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Mapping out the basement membrane
© 2001 Nature Publishing Group http://structbio.nature.com
Robert C. Liddington
Recent structural studies suggest that not only the protein components but also the nature and location of the
protein–protein interaction surfaces in basement membranes are highly conserved throughout the metazoa,
suggesting that their precise ultrastructure will be invariant, and reflecting their critical role in early
development.
The cells of multicellular animals (metazoa) secrete and shape around them a network of proteins called the extracellular
matrix (ECM). One specialized element of
the ECM is called the basement membrane,
a thin (<1,000 Å) sheet of protein and proteoglycan1. The recent sequencing of the
Caenorhabditis elegans genome shows that
the basement membrane is highly conserved throughout metazoan evolution,
much more so than the rest of the ECM2,3.
In the simplest of metazoa such as hydra,
the basement membrane divides the inner
layer of cells (endoderm) from the outer
layer (ectoderm). In more complex organisms, it provides a mechanical support and
barrier in blood vessels and other tissues,
and regulates the growth, differentiation
and migration of the adherent cells. For
example, epithelial cells must be anchored
to the basement membrane to survive; if
contact is lost, they undergo programmed
cell death (apoptosis) in a process that has
been called anoikis, from the Greek word
for homelessness4.
Until recently, our structural knowledge
of the basement membrane was limited to
electron microscopic resolution. Unraveling the atomic resolution network of
interactions and how they are regulated
within the basement membrane is still in its
infancy, but the recent work of Hohenester,
Timpl and colleagues has started to shed
some light. On page 634 of this issue of
Nature Structural Biology, these authors
describe the latest atomic resolution structure of a basement membrane component5,
a ligand-binding module of nidogen, one of
four proteins common to all basement
membranes. They go on to show that a surface of the module that is conserved from
the earliest metazoans provides a high
affinity binding site for another basement
membrane component, perlecan. These
results, together with earlier work from the
same groups, suggest a highly conserved
structure for the basement membrane.
Fig. 1 Cartoon diagram of nidogen. Nidogen contains three globular domains: G1, G2 and G3. G1
is of unknown structure and function. G3 has been predicted to fold into a six-bladed β-propeller7.
G1 and G2 are connected by a flexible linker, whereas G2 and G3 are connected by a rigid rod that
includes EGF-like modules. The structure of G2 is described in the text.
perlecan and nidogen, which form a complex web of interactions. The nonfibrillar
type IV collagen is a very long (∼2,000 Å)
molecule consisiting of a collagen triple
helix with N- and C-terminal globular
domains. Laminin is a large (900 kDa)
trimeric cruciform molecule that is long
enough (∼700 Å) to span the entire width
of the basement membrane. Perlecan is a
five-domain protein (∼400 kDa) that has
three large heparan sulfate chains attached
to its N-terminus. Nidogen is the smallest
of this quartet (150 kDa). One role of
nidogen is to provide a link between
laminin and type IV collagen. In C. elegans
nidogen has also been shown to be
required for the correct positioning of
longitudinal nerves6.
The structure of the second globular
domain (G2) of nidogen revealed in this
issue5 comprises an EGF-like domain
fused to the top of a β-barrel (Fig. 1). The
barrel is 45 Å tall and has a simple shape
but a complex topology, with an α-helix
inserted into the center of the barrel. This
fold has been seen before, in the green fluorescent protein (GFP) from Aequorea
victoria, a brightly luminescent jellyfish8.
Although there was no detectable
sequence identity (∼10%), the structural
Basement membrane components
homology is outstanding (root mean
All basement membranes have four major square (r.m.s.) deviation of 2.5 Å for 195
constituents: type IV collagen, laminin, Cα atoms). The authors reasonably sugnature structural biology • volume 8 number 7 • july 2001
gest that the two proteins have a common
ancestor, as it is hard to imagine such a
complex fold evolving twice. Beyond the
striking similarity in fold, however, there
are no other similarities. Nidogen is not
fluorescent and none of the fluorophore
residues are conserved. GFP is not an
extracellular protein: it is found in intracellular particles called lumisomes that are
2,000 Å in diameter. Sequence searches
with either GFP or nidogen generate only
closely related members of each family.
Jellyfish are amongst the simplest of the
metazoa, on one of the earliest branches of
the metazoan tree. It is very likely that jellyfish have a bona fide nidogen, since the
nematode C. elegans, on the next evolutionary branch, has one. So it seems likely
that nidogen and GFP have diverged from
a common ancestor, and the structural
similarity is just a curiosity at this point.
As new structures are solved, these unexpected structural homologies are becoming increasingly common. One of the
main legacies of the structural genomics
initiatives may be to reveal these evolutionary relationships that are not obvious
by sequence comparisons.
Interactions in the basement
membrane
The assembly of basement membranes
occurs in part in a spontaneous fashion —
573
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laminin and collagen both self-assemble
to form networks in vitro, the former in a
Ca2+-dependent fashion; nidogen and
perlecan spontaneously bind and bridge
between laminin and collagen IV.
Nevertheless, the active participation of
cells is required for proper assembly and
localization of the basement membrane,
and this is dependent on two classes of
transmembrane receptors, the integrins
and the dystroglycan–dystrophin complex, both of which bridge and convey signals to and from the actin cytoskeleton9.
