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Structural elements within the methylation loop (residues 112-117) and EF hands III and IV of calmodulin are required for Lys(115) trimethylation.

ABSTRACT: Calmodulin is trimethylated by a specific methyltransferase on Lys115, a residue located in a six amino acid loop (LGEKLT) between EF hands III and IV. To investigate the structural requirements for methylation, domain exchange mutants as well as single point mutations of conserved methylation loop residues (E114A, Glu114-->Ala; L116T, Leu116-->Thr) were generated. E114A and L116T activated cyclic nucleotide phosphodiesterase (PDE) and NAD+ kinase (NADK) similar to wild-type calmodulin, but lost their ability to be methylated. Domain exchange mutants in which EF hand III or IV was replaced by EF hand I or II respectively (CaM1214 and CaM1232 respectively) showed a modest effect on PDE and NADK activation (50 to 100% of wild-type), but calmodulin methylation was abolished. A third domain exchange mutant, CaMEKL, has the methylation loop sequence placed at a symmetrical position between EF hands I and II in the N-terminal lobe [residues QNP(41-43) replaced by EKL]. CaMEKL activated PDE normally, but did not activate NADK. However, CaMEKL retained the ability to bind to NADK and inhibited activation by wild-type calmodulin. Site-directed mutagenesis of single residues showed that Gln41 and Pro43 substitutions had the strongest effect on NADK activation. Additionally, CaMEKL was not methylated, suggesting that the introduction of the methylation loop between EF hands I and II is not adequate for methyltransferase recognition. Overall the data indicate that residues in the methylation loop are essential but not sufficient for methyltransferase recognition, and that additional residues unique to EF hands III and IV are required. Secondly, the QNP sequence in the loop between EF hands I and II is necessary for NADK activation.

SUBMITTER: Cobb JA

PROVIDER: S-EPMC1220266 | biostudies-other | 1999 Jun

REPOSITORIES: biostudies-other

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Project description:BackgroundCalmodulin (CaM) plays an important role in Ca(2+)-dependent signal transduction. Ca(2+) binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. CaM regulates a Ca(2+)-dependent inactivation process in store-operated Ca(2+) entry, by interacting Orai1. To understand the relationship between Ca(2+)-induced hydrophobicity and CaM/Orai interaction, chimera proteins constructed by exchanging EF-hands of CaM with those of Troponin C (TnC) are used as an informative probe to better understand the functionality of each EF-hand.ResultsANS was used to assess the context of the induced hydrophobic surface on CaM and chimeras upon Ca(2+) binding. The exchanged EF-hands from TnC to CaM resulted in reduced hydrophobicity compared with wild-type CaM. ANS lifetime measurements indicated that there are two types of ANS molecules with rather distinct fluorescence lifetimes, each specifically corresponding to one lobe of CaM or chimeras. Thermodynamic studies indicated the interaction between CaM and a 24-residue peptide corresponding to the CaM-binding domain of Orail1 (Orai-CMBD) is a 1:2 CaM/Orai-CMBD binding, in which each peptide binding yields a similar enthalpy change (ΔH = -5.02 ± 0.13 kcal/mol) and binding affinity (K(a) = 8.92 ± 1.03 × 10(5) M(-1)). With the exchanged EF1 and EF2, the resulting chimeras noted as CaM(1TnC) and CaM(2TnC), displayed a two sequential binding mode with a one-order weaker binding affinity and lower ΔH than that of CaM, while CaM(3TnC) and CaM(4TnC) had similar binding thermodynamics as CaM. The dissociation rate constant for CaM/Orai-CMBD was determined to be 1.41 ± 0.08 s(-1) by rapid kinetics. Stern-Volmer plots of Orai-CMBD Trp76 indicated that the residue is located in a very hydrophobic environment but becomes more solvent accessible when EF1 and EF2 were exchanged.ConclusionsUsing ANS dye to assess induced hydrophobicity showed that exchanging EFs for all Ca(2+)-bound chimeras impaired ANS fluorescence and/or binding affinity, consistent with general concepts about the inadequacy of hydrophobic exposure for chimeras. However, such ANS responses exhibited no correlation with the ability to interact with Orai-CMBD. Here, the model of 1:2 binding stoichiometry of CaM/Orai-CMBD established in solution supports the already published crystal structure.

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Structural elements within the methylation loop (residues 112-117) and EF hands III and IV of calmodulin are required for Lys(115) trimethylation.

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