This document summarizes research on the structural and functional study of fibrillin-1. It describes fibrillin-1 as a large glycoprotein that is the main component of extracellular matrix microfibrils. It has calcium-binding epidermal growth factor (cbEGF) and transforming growth factor β binding protein (TB) domains. The cbEGF domains provide flexibility and stability through calcium binding. Mutations in these domains are associated with Marfan syndrome. The document compares the structures of the cbEGF 12-13 and cbEGF 32-33 domains and how mutations affect their folding and calcium binding. It also describes the structure and function of the TB domain, including its role in cell binding.

1. THE STRUCTRAL AND
FUNCTIONAL STUDY
OF FIBRILLIN-1
Yash Pandya
s2878217
Advance protein science
3002BPS
5TH June 2014

2. 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.

3. 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.

4. 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.

5. 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)”

6. Calcium binding site
(Rao et al.,1997)

7. 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.

8. 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

9. • 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

10. • 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.

11. 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.

12. 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.

13. 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.

14. 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

15. 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

16. 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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