The document discusses the role of calcium-binding proteins, specifically calmodulin and its related proteins, in signal transduction processes within plant cells. It highlights how these proteins sense and respond to transient increases in cytoplasmic Ca2+ levels, their structural features, and their involvement in various physiological and stress-related responses. Calmodulin acts as a pivotal regulator in calcium signaling pathways, influencing processes like gene expression and stress responses by binding to specific target proteins.

1. Signal Transduction:
Calmodulin
Presented By:
Dr. Vibha Khanna
Associate Professor
(Botany)

2. Calcium Binding Proteins
• Transient increase in the cytoplasmic Ca2+ in response to signals is
sensed by an array of Ca2+ sensors.
• Ca2+ sensors are small proteins that bind Ca2+ and change their
conformation in a Ca2+ dependent manner.
• Specificity in the signaling pathway is provided by
• the uniqueness in calcium signatures and
• by plethora of Ca2+ sensors, which can sense the deviation in
Ca2+ concentration, quite precisely.
• Once Ca2+ sensors decode the elevated [Ca2+]cyt, Ca2+efflux into the
cell exterior and/or the sequestration into cellular organelles such as
vacuoles, ER and mitochondria restores its levels to resting state.

3. Calcium Binding Proteins
• Properties essential for Ca2+-binding proteins (CaBPs) to function as
intracellular-Ca2+receptor.
• The Ca2+ receptor/sensor must have Ca2+binding sites essentially unoccupied
in the resting cell and occupied at levels, which are reached upon stimulation.
The affinity constants (Ka) for Ca2+ should be ∼10−6M−1.
• The protein must show selectivity and preference for Ca2+ in the presence of
other cations like Mg2+ and K+.
• After binding to Ca2+, a Ca2+ sensor must undergo a conformational change
that either alters its interaction with other molecules or changes its activity if
it is an enzyme. Formation of Ca2+ protein complex is reversible.
• The kinetics of interaction should be very fast so as to correspond with the
short lived Ca2+ signature.

4. Calcium Binding Proteins:
EF Hand, Structure and Function.
• Most of the Ca2+ sensors bind Ca2+ using a helix-loop-helix motif termed as the ‘EF hand’
motif, which binds a single Ca2+ molecule with high affinity. The Ca2+ sensors utilize the side
chain oxygen atoms of the EF hand motif for Ca2+ coordination.
The properties, structure and function of EF hands:
• The EF hand motifs mostly exist in pairs, which helps in the stabilization of the protein
structure.
• Frequently, EF hand pairs interact through antiparallel β-sheets, which allow cooperation in
Ca2+ binding.
• The EF hand is a highly conserved 29 amino-acid motif consisting of a α helix E (residue 1–10),
a loop (residue 10–21), which binds the Ca2+ ion and a second α-helix F (residue 19–29)
• In EF hand helix-turn-helix motifs, negatively charged oxygen atoms cradle Ca2+ within a ∼12
amino acid loop between two orthogonal α helices
• The Ca2+ ion is coordinated by an oxygen atom or by a bridging water molecule
• In most of the functional EF hand motifs, the first amino acid is Aspartate (Asp) and the
twelfth is Glutamate (Glu), Glu contributes both its side chain oxygen atoms to the metal ion
coordination. Glycine (Gly), present at the apex of the loop is an invariant residue and allows
the loop to take a sharp bend.

5. Calcium Binding Proteins
• The major family of Ca2+ sensors includes;
(1) Calmodulin (CaMs);
(2) Calmodulin like proteins;
(3) Calcium dependent protein kinases (CDPKs);
(4) Calcineurin B-like proteins.

6. Calmodulin (CALcium MODULted proteIN)
• Calmodulin, the Archetypal
Sensor/Adaptor Protein
• Calmodulin (CaM1-4) is a
ubiquitous adaptor protein that
amplifies Ca2+'s diminutive size to
the scale of proteins.
• Calmodulin (CaM) is a small (17
KDa), highly conserved, acidic Ca2+-
binding protein
• CaM is found in the apoplast and in
the cytosol, ER and the nucleus of
plant cells.

7. Ca2+ Sensor and Adaptor Proteins
• It consists of two globular domains, each
containing two EF hand pairs capable of
binding Ca2+, joined by a flexible central
linker region.
• {all 4 EF hands are saturated by Ca2+ ions (4
Ca2+).}
• Thus, Calmodulin is shaped like a dumbbell,
with a flexible joint in its middle.
• When both domains wrap around the
target, the structure compacts into a
globular shape
• Binding of Ca2+ is associated with a large
change in conformation and exposure of
hydrophobic surfaces within each domain,
which triggers calmodulin's Ca2+ sensor
activity (binding to its targets).

