MINIREVIEW
org
Serpins Flex Their Muscle
II. STRUCTURAL INSIGHTS INTO TARGET
PEPTIDASE RECOGNITION,
POLYMERIZATION, AND TRANSPORT
FUNCTIONS *
Published, JBC Papers in Press, May 24, 2010, DOI 10.1074/jbc.R110.141408
James C. Whisstock‡1, Gary A. Silverman§2, Phillip I. Bird‡,
Stephen P. Bottomley‡, Dion Kaiserman‡, Cliff J. Luke§,
Stephen C. Pak§, Jean-Marc Reichhart¶, and James A. Huntington储
From the ‡Department of Biochemistry and Molecular Biology and ARC
Centre of Excellence in Structural and Functional Microbial Genomics,
Monash University, Clayton, Victoria 3800, Australia, the §Departments of
Pediatrics and Cell Biology and Physiology, Children’s Hospital of
Pittsburgh and Magee-Womens Hospital, University of Pittsburgh School
of Medicine, Pittsburgh, Pennsylvania 15201, the ¶Université de
Strasbourg, CNRS UPR 9022, Institut de Biologie Moléculaire et Cellulaire,
67084 Strasbourg Cedex, France, and the 储Department of Haematology,
Cambridge Institute for Medical Research, University of Cambridge,
Cambridge CB2 0XY, United Kingdom
Inhibitory serpins are metastable proteins that undergo a sub-
stantial conformational rearrangement to covalently trap target
peptidases. The serpin reactive center loop contributes a major-
ity of the interactions that serpins make during the initial bind-
ing to target peptidases. However, structural studies on serpin-
peptidase complexes reveal a broader set of contacts on the
scaffold of inhibitory serpins that have substantial influence on
guiding peptidase recognition. Structural and biophysical stud-
ies also reveal how aberrant serpin folding can lead to the for-
mation of domain-swapped serpin multimers rather than the
monomeric metastable state. Serpin domain swapping may
therefore underlie the polymerization events characteristic of
the serpinopathies. Finally, recent structural studies reveal how
the serpin fold has been adapted for non-inhibitory functions
such as hormone binding.
Although amino acid sequence similarity varies from 17 to
95%, key conserved residues facilitate the folding of inhibitory
serpins into a metastable conformation typically comprising
three ␤-sheets, eight to nine ␣-helices, and a solvent-exposed
reactive center loop (RCL)3 (Fig. 1A) (1). The conformational
* This work was supported by National Institutes of Health Grants DK079806
and DK081422 (to G. A. S.) and HL68629 (to J. A. H.); National Health and
Medical Research Council Australia Program Grant 490900 (to P. I. B.,
S. P. B., and J. C. W.); the Medical Research Council (to J. A. H.); The Hartwell
Foundation (to G. A. S.); and the Institut Universitaire de France, CNRS,
Agence Nationale de la Recherche, and Fondation pour la Recherche Medi-
cale (to J.-M. R.). This minireview will be reprinted in the 2010 Minireview
Compendium, which will be available in January, 2011.
1
Australian Research Council Federation Fellow and Honorary National
Health and Medical Research Council Australia Principal Research Fellow.
To whom correspondence may be addressed. E-mail: james.whisstock@
monash.edu.au.
2
To whom correspondence may be addressed. E-mail: gsilverman@upmc.edu.
3
The abbreviations used are: RCL, reactive center loop; ␣1AT, ␣1-antitrypsin; fXa,
factor Xa; s, strand; PCI, protein C inhibitor; HCII, heparin cofactor II; PAI-1, plas-
minogen activator inhibitor 1; PEDF, pigment epithelium-derived factor; CBG,
cortisol-binding globulin; TBG, thyroxine-binding globulin.
AUGUST 6, 2010 • VOLUME 285 • NUMBER 32
This paper is available online at www.jbc.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 32, pp. 24307–24312, August 6, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
“stressed-to-relaxed” transition that classical inhibitory serpins
undergo upon interaction with target peptidases (Figs. 1A and
2A) has been commented on in previous reviews and will not be
discussed extensively here (2–5). Suffice it to say that structural
snapshots are available for native forms with the RCL fully
expelled or partially (e.g. P14)4 inserted into ␤-sheet A and for
inactive forms with the RCL partially (e.g. P12) or fully (e.g.
RCL-cleaved serpins, the final peptidase complex, and the
intact but latent conformer) inserted into ␤-sheet A (6).
Together, these data provide a comprehensive picture of the
range of conformational states that the serpin scaffold adopts,
