Conformational and metal‐binding properties of androcam, a testis‐specific, calmodulin‐related protein from drosophila

Abstract

Androcam is a testis-specific protein of Drosophila melanogaster, with 67% sequence identity to calmodulin and four potential EF-hand calcium-binding sites. Spectroscopic monitoring of the thermal unfolding of recombinant calciumfree androcam shows a biphasic process characteristic of a two-domain protein, with the apo-N-domain less stable than the apo-C-domain. The two EF hands of the C-domain of androcam bind calcium cooperatively with 40-fold higher average affinity than the corresponding calmodulin sites. Magnesium competes with calcium binding @K a~M g! ;3 ϫ 10 3 M Ϫ1 #. Weak calcium binding is also detected at one or more N-domain sites. Compared to apo-calmodulin, apo-androcam has a smaller conformational response to calcium and a lower a-helical content over a range of experimental conditions. Unlike calmodulin, a tryptic cleavage site in the N-domain of apo-androcam remains trypsin sensitive in the presence of calcium, suggesting an altered calcium-dependent conformational change in this domain. The affinity of model target peptides for androcam is 10 3 -10 5 times lower than for calmodulin, and interaction of the N-domain of androcam with these peptides is significantly reduced. Thus, androcam shows calcium-induced conformational responses typical of a calcium sensor, but its properties indicate calcium sensitivity and target interactions significantly different from those of calmodulin. From the sequence differences and the altered calcium-binding properties it is likely that androcam differs from calmodulin in the conformation of residues in the second calcium-binding loop. Molecular modeling supports the deduction that there are significant conformational differences in the N-domain of androcam compared to calmodulin, and that these could affect the surface, conferring a different specificity on androcam in target interactions related to testis-specific calcium signaling functions.

Magnesium Promotes Structural Integrity and Conformational Switching Action of a Calcium Sensor Protein

“…Addition of Mg 2+ to the apo state further diminishes the ANS fluorescence (Figure A), a consequence of the further burial of the hydrophobic core thereby suggesting that the Mg 2+ binding stabilizes the closed conformation of Eh CaBP. This is similar to many EF-hands in CaBPs that have the ability to bind Mg 2+ apart from Ca 2+ , though with lower affinity ( 13 , 14 , − . For example, in calbindin D 9k , a Ca 2+ buffer protein, Mg 2+ binding to the regular EF-hand leads to decrease in interhelical angle between helix III and IV to a more compact form as compared to both the Ca 2+ loaded and metal ion-free forms ().…”

Structure of androcam supports specialized interactions with myosin VI

“…Peak movements fit a single binding site model with K D ¼ 4.1 mM (Fig. 2), 50-fold weaker than the lower limit determined previously (11). The modest spectral changes indicate that Ca 2þ binding has a local effect but does not alter the overall N lobe conformation.…”

“…Androcam binds Ca 2þ at two C-lobe high affinity sites (K D s of 56 nM and 25 nM) and at a weak N-lobe site with a K D lower limit of 80 μM (11). To map the residues at the weak site, we monitored NMR chemical shift changes in a CaCl 2 titration.…”

“…Titrating androcam with this peptide induces small shift changes for C-lobe amides only (Fig. S5), showing that the N lobe does not contact the target and providing an explanation for weak androcam binding to MLCK targets (11). The inability of Ca 2þ or classic helical calmodulin targets like the MLCK and Insert 2 peptides to induce the androcam N lobe to open supports our hypothesis that androcam cannot adopt the archetypal calmodulin 1-14 or 1-10 binding modes (5).…”

“…Sequence alignment (Fig. 1) reveals that the C-terminal lobe (C lobe) is more strongly conserved than the N-terminal lobe (N lobe), and although the androcam C lobe binds two Ca 2þ ions 40-fold more tightly than calmodulin, the N lobe binds weakly to only one Ca 2þ (11). Androcam interacts with both Insert 2 and IQ regions of Drosophila myosin VI in yeast two-hybrid analysis, but binds more weakly than calmodulin to an Insert 2 peptide (8).…”