13 research outputs found
Interaction between Triple-‐Helical Collagens and Human Collagenases
Collagens
are
the
major
structural
proteins
in
animal
tissues.
Their
degradation
is
essential
in
embryogenesis
and
development,
while
unbalanced
collagen
breakdown
is
seen
in
diseases
such
as
arthritis,
atherosclerosis
and
cancer.
Fibril-forming
collagens
I,
II
and
III
consist
of
three
polypeptide
chains
forming
a
triple
helix,
which
is
resistant
to
cleavage
by
most
proteases.
Collagenases
of
the
matrix
metalloproteinase
family
(MMP-1,
MMP-8,
and
MMP-13)
degrade
fibrillar
collagens
by
locally
unwinding
the
helix,
followed
by
cleavage
into
¼
and
¾
fragments.
They
comprise
two
domains,
the
catalytic
(Cat)
domain
and
the
hemopexin
(Hpx)
domain,
which
are
connected
via
a
flexible
linker.
Both
domains
are
essential
for
collagenolysis,
but
the
exact
sites
of
collagenase-collagen
interactions
and
how
they
unwind
collagen
remain
elusive.
This
thesis
addresses
the
roles
of
individual
collagenase
domains,
and
the
sites
in
both
the
enzyme
and
the
substrate
that
are
involved
in
collagen
binding
and
unwinding,
focusing
on
the
fibril-forming
collagens
and
human
MMP-1
as
a
prototype.
MMP-1
bound
to
immobilised
collagen
I
with
markedly
higher
affinity
than
its
Hpx
domain
alone.
The
Cat
domain
alone
failed
to
bind
to
collagen,
but
in
the
full-length
enzyme
it
participated
in
collagen
binding.
Above
25°C
the
two-
domain
binding
involved
the
catalytic
site
cleft.
Triple-helical
peptide
(THP)
Toolkits
of
collagens
II
and
III
were
screened
for
MMP-1
binding,
and
the
collagenase
binding
motif
has
been
established.
It
contains
two
hydrophobic
residues
within
a
9
residue
distance.
Finally,
hydrogen/deuterium
exchange
mass
spectrometry
(H/DXMS)
experiments
indicated
two
potential
collagen
binding
sites:
285-316
and
349-365
in
the
Hpx
domain,
and
suggested
a
possibility
of
a
dynamic
interaction
of
the
collagenase
N-terminus
with
collagen.
These
results
imply
that
the
two
domains
of
collagenase
bind
to
collagen
in
a
cooperative
manner.
Based
on
the
THP
binding
and
H/DXMS
data
a
3D
model
of
collagenase-collagen
interaction
has
been
proposed.
It
assumes
that
collagenase
utilises
hydrophobic
interactions
to
unwind
the
collagen
helix
via
perturbation
of
the
hydrogen-bond
network
which
stabilises
the
helix
Structural insights into how augmin augments the mitotic spindle
Cell division critically requires amplification of microtubules (MTs) in the bipolar mitotic spindle. This relies on the filamentous augmin complex that enables MT branching. Studies by Gabel et al., Zupa et al. and Travis et al. describe consistent integrated atomic models of the extraordinarily flexible augmin complex. Their work prompts the question: what is this flexibility really needed for
Prion strains viewed through the lens of cryo-EM
Mammalian prions are lethal transmissible pathogens that cause fatal neurodegenerative diseases in humans and animals. They consist of fibrils of misfolded, host-encoded prion protein (PrP) which propagate through templated protein polymerisation. Prion strains produce distinct clinicopathological phenotypes in the same host and appear to be encoded by distinct misfolded PrP conformations and assembly states. Despite fundamental advances in our understanding of prion biology, key knowledge gaps remain. These include precise delineation of prion replication mechanisms, detailed explanation of the molecular basis of prion strains and inter-species transmission barriers, and the structural definition of neurotoxic PrP species. Central to addressing these questions is the determination of prion structure. While high-resolution definition of ex vivo prion fibrils once seemed unlikely, recent advances in cryo-electron microscopy (cryo-EM) and computational methods for 3D reconstruction of amyloids have now made this possible. Recently, near-atomic resolution structures of highly infectious, ex vivo prion fibrils from hamster 263K and mouse RML prion strains were reported. The fibrils have a comparable parallel in-register intermolecular β-sheet (PIRIBS) architecture that now provides a structural foundation for understanding prion strain diversity in mammals. Here, we review these new findings and discuss directions for future research
Structural studies of the MMP-3 interaction with triple-helical collagen introduce new roles for the enzyme in tissue remodelling
Abstract: Matrix metalloproteinase-3 (MMP-3) participates in normal extracellular matrix turnover during embryonic development, organ morphogenesis and wound healing, and in tissue-destruction associated with aneurysm, cancer, arthritis and heart failure. Despite its inability to cleave triple-helical collagens, MMP-3 can still bind to them, but the mechanism, location and role of binding are not known. We used the Collagen Toolkits, libraries of triple-helical peptides that embrace the entire helical domains of collagens II and III, to map MMP-3 interaction sites. The enzyme recognises five sites on collagen II and three sites on collagen III. They share a glycine-phenylalanine-hydroxyproline/alanine (GFO/A) motif that is recognised by the enzyme in a context-dependent manner. Neither MMP-3 zymogen (proMMP-3) nor the individual catalytic (Cat) and hemopexin (Hpx) domains of MMP-3 interact with the peptides, revealing cooperative binding of both domains to the triple helix. The Toolkit binding data combined with molecular modelling enabled us to deduce the putative collagen-binding mode of MMP-3, where all three collagen chains make contacts with the enzyme in the valley running across both Cat and Hpx domains. The observed binding pattern casts light on how MMP-3 could regulate collagen turnover and compete with various collagen-binding proteins regulating cell adhesion and proliferation
A microtubule RELION-based pipeline for cryo-EM image processing
Microtubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins
A structural basis for prion strain diversity
