Structural Plasticity Allows Calumenin-1 to Moonlight as a Ca²⁺-Independent Chaperone: Pb²⁺ Enables Probing Alternate...

Human calumenin-1 (HsCalu-1) is an endoplasmic reticulum (ER) and Golgi-resident Ca²⁺-binding protein of the hepta-EF-hand superfamily that plays a vital role in maintaining the cytoplasmic Ca²⁺ concentration below toxic levels by interacting with Sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and ryanodine receptors (RyR), indicating its role in Ca²⁺ homeostasis in the ER. HsCalu-1 seems to be able to exhibit structural plasticity to achieve its plethora of functions. In this study, we demonstrate that HsCalu-1 acts as a chaperone in both its intrinsically disordered state (apo form) and the structured state (Ca²⁺-bound form). HsCalu-1 chaperone activity is independent of Ca²⁺ and Pb²⁺ binding attenuating its chaperone-like activity. Incidentally, Pb²⁺ binds to HsCalu-1 with lower affinity (K_D = 38.46 μM) (compared to Ca²⁺-binding), leading to the formation of a less-stable conformation as observed by a sharp drop in its melting temperature T_m from 67 °C in the Ca²⁺-bound form to 43 °C in the presence of Pb²⁺. The binding site for Pb²⁺ was mapped as being in the EF-Hand-234 domain of HsCalu-1, a region that overlaps with the Ca²⁺-dependent initiator of its functional fold. A change in the secondary and tertiary structure, leading to a less-stable but compact conformation upon Pb²⁺ binding, is the mechanism by which the chaperone-like activity of HsCalu-1 is diminished. Our results not only demonstrate the chaperone activity by a protein in its disordered state but also explain, using Pb²⁺ as a probe, that the multiple functions of calumenin are due to its ability to adopt a quasi-stable conformation.