Molecular recognition and dynamics of linear poly-ubiquitins: integrating coarse-grain simulations and experiments

Poly-ubiquitin chains are flexible multidomain proteins, whose conformational dynamics enable their molecular recognition by a large number of partners in multiple biological pathways. By using alternative linkage, it is possible to obtain poly-ubiquitin molecules with different dynamical properties. This flexibility is further increased by the possibility to tune the length of poly-ubiquitin chains. Characterizing the dynamics of poly-ubiquitins as a function of their length is thus relevant to understand their biology. Structural characterization of poly-ubiquitin conformational dynamics is challenging both experimentally and computationally due to increasing system size and conformational variability. Here, by developing highly efficient and accurate small-angle X-ray scattering driven Martini coarse-grain simulations, we characterize the dynamics of linear M1-linked di-, tri- and tetra-ubiquitin chains. Our data show that the behavior of the di-ubiquitin subunits is independent of the presence of additional ubiquitin modules. We propose that the conformational space sampled by linear poly-ubiquitins, in general, may follow a simple self-avoiding polymer model. These results, combined with experimental data from small angle X-ray scattering, biophysical techniques and additional simulations show that binding of NEMO, a central regulator in the NF-κB pathway, to linear poly-ubiquitin obeys a 2:1 (NEMO:poly-ubiquitin) stoichiometry in solution, even in the context of four ubiquitin units. Eventually, we show how the conformational properties of long poly-ubiquitins may modulate the binding with their partners in a length-dependent manner.SignificanceProtein conformational dynamics plays an essential role in molecular recognition mechanisms. The characterization of conformational dynamics is hampered by the conformational averaging of observable in experimental structural biology techniques and by the limitations in the accuracy of computational methods. By developing an efficient and accurate approach to combine small-angle X-ray scattering solution experiments and coarse-grain Martini simulations, we show that the conformational dynamics of linear poly-ubiquitins can be efficiently determined and to rationalize the role of poly-ubiquitin dynamic in the molecular recognition of the UBAN domain upon binding to the signaling regulator NEMO. The analysis of the conformational ensembles allows us to propose a general model of the dynamics of linear poly-ubiquitin chains where they can be described as a self-avoiding polymer with a characteristic length associated with their specific linkage.