Molecular dynamics simulations elucidate the mode of protein recognition by Skp1 and the F‐box domain in the SCF complex

Polyubiquitination of the target protein by a ubiquitin transferring machinery is key to various cellular processes. E3 ligase Skp1‐Cul1‐F‐box (SCF) is one such complex which plays crucial role in substrate recognition and transfer of the ubiquitin molecule. Previous computational studies have focused on S‐phase kinase‐associated protein 2 (Skp2), cullin, and RING‐finger proteins of this complex, but the roles of the adapter protein Skp1 and F‐box domain of Skp2 have not been determined. Using sub‐microsecond molecular dynamics simulations of full‐length Skp1, unbound Skp2, Skp2‐Cks1 (Cks1: Cyclin‐dependent kinases regulatory subunit 1), Skp1‐Skp2, and Skp1‐Skp2‐Cks1 complexes, we have elucidated the function of Skp1 and the F‐box domain of Skp2. We found that the L₁₆ loop of Skp1_, which was deleted in previous X‐ray crystallography studies, can offer additional stability to the ternary complex via its interactions with the C‐terminal tail of Skp2. Moreover, Skp1 helices H6, H7, and H8 display vivid conformational flexibility when not bound to Skp2, suggesting that these helices can recognize and lock the F‐box proteins. Furthermore, we observed that the F‐box domain could rotate (5°–129°), and that the binding partner determined the degree of conformational flexibility. Finally, Skp1 and Skp2 were found to execute a domain motion in Skp1‐Skp2 and Skp1‐Skp2‐Cks1 complexes that could decrease the distance between ubiquitination site of the substrate and the ubiquitin molecule by 3 nm. Thus, we propose that both the F‐box domain of Skp2 and Skp1‐Skp2 domain motions displaying preferential conformational control can together facilitate polyubiquitination of a wide variety of substrates. Proteins 2016; 84:159–171. © 2015 Wiley Periodicals, Inc.