Detailed Methods for Modeling Studies of Drosophila Integrins
Kishore Shakalya, Daruka Mahadevan
Homology models of PS2m8 and PS2C were generated by threading fly and
mosquito sequences onto the X-ray crystal structure coordinates of human v3
(1JV2) (Xiong et al., 2001). Various models were built including some comprising the
whole protein, the -subunit -propeller domain alone, or this in combination with
the -subunit A/I domain. Modeller (version 7) was used for homology modeling of
aligned sequences and then superimposed (homology aligned using Sybyl 7.2) on the
original crystal structure and merged into a single coordinate file after removing the
v structural coordinates. The models were energy minimized using Sybyl 7.2.
For PS2C, the mosquito sequence was done intially, and the fly structure was then
modeled after this. This sequential approach was taken because of problems caused
by the proline rich loop in the fly structure. For Anopheles gambiae PS2 no cDNA
sequence was available. The gene was found by searching the An. gambiae genome
for the PS2 homolog, constructed by virtual cDNA identification for each exon and
analyzing proposed splicing sequences in detail. The locations and reading frames of
the relevant splice sites are identical to those in Drosophila.
The homology model built by Modeller (version 7) included exon 8 region insertion
of amino acid residues between Gln193 and Gly219.
194 GQTYSIPPDAKFPFKPPLYQPFGTG 218
This insertion is proline rich and was built by Modeller (version 7) as a distorted helix. In a given protein structure the backbone Catoms serve as the corner
attachment point of two different planes, each which can rotate independently of the
other plane. The two planes can twist around the C carbon. The only degrees of
freedom along the backbone are about the NH-C bond called the phi and the C –
C'O bond called the psi angle. They can vary from -180o to +180o. Since proline rich
regions are not -helical we checked the phi-psi angles of the residues in the inserted
exon 8 segment. The ideal bond angles for an extended collagen-like poly-proline
helix are phi = -70o and psi = +138o respectively. These angles were measured using
Sybyl 7.2. It was found that the 4 prolines were modeled with phi/psi angles that
produced a distorted -helix.
Residues
Phi angle
Psi angle
Pro 201
-48.97
-34.81
Pro206
-56.49
-31.37
Pro210
-86.69
49.83
Pro214
-42.14
-39.51
To change the phi-psi angles, one residue on either side of each proline was extracted
and the phi-psi angles were changed to match an ideal collagen-like polyproline helix.
A local energy minimization was performed for a region of around 5Å incorporating
the four prolines in Sybyl 7.2. The final model was superposed on the original model
to obtain a root mean square deviation of <1Å.
The phi-psi angles were then measured after the changes were done. As seen in the
table below, they closely conform to the ideal case. The minor difference in the angles
as compared to the ideal case can be attributed to the surrounding residues and the
overall shape of the structure.
Residues
Phi angle
Psi angle
Pro 201
-47.45
138.68
Pro206
-56.56
158.07
Pro210
-69.27
157.68
Pro214
-37.01
114.26