THE STRUCTRAL AND FUNCTIONAL STUDY

OF FIBRILLIN-1

Yash Pandyas2878217

Advance protein science 3002BPS

5TH June 2014

Introduction:• Fibrillin-1 is a large 350KDa multi domain (calcium

binding) glycoprotein.

• A major structural component of 10-12nm microfibrils located in the extracellular matrix.

• The two predominant domain types in fibrillin-1 are the calcium-binding epidermal growth factor (cbEGF) and transforming growth factor β binding protein –like (TB or 8- cysteine) domain.

Function of Fibrillin-1:• Microfibrils of the extracellualr matrix play important role

in both elastic and non-elastic tissues.• In elastogenesis provides a scaffold for the deposition of

tropo-elastin and form the perifery of the mature elastic fiber.

• Elastin present in the connective tissues is responsible for tissue flexibility upon starching and return to the normal position.

• Non-elastic tissues, like cilliary zonule of the eye and basement membranes, have anchoring function and provide the tensile strength.

• It is involved in some biochemical functions like regulation of growth factors and bone morphogenic proteins.

.

Marfan Syndrome:• MFS is an autosomal dominant heritable disorder of

connective tissues.• Clinical manifestations include aortic dilatation ,

abnormalities in skeletal system, lungs, adipose tissues and skin.

• Researches suggest that clustering of mutations in exons 24-32 are associated with the disease.

• Most frequently, missense mutations on cysteine residue and on calcium binding consensus sequence are observed in the two main domains: cbEGF and TB.

Structure of cbEGF domain:

• In fibrillin-1, 47-EGF domain are present. Of them, 43 are Ca2+ binding (cbEGF) domain,

• It is characterised by six cysteine residues which create disulphide bonds in position 1-3, 2-4, 5-6.

• Calcium binding consensus sequence “(D/N)-x-(D/N)-(E/Q)-Xm-C3-(D/N)*-Xn-(Y/F)”

Calcium binding site

(Rao et al.,1997)

Comparison of active sites of cbEGF domain:

• In fibrillin-1, the cbEGF 12-13 and cbEGF 32-33 are both showing a calcium binding rigid structure.

• Both the domains cbEGF12-13 and cbEGF 32-33 have linear orientations and inter-domain hydrophobic interactions with calcium binding C-terminal region.

• The two domains are maintained by calcium binding to the C-terminal domain and by interdomain hydrophobic packing interactions.

Structure of cbEGF 12-13

• cbEGF12 conserved Tyr is present at the open end of the minor β-sheets, and it packs against the top of the major β-sheets of cbEGF 13.

• Tyr (1101), Gly (1134) and the methyl group of Glu (1133) are involved in the side chain interactions.

• Arg (1083) forms inter-domain interactions against Tyr (1101).

Smallriedge et al.,2003

• Main interaction are observed on side chains of Tyr (2147) with Gly (2186) and two methyl groups Isoleucine (Ile).

• Tyr (2147) plays a key role in the folding of the structure.

• At C-terminal of cbEGF 32-33 the βsheets show a different structure because of the presence of a proline residue.

Structure of cbEGF 32-33

Smallriedge et al.,2003

• The Arg1083 in inter-domain packing may relate to increased calcium binding affinity for cbEGF13 relative to cbEGF33, because of a more stable binding site in cbEGF13.

• Both the structures are similar, but the most significant difference observed is on the N-terminal and C-terminal regions of cbEGF12-13 and 32-33 .

• The cbEGF 13 has a more stable binding with Ca2+ compared to cbEGF 33, because of the disulphide bond between 5-6 cysteine is shorter and because of three proline residues in cbEGF13.

Function of cbEGF domain:

• cbEGF has calcium binding domains, which give more inter-domain flexibility and protect the module against proteolytic cleavage.

• In cbEGF 32-33, N-terminal region has no specific conformation to bind which calcium, reducing its affinity.

• cbEGF 13 has shown a well defined calcium binding site and higher affinity on this domain. In cbEGF 12, an extended loop between cysteines 5-6 was observed to be solvent accessible and unstructured, showing an increased flexibility.

Missense Mutation:• G1127S: it affects the folding of cbEGF 13, because of the

presence of Gly, less flexible on major β-hairpin,

• S1077P: it affects the domain folding because this amino acid change results in a Pro-Pro sequence between the first and second cysteines of cbEGF12, affecting the conformational flexibility of this region.

Structure of TB domain:• Transforming growth factor β- binding protein, also known as

8-cysteine domain,

• Present in Fibrillin1,2,3 and latent TGF-β binding protein family (LTBPs),

• Structural difference between the fibrillins and LTBPs is that the fibrillins are longer in size than LTBPs.

• LTBPs have additional regions at their N-terminal, with no homology with another domain type. It has a unique four cysteine domain.

• Fibrillins have a unique C-terminal region not found in LTBPs.

Structural activity of TB6 domain:

• TB6 has six β-strands from a four stranded of β-sheets shown in B, C, E and F strands.

• These four stranded β-sheets were closely present with helix-1 and form the central part of the globular structure.

• Other two stranded β-sheets A and D and helix-2 pack on either side of the central core.

• Strand E the Tryptophan an aromatic residue plays an important structural role in centre of hydrophobic core.

Downing et al.,1997

Function of TB domain:

• RGD sequence located in fourth TB module of fibrilin1 and 2,

• RGD insertion is positioned in TB6, at the end of the β-hairpin formed by strand B and C,

• RGD is a major cell-binding epitope in the specific interaction between fibrillins and cell surface receptor integrin.

Downing et al.,1997

Conclusion:

• Fibrillin-1 is multidomain protein, cbEGF 12-13 and 32-33 show the information about presence of calcium which provide the stability and flexiblity of structure and it increases the calcium binding affinity. If mutation occurs in specific regions it causes MFS.

• The identification of functional regions of cbEGF and TB domains will facilitate future modelling study for fibrillin and specify the orientation of these domain within microfibils.

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