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Structure-function analysis identifies highly sensitive strigolactone receptors in Striga

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Strigolactones are naturally occurring signaling molecules that affect plant development, fungi-plant interactions, and parasitic plant infestations. We characterized the function of 11 strigolactone receptors from the parasitic plant Striga hermonthica using chemical and structural biology. We found a clade of polyspecific receptors, including one that is sensitive to picomolar concentrations of strigolactone. A crystal structure of a highly sensitive strigolactone receptor from Striga revealed a larger binding pocket than that of the Arabidopsis receptor, which could explain the increased range of strigolactone sensitivity. Thus, the sensitivity of Striga to strigolactones from host plants is driven by receptor sensitivity. By expressing strigolactone receptors in Arabidopsis, we developed a bioassay that can be used to identify chemicals and crops with altered strigolactone levels.
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REFERENCES AND NOTES
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ACK NOW LE DGM EN TS
Arabidopsis plants containing RFP::TUB6 were the kind gift of
C. Ambrose and G. O. Wasteneys. Supported by CREATE and
Discovery grants (L.S. and S.D.M.); Canadian Natural Sciences and
Engineering Research Council doctoral postgraduate scholarships
(Y.W., M.J.M., and L.M.M.); NSF grant MCB-1158372 (D.W.E. and
H.N.C.); and Japan Society for the Promotion of Science KAKENHI
grants 24114002 and 25291062 (T.D.). We thank the technical
support staff of the UBC Bioimaging Facility and R. White for
statistical advice. The pTA7001 plasmid (VP16-GR vector) is
available from N.-H. Chua of The Rockefeller University under a
material transfer agreement; the VND7-GR line is available from
T.D.; and the GFP::CESA3 and YFP::CESA7 VND7-GR lines are
freely available from L.S. or S.D.M. for noncommercial purposes
under a materials transfer agreement on the GVG system with
N.-H. Chua. Further data are reported in the supplementary
materials.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/350/6257/198/suppl/DC1
Materials and Methods
Figs. S1 to S5
Tables S1 and S2
Movies S1 to S6
References (1922)
5June2015;accepted26August2015
10.1126/science.aac7446
PLANT SCIENCE
Structure-function analysis identifies
highly sensitive strigolactone
receptors in Striga
Shigeo Toh,
1
Duncan Holbrook-Smith,
1
Peter J. Stogios,
2,3
Olena Onopriyenko,
2
Shelley Lumba,
1
Yuichiro Tsuchiya,
4
Alexei Savchenko,
2
Peter McCourt
1
*
Strigolactones are naturally occurring signaling molecules that affect plant development,
fungi-plant interactions, and parasitic plant infestations. We characterized the function
of 11 strigolactone receptors from the parasitic plant Striga hermonthica using
chemical and structural biology. We found a clade of polyspecific receptors, including
one that is sensitive to picomolar concentrations of strigolactone. A crystal structure
of a highly sensitive strigolactone receptor from Striga revealed a larger binding pocket
than that of the Arabidopsis receptor , which could explain the increased range of
strigolactone sensitivity. Thus, the sensitivity of Striga to strigolactones from host plants is
driven by receptor sensitivity. By expressing strigolactone receptors in Arabidopsis,we
developed a bioassay that can be used to identify chemicals and crops with altered
strigolactone levels.
S
trigolactones are small molecules that act
as endogenous plant hormones to influence
plant growth and development (1, 2). Unlike
other plant hormones, multiple forms of
bioactive strigolactones exist and vary in
moieties attached to the A and B rings as well
as the orientation of the D ring (fig. S1) (2). Aside
from their hormonal role, strigolactones are also
exuded through roots into the soil, where they act
as exogenous signals to attract mycorrhizal fungi
for a symbiotic interaction that brings nutrients
to the plant (3). Unfortunately, parasitic plants of
the Striga, Phelipanche,andOrobanche genera also
use root-emitted strigolactones to locate a nearby
host (2). Upon perception of strigolactones pro-
duced by host plants, seeds of parasitic plants
germinate. Resulting seedlings attach themselves
to the host to deplete them of nutrients with dev-
astating consequences. In sub-Saharan Africa
alone, Striga has infested up to two thirds of the
arable land and represents the largest challenge
to food securit y on that continent, affec ting more
than 100 million people in 25 countries (4). A
mechanistic understanding of strigolactone syn-
thesis and signaling particularly in Striga could
open avenues for pest control.
Studies from model plants suggest that a fam-
ily of related a/b hydrolases is central to strigo-
lactone perception. For example, D14 (DWARF14)
is a strigolactone receptor important for shoot
branching in rice (57). A related a/b hydrolase
gene, HYPOSENSITIVE TO LIGHT/KARRIKIN
INSENSITIVE2 (HTL/KAI2), herein designated
HTL,hasbeenshowntoplayaroleinArabidopsis
germination (8). Because Striga requires strigolac-
tones to germinate, by inference HTL homologs
may have similar roles in parasitic plant species.
Arabidopsis HTL, however , binds a smoke-derived
germination stimulant, karrikin (9). In contrast,
Striga does not germinate in response to karrikin
but is sensitive to picomolar concentrations of
strigolactones (10). Moreover , Striga appears to dif-
ferentiate among hosts by sensing different com -
binations of strigolactones (10). Because Striga
hermonthica (Striga)isanobligateandoutcrossing
hemiparasite, the functional roles of Striga HTL/KAI2
homologs (ShHTLs) in strigolactone perception
are difficult to elucidate. He r e, we de s crib e the
roles of ShHTLsinstrigolactoneperception.
