Download Functional implications of the modeled structure of maspin

Protein Engineering vol.9 no.7 pp 585-589. 19%
Functional implications of the modeled structure of maspin
Paul A.Fitzpatrick, Daniel T.Wong, Philip J.Barr and
Philip A.Pemberton1
LXR Biotechnology, 1401 Manna Way South. Richmond, CA 94804, USA
'To whom correspondence should be addressed
:
The tumor suppressor maspin (mammary-specific serpin)
is an unstable serpin that does not undergo the stressed to
relaxed transition typical of proteinase inhibitory serpins
and, consequently, is not likely to function as a serine
proteinase inhibitor. This suggests that the positioning and
configuration of the reactive site loop (RSL) of maspin are
likely to resemble those of ovalbumin, the best studied noninhibitory serpin. Accordingly, the tertiary structure of
maspin has been modeled on the crystal structure of native
ovalbumin. Biochemical data and the modeled theoretical
structure of maspin reveal the absence of disulfide bonds
in the molecule and the presence of an unstable RSL that
adopts a distorted helical structure. We confirm that the
RSL is extremely sensitive to limited proteolysis and suggest
that this may provide a structural basis for the proteolytic
inactivation of maspin, a process that is likely to modulate
the activity of maspin in biological systems.
Keywords: homology model/maspin/non-inhibitory/RSL structure/serpin
Introduction
Maspin is a tumor suppressor protein present in normal
mammary epithelial tissue and primary tumors but undetectable
or weakly expressed in metastatic secondary tumors and pleural
effusions. Exogenous recombinant maspin affects directly the
growth and metastatic potential of tumor cells in a nude mouse
model. The molecular basis of maspin function is currently
unknown. However, maspin has been shown to be capable
of blocking tumor cell proliferation, invasion, motility and
metastasis (Sheng et al, 1994; Zou et al, 1994).
Maspin is homologous to the serpin superfamily of proteinase inhibitors but does not possess a classical secretion
signal sequence. Maspin does, however, possess a hydrophobic
amino terminus similar to that present in the secreted, glycosylated serpins ovalbumin and plasminogen activator inhibitor-2
(Paynton et al, 1983; von Heijne et al, 1991). The primary
structure of maspin is most closely related to the equine and
human neutrophil-monocyte elastase inhibitors (43% and 39%
amino acid sequence identity, respectively), the human squamous cell carcinoma antigens 1 and 2 (34% amino acid sequence
identity), plasminogen activator inhibitor-2 (31% amino acid
sequence identity) and chicken ovalbumin (31% amino acid
sequence identity) (Zou et al, 1994). Most serpins function
as proteinase inhibitors by presenting a proteolytically sensitive
sequence on an exposed RSL (for reviews see Carrell et al,
1987; Potempa et al, 1994). Target proteinases attempt to
cleave the PI - P I ' sequence on the RSL but become trapped in
a stable bimolecular complex. Non-target proteinases, however,
© Oxford University Press
can catalytically cleave the RSL resulting in the inactivation
of serpin inhibitory function and a transition from an unstable
stressed (S) form to a more stable relaxed (R) form. In the R
form, the RSL residues amino-terminal to the point of cleavage
become inserted into the A p-pleated sheet, conferring different
degrees of stability depending on the length of peptide inserted
(Schulze et al, 1990). It had been proposed that partial preinsertion of the RSL into the A P-pleated sheet was a
requirement for inhibitory function and the crystal structure
of dimeric antithrombin in provided data to support this
hypothesis (Carrell and Evans, 1992; Lomas et al, 1992;
Carrell et al, 1994). Recently, however, the crystal structure
of a genetically engineered inhibitory a|-antichymotrypsin
has shown that the RSL in this molecule is not inserted,
demonstrating that it is not an absolute prerequisite for inhibitory function in native serpins. Rather, the ability to insert is
likely required for the formation of the serpin-protease complex
(Wei et al, 1994). Clearly, the extent of RSL pre-insertion
will vary from serpin to serpin.
