – the average η_mutants| > 8 sec⁻¹).

Figure 4

Basic switch and hinge region mutations affect Rad50 activity. (a) Bar chart showing the effect of mutation, relative to wildtype activity, for ATP hydrolysis (left - k_cat/K_M), ATP-dependent dimerization (middle - % dimerization), and Mre11 exonuclease activity (right – relative fluorescence). Green, orange, purple, and blue bars represent wildtype, V156M, V160M, and R805E, respectively. The order of the bars follows the general order of peaks that experience significant CSPs upon mutation. For ATP hydrolysis and exonuclease assays, bars represent the average of at least three independent measurements, and the error bars are the standard deviation of the replicate experiments. Dimerization assays were performed once. *^, ** and *** Represent p-values less than 0.05, 0.01, and 0.001, respectively. The inset above shows the CSPs for L47Cδ1. (b) Structure of Mre11^HLH-Rad50^NBD highlighting the side chain methyl groups that have a |mean[R_P,Hydrolysis, R_{P,Dimerization}, R_{P,Exonuclease}]| > 0.65.

Figure 5

Model for Rad50^NBD “activation” to a binding competent state. Left, illustrates the “ground” state Rad50^NBD with stable interactions between the hinge, extended signature helix, basic switch, and Q-loop. This state is in equilibrium (green reaction arrow) with an ensemble of “Dimerization Competent States,” middle, in which the stable interactions are starting to break and increased side chain dynamics are present. The effect of V156M, V160M, and R805E is to increase the population of this state, as denoted by the orange, purple, and blue reaction arrows. ATP binds to this state, and dimerization can subsequently occur, right. The process is reset by ATP hydrolysis and dimer dissociation.