Activation of enzymes by calmodulins containing intramolecular cross-links

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We have reacted calmodulins containing cysteines substituted at positions 3 and 146 or 5 and 146 with bismaleimidohexane (BMH) to generate intramolecularly cross-linked proteins termed BMHCM or BMHCM1, respectively. Reactions were also performed with N-ethylmaleimide (NEM) in place of BMH to generate corresponding S-ethylsuccinimidylated proteins termed NEMCM or NEMCM1. The abilities of these proteins to activate plant NAD kinase, erythrocyte Ca2±ATPase and bovine brain calcineurin activities were assessed. The BMH- or NEM-reacted proteins activate calcineurin activity as does control calmodulin. K act values for Ca2±ATPase activation by BMHCM and BMHCM1 are increased 10-fold relative to the control value, with no corresponding change in V max values. Activation of this enzyme by NEMCM or NEMCM1 is not different from the control. In NAD kinase activation experiments BMHCM and BMHCM1 are associated with a 10 to 20-foid increase in K act values and a 60–75% reduction in V max values relative to the control. NEMCM1 is not associated with any apparent changes in NAD kinase activation, however, NEMCM is associated with a 10-fold increase in the K act value. NEM-reacted calmodulin containing a cysteine only at position 3 is not associated with an increased K act value, implying that this change is due to interactions between S-(ethylsuccinimido)cysteines at positions 3 and 146. In conclusion, cross-linking and associated distortions in the structure of calmodulin appear to have little or no effect on activation of calcineurin enzyme activity. However, bending in the central helix and/or steric restrictions associated with cross-linking increase significantly the K act value for Ca2±ATPase and NAD kinase activation, and dramatically reduce maximal activation of NAD kinase activity.