Coevolution combined with molecular dynamics simulations provides structural and mechanistic insights into the...

Finding the 3D structure of large, multi-subunit complexes is difficult, despite recent advances in cryo-EM technology, due to remaining challenges to expressing and purifying subunits. Computational approaches that predict protein-protein interactions, including Direct Coupling Analysis (DCA), represent an attractive alternative for dissecting interactions within protein complexes. However, they are readily applicable only to small proteins due to high computational complexity and a high number of false positives. To solve this problem, we proposed a modified DCA approach, a powerful tool to predict the most likely interfaces of protein complexes. Since our modified approach cannot provide structural and mechanistic details of interacting peptides, we combine it with Molecular Dynamics (MD) simulations. To illustrate this novel approach, we predict interacting domains and structural details of interactions of two Integrator complex subunits, INTS9 and INTS11. Our predictions of interacting residues of INTS9/INTS11 are highly consistent with crystallographic structure. We then expand our procedure to two complexes whose structures are not well-studied: 1) The heterodimer formed by the Cleavage and Polyadenylation Specificity Factor 100-kD (CPSF100) and 73-kD (CPSF73); 2) The heterotrimer formed by INTS4/INTS9/INTS11. Experimental data supports our predictions of interactions within these two complexes, demonstrating that combining DCA and MD simulations is a powerful approach to revealing structural insights of large protein complexes.