as well as the structural details of the conformational rearrange-
ment that occurs upon RCL cleavage by a target peptidase (Figs.
1A and 2A). Indeed, during peptidase inhibition, it is this latter
event that triggers RCL insertion, which stabilizes the acyl-en-
Downloaded from http://www.jbc.org/ at Univ of St Andrews on March 23, 2015
zyme complex by transposing the enzyme and deforming its
active site before deacylation occurs (4).
Serpins are broadly distributed throughout all major
branches of life; hence, their irreversible inhibitory mechanism
must provide a distinct advantage over standard mechanism
inhibitors when regulating proteolytic circuits. These concepts
are discussed in the accompanying minireview (7). The main
purpose of this minireview is to highlight new structural infor-
mation regarding serpin-peptidase complex formation, hor-
mone binding, and pathologic polymerization.
Serpin Exosite Interactions Enhance Target Peptidase
Recognition
In 2001, the crystal structure of the Michaelis complex
formed between the Manduca sexta P1 Lys serpin 1B with rat
S195A trypsin was published (8). As expected, the RCL is bound
in a substrate-like fashion by the peptidase, poised for attack of
the P1–P1⬘ bond. The core interactions involve residues from
P4 to P3⬘, with no contacts between the body of the serpin and
the peptidase (i.e. no exosite contacts). This structure is con-
sistent with the notion that the RCL is flexible and positioned
away from the body of the serpin as an isolated peptide loop.
Additional evidence suggesting that exosite contacts are not
extensively involved in serpin-peptidase recognition came from
changes in serpin specificity by mutations within the RCL
(principally P1) and an NMR study of the Michaelis complex
between ␣1-antitrypsin (␣1AT) Pittsburgh and trypsin showing
that the each molecule rotated as if in isolation. After 2001,
however, several new crystal structures of serpin-peptidase
Michaelis complexes were solved: two non-physiological
pairings with trypsin, four pairings with thrombin, and one
pairing each with factors Xa (fXa) and IXa. These structures
show that extensive exosite interfaces are a common feature
involved in the recognition of serpins by target peptidases
and involve residues outside P4 –P3⬘ in addition to the RCL
(Fig. 1, B–D) (3).
4
RCL numbering is in accordance with the convention of Schechter and
Berger (Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun.
27, 157–162).
JOURNAL OF BIOLOGICAL CHEMISTRY 24307
MINIREVIEW: Serpin Structural Insights
FIGURE 1. Native serpin structures form docking (Michaelis) complexes
with peptidases using exosites and other factors. A, structure of native/
stressed (left) and cleaved/relaxed (right) ␣1AT. ␤-Sheet A is in red, and the RCL
is in magenta. In the cleaved or covalently complexed form of the serpin (see
Fig. 2A), the RCL forms an extra strand in sheet A. Native, but not cleaved,
serpins are available to form docking complexes. B, superposition of the
structure of the antithrombin-thrombin-heparin ternary complex (with ser-
pin in cyan, the RCL in orange, and thrombin in yellow) with antithrombin-fXa-
pentasaccharide (with serpin in green, the RCL in magenta, and fXa in blue).
The structures are superposed on the serpin. C, structure of ␣2-antiplasmin
(green with magenta RCL). The C-terminal region functions as an exosite for
plasmin; the portion visible in electron density is in cyan, and the approximate
position of a docking peptidase is shown in ghost white. D, structure of protein
Z-dependent inhibitor (green with magenta RCL) in complex with protein Z
(cyan). The approximate position of a docking peptidase is in ghost white.
The RCL of most serpins extends from the P15 residue at the
top of strand (s) 5A to the P3⬘ residue at the beginning of s1C.
There is a high degree of flexibility on the non-prime side, con-
sistent with the need for rapid incorporation in ␤-sheet A, but
the region extending from P1 to P3⬘ is held close to the body of
the serpin. Thus, the peptidase must approach close to the body
of the serpin to engage the P1 residue in the S1 pocket. This
requirement effectively limits the range of possible exosite con-
tacts for typical serpins. To overcome this limitation and to
allow interactions with multiple peptidases, certain serpins, in
particular antithrombin (SERPINC1) and protein C inhibitor
