1. TITLE: Structural Characterization and Structure-Based Pharmacophore Assessment of
Calmodulin, as evinced from Crystallographic Characterization PDB = 1A29
PREPARED BY: Gerald Lushington, principal consultant
Lushington in Silico
http://geraldlushington.com
lushington_insilico@yahoo.com
DRAFT ID: 2013-03-20-zintro01
REVISION OF: n/a

2. Fold Assignment:
The key protein component of protein databank structure 1A29 [1] is calmodulin, a ubiquitous
intermediate messenger protein responsible for modulating various proteins involved in calcium
signalling pathways via Ca2+ transduction. Although calmodulin has a significant amount of
conformational flexibility, the overall tertiary structure in the mammalian protein is dominated by
interactions of six -helices that are typically arranged in two (N-terminal and C-terminal) domains
comprised of three helices per domain. Based on this characterization, approximate fold
assignment can be made as follows:
SCOP [2]: the primary class is all alpha proteins, demonstrating an EF-hand-like fold.
Structurally, this places the protein in the EF-hand superfamily and calmodulin-like family.
CATH [3]: the primary class is mainly alpha, exhibiting orthogonal bundle architecture.
Calmodulin has a topology that is consistent with Recoverin domain 1, with sequence homology
that establishes a clear relationship with the EF-hand family.
PFAM [4]: Calmodulin structure is reminiscent of any of several EF-hand domains, including EF-
Hand, EF-Hand-3 (cytoskeletal regulatory complex), EF-Hand-5 (EF-Hand domain pair) and EF-
Hand-6 (EF-Hand domain pair).
In order to illustrate the EF-Hand fold structure in the specific case of calmodulin and in a more
general sense, Figs. 1 and 2 depict calmodulin [1] and the homologous avian thymine hormone [5]
as secondary-structure cartoons based on the crystallographically resolved backbones of these
proteins. Although calmodulin is larger than its homolog and exhibits one helix (upper left) that is
not represented in avian thymine hormone, the two proteins pack in an analogous manner with a
backbone atom RMSD of less than 3.0 Å across the manifold of aligned homologous residues.
Metal Binding:
Biochemically, the best understood function of calmodulin is its complexation of calcium (Ca2+)
ions, of which the protein may sequester as many as four (one for each of the distinct EF-hand
motifs in the structure; see Fig.3) at any given time. Each Ca2+ is bound in a hexavalent manner,
including one bidentate interaction with a glutamate located on one helix, plus unidentate coupling
with two aspartates, an asparagine and the backbone carbonyl of a neaby threonine, where all of the

3. latter residues are in close proximity within the highly nucleophilic inter-helix coil that is
characteristic of the EF-Hand. Under the crystallographic conditions under which the 1A29
structure was resolved, the equilibrium calcium-oxygen distance for all 24 of the distinct Ca2+
binding interactions was between 2.25 – 2.35 Å, indicated a strong ionic attraction that tends to pull
multiple electronegative moieties into much closer approach than would be observed in the absence
of cationic stabilization. This produces a tangible conformational shift that enhances calmodulin
lipophilic profile by encouraging conformational inversion of multiple methionine residues (see
Fig. 4) whose sidechains rotate outwards from the interdomain region, thus enhancing exposure of
the sidechain's nonpolar epsilon methyl. This conformation is attractive to nonpolar patches on
amphiphilic helices of specific binding target proteins. The resulting protein complexes exploit
calmodulin interactions physiologically for signal transduction and calcium sensing. The significant
conformation differences between the activated (calcium-rich) and inactive forms suggest ion-
dependent variations in pharmacophore profile that might enable selective drug targeting of one
calmodulin conformer relative to the other.
Prospective Pharmacophore:

