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Structure, interaction and design studies involving regulatory peptides and proteins |
| Principal Investigator : Dinakar M Salunke
Project
Associates/Assistants
Technical
Officer
Ph
D Students Collaborator The
theme of the research is to study the structural aspects of molecular
recognition and its applications in analyzing the mechanisms associated with
specific regulatory events and in rational molecular design. The objectives of
the project are (i) understanding the protein architecture, (ii) analysis of
the structural principles of molecular recognition and mimicry, (iii)
structural biology of various regulatory events, and (iv) rational molecular
design studies based on the above. As
part of another on-going activity concerning structural basis of carbohydrate
mimicry, we had determined the crystal structures of concanavalin A and
Jacalin bound to a porphyrin analog, meso-tetrasulphanatophenylporphyrin
(H2TPPS),
and established the mimicry of mannopyranoside and porphyrin in terms of their
interactions with the two lectins. We have now determined and refined the
crystal structure of H2TPPS
in complex with yet another lectin, peanut agglutinin (PNA) at 2.9Å
resolution. PNA co-crystallizes with H2TPPS
in the symmetry packing different from any of its complexes with the
carbohydrate ligands determined earlier. The asymmetric unit in PNA-H2TPPS
binary complex consists of 11 porphyrin molecules associated with a tetramer
of PNA. The ternary complex of PNA with H2TPPS
and lactose crystallizes in the symmetry packing similar to that observed in
the binary complex. The asymmetric unit in the ternary complex consists of 2
lactose molecules in addition to 11 H2TPPS
molecules observed in the case of binary complex associated with a tetramer of
PNA. Only two of the four subunits of PNA therefore, show bound lactose
molecules. Thus, the quaternary structure of PNA and the arrangement of
porphyrin molecules in both complexes are identical. The
carbohydrate-binding sites of PNA are unoccupied in the binary complex. The
two pairs of porphyrins interacting with subunits A and B interact near the
lactose binding site on PNA, however the porphyrin association in the subunits
C and D is farther away from the lactose binding site. In the ternary complex,
lactose molecules occupy the carbohydrate binding sites of subunits C and D,
whereas subunits A and B are still empty. The PNA molecules in the porphyrin
bound state in the binary complex, have a conformational change involving the
residues 95 to 104, as compared to the earlier determined structures of PNA.
The loop encompassing these residues is one of the four loops (91 to 106, 125
to 135, 75 to 83 and 211 to 216) defining the carbohydrate-binding site of the
lectin. The residues of the loop 95 to 104 interact with the porphyrin only in
subunits A and B, but the same conformation of the loop is conserved in
subunits C and D as well. This observation leads us to propose that the
conformation of the loop in the binary complex may in fact represent the
native carbohydrate-free state of the protein. This conformation of the loop
would make binding of the lactose molecule sterically difficult at the
carbohydrate binding sites. The PNA molecule may therefore be required to
undergo conformational changes in this loop when binding to it’s
carbohydrate ligand. This view is reiterated by the fact that subunits C and D
in the ternary complex, when bound to lactose, attain the conformation similar
to that determined from earlier carbohydrate bound structures of PNA. The
studies on peptide-carbohydrate mimicry in the immune response, addressed
using monoclonal antibodies against mannopyranoside were further extended to
include thermodynamic aspects as well. Anti-a-D-mannopyranoside
monoclonal antibodies which were generated earlier have been extensively
characterized for their ligand binding characteristics. Amongst them, mAb 1H7
recognized only the immunizing antigen, a-D-mannopyranoside,
while mAb 2D10 recognized both the carbohydrate and the 12mer peptide.
Comparison of the binding of the two antibodies to different carbohydrate
ligands showed that 1H7 is highly specific and recognizes only the
immunization antigen. On the other hand 2D10 showed indistinguishable binding
to three different sugars, mannopyranoside, glucopyranoside and lactose
implying degeneracy of recognition specificity. Thermodynamic analyses of
binding of the two antibodies to the antigens showed that the affinity of mAb
1H7 to the carbohydrate antigen does not significantly change on increasing
temperature from 10°C to 35°C. On the other hand, significant decrease in
the affinity of the mAb 2D10 was observed over this temperature range.
