Mutations of the EF-Hand Domain, Calcium Ion to Chloride Ion Swap:

Beginnings of ‘Chlormodulin/Chlorbindin’

Daniel C. Chavez

Department of Chemistry, Southern Methodist University, Dallas, Texas, 75205, United States

Abstract:​ The EF-hand motif is ubiquitously found in many eukaryotic cells and a majority of motifs can
seperated in a bi-categorical fashion: motifs functions either as a signal sensor or as a signal modulator.
The most prominent and well known EF-hand motifs are generally associated with the homologous
proteins and calmodulin and calbindin.​1​ The former functions as a biological sensor for the Ca​2+​ ion,
triggering signal transduction to be interpreted by other molecules downstream on the related reaction
pathway, while the latter functions as a modulatory intermediate, inducing conformational changes in
other molecules as a conformationally changing ligand itself, or as a metaphorical timer on how long a
signal might propagate. Here I focus on the binding loop, [the ten to twelve amino acid residues that are
involved in stabilizing and binding to the Ca​2+​ cation], of a canonical EF-hand motif and I successively
mutate it with each iteration building upon the predecessors’ failures; the end goal of said mutations
would be the creation of stable loop that has the capability of semi-tight binding with the Cl​-​ anion.
Conversion of the cation binding EF-hand motif to and anion binding EF-hand motif would provide a new
biological sensor for Cl​-​ ion as well give further insight into the function of chloride in biological systems.
A far-fetched goal would be to also manipulate these developed “chlormodulin” and “chlorbindin”
proteins and fashion them as triggers to initiate conformational changes in other local proteins. This
ongoing project is being conducted in conjunction with the Dodani group at The University of Texas at
Dallas. All mutations were performed via PyMOL V 2.3.2, construction of solvation boxes were made
possible via AMBER’s integrated tleap software, and molecular-dynamics simulations and observations
were made possible via AMBER’s cpptraj and the molecular visualization program VMD V 1.9.3.​2,3,4

Background & Methodology:

The ‘EF-hand’ Ca​2+​ binding motif is characterised by its signature alpha helix-loop-alpha helix
structure and further categorized by the complexing ligand residues that constitute a given hand’s loop
structure. To understand the “why” of my research, I will give more insight into the deductive reasoning
of my structural mutations. The unique structure of loops provides a basis by which conformational
changes can occur or a signal is modulated. Of course mutating chelating residues will change binding
interactions, but it also plays a crucial role in altering the way non-chelating residues interact with each
other. Wildtype canonical EF-hands display crucial Ca-O chelating interactions but just as important are
the hydrogen-bonding interactions occurring between all residues. The hydrogen-bonding interactions
between residues allow a given loop the necessary freedom to form a co-ordination sphere around the
bound Ca​2+​ ion and also provide stabilization for the pentagonal bipyramid stucture, a favored
coordinating geometry among EF-hands.​5​ From Gifford’s, Vogel’s, and Walsh’s work in ‘Structures and
metal-ion-binding properties of the Ca​2+​ -binding helix–loop–helix EF-hand motifs,’ they created a table
which shows the sequence of preference for residues occupying the canonical EF-hand loop. The table,

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(recreated in ​Table 1​), shows the most common, [highest frequency of occurrence] residues that chelate
and those that do not, and how they are oriented in space relative to one another via cartesian coordinates.

Table 1
EF-loop position 1 2 3 4 5 6 7 8 9 10 11 12

Coordinating ligand* X Y Z -Y -X -Z
sc sc sc bb sc** sc​2

Most common Asp Lys Asp Gly Asp Gly Thr Ile Asp Phe Glu Glu
100% 29% 76% 56% 52% 96% 23% 68% 32% 23% 29% 92%

Also frequently observed Ala Asn Lys Ser Phe Val Ser Tyr Asp Asp
Gln Arg Asn Lys Leu Thr Ala Lys
Thr Asn Gln Glu Thr Ala
Val Tyr Asn Leu Pro
Ile Glu Gly Glu Asn
Ser Arg Gln Lys
Glu
Arg