(PCI/SERPINA5), have 3– 4-residue extensions on the P⬘ side
(9, 10). Antithrombin recognizes thrombin by engaging exten-
sive exosites accessible only by stretching the P⬘ side toward the
front of the serpin (10), whereas the fXa recognition site
requires stretching of the non-prime side (Fig. 1B) (11). Simi-
larly, PCI forms different complexes with thrombin and
activated protein C by the apparent twisting of the peptidase
relative to the serpin (9). By contrast, serpins with only one
physiological target such as heparin cofactor II (HCII/
SERPIND1) and ␣1AT have short P⬘ sides (P2⬘ and P3⬘ of ␣1AT
are conformationally restrictive prolines) (12). The hallmark of
24308 JOURNAL OF BIOLOGICAL CHEMISTRY
multispecific (not promiscuous) serpins thus appears to be the
extension of the P⬘ region.
By calculating total interaction surface areas, with and with-
out the RCL, we can assess the relative importance of RCL and
exosite contacts in determining serpin specificity (Table 1). The
surface area buried in non-physiological complexes (e.g. the two
involving trypsin) is typically ⬍1000 Å2, 90% of which involves
the RCL. By contrast, the physiologically relevant pairings all
bury ⬎1000 Å2 and rely to varying degrees on exosite contacts.
Indeed, there appears to be a tradeoff between the quality of the
RCL sequence and the dependence on exosite contacts. This
analysis is most interesting with respect to thrombin recogni-
tion by various serpins. For example, the disfavored P1 Leu of
HCII and the P2 Gly of antithrombin necessitate large exosite
contacts of over 1000 and 500 Å2, respectively, whereas the
favorable P2 Pro and P1 Arg sequence of PCI requires an exosite
contact of only 150 Å2 for efficient recognition by thrombin.
A second important finding from these structural data is the
Downloaded from http://www.jbc.org/ at Univ of St Andrews on March 23, 2015
demonstration that exosites on the serpin scaffold play a crucial
role in facilitating initial serpin-peptidase interactions (Table
1). Interestingly, different peptidases appear to rest in different
ways on the top of the serpin scaffold (even where the serpin
component is the same). In several complexes, the peptidase
lies far over the “front” of the serpin scaffold and predominantly
forms contacts with a conserved single turn helix that precedes
s4C as well as surrounding residues (Fig. 1B) (10). Conversely,
in other complexes, the peptidases dock “on the back foot” (e.g.
the PCI-thrombin-heparin complex) by forming interactions
with residues on ␤-sheet B/s1C, the N-terminal end of s2C, and
the C-terminal portion of s3C (9).
Finally, it is interesting to note that three human serpins use
protein sequences outside the serpin scaffold as key exosites.
HCII utilizes an N-terminal extension to bind to exosite I of
thrombin (12), and similarly, ␣2-antiplasmin contains an exten-
sive C-terminal extension that functions to bind the Kringle
domains of plasmin (13). The x-ray crystal structure of ␣2-anti-
plasmin reveals that the C terminus is positioned appropri-
ately near the RCL to bind to the peptidase (Fig. 1C). Finally, the
crystal structure of the protein Z-dependent inhibitor (SER-
PINA10) of fXa reveals that the serpin recruits protein Z via a
binding site centered on sheet C (14). Whereas the full ternary
complex (SERPINA10-protein Z-fXa) remains to be determined,
modeling studies suggest that the epidermal growth factor domain
of protein Z is placed to assist in recruiting fXa (Fig. 1D).
Taken together, these results show that the RCL sequence
itself provides insufficient information for determining actual
peptidase target(s), as some targets poorly recognize certain
RCL sequences in the absence of regulatory cofactors. Thus, the
identification of specificity-determining exosites is of thera-
peutic interest because these sites represent potentially impor-
tant new targets for development of molecules that block or
enhance serpin-peptidase interactions.
Domain-swapped Model of Serpin Polymerization
The use of serpins to modulate diverse molecular pathways
predicts that serpin mutations will yield a wide range of disease
phenotypes. Indeed loss-of-function mutations associated with
␣1AT, antithrombin, and C1 esterase inhibitor (SERPING1)
VOLUME 285 • NUMBER 32 • AUGUST 6, 2010
 MINIREVIEW: Serpin Structural Insights