8. Role of CaM:
• It is involved in many physiological processes like response to light,
gravity, mechanical stress, phytohormones, pathogens, osmotic
stress, heat shock and chilling.
• CaM appears to be regulatory protein and induces large changes in
inter-helical angles as Ca2+ is bound.
• The affinity of CaM for Ca2+ is influenced by the presence of particular
target proteins.
• CaM can also regulate gene expression by binding to specific
transcription factors.

9. Calcium – calmodulin Complex
• When Ca2+ binds, the shape of the calmodulin domains change,
triggering their ability to
• relieve protein autoinhibition,
• remodel active sites, and
• dimerize proteins
• Calmodulin also extends the reach of Ca2+ by activating
phosphorylation pathways.

10. For eg. :-
• Ca2+/calmodulin binding
relieves autoinhibition of
the catalytic domain of
calmodulin kinase (CaMK)
family enzymes.
• CaMKIIs multimerize,
leading to auto- and
interphosphorylations that
prolong kinase activity.

11. EF Hand in Calmodulin
• The protein chelator of Ca2+ is the EF hand
domain (named after the E and F regions of
parvalbumin, a calcium-binding protein with
low molecular weight.)
• In EF hand helix-turn-helix motifs, negatively
charged oxygen atoms cradle Ca2+ within a
∼12 amino acid loop between two α helices
which are at right angles to each other
(Orthogonal)
• The EF hands of calmodulin have distinct
affinities for Ca2+, and their binding affinities
are often increased by interaction with target
proteins.
• Hydrophobic residues, usually containing
methionine, wrap around amphipathic
regions of target proteins, such as the α
helices in myosin light chain kinase (MLCK)
and calmodulin dependent kinase II
(CaMKII).

12. CaM and CaM-Like Proteins in Plants
• The “typical CaM” are identical with CaM found in vertebrates or
insect such as drosophila
• CaM-related proteins (CMLs) exhibit significant structural divergences
with the typical CaM
• CMLs are defined
• by the presence of 2 to 6 predicted EF-hands motifs,
• by the absence of any other identifiable functional domain and
• at least by 15% amino acid identity with CaMs.
• CaM and CML may exert different functions through their binding
targets that can be located in different cellular compartments.
• Cytosol, nucleus, peroxysomes, cell membrane, extra-cellular matrix

13. Physiological Functions of Calmodulins and CMLs:
Plant Development
• Cytochrome P450 like enzyme catalyzes an early step in the biosynthesis
of brassinosteroid, a plant-specific steroid hormone which is essential for
plant growth.
• Presence of CaM binding domain, suggests that in plants this protein is regulated
by Ca2+/CaM .
• NtCBK1 gene is expressed in the shoot apical meristem during vegetative
growth, but its expression in the meristem is dramatically decreased
after floral determination, suggesting a role of this protein kinase in the
transition to flowering.
• Ca2+/CaM binding protein kinase (NtCBK1) was reported to function as a negative
regulator of flowering.

14. Physiological Functions of Calmodulins and CMLs
Stress Physiology
• Changes in intracellular Ca2+ levels have been reported as an early response
to diverse abiotic signals including mechanical stimuli, osmotic and salt
treatments, cold and heat shocks.
• Ca2+ signal has been shown to be essential for the activation of defense
responses such as the induction of defense-related genes and
hypersensitive cell death.
• Establishment of the legume/rhizobia symbiosis requires a signaling
molecule termed Nod factor that is produced by the Rhizobium bacteria
and recognized by the root hair cells of the host plant. Early events in this
recognition include Ca2+ responses that are separated both spatially and
temporally.
• An initial Ca2+ flux occurs at the tip of the root hair, then repetitive cytosolic
oscillations of Ca2+ or Ca2+ spiking appears in the region surrounding the nucleus.

15. Calcium signaling with Calmodulins and CMLs
Environmental and Developmental stimuli
Elevation of Ca2+ concentration
Ca2+ variations decoded by a wide range of
Ca2+ sensors (CaM and CMLs proteins)
Interact and modulate the activity of
downstream target proteins
initiate biochemical, cellular and physiological
responses