Although the data are sketchy, it may not
be too early to review what is known about
these interactions and speculate on how
they are regulated during biological
processes such as development, wound
healing, and angiogenesis, which require
modeling or remodeling of the basement
membrane. In 1996 Timpl, Huber and colleagues described the first crystal structure
of a laminin module, comprising three
tandem repeats of an EGF-like domain
(LE domain)10. By mutagenesis, they
showed that the binding site for nidogen
involves a short loop containing the conserved sequence Asp-Pro-Asn-Ala-Val in a
single LE domain11. This high affinity
interaction (Kd = 0.5 nM) is critically
dependent on three residues in a conserved
motif — Asp, Asn and Val. These data suggest a highly specific interaction in which a
ridge on laminin fits perfectly into a
groove on the complementary region of
nidogen.
In 1999 Hohenester, Timpl and colleagues described a second laminin module, the LG (for laminin globular)
domain12. This module connects the basement membrane to the dystroglycan–dystrophin complex. A metal ion site found
on the surface, coordinated by conserved
acidic groups on the domain surface and a
sulfate ion, was suggested to resemble the
574
interaction of a sulfated dystroglycan carbohydrate, in which the Ca2+ ion forms an
integral part of the interaction — that is, a
metal bridge of the kind seen in
integrin–ligand interactions13.
The latest structural study by
Hohenester and colleagues5 suggests a
more conventional kind of protein–protein interaction. The authors show that a
patch on the barrel surface of the nidogen
G2 domain is strikingly conserved in all
metazoan nidogens. They further show by
mutagenesis that this surface is very likely
the binding surface for one of the
immunoglobulin (Ig)-like domains of
perlecan. This is a high affinity interaction
and appears to involve a large surface on
the barrel, which presumably binds a concave surface on the Ig domain.
Is the binding of nidogen to perlecan
regulated? The G2 domain binds transition metals strongly, and metal binding
inhibits perlecan binding. Although no
metal ion was found in the crystals (zinc
was included in the crystallization buffer),
owing perhaps to the low pH of crystal
growth, the authors suggest that a loop
adjacent to the binding surface has the
potential for zinc binding. Metal ion binding could provide a credible mechanism of
inhibition by distorting the binding surface. Curiously, Zn2+ has been shown to
enhance the interactions between laminin
and both nidogen and collagen IV, perhaps via the formation of zinc finger-like
structures14.
Could metal ions play a regulatory role
in the assembly of basement membranes?
There are no data on changes in extracellular Zn2+ concentration. However, Ca2+
and Mg2+, which are normally present at a
high and constant level (∼mM), are
known to undergo rapid changes in cation
concentrations at wound sites, for example, where basement membrane remodel-
ing is required15. These circumstances
could potentially regulate the protein–
protein interactions discussed above, if
the metal dissociation constants are commensurate.
Perspective
Now that high resolution structures of
parts of the basement membrane components are available, the exciting challenges
for the future clearly lie in the study of
these parts in the context of the full length
proteins, as well as in the protein complexes within the basement membrane.
Integration of such information into
lower resolution images of intact basement membranes derived from electron
microscopy should allow detailed models
of these important ECM elements to be
built.
Robert C. Liddington is Director of the
Program on Cell Adhesion, The Burnham
Institute, 10901 North Torrey Pines Road,
La Jolla, California 92037, USA. email:
[email protected]
1. Yurchenco, P.D. & O’Rear, J.J. Curr. Opin. Cell Biol. 6,
674–81 (1994)
2. Hutter, H. et al. Science 287, 989–94 (2000)
3. Hynes, R.O. & Zhao, Q. J. Cell. Biol. 150, F89–96
(2000)
4. Frisch, S. & Ruoslahti, E. Curr. Opin. Cell Biol. 9, 701706 (1997).
5. Hopf, M., Göhring, W., Ries, A., Timpl, R. &
Hohenester, E. Nature Struct. Biol. 8, 634–640
(2001)
6. Kim, S. & Wadsworth, W.G. Science 288, 150–154
(2000)
7. Springer, T.A. J. Mol. Biol. 283, 837–862 (1998)
8. Ormö, M. et al. Science 273, 1392–1395 (1996).
9. Lohikangas, L., Gullberg, D. & Johansson, S. Exp.
Cell Res. 265, 135–144 (2001).
10. Stetefeld, J., Mayer, U., Timpl, R. & Huber, R. J. Mol.
Biol. 257, 644–657 (1996).
11. Pöschl, E. et al. EMBO J. 15, 5154–5159 (1996)
12. Hohenester, E., Tisi, D., Talts, J.F. & Timpl, R. Mol.
Cell 4, 783–792 (1999)
13. Emsley, J., Knight, C.G., Farndale, R.W., Barnes, M.J.
& Liddington, R.C. Cell 101, 47–56 (2000)
14. Ancsin, J.B. & Kisilevsky, R. J. Biol. Chem. 271,
6845–6851 (1996)
15. Brown, E.M., Vassilev, P.M. & Hebert, S.C. Cell 83,
679–682 (1995)
nature structural biology • volume 8 number 7 • july 2001