Downloaded from http://www.jbc.org/ at Univ of St Andrews on March 23, 2015

FIGURE 2. Serpin mechanism, folding, and misfolding. A, active serpins fold into a metastable state. Following the initial interaction with a target peptidase
and RCL cleavage, the serpin undergoes a radical conformational change (RCL/s4A incorporation into ␤-sheet A) that culminates in peptidase inhibition via
distortion of the catalytic residues. B, loop-sheet model of serpin polymerization. The RCL of one molecule is inserted into open ␤-sheet A of another.
C, domain-swapped model of serpin polymerization. The model is based on the structure of a domain-swapped antithrombin dimer, where s5A and s4A (RCL)
of a donor molecule insert into ␤-sheet A of a recipient. D, normal serpins may fold through a polymerogenic intermediate that is stabilized by certain
mutations. The black dotted arrow indicates a gap in ␤-sheet A that accommodates s5A to form the s4A (RCL) exposed native form in A or the s5A and s4A
domain-swapped structure in C.

result in emphysema, thrombosis, and angioedema, respec-
tively. Historically, these loss-of-function mutations underpin
the clinical deficiencies that are classified as either type 1
(absent or decreased circulating levels below a critical threshold
of a functionally normal protein) or type 2 (normal circulating
levels of a dysfunctional protein). A spectrum of genetic muta-
tions (missense, nonsense, indels) leads to these classes of defi-
ciencies (15). However, several of the missense mutations also
induce toxic gain-of-function phenotypes by encoding full-
length molecules that are prone to misfolding and/or polymer
formation. For example, the Z mutation (E342K) of ␣1AT leads
to the accumulation of misfolded and polymerized protein
within the endoplasmic reticulum of hepatocytes (16). The
marked decrease in circulating levels of ␣1AT (the major anti-
peptidase in extracellular fluids) predisposes to emphysema, a
loss-of-function phenotype (17). In contrast, the accumulation
of misfolded or aggregated ␣1AT in hepatocytes probably leads
to overloading of protein quality control systems, resulting in
cellular injury and the development of cirrhosis, a toxic gain-
of-function phenotype. Collectively, toxic gain-of-function
phenotypes observed with destabilizing mutations have been
termed the serpinopathies (18). Because full-length serpins
associated with the serpinopathies retain some functional
inhibitory activity, a better understanding of the mechanism of
serpin polymer formation could lead to therapeutic strategies
designed to block serpin accumulation and to enhance secre-
tion and thereby treat both disease phenotypes.
The early observation that inhibitory serpins accept their
RCL as an additional strand in ␤-sheet A led to the suggestion
that RCL insertion in trans may represent the physiological
basis for serpin polymerization. Although alternative models
have been suggested, the “loop-sheet A” model of serpin poly-
merization has been generally accepted until recently (Fig. 2B).
Specifically, it was suggested that mutations might destabilize
␤-sheet A of the native serpin and enhance the ability of this
region to accept another serpin RCL in trans. It was therefore
suggested that serpin metastability and the requirement to
undergo the stressed-to-relaxed conformational change as part
of function thus represented a key weakness of the serpin scaf-
fold. However, despite the appealing rationale of this proposal,
it has remained challenging to reconcile the loop-sheet A
polymerization model with the biophysical data, suggesting
that polymerogenic serpin mutations result in the stabilization
of a serpin folding intermediate that is polymerogenic (termed
M*) rather than subsequent polymerization of the native folded
state. Indeed, comparisons between a fully folded native poly-

