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  • In the present study we focused on a role

    2021-10-03

    In the present study, we focused on a role of the C-terminal segment in GCAP2 using two chimeric proteins, in which the C-terminal segment of GCAP2 was either replaced by the corresponding sequence of recoverin (chimera GR) or transferred into recoverin (chimera RG). We have monitored Ca-dependent conformational rearrangements and functional features of GCAP2 displaying a critical participation of its C-terminal segment, for example in membrane binding of the protein under control of phosphorylation. Furthermore we suggest a regulatory role of this segment which while undergoing Ca-dependent conformational transition modulates the accessibility of exposed hydrophobic cavities in GCAP2 thereby ensuring the specificity of target(s) recognition.
    Mateials and methods
    Results and discussion GCAP2 and recoverin belong to different subgroups of the NCS family and display ~60% of sequence similarity, which is maximal in the EF-hand containing core region and minimal at the N- and C-terminus (Fig. 1A). The variable C-terminal region of NCS proteins downstream EF-hand 4 (“C-terminal segment”) corresponds to regions Asp177-Phe204 in GCAP2 and to Asn179-Leu202 recoverin (see box in Fig. 1). Since the truncation of more than 15 C-terminal BKT140 results in critical disturbance of the NCS structure as observed for recoverin and NCS1 [15], [20], we employed chimeric proteins instead of a truncated mutant of GCAP2. Chimeric proteins named GR and RG were constructed on the base of GCAP2 and recoverin. In the GR chimera, recoverin fragment Asn179-Leu202 substitutes for Asp177-Phe204 in GCAP2 while in the RG chimera GCAP2 fragment Asp177-Phe204 substitutes for Asn179-Leu202 in recoverin (Fig. 1A). The structural and functional analysis of such constructs allowed one (i) determining the impact of replacing the C-terminal segment on conformation and activity of GCAP2, (ii) characterizing the BKT140 behavior of GCAP2 C-terminal segment in the structural environment of a homologous NCS protein and (iii) investigating in more detail the regulatory function of recoverin´s C-terminal segment established in our previous studies [15], [16]. The GR and RG constructs were co-expressed in Echerichia coli with yeast N-myristoyl transferase from Saccharomyces cerevisiae yielding N-terminally myristoylated proteins after purification following the standard protocols for GCAP2 and recoverin, respectively (Fig. 1B). The degree of myristoylation of both purified chimeras exceeded 90%. Purified myristoylated GR exhibited the same electrophoretic mobility shift in 15% gel as WT GCAP2 indicating that the chimera retains ability to bind Ca (Fig. 1B) [12]. Thus, the genetic exchange of C-terminal segments between GCAP2 and recoverin allowed us to obtain folded Ca-sensitive chimeric proteins that were applied for the further characterization including comparison of their secondary structure, thermal stability and Ca-sensitivity, monitoring of their Ca-induced conformational changes as well as examining of their membrane association and regulatory activity.
    Conclusions In the present study, we propose a new role of the C-terminal segment of GCAP2 as a structural element undergoing Ca-dependent conformational changes. Although the exchange of C-terminal segments between GCAP2 and recoverin does not significantly affect the overall structural integrity of both proteins and the Ca-sensitivity of GCAP2, within the latter this structural region participates in conformational rearrangements associated with Ca-binding, is controlling the exposure of hydrophobic regions upon Ca-binding and is involved in membrane binding and specifying target recognition, A further hallmark of the C-terminal segment of GCAP2 is that it provides sensitivity to phosphorylation by signaling kinases thereby coupling regulatory activity of the Ca-sensor to Ca-independent signaling pathways. Based on available data concerning structural and functional properties of the C-terminal segments in NCS proteins, we conclude that this structural element represents a general structural motif exhibiting high flexibility for serving as a Ca-dependent control module. Future studies are necessary to validate the regulatory function of the C-terminal segment of NCS proteins under physiological in vivo conditions.