4. The interdomain complex exhibits a single, marginal hydrogen bond (3.2 Å) between the
piperazinium nitrogen atom and the Glu 120 side chain, while the remainder of the pharmacophore
is dominated by lipophilic interactions. The intradomain complex appears to sustain no obvious H-
bonds, with the binding mediated almost entirely by a range of lipophilic interactions, the strongest
of which are derived from situation of the trifluoromethyl group in a small, sterically compatible
hydrophobic cavity.
While trifluoroperazine is an approved antipsychotic, its primary mechanism of action has been
postulated to occur through antagonism of the 1-andrenergic [7], D1 and/or D2 dopamine [8]
receptors. Despite, well characterized binding kinetics, the viable therapeutic exploitation of its
calmodulin antagonism has not been definitively demonstrated, however the pharmacophore
evident from the crystal structure suggests possible avenues for optimization that may provide the
basis for practical targeting. For example, the ample array of H-acceptor features within close
proximity of the trifluoroperazine binding sites suggests that judicious augmentation of polar
contacts (primarily H-donors) might produce stable H-bonding interactions while simultaneously
augmenting the ADME profile by enhancing ligand solubility. Secondly, the receptor lipophilic
interactions with the ligand phenothiazine are produced nearly entirely by alkyl and thioalkyl
residues (alanine, valine and methionine), thus analogous heterocyclic systems with greater SP3
hybridization might prove more compatible with the receptor. Furthermore, from Fig. 7, one can
intuit effective avenues for formulation of chimeric ligands that simultaneously exploit one or more
of the trifluoroperazine sites and spatially distinct sites other known calmodulin modulators such as
KAR-2, as derived from crystallographic analysis [9].
References:
[1] Vertessy, BG, Harmat, V, Bocskei, Z, Naray-Szabo, G, Orosz, F, Ovadi, J (1998). Simultaneous
binding of drugs with different chemical structures to Ca2+-calmodulin: crystallographic and
spectroscopic studies. Biochemistry 37: 15300-15310
[2] Murzin AG, Brenner SE, Hubbard T, Chothia C (1995). SCOP: a structural classification of
proteins database for the investigation of sequences and structures. J. Mol. Biol. 247: 536-540.
[3] Sillitoe I, Cuff AL, Dessailly BH, Dawson NL, Furnham N, Lee D, Lees JG, Lewis TE, Studer
RA, Rentzsch R, Yeats C, Thornton JM, Orengo CA (2013). New functional families (FunFams) in

5. CATH to improve the mapping of conserved functional sites to 3D structures. Nucleic Acids Res.
41: D490-D498.
[4] M. Punta, M, Coggill, PC, Eberhardt, RY, Mistry, J, Tate, J, Boursnell, C, Pang, N, Forslund, K,
Ceric, G, Clements, J, Heger, A, Holm, L, Sonnhammer, ELL, Eddy, SR, Bateman, A, Finn, RD
(2012) The Pfam protein families database. Nucleic Acids Research. 40: D290-D301.
[5] Schuermann, JP, Tan, A, Tanner, JJ, Henzl, MT (2010). Structure of avian thymic hormone, a
high-affinity avian beta-parvalbumin, in the Ca2+-free and Ca2+-bound states. J. Mol. Biol. 397:
991-1002
[6] Marvin calculator plug-ins, version 5.11. Chemaxon, Inc. Budapest Hungary, 2012.
[7] Huerta-Bahena J, Villalobos-Molina R, García-Sáinz JA (1983). "Trifluoperazine and
chlorpromazine antagonize alpha 1- but not alpha2- adrenergic effects". Molecular Pharmacology
23: 67–70.
[8] Seeman P, Lee T, Chau-Wong M, Wong K (1976). "Antipsychotic drug doses and
neuroleptic/dopamine receptors". Nature 261: 717–719.
[9] Horvath, I, Harmat, V, Perczel, A, Palfi, V, Nyitrai, L, Nagy, A, Hlavanda, E, Naray-Szabo, G.,
Ovadi, J (2005). The structure of the complex of calmodulin with KAR-2: a novel mode of binding
explains the unique pharmacology of the drug. J. Biol. Chem. 280: 8266-8274