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Structure, interaction and design studies involving regulatory peptides and proteins |
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Principal Investigator : Dinakar M Salunke Project
Associates Technical
Officer PhD
Students
Collaborators
The
structural aspects of molecular recognition and its applications in analyzing
the mechanisms associated with specific regulatory events and in rational
molecular design, is the main theme of research. The specific objectives of
the project are aimed at understanding the protein architecture, analysis of
the structural principles of molecular recognition and mimicry, structural
biology of various regulatory events and rational molecular design studies. The
sugar-peptide mimicry, established on the basis of polyclonal antibody
cross-reactivity and structural similarity earlier, was further addressed with
the help of a panel of monoclonal antibodies. Monoclonal antibodies were
generated against a-D mannopyranoside by using the sugar conjugated to keyhole limpet
hemocyanin as the immunogen. The hybridoma clones were selected by screening
the supernatant for binding to the sugar as well as the carbohydrate mimicking
decapeptide in an ELISA based assay. Three IgM and five IgG clones have so far
been stabilized after several levels of subcloning from the primary and the
secondary immune responses, respectively. All
the three IgM clones show cross reactivity with the carbohydrate mimicking
peptides. Also, they show binding to other sugars like glucose and lactose
with affinities comparable to those of the peptides. This implies that the
sugar recognition of the antibodies in the primary response is not fine-tuned
to specifically recognize a-D-mannopyranoside. Of the 5 IgG clones that have
been stabilized, two have been characterized for their binding specificities.
One of them (1H7) binds to a-D mannopyranoside but does not
recognize the carbohydrate mimicking peptides. On the other hand, the second
clone (2D10) recognizes the peptide mimics with varying affinities. The
antibody 1H7 is highly specific to mannose as it shows only a small
cross-reactivity with glucose but no cross-reactivity with lactose. The
antibody 2D10, which recognizes peptide as well as the sugar shows much
broader specificity exhibiting significant cross-reactivity with the other
sugars. Epitope
mapping using combinatorial library of peptides on pins was carried out for
IgG clone 2D10. The antibody 2D10 binds to the end-protected hexapeptide with
higher affinity compared to the other three peptides as is the case with ConA.
Combinatorial library was designed to have the diverse peptides with glycine
substitution at each residue in four different carbohydrate mimicking
peptides. Binding of 2D10 to these peptide analogs in an ELISA led to
recognition of distinct pattern of interacting residues in case of each of the
carbohydrate mimicking peptides. Based on this pattern, it could be inferred
that the anti-mannopyranoside antibody, 2D10 resembles ConA in binding to
these peptides. While
broadening the molecular mimicry applications, we addressed if the principles
of retro-inverso mimicry were applicable to immune epitopes. Structural
rationale for the molecular mimicry involving retro-inverso design was
analyzed using T-cell epitopes derived from vesicular somatitis virus protein
(VSVp) and the ovalbumin protein (OVAp) and the B-cell epitope PS1 the
interactions with various monoclonal antibodies of which have been
investigated in this laboratory. It
was expected that a retro-inverso peptide analog corresponding to a given
sequence should adopt a conformation similar to that of the corresponding L
peptide. Major difference, however, being that the N and C termini of the
retro-inverso peptides would be interchanged with respect to the native
peptide. Particularly, the terminal functional groups of VSVp and OVAp have
been known to play a significant role in binding to MHC class I molecule H-2Kb (MHCI). The possible repulsive interactions,
arising from the charge reversal were reduced by neutralizing the charges on
the end groups. The conformational models of retro-inverso analogs of VSVp and
OVAp (called rVSVp and rOVAp) suggested that the exact superimposition of the
retro-inverso model with the native peptide was not achievable. However,
the backbone dihedral angles, f and j,
for the native and retro-inverso versions were localized in the corresponding
equivalent regions of the Ramachandran plot. The molecular dynamics analyses
show that the distribution of the backbone rmsd values is comparable in case
of both native L and retro-inverso versions of each of the T cell epitopes.
The extent of change with respect to the starting conformation that the two
versions undergo in solution is same, indicating an inherent similarity in
conformational flexibilities. Thus, the analyses of dihedral angles and
conformational propensities showed that retro-inverso analogs could
potentially mimic the T cell epitope peptides. The functional assays carried
out on using the retro-inverso peptides as per above design, in collaboration
with the Immunobiology Laboratory, indeed confirmed this surmise. Comparison
of the computer-docked models of the retro-inverso peptide-H-2Kb complexes and the crystal structures of the
corresponding native L peptides complexed with H-2Kb reveal that the same peptide residues are
present in the corresponding structural pockets. The mimicry between the
retro-inverso peptides and their corresponding native L counterparts is
evident from the contacts between the MHCI molecule and the bound peptides.
The solvent accessible surface area buried on complexation is similar in case
of the two versions of both the T cell epitopes. The almost precise
correspondence of the surface features between the two cases is evident. The
complexes of H-2Kb with native and retro-inverso versions of OVAp
would thus present a similar surface for the cognate T cell receptors to
recognize. The topological features and the localization of the regions of
similar hydrophobicity are remarkably alike for the two versions of each
peptide. Hence, it was anticipated, and subsequently observed in the
functional assays carried out in the collaborating laboratory, that the H-2Kb:OVAp and H-2Kb:rOVAp
complexes will bind to the same T cell receptor with comparable affinity. To
test whether the mimicry observed in case of the T-cell epitopes also exists
in case of the B-cell epitopes, analogous computational and biochemical
experiments were carried out using PS1. Unlike the T cell epitopes, PS1 does
not adopt an extended conformation when bound to any of the three independent
monoclonal antibodies. The stretch DPAF, which also represents an
immunodominant epitope, adopts a b-turn conformation.
