Significance Heat shock protein 70 (Hsp70) molecular chaperones help maintain protein homeostasis. Hsp70 functions require regulated promiscuous binding and release of a wide range of protein substrates. ATP binding to the Hsp70 nucleotide-binding domain (NBD) regulates the affinity and kinetics of substrate binding to their substrate-binding domain (SBD). Our work sought deeper understanding of the role of conformational dynamics for allosteric signaling in Hsp70s: The SBD undergoes a seesaw-like conformational change from a high substrate affinity state to one with lower substrate affinity, and we show that this conformational change results in drastic changes in conformational flexibility for the SBD that are essential for efficient substrate binding and release. These insights will help efforts to use Hsp70s as therapeutic targets.