Conformational and metal-binding properties of androcam, a testis-specific, calmodulin-related protein from Drosophila.
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 calcium-free 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 [Ka(Mg) approximately 3 x 10(3) M(-1)]. 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 alpha-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.
Martin
S R
SR
Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London, United Kingdom.
Lu
A Q
AQ
Xiao
J
J
Kleinjung
J
J
Beckingham
K
K
Bayley
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry*; D002942:Circular Dichroism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013737:Testis
C090181:Acam protein, Drosophila; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D029721:Drosophila Proteins; D010446:Peptide Fragments; D002118:Calcium