Several serpins, including angiotensinogen, ovalbumin and
pigment epithelium-derived factor, have no apparent inhibitory
function, and the RSL sequences of these proteins are catalytically cleaved with no concomitant increase in stability
(Stein et al, 1989; Becerra et al, 1995). The crystal structures
of both native and cleaved ovalbumin have been solved and
reveal no insertion of the RSL into the A P-pleated sheet
(Wright et al, 1990; Stein et al, 1991).
Recent studies have indicated that maspin also is unlikely
to function as a proteinase inhibitor (Hopkins and Whisstock,
1994; Pemberton et al, 1995). For example, the 'hinge' region
of maspin is highly divergent from those of reported inhibitory
serpins. This region comprises an RSL peptide stretch 9-15
residues amino-terminal to the reactive site peptide bond that
helps present the reactive site in an optimal configuration for
docking, binding and subsequent interaction with a proteinase.
Furthermore, maspin is incapable of undergoing the stressed
(S) to relaxed (R) transition, and both native maspin and RSLcleaved maspin can undergo polymerization. The latter two
points indicate that, after cleavage, the RSL residues do not
insert into the A p-pleated sheet of maspin. Maspin function
is, however, critically dependent upon the integrity of the RSL,
as proteolysis, or antibody neutralization, of the RSL abolishes
the ability of maspin to block tumor cell invasion through
basement membranes (Sheng et al, 1994). These data suggest
that the positioning and configuration of the RSL in maspin
are likely to be similar to those of the non-inhibitory serpin
ovalbumin. Therefore, the tertiary structure of maspin has been
modeled on the structure of native ovalbumin. The resulting
theoretical model provides an explanation for the observed
sensitivity of the maspin RSL to limited proteolysis and a
molecular basis for the proteolytic inactivation of maspin.
Materials and methods
Maspin production
Biologically active recombinant maspin was expressed in and
purified from Saccharomyces cerevisiae strain BJ2168 as
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P.A.FItzpatrick et al
- Helix A
Table I. Refinement of homology model of maspin
Refinement
method
Number Backbone
of steps potential
Steepest descents
100
Conjugate gradients 5974
Conjugate gradients 8000
Conjugate gradients 1187
Steepest descents
2000
Conjugate gradients 1865
Conjugate gradients 2926
Fixed
Fixed
Tethered
(500-100 kcal/A2)
Tethered
(50 kcal/A2)
Tethered
(25 kcal/A2)
Unrestrained
Unrestrained
Morse Cross Solvation
terms
N
N
N
N
N
N
N
N
N
Y
Y
N
N
N
Y
N
Y
N
Y
Y
Y
-Hetix B-
Homology modeling of maspin
Modeling of maspin was based on its homology to native
ovalbumin, the x-ray crystallographic structure of which is
known (Stein et al, 1991). The sequences were aligned as
described by Zou et al. (1994). The program Homology
(Biosym) was used to determine approximate coordinates for
the maspin structure and the program Discover (Biosym) used
to refine the coordinates. The unrefined native maspin model
was constructed by using residue for residue replacement of
the aligned maspin sequence upon the native ovalbumin
sequence. Gaps in the maspin sequence were simply spliced
together while insertions were accommodated for using the
Biosym 'loop search' feature within the Homology module.
Steric interferences between atoms (overlap parameter >0.8)
were manually corrected prior to refinement.
For the refinement of the native maspin structure a combination of steepest descent and conjugate gradient refinement
steps was used with the cvff force field (Biosym). The backbone
of the structure was first held fixed and then gradually relaxed
by tethering it with a decreasing force constant until the final
steps of refinement, when the backbone was not restrained.
Initially, the refinement was calculated in vacuo with a dielectric
constant of 1, but after partial refinement, an 8 A layer of
water molecules was added. Note that no cut-off distances
were applied. Table I provides details of the refinement process.
