Download Molecular dynamics of serpins

What are the serpins?
• It is a family of proteins characterised by a common molecular
architecture
• Most of the serpins are serine protease inhibitors, but some of
them have other functions
• Today, more than 500 serpins have been identified in animals,
plants, bacteria and viruses
• Serpin structure
• Inhibitory mechanism of serpins
• Serpin polymerisation
PAI-1 (plasminogen activator inhibitor type 1),
the only serpin which spontaneously converts to the
latent form
• inhibits the plasminogen activators, uPA and
tPA
• regulates fibrinolysis (dissolving of blood
clots) and cell migration
• the specificity and stability of PAI-1 is
regulated by cofactors such as heparin and
vitronectin
• lacks cysteine residues
(from Sharp et al. 1999)
Why PAI-1 spontaneously converts to latent form?
Distance measurement using donor-donor
energy migration (DDEM)
Time
Localisation of the RCL in PAI-1 by
intramolecular distance measurements
Distances
measured
P1’ - 313
P3 - 313
Distances in stable PAI-1
mutant (X-ray )
(Å)
69
68
Distances determined
by the DDEM method
(Å)
55  2
55  2
Preinsertion of the RCL studied by the ability to
form intramolecular disulfide bonds
Complex
Intact
Cleaved
Oxidized
Conclusion: formation of disulfide bonds between the cysteines in RCL
and cysteines in the A--sheet suggests that the RCL in active PAI-1 can
be preinserted.
Conclusion
In contrast to other serpins, active
PAI-1 has RCL located close to the
core and preinserted. This may be a
reason why PAI-1 spontaneously
converts to latent form.
P. Hägglöf et al., J. Mol. Biol. 2003.
Inhibitory mechanism of serpins
What was known:
•serpins form very stable/irreversible complexes with their target proteases
• when the complexes were analysed by SDS-PAGE or amino acid sequencing,
the serpins were cleaved
Major questions:
• Are serpins cleaved in the native
complexes or the cleavage is an artifact of
the analyses?
• How look the serpin/protease complex?
Quantification of free N-terminals in native
serpin/protease complexes
PCF
Result: in native serpin-protease
complexes the N-terminus of
PCF is blocked to the same extent as
the other N-termini
Conclusion: in the native serpin/protease complex the reactive centre of
serpin is cleaved and the protease covalently bound to the serpin
M. Wilczynska, et al., J. Biol. Chem. 1995.
What is the conformation of
serpin/protease complex?
Hypothetical conformations of stable
serpin/protease complex
Distance measurement in the
PAI-1/uPA complex
Conclusion: the distance data exclude the “docking
conformation” of the PAI-1/uPA complex but does
not distinguish between full and partial-insertion
models
X
M. Wilczynska, et al., Nat. Struct. Biol. 1997.
Structural analysis of PAI-1/uPA complex by
distance measurement and triangulation
Model of the complex
P3-266
Distances (Å)
P3-185 P3-P1’
P3-313
43,6
34,2
60,3
39,2
49,8
52,1
60,3
8,6
52
52
60
Partial-insertion model
Full-insertion model
Distances measured by DDEM
<30
Conclusion: the distances measured are compatible with full-insertion model
M. Fa, et al., Struct. Fold. and Des. 2000.
Serpin inhibitory mechanism is
driven by serpin metastability
Serpin inhibition involves reactive
center cleavage and full loop
insertion, so the covalently linked
protease is translocated from the
initial docking site to distal end of
serpin.
Loop-sheet polymerisation of serpins
 Wild-type serpins polymerise only under mild
denaturing conditions.
 Some of natural serpin mutants spontaneously
polymerise in vivo. This results in diseases
like cirrhosis and emphysemia (polymerisation
of 1-antitrypsin), angioedema
(polymerisation of C1-inhibitor), and
dementia (polymerisation of neuroserpin).
 The polymerisation is accompanied by loss of
inhibitory activity.
