Jos A. Cox

Muscle of amphioxus contains large amounts of a four EF‐hand Ca²⁺‐binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N‐CaVP) and carboxy‐terminal halves (C‐CaVP). The interactive properties of recombinant and wild‐type CaVP are very similar, despite three post‐translational modifications in the wild‐type protein. N‐CaVP does not bind Ca²⁺, shows a well‐formed hydrophobic core, and melts at 44°C. C‐CaVP binds two Ca²⁺ with intrinsic dissociation constants of 0.22 and 140 μM (i.e., very similar to the entire CaVP). The metal‐free domain in CaVP and C‐CaVP shows no distinct melting transition, whereas its 1Ca²⁺ and 2Ca²⁺ forms melt in the 111°–123°C range, suggesting that C‐CaVP and the carboxy‐ domain of CaVP are natively unfolded in the metal‐free state and progressively gain structure upon binding of 1Ca²⁺ and 2Ca²⁺. Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca²⁺ and 2Ca²⁺ states of the carboxy‐domain destabilize to different degrees the amino‐domain. Only C‐CaVP forms a Ca²⁺‐dependent 1:1 complex with CaVPT. Our results suggest that the carboxy‐terminal domain of CaVP interacts with CaVPT and that the amino‐terminal lobe modulates this interaction.

Parvalbumin (PV) and the homologous protein oncomodulin (OM) contain three EF‐hand motifs, but the first site (AB) cannot bind Ca²⁺. Here we aimed to recreate the putative ancestral proteins [D19‐28E]PV and [D19‐28E]OM by replacing the 10‐residue‐long nonfunctional loop in the AB site by a 12‐residue canonical loop. To create an optical conformational probe we also expressed the homologs with a F102W replacement. Unexpectedly, in none of the proteins did the mutation reactivate the AB site. The AB‐remodeled parvalbumins bind two Ca²⁺ ions with strong positive cooperativity (n_H = 2) and moderate affinity ([Ca²⁺]_0.5 = 2 µm), compared with [Ca²⁺]_0.5 = 37 nm and n_H = 1 for the wild‐type protein. Increasing Mg²⁺ concentrations changed n_H from 2 to 0.65, but without modification of the [Ca²⁺]_0.5‐value. CD revealed that the Ca²⁺ and Mg²⁺ forms of the remodeled parvalbumins lost one‐third of their α helix content compared with the Ca²⁺ form of wild‐type parvalbumin. However, the microenvironment of single Trp residues in the hydrophobic cores, monitored using intrinsic fluorescence and difference optical density, is the same. The metal‐free remodeled parvalbumins possess unfolded conformations. The AB‐remodeled oncomodulins also bind two Ca²⁺ with [Ca²⁺]_0.5 = 43 µm and n_H = 1.45. Mg²⁺ does not affect Ca²⁺ binding. Again the Ca²⁺ forms display two‐thirds of the α‐helical content in the wild‐type, while their core is still strongly hydrophobic as monitored by Trp and Tyr fluorescence. The metal‐free oncomodulins are partially unfolded and seem not to possess a hydrophobic core. Our data indicate that AB‐remodeled parvalbumin has the potential to regulate cell functions, whereas it is unlikely that [D19‐28E]OM can play a regulatory role in vivo. The predicted evolution of the AB site from a canonical to an abortive EF‐hand may have been dictated by the need for stronger interaction with Mg²⁺ and Ca²⁺, and a high conformational stability of the metal‐free forms.

NSCP, a sarcoplasmic Ca²⁺/Mg²⁺-binding protein from Nereis diversicolor, shows an allosteric change during Ca²⁺ binding and a high positive cooperativity for Mg²⁺ binding. Here we report the results of CD and NMR experiments aiming to characterize the apo state and the Ca²⁺-induced conformational changes in this protein. Circular dichroism spectra of the apo form are indicative of a reduced helical structure. In contrast, NMR spectra show no element of regular secondary or tertiary structure. Addition of one Ca²⁺ determines large spectral changes bringing the molecule in a conformation which is very close to the native three Ca ²⁺ state. Addition of the second and third Ca ²⁺ shifts this equilibrium progressively towards the liganded conformation but affects only minimally the spectrum of the liganded species.