AUGUST 6, 2010 • VOLUME 285 • NUMBER 32 JOURNAL OF BIOLOGICAL CHEMISTRY 24309

MINIREVIEW: Serpin Structural Insights
TABLE 1
Structures of serpin-peptidase complexes
PDB, Protein Data Bank.
PDB code Complex
2GD4 (3.3 Å) and 1SR5 Antithrombin-S195A factor
(3.1 Å) Xa-pentasaccharide complex
1OPH (2.3 Å) ␣1-AT (Pittsburgh) in complex
with S195A trypsin
1JMO (2.2 Å) Crystal structure of heparin
cofactor II-S195A thrombin
complex
1K9O (2.75 Å) Insect serpin (Ala serpin) in
complex with trypsin
(non-physiological paring)
3B9F (1.6 Å) PCI-thrombin-heparin
complex
1TB6 (2.5 Å) (also 1SR5 at Antithrombin-thrombin-
lower resolution) heparin ternary complex
merogenic serpin variant and the wild-type counterpart reveal
only modest differences in thermal stability or inhibitory activ-
ity (19). Finally, it has proven difficult to build physiochemically
reasonable loop-sheet A polymer models that contain both an
RCL linkage and completed ␤-sheet A hydrogen bonding. (In
the illustrative model shown in Fig. 2B, the top of ␤-sheet A is
open.)
Recently, the x-ray crystal structure of a domain-swapped
antithrombin dimer suggested a new model for serpin poly-
merization (20). Strikingly, this structure reveals that both s5A
and the RCL are incorporated into ␤-sheet A of another serpin
molecule (Fig. 2C). Although the crystal structure was that of a
self-terminating antithrombin dimer, and thus not able to
propagate further, it was easy to open this model to form long
chain polymers. Limited proteolysis data, together with disul-
fide trapping experiments, support the formation of such poly-
mers in vitro in response to chemical denaturants and heat.
The domain-swapped serpin dimer has a number of implica-
tions for the mechanism of serpin polymerization. In particular,
this model suggests that polymerogenic serpin variants, which
generally cluster on and around s5A and s6A, interfere with the
final stages of ␤-sheet A assembly and permit the domain-
swapping event (Fig. 2, C and D). Thus, the polymerogenic M*
intermediate may resemble a serpin with a substantially incom-
plete or disordered ␤-sheet A (Fig. 2D). A key question is
whether similar domain-swapped serpin polymers of polymeric
variants form in the endoplasmic reticulum in vivo.
Serpins from Thermophilic Organisms Provide New
Insights into Function and Dysfunction
Serpins are sporadically distributed in Bacteria and Archaea
(21, 22), suggesting an ancient origin for the serpin fold.
24310 JOURNAL OF BIOLOGICAL CHEMISTRY
Buried surface area (Å2) at
RCL
Serpin exosite interface (% contributed by
contacts
exosite)
Interactions between thrombin “140 P4–P6⬘ 1257 (29.6% exosite) and 1031
loop” and helical turn 231–234 (12.9% exosite)
(preceding s4C), s4C, and s3C; also
minor interaction between thrombin
“30 loop” and C-terminal end of s1B.
Few exosite interactions: one contact P3–P2⬘ 788 (10.3% exosite)
between Asn97 (trypsin) and Asp280
(antitrypsin, on loop between helix H
and s2C).
Interactions between thrombin 140 loop P4–P3⬘ 1834 (59.3% exosite)
and helical turn 285–289 (preceding
s4C), s4C, and s3C; also minor
interaction between thrombin 30 loop
and C-terminal end of s3B and minor
interaction between thrombin “60
loop” and s2C; also major interactions
between thrombin and flexible N-
terminal “tail” of HCII (this region is
Downloaded from http://www.jbc.org/ at Univ of St Andrews on March 23, 2015
sandwiched between the serpin and
the peptidase).
Interaction between trypsin 140 loop P4–P4⬘ 999 (12.9% exosite)
and helical turn 189–193 (preceding
s4C).
Thrombin 60 loop and N-terminal end P4–P4⬘ 1183 (12.9% exosite)
of s3C.
Multiple interactions with thrombin and P4–P5⬘ 1500 (32.7% exosite)
helical turn 231–234 (preceding s4C);
also interactions with s1C, s2C, s3C,
and s4C.
Although the role of most prokaryote serpins is not understood,
some of these molecules (serpins from Clostridium thermocel-
lum) localize to the cellulosome, a multiprotein extracellular
complex that digests material such as cellulose (23). These ser-
pins may protect the cellulosome against unwanted peptidase
activity.
Considering the conformational lability of most serpins in
eukaryotes, it was surprising to find a large number of inhibi-
tory serpins encoded in extremophilic organisms. In addition to
C. thermocellum serpin, exemplars include thermopin (from
Thermobifida fusca; 55 °C), tengpin (from Thermoanaer-
obacter tengcongensis; 75 °C), and aeropin (from Pyrobaculum
aerophilum; 100 °C). Biophysical studies reveal that both the
native and cleaved states of these latter molecules have substan-
tially elevated stability and that these serpins still function as
metastable inhibitors in essentially the same way as their meso-
philic counterparts. Interestingly, however, one of these mole-
cules, thermopin, employs a novel strategy to fold at elevated
temperatures. Structural studies reveal that thermopin pos-
sesses an extreme C-terminal sequence that folds across the
front of the molecule and interacts with the top of sheet A (Fig.
3A) (21, 24). Biophysical studies show that the C-terminal
sequence plays no detectable role in influencing the stability of
the native fold but instead appears to be important for stabiliz-
ing a folding intermediate. This concept is intriguing in light of
the recent discovery of how native serpins may fold and domain
swap (20). As suggested above, the final stage of serpin folding is
most likely the assembly of s5A into ␤-sheet A (Fig. 2D). Thus,
the C-terminal sequence of thermopin could provide an extra
set of interactions that assist in efficient recruitment of s5A to
the folding serpin. An alternative explanation is that the C-ter-
VOLUME 285 • NUMBER 32 • AUGUST 6, 2010

