Crystal structures of the regulatory subunit of Thr-sensitive aspartate kinase (AK; EC 2.7.2.4) from Thermus thermophilus (TtAKbeta) were determined at 2.15 A in the Thr-bound form (TtAKbeta-Thr) and at 2.98 A in the Thr-free form (TtAKbeta-free). Although both forms are crystallized as dimers, the contact surface area of the dimer interface in TtAKbeta-free (3200 A(2)) is smaller than that of TtAKbeta-Thr (3890 A(2)). Sedimentation equilibrium analyzed by ultracentrifugation revealed that TtAKbeta is present in equilibrium between a monomer and dimer, and that Thr binding shifts the equilibrium to dimer formation. In the absence of Thr, an outward shift of beta-strands near the Thr-binding site (site 1) and a concomitant loss of the electron density of the loop region between beta3 and beta4 near the Thr-binding site are observed. The mechanism of regulation by Thr is discussed on the basis of the crystal structures. TtAKbeta has higher thermostability than the regulatory subunit of Corynebacterium glutamicum AK, with a difference in denaturation temperature (T(m)) of 40 degrees C. Comparison of the crystal structures of TtAKbeta and the regulatory subunit of C. glutamicum AK showed that the well-packed hydrophobic core and high Pro content in loops contribute to the high thermostability of TtAKbeta.