Elucidating the mechanics and mechanism of two prototypical ring shaped proteins using modeling and simulation

Ring-shaped proteins are involved in a diverse set of essential cellular processes. The topology of these proteins allow them to encircle the substrate with which they associate. This quaternary arrangement is observed in a myriad of proteins involved in DNA metabolism, where a sterically loose but topologically tight linkage allows them to localize on their track, but provides for a dynamic association that often underlies their function. Here we present work on two prototypical ring-shaped proteins, the transcription termination factor Rho and the proliferating cell nuclear antigen (PCNA). Rho is a homohexameric ATPase that translocates along single-stranded RNA, while PCNA acts as the processivity factor for the Eukaryotic replisome. These proteins are selected in order to examine two important questions related to ring-shaped proteins: (1) How do ring-shaped enzymes with multiple ATP catalytic sites coordinate their hydrolysis cycles to translocate on nucleic acid substrates? (2) How do the mechanical properties of a ring-like protein impact its ability to load on its nucleic acid substrate?The first question is examined by constructing a physically motivated kinetic model of Rho from biochemical measurements and structural data in order to understand how this motor coordinates ATP hydrolysis with nucleic acid translocation. This model allows us to globally fit a number of steady and pre-steady state kinetic experiments as well as successfully reproduce the behavior of a RNA binding deficient mutant. This work suggests the role of non-nearest neighbor coordination between individual catalytic sites within the protein, and may indicate the necessity of allosteric communication between subunits in a subset of ring-shaped ATPases.We then use atomistic and coarse-grained models and simulation to examine the conformational flexibility of PCNA in order to determine how its mechanical properties impact its role in loading onto DNA. PCNA must interconvert from a closed planar ring to an open structure that can accommodate the passage of DNA through a gap formed by disrupting one of its subunit-subunit interfaces. From these simulations, we are able to predict the conformational flexibility of PCNA and estimate the energetic cost of deforming this molecule in the in- and out-of-plane directions. These structural studies place important mechanistic constraints on the clamp loading process, and provide a foundation for studying other ring-like proteins that are pre-assembled and then must be loaded onto DNA.