Abstract
We have recently discovered that the ZZ zinc finger domain represents a novel small ubiquitin-like modifier (SUMO) binding motif. In this study we identify the binding epitopes in the ZZ domain of CBP (CREB-binding protein) and SUMO1 using NMR spectroscopy. The binding site on SUMO1 represents a unique epitope for SUMO interaction spatially opposite to that observed for canonical SUMO interaction motifs (SIMs). HADDOCK docking simulations using chemical shift perturbations and residual dipolar couplings was employed to obtain a structural model for the ZZ domain-SUMO1 complex. Isothermal titration calorimetry experiments support this model by showing that the mutation of key residues in the binding site abolishes binding and that SUMO1 can simultaneously and non-cooperatively bind both the ZZ domain and a canonical SIM motif. The binding dynamics of SUMO1 was further characterized using (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersions, which define the off rates for the ZZ domain and SIM motif and show that the dynamic binding process has different characteristics for the two cases. Furthermore, in the absence of bound ligands SUMO1 transiently samples a high energy conformation, which might be involved in ligand binding.
Highlights
Only a subset of the residues affected by the binding of the SIM peptide is within 5 Å of SIMPX, as gauged from the SIM-SUMO1 structure (Protein Data Bank code 2ASQ). This result indicates that the binding of SIMPX induces a conformational change in SUMO1
The fourth row shows the data for the binding of SIMPX to the preformed complex between the ZZ domain (ZZwt) and SUMO1
Upon the addition of SIMPX to SUMO1, the exchange is quenched for many residues, but 20 residues show exchange in both apo- and SIMPX-bound SUMO1
Summary
Molecular Biology—DNA sequences for the ZZ domain from CBP (UniProtKB ϭ Q92793) and SUMO1 (UniProtKB ϭ P63165) were synthesized by Geneart and subcloned into a pNIC28-BsaI vector using ligation independent cloning [14]. RDCs were measured for each component of the ZZ domain-SUMO1 complex using two separate samples, in which one protein was labeled with 13C,15N and the other unlabeled. The final NMR samples of the ZZ domain-SUMO1 complex contains 4% unbound SUMO1, as calculated from the Kd and protein concentrations. Residues in the N and C termini were considered flexible [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 95–103] based on R20 values from the CPMG relaxation dispersion experiments and were excluded from the structure calculations. Errors in the fitted parameters were estimated from 1000 synthetic data sets created using Monte-Carlo simulations [41, 42]
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