Recent cryogenic electron microscopy (cryo-EM) studies of infectious, ex vivo, prion fibrils from hamster 263K and mouse RML prion strains revealed a similar, parallel in-register intermolecular β-sheet (PIRIBS) amyloid architecture. Rungs of the fibrils are composed of individual prion protein (PrP) monomers that fold to create distinct N-terminal and C-terminal lobes. However, disparity in the hamster/mouse PrP sequence precludes understanding of how divergent prion strains emerge from an identical PrP substrate. In this study, we determined the near-atomic resolution cryo-EM structure of infectious, ex vivo mouse prion fibrils from the ME7 prion strain and compared this with the RML fibril structure. This structural comparison of two biologically distinct mouse-adapted prion strains suggests defined folding subdomains of PrP rungs and the way in which they are interrelated, providing a structural definition of intra-species prion strain-specific conformations
Prion propagation is dependent on key amino acids in Charge cluster 2 within the prion protein
To dissect the N-terminal residues within the cellular prion protein (PrPC) that are critical for efficient prion propagation, we generated a library of point, double, or triple alanine replacements within residues 23-111 of PrP, stably expressed them in cells silenced for endogenous mouse PrPC and challenged the reconstituted cells with four common but biologically diverse mouse prion strains. Amino acids (aa) 105-111 of Charge Cluster 2 (CC2), which is disordered in PrPC, were found to be required for propagation of all four prion strains; other residues had no effect or exhibited strain-specific effects. Replacements in CC2, including aa105-111, dominantly inhibited prion propagation in the presence of endogenous wild type PrPC whilst other changes were not inhibitory. Single alanine replacements within aa105-111 identified leucine 108 and valine 111 or the cluster of lysine 105, threonine 106 and asparagine 107 as critical for prion propagation. These residues mediate specific ordering of unstructured CC2 into β-sheets in the infectious prion fibrils from Rocky Mountain Laboratory (RML) and ME7 mouse prion strains
Microtubule structure by cryo-EM: snapshots of dynamic instability
The development of cryo-electron microscopy (cryo-EM) allowed microtubules to be captured in their solution-like state, enabling decades of insight into their dynamic mechanisms and interactions with binding partners. Cryo-EM micrographs provide 2D visualization of microtubules, and these 2D images can also be used to reconstruct the 3D structure of the polymer and any associated binding partners. In this way, the binding sites for numerous components of the microtubule cytoskeleton - including motor domains from many kinesin motors, and the microtubule-binding domains of dynein motors and an expanding collection of microtubule associated proteins - have been determined. The effects of various microtubule-binding drugs have also been studied. High resolution cryo-EM structures have also been used to probe the molecular basis of microtubule dynamic instability, driven by the GTPase activity of β-tubulin. These studies have shown the conformational changes in lattice-confined tubulin dimers in response to steps in the tubulin GTPase cycle, most notably lattice compaction at the longitudinal inter-dimer interface. Although work is ongoing to define a complete structural model of dynamic instability, attention has focused on the role of gradual destabilization of lateral contacts between tubulin protofilaments, particularly at the microtubule seam. Furthermore, lower resolution cryo-electron tomography 3D structures are shedding light on the heterogeneity of microtubule ends and how their 3D organization contributes to dynamic instability. The snapshots of these polymers captured using cryo-EM will continue to provide critical insights into their dynamics, interactions with cellular components, and the way microtubules contribute to cellular functions in diverse physiological contexts
Mapping the Binding Sites of MMPs on Types II and III Collagens Using Triple-Helical Peptide Toolkits
Libraries of triple-helical collagen-like peptides (Collagen Toolkits) have helped to define collagens II and III binding specificities of numerous collagen-binding proteins. Here I describe a simple solid-phase binding assay utilizing a biotin–streptavidin system to screen the Collagen Toolkits for binding of two distinct matrix metalloproteinases (MMPs) implicated in cancer: the collagenolytic MMP1 (collagenase 1) and the non-collagenolytic MMP3 (stromelysin 1). The screening revealed markedly disparate binding footprints of these MMPs on collagens II and III, in line with their distinct biological activities. Analogous screening of other potentially collagen-binding proteases may shed light on their inherent tissue retention capabilities and their pro- or anti-metastatic potential
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Structural studies of the MMP-3 interaction with triple-helical collagen introduce new roles for the enzyme in tissue remodelling.
Matrix metalloproteinase-3 (MMP-3) participates in normal extracellular matrix turnover during embryonic development, organ morphogenesis and wound healing, and in tissue-destruction associated with aneurysm, cancer, arthritis and heart failure. Despite its inability to cleave triple-helical collagens, MMP-3 can still bind to them, but the mechanism, location and role of binding are not known. We used the Collagen Toolkits, libraries of triple-helical peptides that embrace the entire helical domains of collagens II and III, to map MMP-3 interaction sites. The enzyme recognises five sites on collagen II and three sites on collagen III. They share a glycine-phenylalanine-hydroxyproline/alanine (GFO/A) motif that is recognised by the enzyme in a context-dependent manner. Neither MMP-3 zymogen (proMMP-3) nor the individual catalytic (Cat) and hemopexin (Hpx) domains of MMP-3 interact with the peptides, revealing cooperative binding of both domains to the triple helix. The Toolkit binding data combined with molecular modelling enabled us to deduce the putative collagen-binding mode of MMP-3, where all three collagen chains make contacts with the enzyme in the valley running across both Cat and Hpx domains. The observed binding pattern casts light on how MMP-3 could regulate collagen turnover and compete with various collagen-binding proteins regulating cell adhesion and proliferation