Plasminogen activator inhibitor type 2, PAI-2, the
only serpin which polymerises as wild-type protein
• PAI-2 exists as:
* extracellular glycosylated form
* intracellular non-glycosylated form
• PAI-2 has the largest CD-loop in the serpin family
What are the molecular determinants
of PAI-2 polymerisation?
Comparison between PAI-2 and 1-AT
Breach
region
Conclusion: the breach region does
not determine the polymerisation
ability of PAI-2
M. Wilczynska et al., Febs Lett. 2003
Polymerisation of native and DTT-reduced PAI-2
Non-denaturing PAGE
Native
PAI-2
Reduced
PAI-2
Conclusion: reduction of PAI-2 makes the protein resistant to polymerisation.
Identification of a cysteine which is
important for polymerisation ability of PAI-2
Non-denaturing PAGE
Conclusion: Substitution of C79 or C161 to serine makes PAI-2
resistant to polymerisation.
Analysis of trypsin-degraded wt PAI-2
by Maldi-tof mass spectrometry
Cysteines 79 and 161 form disulfide bond
Polymerisation of PAI-2 mutant with two cysteines only
(C79 and C161) under different redox conditions
2
Oxidation
Polymerogenic form
?
Conclusions:
The polymerogenic form PAI-2
is stabilised by the C79/C161
disulfide bond.
The polymerogenic and stable
monomeric forms of PAI-2 are
interconvertible.
Stable monomerogenic form
Triangulation of the C79 in stable monomeric PAI-2
by intramolecular distance measurements using DDEM
Conclusion: Stable monomeric form of PAI-2 has the CD-loop
folded on a side of the molecule
Is the translocation of CD-loop in PAI-2 linked to
conformational changes in the A-β-sheet of the
inhibitor?
+
Non-annealed
+
uPA
RCL
peptide PAI-2
Annealed
*SDS/PAGE
Complex *Western blot
*Quantification of
cleaved PAI-2 by
phosphorimager
Annealing
[%]
Annealing of synthetic RCL-peptide into wt PAI-2 and
its mutants to compare the opening of the A--sheet
Wt PAI-2
C5S PAI-2
C145S PAI-2
C79S PAI-2
C161S PAI-2
Nonsecific peptide
30
20
10
Cleaved =
annealed
0
0 25 50 75 100
Peptide excess
Conclusion: the A--sheet of PAI-2 is more open in the polymerogenic form than
in the stable monomeric form of the inhibitor.
Conclusion
Reduction
Oxidation
50 Å
Polymerogenic
form of PAI-2
Stable monomeric
form of PAI-2
Conversion of PAI-2 from the polymerogenic form to the stable
monomeric form is accompanied by closing of the -sheet A and
by translocation of the CD-loop from the bottom to the side of the
molecule.
Do the polymerogenic and stable monomeric
forms of PAI-2 exist in nature?
Polymerisation of PAI-2 from the cytosol (wt PAI-2)
and from the secretory pathway (SP-PAI-2) of CHO cells
Conclusions:
• in the cytosol, PAI-2 exists mainly in the stable monomeric form
• in secretory pathway, PAI-2 is in the polymerogenic form.
Interaction of PAI-2 with vitronectin
Conclusion: PAI-2 can form disulfide-bond to vitronectin via the C79 in CD-loop
Conclusions
• PAI-2 is a unique serpin with two mobile loops: the RCL and the CD-loop
• The CD-loop of PAI-2 is a redox-sensitive molecular switch that regulates
conversion between the polymerogenic and the stable monomeric forms of
PAI-2.
Reduction
polymerisation
Oxidation
Polymerogenic
form of PAI-2
50 Å
Stable monomeric
form of PAI-2
• Polymerisation of PAI-2 in vivo may be regulated by redox status of the cell.
• Disulfide-binding of vitronectin to the C79 in the CD-loop of PAI-2 may
stabilise the active PAI-2 in extracellular compartments.
M. Wilczynska et al., EMBO J. 2003;
S. Lobov et al., J. Mol. Biol. 2004.