protein (GFP) tag (fig. S12). Consistent with the
irrelevance of P1-Lys for cleavage site recognition
(Fig. 4, C and D), the P1-Lys/Ala mutant (K/A in
Fig. 4E) complemented the abscission defect of
ida to almost the same extent as WT IDA-GFP.
Substitution of Pro (P) in P2 in addition to P1-Lys
(PK/A) reduced the ability to complement the
mutant phenotype, and the P2-P4 double mutant
(YP/A; Y, Tyr) was essentially inactive (Fig. 4E).
Similarly, an ePIPP expression construct genetically complemented the ida mutant, and activity
was impaired when Pro in P2 was substituted by
Ala (P/A) (fig. S13).
Failure of the SBT-resistant precursor variants
to restore abscission in the ida mutant confirms
the IDA precursor as a physiological substrate of
SBTs and indicates a role for SBTs in signal maturation. We conclude that precursor processing
at the Lys/Gly bond is a prerequisite for the biogenesis of the abscission signal in Arabidopsis
flowers, implicating the 14-aa peptide mIDA as
the endogenous signal. Previous studies favored
the 12-aa PIPP peptide (Fig. 4A) as the abscission
signal (8, 18). We cannot exclude further trimming of the N terminus and the generation of
PIPP by an unidentified aminopeptidase after
initial cleavage of the Lys/Gly bond. However,
considering the reduced activity of PIPP as compared with mIDA (Fig. 4B), this would decrease
rather than increase signal intensity.
The close correlation observed between the
amino acid residues required for the biogenesis
of mIDA in vivo and for substrate recognition by
SBT4.13 in vitro implicates SBT4.13 in the maturation process. However, abscission is normal in
SBT4.13 single-gene loss-of-function mutants (fig.
S5); therefore, SBT4.13 cannot be the only protease responsible. SBT4.12 and SBT5.2 are also
involved, as they are coexpressed with IDA and
SBT4.13 in abscission zones (fig. S2) and show
the same cleavage specificity with respect to proIDA
processing (fig. S10). The activity of several redundant subtilases is thus responsible for the
biogenesis of the abscission signal.
Although AtSBT6.1, which is one of two intracellular SBTs and the ortholog of human site-
1 protease, has previously been implicated in
precursor processing (15, 21–24), peptide hormone
maturation by cognate SBTs was hitherto obscured
by functional redundancy in the large SBT family. Using EPI inhibitors as a tool to overcome
this functional redundancy, we demonstrated
a role for SBTs in IDA maturation and abscission. Tissue-specific expression of EPI inhibitors
will be instrumental for further analysis of SBTs
in peptide hormone biogenesis. Tissue-specific
expression of enzyme inhibitors may also be useful in other systems for the analysis of functionally redundant enzymes.
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We thank S. Kamoun (The Sainsbury Laboratory) for EPI1a
and EPI10 expression constructs and R. Aalen (University of
Oslo) for seeds of the ida mutant and the PGAZAT reporter
line. We also thank them for fruitful discussions, as well as
U. Glück-Behrens and J. Babo for technical assistance. This work
was supported in part by grants from the German Research
Foundation (Deutsche Forschungsgemeinschaft) to A.Sc. (SCHA
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Materials and Methods
Figs. S1 to S14
22 August 2016; accepted 22 November 2016
Published online 8 December 2016
Crystal structure of unlinked
NS2B-NS3 protease from Zika virus
Zhenzhen Zhang,1,2 Yan Li,3 Ying Ru Loh,3 Wint Wint Phoo,1,2,4 Alvin W. Hung,3
CongBao Kang,3† Dahai Luo1,2†
Zika virus (ZIKV) has rapidly emerged as a global public health concern. Viral NS2B-NS3
protease processes viral polyprotein and is essential for the virus replication, making it
an attractive antiviral drug target. We report crystal structures at 1.58-angstrom resolution
of the unlinked NS2B-NS3 protease from ZIKV as free enzyme and bound to a peptide
reversely oriented at the active site. The unlinked NS2B-NS3 protease adopts a closed
conformation in which NS2B engages NS3 to form an empty substrate-binding site. A second
protease in the same crystal binds to the residues K14K15G16E17 from the neighboring NS3 in
reverse orientation, resisting proteolysis. These features of ZIKV NS2B-NS3 protease may
accelerate the discovery of structure-based antiviral drugs against ZIKV and related
Zika virus (ZIKV) has spread across the world rapidly and is becoming a serious public health concern owing to its link to severe neurological diseases such as fetal micro- cephaly and Guillain-Barré syndrome in
adults (1, 2). Specific antiviral therapeutics against
ZIKV are urgently needed to fight this pandemic.
ZIKV belongs to the Flaviviridae family, Flavivirus
genus, which contains important human pathogens including dengue virus (DENV), West Nile
virus (WNV), yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus (3–5).
The genomes of these viruses encode a polyprotein that is processed into three structural
proteins (capsid, membrane, and envelope proteins) and seven nonstructural (NS) proteins (NS1,
NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by both
the host proteases and the viral NS2B-NS3 protease. As such, the NS2B-NS3 protease is an attractive target for antiviral drug development
(6, 7). NS3 contains a trypsin-like fold and carries the conserved catalytic triad (H51, D75, and
S135). The small transmembrane protein NS2B
anchors NS3 to the endoplasmic reticulum membrane, and together they form an active enzyme
for substrate recognition and catalysis (7–10). The
minimal cofactor region of the NS2B comprises
the hydrophilic residues 49 to 97 (11). The N-terminal 18 residues (49 to 67) of NS2B cofactor
support the proper fold of NS3 protease by
forming a beta strand inserted into the protease
domain (12, 13). The C-terminal part of NS2B
cofactor (residues 68 to 96) forms a b-hairpin to
create the S2 and S3 pockets in the substrate-binding site of NS3 protease (13–15).
Crystal structures of the proteases from DENV
(13, 14, 16), WNV (13, 15, 17, 18), Murray Valley
SCIENCE sciencemag.org 23 DECEMBER 2016 • VOL 354 ISSUE 6319 1597
1Lee Kong Chian School of Medicine, Nanyang Technological
University, Experimental Medicine Building 03-07, 59
Nanyang Drive, Singapore 636921. 2NTU Institute of
Structural Biology, Nanyang Technological University,
Experimental Medicine Building 06-01, 59 Nanyang Drive,
Singapore 636921. 3Experimental Therapeutics Centre,
Agency for Science, Technology and Research (A*STAR), 31
Biopolis Way, Nanos, #03-01, Singapore 138669. 4School of
Biological Sciences, Nanyang Technological University, 60
Nanyang Drive, Singapore 637551.
*These authors contributed equally to this work. †Corresponding
author. Email: email@example.com (C.K.); luodahai@ntu.
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