Analysis of the thermodynamic data along with the kinetics of binding
suggested that 1H7 has well-defined relatively rigid antigen combining site
and 2D10 may have substantial flexibility in the CDRs providing plasticity of
interaction in the antigen combining site. It is attractive to propose that
the plasticity in the antigen combining site of the mimicry-recognizing
antibody is linked with the observed molecular mimicry. Screening of random
peptide libraries displayed on bacteriophage indicated that mAb 2D10
recognized a wide range of peptides. However, the peptide repertoire that
binds to mAb 1H7 was found to be restricted in the sequence, confirming that
mAb 2D10 exhibited degenerate specificity while mAb 1H7 did not. Apparently,
the functional mimicry between the carbohydrate and the peptide, as seen by
the immune response, reflected in the recognition specificities with regard to
screening of random peptide libraries. These observations also strengthen the
proposed link between molecular mimicry and plasticity of antigen-antibody
interaction. In
the context of the antibody flexibility and degenerate specificity, maturation
of the specificity of recognition in the humoral immune response is being
analysed by crystallographic studies involving germ line antibodies.
Degenerate specificity in germline antibodies has been demonstrated earlier
using phage displayed peptides and identifying diverse peptides which can bind
to the antibody. This has been attributed to flexibility in the paratope. We
set out to structurally image the correlation of the degenerate specificity
with conformational flexibility by determining crystal structures involving
antibody and peptide. We have determined structure of the Fab fragment of
anti-arsonate antibody 36-65 at 2.7Å resolution. The crystals contain two
molecules in the asymmetric unit with end to end packing providing psedio-antigen
bound environment at the antigen combining sites of the two molecules. The
conformational differences and the packing interactions involving paratopes of
the two molecules in the asymmetric unit shed light on the correlation of the
plasticity of interaction with conformational variability. The paratopes of
the two molecules show differential interactions with the loops of the
symmetry related Fab molecules, with corresponding variations in the
conformations of the CDR’s L3, H2 and H3 providing structural evidence,
relating degenerate specificity with conformational flexibility. We
are presently expanding our studies to include allegy-related functions. We
had effectively exploited plant lectins for addressing structural basis of
molecular mimicry. A variety of other plant seed proteins with potential
allergenic activities have been identified; we are exploring the
structure-activity correlation among these proteins. A tropical legume, Vigna
unguiculata, has been subjected to structural proteomics as a part of
these efforts. A 25kDa protein was purified by subjecting the seed extracts
first to ammonium sulphate fractionation and then gel filteration as well as
ion-exchange chromatography. Initial attempts at sequencing the N-terminal
region of the 25KDa protein for its characterization suggested that the
N-terminus was blocked. Trypsin digestion subsequently enabled sequencing of
four different internal fragments which were used to identify the protein by
database searches. The protein showed more than 85% homology to mung bean seed
albumin. Legume albumins are known to be intrinsically allergenic. Structures
of none of the protein homologous to Vigna unguiculata albumin is been
determined so far. The CD profiles suggested the protein to have relatively
high b
sheet content. When the purified protein was subjected to crystallization
attempts, rhombic shaped crystals which diffracted to 2.1Å resolution, were
obtained. The crystals belong to the space group C2 with cell parameters,
a=124.9Å,b= 60.1Å,c= 67.5Å and b=111.1°.
Intensity data has been obtained and attempts at determining structure are in
progress.
Publications Original peer-reviewed articles 1. Chakrabarty P, Sethi DK, Padhan N, Kaur KJ, Salunke DM, Bhattacharya S and Bhattacharya A (2004) Identification and characterization of EhCaBP2: a second member of the calcium-binding protein family of the protozoan parasite Entamoeba histolytica. J Biol Chem (in press). 2. Jha BK, Mitra M, Rana R, Surolia A, Salunke DM and Datta K (2004) pH and cation induced thermodynamic stability of human hyaluronan binding protein 1 regulates its hyaluronan affinity. J Biol Chem (in press). 3. Goel M, Anuradha P, Kaur KJ, Maiya BG, Swamy MJ and Salunke DM (2004) Porphyrin binding to jacalin is facilitated by the inherent plasticity of the carbohydrate-binding site: novel mode of lectin-ligand interaction Acta Crystallogr D Biol Crystallogr 60:281-288. 4.
Jha BK, Salunke DM and Datta K (2003) Structural flexibility of
multifunctional HABP1 may be important for regulating its binding to different
ligands. J Biol Chem 278:27464-27472.
Reviews/Proceedings 1.
Basu SK, Batra JK and Salunke DM (Eds) (2004) Deep roots, open skies: New
biology in india. Narosa Publishing House, New Delhi, 200p. |