1. (This is an abstract of Ph.D thesis work carried out (Jan 2007- Jan 2014) by V
Prabahar and submitted to Indian Institute of Technology Madras)
Drosophila Nucleobindin: Characterization of an Evolutionarily Conserved Ca2+
-,
Zn2+
- and G protein-Binding Protein
Vadivel Prabahar
1
Department of Biotechnology, Indian Institute of Technology Madras, Chennai-
600036. India
Abstract
Drosophila nucleobindin (Dmnucb) is a calcium and zinc-binding protein (569 aa)
and undergoes distinct conformational changes upon binding to them. The protein has
a caspase cleavage site in the middle, which is flanked by zinc-binding motifs
(inverzincins and zincin) at the N-terminal and two canonical EF- hand motifs
(calcium sensor and calcium buffer type) at the C-terminal end of the protein.
Spectroscopic studies involving stains-all binding, intrinsic, extrinsic fluorescence,
circular dichroism and isothermal titration calorimetry using near-full length
recombinant protein (Dmnuc53, 53 kDa) and its variants (Dmnuc23, Dmnuc28,
Dmnuc30) revealed its ion-specific interaction, structural changes (Tryptophan
mobility, secondary, tertiary) and binding affinity for Zn2+
and Ca2+
. The apo form of
Dmnucb protein reveals all the tryptophan residues are exposed to the surface but
Zn2+
and Ca2+
- binding modulate the protein structure differently. Zn2+
-binding causes
a change in the surface hydrophobicity that depends on the type of zinc-binding motif
whereas, Ca2+
-binding showed no influence on the surface hydrophobicity. The
conformational behaviour of Zn2+
bound protein is detected by the mobility of its
tryptophan residue positioned at 227. In contrast, Ca2+
induced conformational
changes trigger mobility of Trp 255 towards hydrophobic core of the protein.
Dmnuc30, a variant of Dmnucb binds to Gαi1 (GDP bound) protein under Ca2+
free
condition through the conserved G protein binding and activation (GBA) motif, which
is embedded in the Ca2+
binding loop of EF-hand2. Apo Dmnuc30, which is
relatively unstructured, adopts more helical structure upon binding to Ca2+
and Zn2+
.
Isothermal titration calorimetry reveals Dmnuc30 and Gαi1.GDP interaction is
determined by Ca2+
confirming the highly conserved GBA motif in Drosophila
melanogaster.
Key Words
Drosophila nucleobindin (Dmnucb), Characterization of Dmnucb1, Calcium and Zinc
binding motif, GBA motif in Drosophila melanogaster, Glutamine rich protein.

2. 1. Introduction
2 MATERIALS AND METHODS
2.1 Cloning
2.1.1 Generation of Dmnucb1 Fragments for the GST tagged expression
2.2 Protein Expression and Purification
2.2.1 Expression of GST fused Dmnucb near-full length protein and its fragments
2.3 Spectroscopy
2.3.1 Fluorescence Spectroscopy
2.3.2 Monitoring Changes in the Surface hydrophobicity of Dmnucb using Extrinsic
fluorophore
2.4 CD Spectroscopy
2.4.1 Far UV and Near UV CD spectroscopy
2.5 Isothermal Titration Calorimetry
2.6 Results and Discussion
2.6.1 BIOINFORMATIC ANALYSIS OF DMNUCB
2.6.1.1 Primary structure of the Dmnucb protein
2.6.1.2 EF-hand motifs in Dmnucb is a sensor and buffer type
2.6.1.3 Zn2+
- binding motifs (ZBM) in Dmnucb are unique and clustered at the N-
terminus
2.6.1.4 G Protein Binding and Activation Motifs (GBA) in Dmnucb
2.6.1.5 Glutamine (Q) Rich C-Terminal in Dmnucb
2.7 The Structural Changes Induced By Ca2+
and Zn2+
2.7.1 Structural differences due to ligand binding is responsible for the
metachromatic behaviour of Dmnucb protein towards stains-all dye.
2.7.1.1 Dmnuc30 induces J band (apo state) and γ band (upon addition of Ca2+
)

3. 2.7.1.2 Dmnuc23, Dmnuc28 induces only J band and Dmnuc30CT-Q yields no
signature
2.7.2 Monitoring Changes in the Tryptophan Fluorescence Signature: Ca2+
and
Zn2+
binding triggers a distinct conformational changes
2.7.2.1 Perturbed Tryptophans (W227 and 255
) suggests a distinct structural changes
induced by Ca2+
and Zn2+
binding
2.7.2.2 Influence of Zn2+
and Ca2+
on the surface hydrophobicity of Dmnucb1
2.7.2.3 Ca2+
do not modulate the surface hydrophobicity
2.7.2.4 Zn2+
and the type of Zn2+
binding site modulate the surface hydrophobicity
differently.
2.7.3 Secondary Structure of Dmnucb Reveals Unstructured C-terminal Region
2.7.4 Tertiary Structure of Dmnucb is specifically influenced upon Ca2+
-binding.
2.7.5 Ion binding studies using Isothermal Titration Calorimetry (ITC)
2.7.6 Embedded GBA motif in the loop region of EF-hand2 check the Dmnucb-Gαi1
interaction
Conclusion
In the present work, we have shown that Dmnucb is a Ca2+
and Zn2+
-binding protein
having distinct motifs to coordinate their ligands. The Drosophila homolog of
mammalian nucleobindin is named as Dmnucb, since only single copy of
nucleobindin is found in invertebrates and its identity with human nucb1 (45 %) and
nucb2 (47 %) are very close. Despite the fact that Dmnucb (in general, nucleobindins)
do not share homology with other known CaBPs, significant commonness were found
with the members of CREC (Cab45, Reticulocalbin, ERC-45 and Calumenin) family,
such as, their presence in the secretory pathway, conserved proline residue as ER
export signal, calumenin type loop region in EF-hand2, enriched presence in Golgi
(like Cab45), conserved weak Golgi retention signal HEEF (RGEF in Dmnucb),
presence of Ca2+
binding motif etc. The phylogeny of nucleobindin is interesting that
invertebrates possess a single copy of nucleobindin and vertebrates have two
homologs, nucb1 and nucb2 (Data not shown). They are diverged from actinopterygii
(ray finned fishes), a dominant class of vertebrates. Nucb1 is found missing in

4. sarcopterygii (lobe finned fishes) and aves but present in actinopterygii, amphibians,
reptiles and mammals. We have demonstrated the structural changes associated with
ion binding and their affinity for divalent cations, especially Ca2+
and Zn2+
.
The work deals with the Ca2+
-, Zn2+
- and the structural changes associated with ion
binding and the specific role of Ca2+
in determining Dmnucb-Gαi1 protein interaction.
In our work, we have provided evidence detailing the different types of Zn2+
binding
motif, Ca2+
binding motif and the distinct structural changes associated with Ca2+
and
Zn2+
-binding using fluorescence, CD spectroscopy and Isothermal titration
calorimetry. We conclude here that based on spectroscopic studies that the Ca2+
and
Zn2+
bound states are structurally distinct and affects the local structure around the
aromatic residues especially W227
and W327
. The C-terminus of Dmnucb is relatively
unstructured and undergoes no structural arrangement in the presence of Ca2+
and
Zn2+
.