Charge/Polarity of most δ​- δ​- δ​- Back

bone δ​- 2 δ​-
common ligands C=​O

Table 1: The first 4 rows are taken from Gifford’s, Vogel’s, and Walsh’s work in ‘Structures and metal-ion-binding
properties of the Ca​2+​ -binding helix–loop–helix EF-hand motifs.’ The last row is my own, a visual intended to
showcase the chelation pattern of the residues as well their [chelating residues] respective charges.
* sc = sidechain oxygen, bb = backbone interaction, sc​2​ = 2 coordinating bonds from the same sidechain
** A water molecule’s [held in place by hydrogen-bonding] oxygen at position nine [via Asp] creates a coordinating bond

Analyzing Gifford’s, Vogel’s, and Walsh’s findings, an interesting pattern emerges: every other
position serves as a chelation site for the Ca​2+​ ion, seven in total, [Glu at position eleven counts as two
because of its carboxylate end, where the electron density is spread equally between both oxygen atoms],
so every residue that does not chelate, must be of structural importance. It can also be seen that the most
common chelating residue is aspartic acid, and any chemists generalized intuition would undoubtedly say
this makes sense, negative residues will complex with positive ions; however, carrying over this inductive
reasoning in terms of ion swapping, (Ca​2+​ to Cl​-​), and residue swapping, (negative to positive), is very
difficult. The only residues with full positive charges at cytosolic pH values are Arginine and Lysine, and
both are decently larger is size, meaning stabilizing everything between the α-carbon and the positive
charge localized on the nitrogen becomes increasingly problematic.

The greatest challenge of this project was starting. All discovered chloride ion sensors and
tight-binders, organic and inorganic, do not rely on amino acids; rather, they are elaborately structured
molecules that require complex inorganic synthesis methods to reproduce/replicate. Retrospectively, a
logical starting point would have been to mutate all chelating residues, [most commonly aspartic acid], to
positively charged residues and observed the loop’s response, but this I will visit this idea at a later date.
To begin, I rid my canonical loop of its negatively charged tail residue [glutamic acid] and arbitrarily

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chose a chelating residue location and mutated it to proline. The logic behind these mutations is twofold:
first, the choice of proline, and second, ridding the loop of outright negative charges and including more
polar uncharged residues. To my knowledge, proline is the only amino acid with any association to the Cl​-
ion; interestingly, a relatively large number of plant species develop high concentrations of proline in
response to increasing salinity stress, (namely sodium chloride).​6​ To see how the Cl​-​ ion interacted with
the proline and asparagine residues, I performed an experimental simulation; however, this simulation
showed a minor change in coordinating bond affinity, (from the canonical loop to the experimental loop),
in the amount of time the Cl​-​ ion stayed within the loop. From here I began the process of successively
mutating the loops based off previous failures and observations as seen in ​Table 2​.