FIGURE 3. Recent serpin structures. A, structure of native thermopin (24).
The serpin is in green, with the RCL in magenta (part of the RCL is not visible in
electron density; the dotted line shows connectivity). The C-terminal
sequence is in addition to the serpin domain and folds across the front of
␤-sheet A. B, structure of native tengpin (26). The serpin is in green, with the
RCL in magenta and helix E/s1A in dark blue. The N-terminal sequence (orange
coil) is crucial for maintaining the native metastable state. C, structure of PAI-1
in complex with the somatomedin B domain of vitronectin (orange). The ser-
pin is in green, with the RCL in magenta and helix E/s1A in dark blue. D, struc-
ture of native TBG in complex with thyroxine (28). The serpin is in green, the
RCL is in magenta, and thyroxine is in cyan spheres.
minal sequence contributes to folding by making the interme-
diate less likely to undergo a domain-swapping event. Bioinfor-
matic studies on aeropin also reveal the presence of a
C-terminal extension; however, the role of this region remains
to be investigated (25).
Another interesting insight from the study of bacterial ser-
pins is the discovery that tengpin contains an N-terminal
sequence that makes extensive contacts with the first ␤-strand
of sheet A (s1A) and helix E (Fig. 3B) (26). Deletion of this
region results in a protein that initially folds to the native state
but rapidly undergoes conformational change to adopt the
latent conformation. The N-terminal region thus functions to
stabilize the native state. Interesting parallels can be drawn
between this situation and that of mammalian plasminogen
activator inhibitor 1 (PAI-1/SERPINE1), which, in the absence
of the cofactor vitronectin, rapidly switches from the native to
the latent conformation. The x-ray crystal structure of PAI-1 in
complex with vitronectin reveals that the cofactor similarly
interacts with s1A and helix E (Fig. 3C) (27). Although the folds
of the N terminus of tengpin and vitronectin are different, these
data nonetheless point toward a common means of stabilizing
the metastable serpin fold.
Structural Studies on Non-inhibitory Serpins:
Hormone-binding Serpins, Pigment Epithelium-derived
Factor, and Maspin
Of the 36 known human serpins, six have evolved to perform
homeostatic non-inhibitory functions: maspin (SERPINB5),
pigment epithelium-derived factor (PEDF/SERPINF1), HSP47
(SERPINH1), cortisol-binding globulin (CBG/SERPINA6), thy-
roxine-binding globulin (TBG/SERPINA7), and angiotensino-
gen. Structural studies on the hormone-binding serpins CBG
and TBG have provided seminal insights into how these mole-
cules interact with and transport the hormones cortisol and
thyroxine, respectively. For both serpins, the native conforma-
tion has a higher affinity (CBG, ⬃10-fold; and TBG, ⬃3-fold)
for ligand than the cleaved conformation. Until recently, the
precise molecular mechanism of ligand binding and release was
obscure, although it was postulated that hormone release may
be mediated via RCL cleavage by promiscuous proteolytic
AUGUST 6, 2010 • VOLUME 285 • NUMBER 32
MINIREVIEW: Serpin Structural Insights
activity at the site of delivery. Recent structures of both serpins
reveal the unexpected finding that cortisol and thyroxine bind
into a pocket formed by helix H, s3B, s4B, s5B, and the loop
between s4B and s5B (Fig. 3D) (28, 29). The structures also
reveal how RCL cleavage results in substantial mobility in a loop
at the top of helix D, which forms extensive contacts with resi-
dues in ␤-sheet B that form the ligand-binding site. This finding
suggests that the enhanced flexibility of this region results in a
hormone-binding region less able to adopt the conformation
required for high affinity binding.
