Basic interdomain boundary residues in calmodulin decrease calcium affinity of sites I and II by stabilizing helix-helix interactions.


Calmodulin is an EF-hand calcium-binding protein (148 a.a.) essential in intracellular signal transduction. Its homologous N- and C-terminal domains are separated by a linker that appears disordered in NMR studies. In a study of an N-domain fragment of Paramecium CaM (PCaM1-75), the addition of linker residues 76 to 80 (MKEQD) raised the Tm by 9 degrees C and lowered calcium binding by 0.54 kcal/mol (Sorensen et al., [Biochemistry 2002;41:15-20]), showing that these tether residues affect energetics as well as being a barrier to diffusion. To determine the individual contributions of residues 74 through 80 (RKMKEQD) to stability and calcium affinity, we compared a nested series of 7 fragments (PCaM1-74 to PCaM1-80). For the first 4, PCaM1-74 through PCaM1-77, single amino acid additions at the C-terminus corresponded to stepwise increases in thermostability and decreases in calcium affinity with a net change of 13.5 degrees C in Tm and 0.55 kcal/mol in free energy. The thermodynamic properties of fragments PCaM1-77 through PCaM1-80 were nearly identical. We concluded that the 3 basic residues in the sequence from 74 to 77 (RKMK) are critical to the increased stability and decreased calcium affinity of the longer N-domain fragments. Comparisons of NMR (HSQC) spectra of 15N-PCaM1-74 and 15N-PCaM1-80 and analysis of high-resolution structural models suggest these residues are latched to amino acids in helix A of CaM. The addition of residues E78, Q79, and D80 had a minimal effect on sites I and II, but they may contribute to the mechanism of energetic communication between the domains. Study holds ProTherm entries: 16184, 16185, 16186, 16187, 16188, 16189, 16190, 16191 Extra Details: (i) 5mM NTA and 0.05mM EGTA were added in the experiment,(ii) fragment 1-74 thermodynamics; domain organization; allosteric interactions; calcium binding; interdomain communication; protein stability; calcium affinity; tertiary constraints

Submission Details

ID: gpBTvrxX3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Faga LA;Sorensen BR;VanScyoc WS;Shea MA,Proteins (2003) Basic interdomain boundary residues in calmodulin decrease calcium affinity of sites I and II by stabilizing helix-helix interactions. PMID:12557181
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Calmodulin P07463 CALM_PARTE
96.6 Calmodulin P02598 CALM_TETPY
95.3 Calmodulin P27166 CALM_STYLE
94.6 Calmodulin A4UHC0 CALM_ALEFU
94.6 Calmodulin A3E4F9 CALM_KARVE
94.6 Calmodulin A3E3H0 CALM_PFIPI
94.6 Calmodulin A3E4D8 CALM_PROMN
94.0 Calmodulin A8I1Q0 CALM_HETTR
91.3 Calmodulin A8CEP3 CALM_SACJA
90.6 Calmodulin P11120 CALM_PLECO
91.3 Calmodulin P27163 CALM2_PETHY
91.9 Calmodulin P27161 CALM_SOLLC
91.3 Calmodulin P13868 CALM1_SOLTU
91.5 Calmodulin P62146 CALMA_ARBPU
97.1 Calmodulin O96081 CALMB_HALRO
94.4 Calmodulin Q9U6D3 CALM_MYXGL
95.7 Calmodulin O97341 CALM_SUBDO
91.4 Calmodulin P62203 CALM_PLAF7
91.4 Calmodulin P24044 CALM_PLAFA
94.0 Calmodulin Q95NI4 CALM_HALOK
96.7 Calmodulin P05932 CALMB_ARBPU
93.1 Calmodulin Q9HFY6 CALM_BLAEM
96.2 Calmodulin P02594 CALM_ELEEL
94.3 Calmodulin P11118 CALM_EUGGR
95.7 Calmodulin P02595 CALM_PATSP
95.7 Calmodulin P11121 CALM_PYUSP
95.7 Calmodulin P62184 CALM_RENRE