Ramachandran plot of PS1 shows that the distribution of f
and j angles is localized to distinctly different regions for native and
retro-inverso residues. While the native peptide occupies the core and allowed
regions corresponding to a-helix, some residues of retro-inverso
occupy the allowed and generously allowed regions corresponding to the left
handed helices while others are found in the disallowed regions. Thus, in case
of the rPS1 peptide, the conformation which would be a topochemical equivalent
of the bound PS1 conformation is energetically unfavorable. Hence the plots
indicate that there might be a drastic difference between the functional
properties of the native and retro-inverso versions of PS1. The MD simulations
of the peptides carried out suggest a considerable difference in the
structural plasticities of the two versions of the peptide. The retro-inverso
analog exhibits a higher variation in its conformation than the native L
peptide. It is stereo-chemically not possible for rPS1 to adopt a conformation
similar to that of the bound PS1. Hence, it is possible that the retro-inverso
analog of PS1 might not be functionally active. Indeed, the retro-inverso
version of the B-cell epitope, PS1, does not bind to the anti-PS1 monoclonal
antibodies, consistent with their structural differences. Thus, the retro-inverso
version of the B-cell epitope PS1 is not functionally active unlike the T cell
epitopes VSVp and OVAp. The
correlation of conformational propensities of the native L peptides and their
retro-inverso counterparts depends on the inherent structural properties of
the individual peptides. While the T cell epitopes VSVp and OVAp, which both
adopt the extended conformation, exhibit similar conformational propensities
in L and retro-inverso forms, the B-cell epitope PS1 shows distinct
conformational propensities in the retro-inverso version. The retro-inverso
analogs of VSVp and OVAp are in fact functionally active through similar
molecular interactions with their receptor. However, in case of the B-cell
epitope, the retro-inverso analog cannot bind to anti-PS1 antibodies
indicating different conformational preferences than the native peptide. The
application of retro-inverso logic for molecular mimicry was exploited in yet
another immunologically relevant system. Antibacterial peptides expressed as a
result of bacterial infection form an important component of innate immune
system. Among them, indolicidin is a cationic peptide produced by bovine
neutrophils which specifically binds to the bacterial endotoxin,
lipopolysaccharide (LPS). We have shown that the retro-inverso analog of
indolicidin is functionally similar to the native peptide. Monoclonal
antibodies were generated against indolicidin and screened for binding to
retro-inverso analog. The monoclonal antibody C6H6G2, recognizes retro-inverso
indolicidin with the affinity comparable to that with the native peptide. The
comparison of binding profiles of indolicidin, retro-inverso indolicidin and
other analogs with the antibody and LPS indicates that the antibody and LPS
were equivalent in terms of their binding to indolicidin and its analogs. We
have shown that indolicidin binds to LPS in a pattern-based manner exploiting
plasticity of conformation and interactions. It appears that the antibody also
binds to indolicidin similarly. Thus, the C6H6G2 seems to be a close mimic of
LPS since it shows similar peptide binding profiles as LPS. Publications Original
peer-reviewed articles 1.
Nair DT, Singh K, Siddiqui Z, Nayak BP, Rao KVS and Salunke DM (2002)
Epitope recognition by diverse antibodies suggests conformational convergence
in an antibody response. J Immunol 168:2371-2382. 2.
Goel M, Jain D, Kaur KJ, Kenoth R, Maiya BG, Swamy MJ and Salunke DM
(2001) Functional equality in the absence of structural similarity: an added
dimension to molecular mimicry. J Biol Chem 276: 39277-39281. 3.
Jain D, Nair DT, Swaminathan GJ, Abraham EG, Nagaraju J and Salunke DM
(2001) Structure of the induced antibacterial protein from Tasar silkworm, Antheraea
mylitta: Implications to molecular evolution. J Biol Chem 276:41377-41382. 4.
Jain D, Kaur KJ and Salunke DM (2001) Enhanced binding of a rationally
designed peptide ligand of concanavalin A arises from improved geometrical
complementarity. Biochemistry 40:12059-12066. 5.
Nayak SK, Bagga S, Gaur D, Nair DT, Salunke DM and Batra JK (2001)
Mechanism of specific target recognition and RNA hydrolysis by ribonucleolytic
toxin restrictocin. Biochemistry 40:9115-9124. 6.
Hemachand T, Gopalakrishnan B, Salunke DM, Totey SM and Shaha C (2002)
Sperm plasma membrane associated glutathione S-transferases as gamete
recognition molecules. J Cell Sci (in press). 7.
Kumar R, Choudhury NR, Salunke DM and Datta K (2001) Evidence for
clustered mannose as a new ligand for hyaluronan-binding protein (HABP1) from
human fibroblasts. J Biosci 26:325-332. 8.
Jha BK, Salunke DM and Datta K (2002) Enhancement of ligand affinity of
trimeric hyaluronan binding protein 1 (HABP1)/p32/gC1qR is facilitated by
disulphide bond formation through cystein186 Eur J Biochem 269:298-306. 9. *Kaur KJ, Jain D, Goel M
and Salunke DM (2001) Immunological implications of structural mimicry between
a dodecapeptide and a carbohydrate moiety. Vaccine 19:3124-3130
(*in press last year, since published). 10.
*Jain D,
Kaur KJ and Salunke DM (2001) Plasticity in protein-peptide recognition:
Crystal structures of two different peptides bound to concanavalin A. Biophys
J 80:2912-2921 (*in
press last year, since published). |