Unfortunately, determination of the structures of PEDF and
maspin has not provided insights into their mechanisms of
action, as their partners and molecular functions remain some-
what enigmatic. Compelling evidence from cell and animal
models implicate PEDF (an extracellular glycoprotein) in the
maintenance of tissue homeostasis via cell-surface receptors
and interaction with extracellular matrix components (30). For
example, it inhibits angiogenesis by suppressing the migration
Downloaded from http://www.jbc.org/ at Univ of St Andrews on March 23, 2015
and proliferation of endothelial cells and by promoting apopto-
sis. Although maspin is also implicated in tissue development
and homeostasis, as evidenced by early developmental failure of
null mice and its loss in carcinomas (31, 32), it is a nucleocyto-
plasmic protein (33), which may bind transcription factors (34).
The only structure of the human non-inhibitory serpins that
remains to be determined is HSP47, and this is perhaps the
most tantalizing, as it may shed light on how this serpin per-
forms its critical intracellular chaperone function in collagen
biosynthesis (35).
Conclusion
Taken together, these studies underscore the versatility of
the large complex metastable serpin scaffold in regulating pep-
tidase and non-proteolytic functions. In addition to the speci-
ficity afforded by the primary amino acid sequence of the RCL,
inhibitory serpins achieve fine-tuning of specificity via the use
of specific exosites present both inside and outside the serpin
domain. Accordingly, a single serpin is able to effectively mod-
ulate the function of more than one target peptidase and, fur-
thermore, ensure tight physiological specificity even when con-
fronted by a group of peptidases whose cleavage specificity is
similar. The cost of complexity is, however, that serpins are
unusually vulnerable to mutations that impact their folding
pathway. As a consequence, and in common with many other
proteins that cause conformational disease (36), serpins are
able to domain swap. These events most likely underlie the
physiological basis for serpin polymerization.
Acknowledgments—We are indebted to colleagues in the serpin com-
munity for knowledge and data and whose collective insights under-
pin the concepts presented here.
REFERENCES
1. Irving, J. A., Pike, R. N., Lesk, A. M., and Whisstock, J. C. (2000) Genome
Res. 10, 1845–1864
2. Gettins, P. G., and Olson, S. T. (2009) J. Biol. Chem. 284, 20441–20445
3. Huntington, J. A. (2006) Trends Biochem. Sci. 31, 427– 435
4. Huntington, J. A., Read, R. J., and Carrell, R. W. (2000) Nature 407,
923–926
JOURNAL OF BIOLOGICAL CHEMISTRY 24311
MINIREVIEW: Serpin Structural Insights
5. Whisstock, J. C., and Bottomley, S. P. (2006) Curr. Opin. Struct. Biol. 16,
761–768
6. Gooptu, B., and Lomas, D. A. (2009) Annu. Rev. Biochem. 78, 147–176
7. Silverman, G. A., Whisstock, J. C., Bottomley, S. P., Huntington, J. A.,
Kaiserman, D., Luke, C. J., Pak, S. C., Reichhart, J. M., and Bird, P. I. (2010)
J. Biol. Chem. 285, 24299 –24305
8. Ye, S., Cech, A. L., Belmares, R., Bergstrom, R. C., Tong, Y., Corey, D. R.,
Kanost, M. R., and Goldsmith, E. J. (2001) Nat. Struct. Biol. 8, 979 –983
9. Li, W., Adams, T. E., Nangalia, J., Esmon, C. T., and Huntington, J. A.
(2008) Proc. Natl. Acad. Sci. U.S.A. 105, 4661– 4666
10. Li, W., Johnson, D. J., Esmon, C. T., and Huntington, J. A. (2004) Nat
Struct. Mol. Biol. 11, 857– 862
11. Johnson, D. J., Li, W., Adams, T. E., and Huntington, J. A. (2006) EMBO J.
25, 2029 –2037
12. Baglin, T. P., Carrell, R. W., Church, F. C., Esmon, C. T., and Huntington,
J. A. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11079 –11084
13. Law, R. H., Sofian, T., Kan, W. T., Horvath, A. J., Hitchen, C. R., Langen-