Table 2
EF-loop position 1 2 3 4 5 6 7 8 9 10 11 12

Wildtype Ca​2+ Asn Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu
EF-Loop

Experimental run Asn Ile PRO Glu SER Gly Gln LEU ASP Val Asp ASN

Mutation 1: Asn Ile PRO Glu SER Gly Gln VAL HIS Val Asp ASN

Mutation 2: Asn Ile SER Glu PRO Gly Gln LEU TYR Val Asp ASN

Mutation 3: Asn Ile PRO Glu SER Gly Gln ISO HIS Val Asp ASN

Mutation 4: Asn Ile PRO Glu SER Gly Gln LEU PHE Val Asp ARG

Mutation 5: Asn Ile PRO Glu ALA Gly Gln LEU PHE Val Asp ARG

Mutation 6: Asn Ile PRO Glu SER Gly Gln VAL PRO Val Asp ASN

Mutation 7: Asn Ile SER Glu PRO Gly Gln LEU PRO Val Asp ASN

Mutation 8: Asn Ile SER Glu PRO Gly Gln LEU PRO Val Asp ARG

Mutation 9: Asn Ile PRO Glu SER Gly Gln LEU PRO Val Asp ASN

Mutation 10: Asn Ile PRO Glu SER Gly Gln LEU PRO Val Asp ARG

Mutation 11: Asn Ile SER Glu ALA Gly Gln LEU PRO Val Asp ARG

Mutation 12: Asn Ile SER Glu PRO Gly Gln LEU PRO Val Asp ARG

Mutation 13: Asn Ile ALA Glu ALA Gly Gln LEU PRO Val Asp ARG

Mutation 14: Asn Ile SER Glu ALA Gly Gln LEU ALA Val Asp ARG

Table 2: This table showcases the starting canonical EF-hand ion-binding loop and each successive that was performed
on it. The original canonical loop is represented in bold-red font, and hence, every red residue that exists within a given
mutated loop is a residue that remained completely unchanged from the wildtypes skeletal structure. Every yellow
highlight serves as a guide to show where each mutation occurred [from its predecessor]. ​Mutation 5​, ​Mutation 8​, and
Mutation 11​, are highlighted because they were the three most successful loops; ​Mutation 11​ was the most successful
mutation.

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Results and Discussion:
The first five mutations were inspired by proline’s connection with the Cl​-​ ion, introducing a
polar uncharged residue into the chain to see if a H-Cl​-​ bond [analogous to a pseudo hydrogen-bond]
would be induced, seeing if hydrophobic residues might protect the ion itself from the multitude of waters
surrounding it, and toying around with the idea that an aromatic ring with delocalized electron density
around the ring, (forming a partial positive charge in the center of the ring), would act as a “lid on the
loop.” Other mutations were coupled for easy analysis such as seven and eight, nine and ten, and eleven
and twelve; all are nearly identical except for one or two residues locations being swapped. From
mutations seven through fourteen, it was deduced that serine preferred residue location number three,
alanine number five, and arginine was favored over asparagine as a tail residue [most likely due to its full
positive charge]. Presented in ​Table 3 a​ re the mutations and the corresponding occupancy times of the Cl​-
ion in each of their loops. Again, the three most successful mutations have been highlighted.

Table 3
Mutation # 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Time Cl​-​ ion 0 0.1 0.06 0.05 0.26 0.05 0.12 0.42 0.03 0.24 0.63 0.25 0.07 0.16
spent in loop (ns)

Table 3: As seen, ​Mutation 11​ stands above the rest in terms of success. This success was not by chance; rather, it was
manifested through iterative deductive reasoning.

The idea that varying the chloride concentration might affect occupancy time within the loop was
investigated as well. Taking the three most successful mutations, I created three new water boxes for
each, varying the concentration with 10 mM being the lowest concentration, 100 mM the middle, and 200
mM being the max concentration. These results are shown in ​Figure 1​. So far, there appears to be no
correlation between varying the concentration of chloride and the amount of time the Cl​-​ ion stays within
the loop; however, to my delight, the 100 mM concentration boosted the occupancy time of the Cl​-​ ion by
~ 214%. This paper will now focus completely on the mutation 11 in its concentrated [100 mM]
environment.

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 Every canonical Ca​2+​ binding EF-hand loop finds lowest energy stability in the pentagonal
bipyramidal structure that provides coordination points for the Ca​2+​ ion itself.​5​ To see if my mutation
behaved in a similar fashion, I analyzed the geometry every 0.25 nanoseconds [1,250 frames] after the
equilibration phase, which lasts 0.02 nanoseconds [100 frames]. During the equilibration phase in every
simulation the Cl​-​ ion stays in the loop, while the system heats from 0 K to 300 K. Once room
temperature is reached, the thermodynamic ensemble switches from NVT to NPT and the results in terms
of Cl​-​ ion loop occupancy can be seen to vary based off the data I have presented. Other ensemble setups
were tested, but the trends in terms of occupancy held constant, (this is most likely due to the system size;
to enhance computational efficiency, only the EF-hand was put under simulation). Of course every
mutation has its own evolving geometry, and mutation 11 showcases a geometry that, although
incomplete, eerily mimics that of a wildtype canonical Ca​2+​ loop. In ​Table 4 ​I bring back Gifford’s,
Vogel’s, and Walsh’s “most common” loop and contrast it with Mutation 11 in an effort to showcase the
differences in coordinating residues.

