We obtained the 3D structure of Gal10p from
Kluyveromyces lactis by
swiss model software [
11]
via homology modeling (). We
subjected the protein sequence of Gal10p through SWISS MODEL by
using default parameters. This software developed the 3D structure of
Gal10p from
K. lactis by using chain A of 1Z45 as the template protein
whose 3D structure is known and submitted in protein data bank (
Table 1Table 1).
The template protein 1Z45 is Gal10p from
S. cerevisiae. The protein
sequence of Gal10p from
K. Lactis showed sequence identity of 54.191%
and e-value of 0.00e-1 with 1Z45 protein (Gal10p of
S.cerevisiae). The
modeled structure is made up of mixture of Helix and sheets ( see
Table 1).
Ramachandran plot analysis via procheck estimated the model quality and
confirmed that overall accuracy of the developed model was 98.20% where
majority of the amino acid residues were in favored [A,B,L]+ additionally
allowed [a,b,l,p] regions. The numbers of bad contacts per 100 residues
were measured to be only one. ProSA-Web server analysis revealed that
the modeled structure occu]pied region of X-ray predicted native protein
structures of same size with Z score of -9.78 (). Energy
minimization by Gromos96 (Via Swiss Pdb Viewer) stabilized the Gal10p
modeled structure from energy of 3281.895 KJ/mol to -23676.174 KJ/mol.
The structures of Gal10p (Epimerase enzymes) from (Modeled structure)
K. lactis, Gal10p (1A9Z) from
E.coli and Gal10p (1Z45) from
S.
cerevisiae were subjected to functional sites prediction serves like
PINTS(12), PROFUNC(13) and Q-SITEFINDER(14) for finding of
putative active sites residues domain. The DALI server provided
significant match for Gal10p from
K. lactis with 1Z45 (Z score 60.5,
RMSD=0.7). DALI server template pdb also matched with the SWISS
MODEL. The functional site prediction servers predicted following active
site residues in Gal10p of
E.coli Tyr11, Ile12, Tyr177, Lys84, Ala9, Gly10,
Phe80, Ser122, Gly7, Lys153, Ser123, Pro180, in Gal10p (1Z45) of
S.
cerevisiae Asn445, His471, Tyr435, Ile36, Lys108, Tyr205, Phe104,
Gly31, Ser146, Ser147. Gly34. Pro208, Tyr22, Ile23, Asp42, Asn43,
Leu44, Ser45, Asn46, Ser47, Leu70, Phe91, Asn110, Ser135, Tyr163,
Lys167 and in Gal10p of
K.lactis Tyr15, Ile16, Tyr184, Phe84, Ser126,
Ser127, Lys88, Gly14, Pro187, Gly12, Gly11, Tyr156, Lys160 with
significant match. The functional sites predicted by Q-SITEFINDER [
10]
server also matched with the Profunc server. The sequence (by BLAST
method) and structure (by swiss pdb viewer method) similarity have been
estimated between the Gal10p of
S.cerevisiae, K.lactis and
E.coli. The
Gal10p from
S.cerevisiae (1Z45),
K.lactis and
E.coli (1A9Z) did not show
any nucleotide sequence similarity with each other but the protein
sequence produced significant sequence similarity with each other. The
Gal10p protein of
S. cerevisiae produced sequence identity of 56% and
evalue of 0.00, score 799 with Gal10p protein of
K. lactis. But in case of
matching with
E.coli Gal10p the sequence identity was 49%, e-value 6e-
97, score 337 which is less then homology of Gal10p of
S. cerevisiae with
Gal10p of
K. lactis. Gal10p of
K. lactis with Gal10p of
E. coli produced
sequence identity of 47%, e-value 3e-89, score 311. The protein sequence
identity was also reflected by Dot matrix plot where among all Gal10p
proteins, the Gal10p of
S. cerevisiae and Gal10p of
K. lactis are diagonally
align with each other ().
We have done the structure-structure superposition by swiss pdb viewer
and calculated the Root Mean Square Deviation (RMSD) value for finding
the structure similarity among Gal10p proteins. Superposition of Gal10p
of
S.cerevisiae produced low RMSD with the Gal10p of
K. lactis (RMSD = 0.28Å)
as compare to Gal10p of
E.coli (RMSD= 0.98 Å) and between
Gal10p of
K. lactis & Gal10p of
E.coli RMSD was 1.03 Å
( see
Table 2). We have obtained the putative protein-protein
interaction network for Gal10p proteins in
S. cerevisiae, K. lactis and
E.
coli via string (version 8.2) (
http://string.embl.de/)
software (15) ().
The D-galactose pathway is regulated by several proteins which are known
to interact with each other and regulate the synthesis of galactose
metabolizing enzyme. The Gal10p may also interact with its nearest
proteins to carry out its function therefore we determined the affinity
between the Gal10p with other GAL proteins present in the
K. lactis ,
S.cerevisiae and
E.coli. In order to estimate the strength of interaction
affinity between the Gal10p and other Gal proteins within genome of
S.
cerevisiae, K. lactis and
E. coli, we used patchdock software for proteinprotein
interaction study. Before estimating the interaction, we also
developed the modeled 3D structures of Gal1p, Gal3p, Gal4p, Gal7p, and
Gal80p by SWISS MODEL software for
S. cerevisiae, K.lactis and
E.coli
( see
Table 1). The Gal10p of
S.cerevisiae
produced greater affinity for its Gal3p protein with patch dock score 18718
as compared to its other Gal proteins (see
Table 3).
On the other hand, Gal10p of
K.lactis produced greater affinity
for its Gal7p with patch dock score 14986. The Gal10p of
E. coli
showedgreater interaction for Gal1 (galK) with patchdock score 16562
(see
Table 3).