dorf, C. G., Buckle, A. M., Whisstock, J. C., and Coughlin, P. B. (2008)
Blood 111, 2049 –2052
14. Wei, Z., Yan, Y., Carrell, R. W., and Zhou, A. (2009) Blood 114, 3662–3667
15. Stein, P. E., and Carrell, R. W. (1995) Nat. Struct. Biol. 2, 96 –113
16. Perlmutter, D. H. (2002) J. Clin. Invest. 110, 1579 –1583
17. Gooptu, B., Ekeowa, U. I., and Lomas, D. A. (2009) Eur. Respir. J. 34,
475– 488
18. Lomas, D. A., Evans, D. L., Finch, J. T., and Carrell, R. W. (1992) Nature
357, 605– 607
19. Yu, M. H., Lee, K. N., and Kim, J. (1995) Nat. Struct. Biol. 2, 363–367
20. Yamasaki, M., Li, W., Johnson, D. J., and Huntington, J. A. (2008) Nature
455, 1255–1258
21. Irving, J. A., Cabrita, L. D., Rossjohn, J., Pike, R. N., Bottomley, S. P., and
Whisstock, J. C. (2003) Structure 11, 387–397
22. Irving, J. A., Steenbakkers, P. J., Lesk, A. M., Op den Camp, H. J., Pike, R. N.,
24312 JOURNAL OF BIOLOGICAL CHEMISTRY
and Whisstock, J. C. (2002) Mol. Biol. Evol. 19, 1881–1890
23. Kang, S., Barak, Y., Lamed, R., Bayer, E. A., and Morrison, M. (2006) Mol.
Microbiol. 60, 1344 –1354
24. Fulton, K. F., Buckle, A. M., Cabrita, L. D., Irving, J. A., Butcher, R. E.,
Smith, I., Reeve, S., Lesk, A. M., Bottomley, S. P., Rossjohn, J., and Whiss-
tock, J. C. (2005) J. Biol. Chem. 280, 8435– 8442
25. Cabrita, L. D., Irving, J. A., Pearce, M. C., Whisstock, J. C., and Bottomley,
S. P. (2007) J. Biol. Chem. 282, 26802–26809
26. Zhang, Q., Buckle, A. M., Law, R. H., Pearce, M. C., Cabrita, L. D., Lloyd,
G. J., Irving, J. A., Smith, A. I., Ruzyla, K., Rossjohn, J., Bottomley, S. P., and
Whisstock, J. C. (2007) EMBO Rep. 8, 658 – 663
27. Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J.
(2003) Nat. Struct. Biol. 10, 541–544
28. Zhou, A., Wei, Z., Read, R. J., and Carrell, R. W. (2006) Proc. Natl. Acad.
Sci. U.S.A. 103, 13321–13326
29. Zhou, A., Wei, Z., Stanley, P. L., Read, R. J., Stein, P. E., and Carrell, R. W.
(2008) J. Mol. Biol. 380, 244 –251
30. Filleur, S., Nelius, T., de Riese, W., and Kennedy, R. C. (2009) J. Cell.
Biochem. 106, 769 –775
31. Bailey, C. M., Khalkhali-Ellis, Z., Seftor, E. A., and Hendrix, M. J. (2006)
Downloaded from http://www.jbc.org/ at Univ of St Andrews on March 23, 2015
J. Cell. Physiol. 209, 617– 624
32. Gao, F., Shi, H. Y., Daughty, C., Cella, N., and Zhang, M. (2004) Develop-
ment 131, 1479 –1489
33. Teoh, S. S., Whisstock, J. C., and Bird, P. I. (2010) J. Biol. Chem. 285,
10862–10869
34. Bailey, C. M., Khalkhali-Ellis, Z., Kondo, S., Margaryan, N. V., Seftor, R. E.,
Wheaton, W. W., Amir, S., Pins, M. R., Schutte, B. C., and Hendrix, M. J.
(2005) J. Biol. Chem. 280, 34210 –34217
35. Nagata, K. (2003) Semin. Cell Dev. Biol. 14, 275–282
36. Bennett, M. J., Sawaya, M. R., and Eisenberg, D. (2006) Structure 14,
811– 824
VOLUME 285 • NUMBER 32 • AUGUST 6, 2010
Minireviews:
Serpins Flex Their Muscle: II.
STRUCTURAL INSIGHTS INTO
TARGET PEPTIDASE RECOGNITION,
POLYMERIZATION, AND TRANSPORT
FUNCTIONS

James C. Whisstock, Gary A. Silverman,

Phillip I. Bird, Stephen P. Bottomley, Dion
Kaiserman, Cliff J. Luke, Stephen C. Pak,

Downloaded from http://www.jbc.org/ at Univ of St Andrews on March 23, 2015

Jean-Marc Reichhart and James A.
Huntington
J. Biol. Chem. 2010, 285:24307-24312.
doi: 10.1074/jbc.R110.141408 originally published online May 24, 2010

Access the most updated version of this article at doi: 10.1074/jbc.R110.141408

Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.

Alerts:
• When this article is cited
• When a correction for this article is posted

Click here to choose from all of JBC's e-mail alerts

This article cites 36 references, 15 of which can be accessed free at

http://www.jbc.org/content/285/32/24307.full.html#ref-list-1