The model was considered to have converged when the
maximum derivative reached less than 10~3 kcal/mol.A (the
final maximum derivative was 0.00089 kcal/mol.A). All
modeling and refinements were performed on a Silicon Graphics Indigo2 XZ with an R4400 MIPS CPU. Crystallographic
coordinates for native ovalbumin were obtained from the
E[LJK VHH AN Egjjl F Y C | P ]
Hefix C
MASPIN
OVALB.
MASPIN
OVALB...
D
IPFGFQTVTSDVNp
GFGDSIEAQCGTSVNVHSSLRDILNQI
Str. A2
Helx E
MASPIN
OVALB...
LSS
PND
P K S L N L S TfplF I S S T(K]R PFY|A K
R Y P I L P{E)Y L Q C V | K | E L|YJRG
-Str. A1
described previously (Pemberton et al., 1995). This material
is >95% pure as judged by SDS-PAGE and migrates as a
single peak on reversed-phase high-performance liquid chromatography.
Free thiol analysis
The quantitation of free thiols in recombinant maspin was
performed with Ellman's reagent according to Riddles et al.
(1983). The total protein content of the sample was calculated
by standard Bradford assay using a bovine serum albumin
standard (Bio-Rad) . The accuracy of this measurement was
confirmed by mass balance analysis. Maspin in TE buffer was
lyophilized, the powder weighed and the salt content of the
powder calculated. The water content of the freeze-dried
powder was determined with a Karl Fischer apparatus (Fischer
Coulomatic Titrimeter, Model 447). The protein present in the
freeze-dried sample was then calculated.
586
3[Dc E K E P L GRlV L FS|Pl
MASPIN
OVALB
Hefix F-
MASPIN
OVALB...
MASPIN
OVALB...
MASPIN
OVALB
MASPIN
OVALB...
211
MASPIN
OVALB
243
MASPIN
OVALB...
275
2B6
MASPIN
OVALB...
307
316
MASPIN
OVALB...
340
225
253
Str. B5 — ^
MASPIN
OVALB..
I IF F Q]K F C|S~P|
UFFQIRC vb PI
Fig. 1. Sequence alignment between maspin and ovalbumin. Identical
residues are boxed and conserved residues are shaded. Secondary structure
is indicated with arrows and labels.
Brookhaven Data Base crystal coordinate file (Bernstein
etal., 1977).
Limited proteolysis of maspin
Purified recombinant maspin was incubated with trypsin at a
molar ratio of 1:7000 (proteinase:maspin) in 10 mM Tris-1
mM EDTA-150 mM NaCl (pH 8.0) at 37°C. Samples were
taken during the incubation, PMSF added to inhibit trypsin
(final concentration 0.1 mM) and maspin proteolysis analysed
by 12.5% SDS-PAGE.
Results and discussion
Modeling results
Modeling of the native structure of maspin was accomplished
by using the crystallographic coordinates of native ovalbumin
as a structural template. Ovalbumin was chosen as the template
because of its high level of amino acid sequence identity to
maspin (Figure 1) and because of the structural similarities
between the two proteins. Unlike most serpins, neither maspin
nor ovalbumin has known protease inhibitory activity and
neither undergoes the stressed to relaxed transition upon RSL
cleavage that is characteristic of many serpins (Stein et al,
Modeling maspin on the crystallographic structure of native ovalbumln
Fig. 2. Completely refined structure of maspin. Carbons are displayed as green, nitrogens as blue, oxygens as red, sulfur as yellow and the reactive site loop
as solid purple
Table II. Location of cysteine residues in refined maspin structure
Cysteine residue number
Cysteine location
20
34
183
205
214
287
323
373
C-terminus of O-helix A
N-terminus of a-helix B
Middle of (5-pleated sheet strand C4
Between fi-pleated sheet strands C3 and B1
Start of p-pleated sheet strand B2
Middle of a-helix I
Middle of fi-pleated sheet strand A5
C-terminus of fi-pleated sheet strand B5
1989, Pemberton et al, 1995). It should also be noted that
maspin has a deletion of several residues within the RSL,
making it one of the shortest loops present in a serpin.
