SUNDAY, DECEMBER 4- Late Abstracts 1 - Molecular Biology of the ...

SUNDAY
the retina, in which AII amacrine cells make their gap junctions. Antibodies to PSD95 did not
label the inner synaptic layer, but did label the outer synaptic layer, where photoreceptor
synapses are located. In the inner synaptic layer, approximately 50% of the Cx36 gap junctions
were co-localized with either SAP97 or SAP102, and 15% co-localized with both. SAP102 colocalized
with about twice as many Cx36 gap junctions as did SAP97. Furthermore, of the
SAP102 puncta in the inner synaptic layer, fully one third were associated with Cx36 gap
junctions. In the outer synaptic layer, some PSD95 immunoreactivity was closely associated
with Cx36. Our data show that synaptic scaffolding proteins are associated not only with
conventional synapses, but also with electrical synapses. These scaffolding proteins may
facilitate assembly of non-synaptic neurotransmitter receptors, such as the NMDA receptors on
AII amacrine cells, with Cx36 gap junctions. They may alternatively, or in addition, facilitate the
association of conventional synapses and gap junctions to form mixed synapses.
2040
A structural biology approach to predict proteolytic sites of accessory gland proteins in
D. Melanogaster.
C. A. Del Carpio 1 , M-T. Yamamoto 1 ; 1 Drosophila Genetic Resource Center (DGRC), Kyoto
Institute of Technology, Kyoto, Japan
Extra and intra-cellular protein processing and degradation accounts for one of the main
biochemical processes involved in series of signal transduction and regulation pathways.
Moreover, genome-wide studies of higher organisms have unveiled that a large proportion of
encoded proteins are enzymes oriented to the cleavage of other proteins. Thus predicting the
cleavage sites of substrate proteins and the enzyme involved is highly required in functional
genome analyses including the elucidation of intra and extra-cellular mechanisms at the origin
of cellular malfunction and diseases. Sequence based inference of proteolytic cleavage sites in
polypeptides frequently leads to several sites that are located in the core of the molecule or are
sterically hindered and hardly accessible to the cleaving enzyme. Catering to the need of
including 3D structural factors in the assessment of cleavage sites in polypeptides and reduce
the number of candidates output at the primary structure level, we propose a bio-computational
strategy to firstly evaluate substrate-enzyme affinity and subsequently infer putative scissible
bonds. Affinity is evaluated by docking the 3D structures of the substrate and the enzyme, and
molecular dynamics (MD) simulation is performed to assess the formation of the transition state
(TS) complex triggering the proteolytic reaction. The cleavage sites are then inferred by tracing
the main atomic interactions throughout the MD simulation leading to formation of the TS
species. The methodology involves protein structure prediction, as well as protein docking to
assess substrate-enzyme interaction. As a case study we applied the methodology to the
prediction of cleavage sites for Acp36DE, a D. Melanogaster seminal fluid protein that plays an
important role in sperm storage and competition in multiply mated females. The results show
that among several candidate regions amenable to protease attack, the region LEU514-
ASP517, located at a disordered region between two alpha helices shows geometrical
complementarity and energetic affinity for an astacin like protease. The TS species derived
through the MD simulation shows the metal (Zn) in the astacin pulling the LYS515.O, and the
contraction of the distance between the SER516.N and GLU101.OE2 in astacin suggests the
scissible peptide bond. Repeating the calculations with serine proteases reported to be
expressed at copulation shows a higher affinity region for the hydrophobic region that includes
amino acids SER21-GLU22-SER23-PHE24 the cleavage bond being GLU22-SER23. Cleavage
of Acp36DE at LYS515-SER516 generates fragments of Acp36DE of molecular weights that
compare well with reported experimental findings