Table 4
EF-loop position 1 2 3 4 5 6 7 8 9 10 11 12

Coordinating ligand X Y Z -Y -X -Z
Gifford’s, Vogel’s, and Walsh’s Loop sc sc sc bb sc sc​2

Most common Asp Lys Asp Gly Asp Gly Thr Ile Asp Phe Glu Glu
Gifford’s, Vogel’s, and Walsh’s Loop

Charge/Polarity of most δ​- δ​- δ​- Back

bone δ​- δ​-
common C=​O

Researched Canonical Loop Asn Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu
Ca​2+​ binding

Mutation #11* Asn Ile Ser Glu Ala Gly Gln Leu Pro Val Asp Arg
Cl​-​ binding bb -NH sc -OH bb -NH bb -NH bb -NH bb -NH bb -NH sc -CH sc -N​+​H​2

Table 4: Canonical Ca​2+​ EF-hand loops demonstrate a constant binding geometry. Release of the Ca​2+​ ion in wildtype
loops depends on how quickly molecules such as calmodulin or calbindin can find another protein to bind with. Every
residue that demonstrates chelation in wildtype loops contains a negative charges that complexes with the Ca​2+​ ion with
fascinating precision. Mutation 11 remains stable for 1.35 ns in 100 mM Cl​-​ aqueous solution with an evolving
geometry that is not time dependent but dependent on the force-field environment and residues that comprise the loop.
Residues highlighted in green coordinate with Cl​-​ for almost the entire duration of Cl​-​ occupancy, while residues
highlighted in yellow show coordination interests, but seem to have commitment issues. Residues highlighted in red
showed no coordination interest and may or may not have caused the loop to destabilize.
* The small font script below each residue in Mutation 11 show which atoms the the Cl​-​ coordinated with and where on the
residue it occured.

As shown in ​Table 5 ​figures ​(A)​ and ​(B)​, there are seven coordinating residues within 3.5 Å of
-​
the Cl​ ion which appear to be arranged in a warped pentagonal bipyramidal structure. I would like to
highlight the coordination of leucine, glutamine, and glycine. All three show promising interactions
between the Cl​-​ ion [represented in orange] and nitrogen bound hydrogens located on the backbone of the
loop. This discovery is reassuring and other research groups seem to have found similar results when

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designing chloride sensors. For example a synthetically designed c​hloride selective calix[4]arene optical
sensor​ provides two cavities in which Cl​-​ ions may bind with; both cavities contain four coordinating
backbone nitrogen bound hydrogens, all interacting with the Cl​-​ ion.​7​ Another interesting find was the
coordination of the Cl​-​ ion and hydrogens from different water molecules. This is analogous to the
chelation between a water molecule’s oxygen and the Ca​2+​ ion at position nine in Gifford’s, Vogel’s, and
Walsh’s “most common” loop.​5​ The hydrogen [from water] in figure ​(A) ​is shown in green and color
matters here; as shown, a new water molecule swaps out with the original and this new hydrogen is
colored yellow in figure ​(B)​. Figure ​(C) m
​ aintains this same pseudo hydrogen-bond and figure ​(D)
showcases the final water-swap [red]. This dynamic scaffolding might be unique to this mutation and I
will further investigate this water-swapping action in both old and new mutations. Figures ​(C) ​and
(D)/(E)​ exhibit square bipyramidal and incomplete pentagonal bipyramidal geometries respectively,
which leads me to believe that Cl​-​ ion’s stability within the loops is deeply dependent upon an amino acid
sequence whose backbone can provide equatorial stability, whose coordinating sidechain(s) can provide
axial stability from underneath, and provide room in which a water molecule can stably hydrogen-bond
and coordinate with the Cl​-​ ion axially on top. This crucial multiplanar geometric stabilization is even
shown in figure ​(F)​ where the loop forms a relatively rudimentary tetrahedral geometry around the Cl​-
ion.