The completely refined theoretical model of native maspin
is shown in Figure 2. Generally, this structure of maspin shows
the same gross structure typical of serpins. Biochemical
titrimetric analyses demonstrate that recombinant maspin does
not possess any intramolecular disulfide bonds despite the
presence of eight cysteine residues at positions 20, 34, 183,
205, 214, 287, 323 and 373. From the modeled structure it
can be seen that these cysteines are either in different (ipleated sheet strands or in different a-helices and are either
distant from each other or improperly oriented to form disulfide
bonds (Table II). Recombinant maspin was produced in a
reducing environment intracellularly in yeast. Nonetheless,
purified maspin is biologically active in in vitro and in vivo
model systems, indicating that maspin is folded correctly
(Sheng et al, 1994; P.A.Pemberton et al, unpublished data).
Thus, disulfide bonds do not appear to be involved in stabilizing
the tertiary structure of the molecule. Indeed, the lack of
disulfide bonds may partly explain why maspin is less stable
than ovalbumin, which possesses one intramolecular disulfide
bond. Maspin denatures readily at temperatures above 40°C
while ovalbumin is stable at higher temperatures (Stein et al,
1989; Pemberton et al, 1995). Nonetheless, the presence and
surface topology of these cysteines in maspin may signify
some role in the biological function of maspin. A precedent
for this is the archetypal inhibitory serpin human <X|-antitrypsin.
This protein has been shown to form bimolecular disulfide
bonded complexes with IgA via Cys232 (Dawes et al, 1987).
A comparison of the structures of native ovalbumin and
maspin is shown in Figure 3. There is a striking similarity in
structures with the significant exception of the reactive site
loops. In Figure 4 is displayed the overlap between the known
structure of ovalbumin and the theoretical structure of maspin.
The root mean square (r.m.s.) shift between the paired (includes
all residues not part of a gap or deletion) backbone atoms
(excluding hydrogens) of the ovalbumin and maspin structures
is 1.74 A, indicating the close preservation of basic structure.
In contrast, the active site region is not highly conserved.
Indeed, the greatest r.m.s. shift between paired a-carbons is
in the region of the RSL (maspin residues 330-345). The
deletion within the active site region of maspin leads to the
degradation of the helical structure observed for ovalbumin
such that the RSL of maspin more closely resembles a loop
rather than a helix. Careful examination of this region reveals
the presence of slight helical character, but the helix clearly
has been stretched to accommodate the deletion of residues.
The distortions within the RSL would indicate that it is
relatively unstable and extremely sensitive to limited proteolysis. We have shown previously that trypsin:maspin ratios of
greater than 1:1000 can inactivate maspin's biological tumorsuppressing functions by cleavage within the RSL (Sheng
et al, 1994; Pemberton et al, 1995). Here we extend these
observations to show that maspin is extremely sensitive to
limited proteolysis by trypsin at a molar ratio of 1:7000
(proteinase:maspin) at physiological temperatures (Figure 5).
Indeed, after 40 min of incubation >50% of the maspin has
been cleaved within the RSL. These results are in keeping
with the modeled theoretical structure of maspin presented
here, and suggest that limited proteolysis might be one physiological way of regulating maspin activity. In addition, the
trypsin-cleaved form and native form of maspin have different
crystallization characteristics (unpublished results), indicating
587
P.A.Fitzpatrick et aL
Fig. 3. Display of a-carbon traces of ovalbumin and maspin side by side (3-Pleated sheets A are colored blue and the reactive site loops are yellow.
Fig. 4. Stereoscopic display of ovalbumin a-carbon trace (light lines) superimposed on that of maspin (dark lines).
Mr
(kDa)
1.
2.
3. 4.
5.
6.
7.
8.
Fig. 5. Limited proteolysis of maspin. Lanes 1-8 represent a time course of
maspin proteolysis by trypsin. The molar ratio is 1:7000 trypsin:maspin.
Lanes: I, native maspin; 2, 5 min; 3, 20 min; 4, 40 min; 5, 60 min; 6,
120 min; 7, 180 min; 8, 240 min.
structural and/or energetic differences between the two forms.
