Scripts containing all the analyses.

Proteins carry out cellular functions by changing their structure among a few conformations, each characterised by a different energy level. Therefore, structural changes, energy transformations, and protein function are intimately related. Despite its central importance, this relationship remains elusive. For example, while many hexameric ATPase motors are known to function using a hand-over-hand alternation of subunits, how energy transduction throughout the assembly’s structure drives the hand-over-hand mechanism is not known. In this work, we unravel the energetic basis of hand-over-hand in a model AAA+ motor, RuvB. To do so, we develop a general method to compute the residue-scale elastic pseudoenergy due to structure changes and apply it to RuvB structures, recently resolved through cryo-EM. This allows us to quantify how progression through RuvB’s mechanochemical cycle translates into residue-scale energy transduction. In particular, we find that DNA binding is associated with overcoming a high energy barrier. This is possible through inter-subunit transmission of energy, and ultimately driven by nucleotide exchange. Furthermore, we show how this structure-inferred energetic quantification can be integrated into a non-equilibrium model of AAA+ assembly dynamics, consistent with single-molecule biophysics measurements. Overall, our work elucidates the energetic basis for the hand-over-hand mechanism in RuvB’s cycle. Besides, it presents a generally applicable methodology for studying the energetics of conformational cycles in other proteins, allowing to quantitatively bridge data from structural biology and single-molecule biophysics.