Table 5*

A [Frame: 100] ​B [Frame: 1,350]

Table 5: Figures (A) and (B) both demonstrate a warped pentagonal bipyramidal geometry, but the residues showing
coordination differ from figure to figure. Even though arginine has an outright positive charge, its coordination with
the Cl​-​ ion is limited to less than the first 0.25 ns of simulation. Proline behaved similarly. Alanine’s backbone -NH
showed strong coordination further solidifying my resolve that backbone nitrogen bound hydrogens will play a crucial
role in how I mutate the loop in the future. Serine’s sidechain hydroxyl group briefly showed coordination interest, but
it was pulled away by more electropositive residues.

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C [Frame: 2,600] D [Frame: 3,850]

E [Frame: 5,100] F [Frame: 6,350]

Table 5: Figures (C), (D), (E), and (F), all display the aforementioned crucial Cl​-​-HN bonds. Starting with figure (C),
asparagine’s sidechain amino group coordinates with the Cl​-​ ion in an axial position from underneath. In figures (C),
(D), and (E), leucine, glutamine, glycine, and alanine all lend their hydrogens [from backbone nitrogens] illustrating
the importance of equatorial stability derived from backbone structure. Residue locations two through four, ten, and
eleven showed little to no binding interests.
* All images were rendered from VMD 1.9.3 and labels/coordinating bonds were made using Adobe Photoshop 14.0.0.

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Conclusion:
To the extent that I have researched mutation methods, successively creating new mutations
taking into account predecessors failures seems [for right now] the best method. With regard to the
stability of the loop, it seems that the stable preferred binding geometry for the loop is one in which
waters can frequently swap out and axially coordinate from the top, sidechain amino group hydrogens can
axially coordinate from the bottom, and backbone peptidyl hydrogens can provide stable equatorial
coordination spots for the Cl​-​ ion. I find it fascinating how backbone carbonyl oxygens in the canonical
EF-hand loops chelate with the Ca​2+​ ion and analogously the backbone -NH bonds coordinate with the Cl​-
ion. The similarities in geometry and backbone behavior between canonical EF-hands and my Mutation
11 are slightly uncanny, but are most definitely welcome; I will continue to use the former to drive
forward the progress of the latter. Addressing the issue of varying Cl​-​ concentration I can firmly say that
so far, I have found no correlation between Cl​-​ concentration and the occupancy time of the Cl​-​ ion within
the loop, but I will continue to collect more data off other mutations to see if trends manifest in a
correlative fashion. Looking to the future, I will be able to improve the stability and binding time of the
loop to the Cl​-​ ion greatly via more efficient geometrical and coordination bond analysis. In the near
future I will also use a water-swap reaction coordinate system to calculate the absolute protein-ligand
binding free energies of the most successful loops.

Acknowledgements:
I would like to thank my mentor, PhD student Francesco Trozzi, for helping me understand the
coding behind molecular dynamics software and for helping me write the scripts necessary to produce
simulations. He patiently waited for me to climb the learning curves of AMBER and VMD and has shown
me nothing but kindness. I would also like to thank Dr. Peng Tao for giving me the opportunity to do this
research; his constant support and optimism coupled with his feverish work ethic have proven invaluable
and inspirational to my own growth as a scholar of the sciences.

Looking Forward:
Computational chemistry is a relatively new field in the scientific community and its useful
capacity grows every day. If anything, I’ve learned the value of its predictive nature. It’s alluring to me
because I like to watch the gears of nature turn, picosecond by picosecond, so naturally I can see myself
continuing to do research in this field. While costly from a computational perspective, the time advantage
that running multiple simulations gives is invaluable, and coupled with experimental results… that is a
powerful tool. I’m also drawn to this area because of the freedom a computer provides, I’ve never been
restricted to one physical locality; my ideas can be tested from anywhere, on the fly. In the long run, I
seek to become an MD; however, I see no reason to believe that my research can’t be tied to the medical
field in a useful way.

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References:
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domain: A globally cooperative structural unit. Protein Science, 11: 198-205.
doi:​10.1110/ps.33302
(2) The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.
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Optical Sensor Combining Urea Functionality with Pyrene Excimer Transduction. Journal of the
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