These differences must account for the observed loss of activity
upon proteolysis and, although the conformation of the loop
in trypsin-cleaved maspin is not known, it probably differs
significantly from that present in native maspin. This may be
sufficient to destroy any inherent receptor-ligand binding
function.
588
Proteolysis of the RSL is common amongst members of the
serpin superfamily and may occur through the action of
endogenous inflammatory proteases or exogenous infectious
bacterial proteases (Carrell et aL, 1987; Pemberton et aL,
1988; Desrochers et aL, 1992). We have suggested previously
that proteolytic inactivation of maspin might occur physiologically at regions of active cell proliferation and may be induced
by a trypsin-like protease such as hepsin (Pemberton et aL,
1995). Other possible candidates for maspin inactivation might
include such proteinases as stromelysin or cathepsin D (Basset
et aL, 1990; Tandon et aL, 1990). These and other proteinases
are overexpressed in tumors and may well enhance the growth,
invasiveness and metastatic capability of tumor cells that
express maspin by switching off its tumor-suppressing activity.
Maspin does not undergo the S to R transition typical of
inhibitory serpins and both native and cleaved forms of the
molecule can polymerize in response to changes in pH and
ionic strength (Pemberton et aL, 1995). The inability to undergo
the S to R transition indicates that maspin is probably not a
proteinase inhibitor. The observation that RSL-cleaved maspin
can polymerize indicates that after cleavage the RSL probably
does not insert into fi-pleated sheet A. Structurally, it is possible
Modeling maspin on the crystallographic structure of native ovalbumin
that the proline residue at position P8 might function to block
insertion but that present in the RSL of a mutant form of a r
antichymotrypsin apparently does not (Wei et al., 1994). The
evidence for non-insertion also derives from the two published
mechanisms by which serpins polymerize. Both rely on the
RSL of one molecule being available for insertion into either
the (3-pleated A sheet or C sheet structure present in another
molecule (Lomas et al, 1992; Carrell et al., 1994). Clearly, if
the RSL of one maspin molecule had inserted into its own (3pleated sheet A following proteolysis then it would be unavailable for insertion into either fi-pleated sheet A or fj-pleated sheet
C of another maspin molecule, thus precluding polymerization.
Similarly, since the cleaved RSL of one maspin molecule does
not insert into its own (3-pleated sheet A it is unlikely that it
will insert into the p-pleated sheet A of another maspin
molecule. We therefore propose that RSL insertion into fipleated sheet C seems more likely. The present theoretical
model of maspin will allow us to examine potential interactions
between the RSL and the C-sheet of maspin and to examine
the structural constraints that preclude RSL insertion into f3pleated sheet A.
In conclusion, the function of maspin is critically dependent
on the RSL. Thus, the configuration that the loop adopts to
confer biological activity is of paramount importance in
understanding the molecular basis of maspin function. Because
maspin is structurally similar to ovalbumin it implies that RSL
proteolysis will result only in structural alterations within the
loop; hence the structure of the RSL must be sufficient to
account for the biological activity and tumor suppressor
functions of maspin. We have suggested previously that the
RSL may constitute a receptor binding domain or directly bind
and modulate elements responsible for cell growth, motility
and invasiveness. We have now identified proteins involved
in cell proliferation and motility, that bind either directly or
indirectly to maspin, and are examining the role of the RSL
in binding (unpublished data). In the absence of a solved
crystallographic structure, the present theoretical model of
maspin will help delineate the domains involved in binding
and allow a more complete understanding of the molecular
mechanism of maspin action. This is of particular importance,
given its recent finding in prostate, thymic and intestinal
epithelial tissues (Sager etal, 1996; P.A.Pemberton, N.Pavloff,
W.-C.A.Chen, D.T.Wong, M.Shih, X.-D.Ji, R.Sager and
P.J.Barr, submitted for publication). Thus, in addition to tumor
suppressor activity in metastatic breast cancer, maspin may
also have clinical significance in the control of tumors arising
from a number of other tissue types.
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