Phylogenetic analysis of public databases led
us to identify a nested clade of 11 HTL homologs
in Striga (Fig. 1A). To analyze the function of
these ShHTL genes, we expressed each of the
11 ShHTLs individually in an Arabidopsis loss-
of-function HTL/KAI 2 mutant (htl-3)background
and assayed for germination phenotypes. Seeds of
the Arabidopsis ecotype Columbia germinate
poorly after exposure to high temperature, but
this thermoinhibition is alleviated by strigolac-
tone addition (11). The strigolactone-dependent
rescue of thermoinhibited seeds requires a func-
tional AtHTL gene, which provided the basis
for introducing ShHTL genes into an htl-3 ge-
netic background. We monitored the functional
activity of these genes by assaying for suppres-
sion of thermoinhibited seeds. Thermoinhibited
htl-3 seed constitutively overexpressing AtHTL
driven by the 35S promoter germinated upon
addition of a synthetic strigolactone, GR24 (fig.
S2). Similarly, thermoinhibited htl-3 seed con-
taining different ShHTL transgenes germinated
on GR24 to varying degrees, depending on the
transgene (fig. S2). To compare lines, we de-
termined the effective concentration of GR24
required for 50% germinat io n (EC
50
)oftransgen-
ic seed (fig. S3).TheEC
50
of a transgenic line
overexpressing AtHTL is lower than that of the
wild type, indicating increased strigolactone re-
sponsiveness (Fig. 1B). In contrast, ShHTL1,
ShHTL2,andShHTL3 expression lines were less
responsive to GR24 compared with either wild
type or misexpres sed AtHTL seed (Fig. 1B).
ShHTL8 and ShHTL9 transgenics were less
SCIENCE sciencemag.org 9OCTOBER2015 VOL 350 ISSUE 6257 203
1
Cell and Systems Biology, University of Toronto, 25
Willcocks Street, Toronto M5S 3B2, Canada.
2
Department of
Chemical Engineering and Applied Chemistry, Banting and
Best Department of Medical Research, University of Toronto,
200 College Street, Toronto M5S 3E5, Canada.
3
Center for
Structural Genomics of Infectious Diseases, contracted by
National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, MD, USA.
4
Institute of
Transformative Bio-Molecules, Nagoya University, Japan,
Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan.
*Corresponding author. E-mail: peter.mccourt@utoronto.ca
RESEARCH | REPORTS
effective, whereas ShHTL4, ShHTL5,andShHTL6
were more responsive compared with AtHTL
(Fig. 1B). Complementation results from a study
in which four ShHTL genes were transformed
into an Arabidopsis htl mutant were consistent
with our fin dings (12). ShHTL7 transgenic seed
were four t o five order s o f m agnitude more re-
sponsive than other lines (Fig. 1C). This pico-
molar level of sen si ti vi ty is in the rea lm of Striga
strigolactone sensitivity, which was unexpected
because the remainder of the strigolactone sig-
naling machinery in our transgenic plants is
204 9OCTOBER2015 VOL 350 ISSUE 6257 sciencemag.org SC IENCE
Fig. 1. Striga ShHTL genes.
(A)Phylogenetictreeof
ShHTL genes from Striga
hermonthica and a variety of
nonparasitic plants. The
circle represents Arabidopsis
HTL/KAI2. Bootstrap values
of 100 replicates are indi-
cated by each node. (B)EC
50
of GR24
rac
that germinates
50% of htl-3 seeds
containing an AtHTL or
ShHTL transgene. Numbers
represent biological repli-
cates from three inde-
pendent transformants. EC
50
was calculated from fig. S3.
(C)Representativepictureof
thermoinhibited wild-type or
htl-3 35S::ShHTL7 seed
exposed to different concen-
trations of GR24
rac
.
EC50 (µM)
wild type
0.90
htl-3
AtHTL
0.05
ShHTL1
8.79
ShHTL2
3.43
ShHTL3
10.91
ShHTL4
0.01
ShHTL5
0.01
ShHTL6
0.03
ShHTL7
17.5 x 10
-6
ShHTL8
0.10
ShHTL9
0.15
Striga
hermonthica
Fern
Moss
D14
ShHTL2
ShHTL4
ShHTL3
ShHTL7
ShHTL8
ShHTL11
Castor HTL/K
AI2
Sorghum HTL/KAI2
HTL/KAI2
Ppa:Pp1s150_116V6.2
Isotig05061
35
100
100
100
100
100
100
64
42
68
91
100
66
94
100
100
0.350
wild type
ShHTL7
.001
.01
1
10
GR24 (µM)
.1
DMSO
Fig. 2. Chemical responsiveness
of ShHTL transgenics. (A)Ger-
mination rate of htl-3 , AtHTL,and
ShHTL seed on strigolactones (1 mM)
and strigolactone agonists (10 mM).
(Inset) An expansion of strigolactone
concentrations tested on ShHTL7
seed. Genes were clustered on
the basis of their similarity of
response to all chemicals. Strigol
(STR), 5-deoxy strigol (5DS),
2-epi-5-deoxy strigol (Epi-DS), and
Sorgolactone (SOR) experiments
were performed by using the
thermotoleranceassay.Karrikin1
(KAR) and agonists 1 to 7 experiments
were performed by using the
primary dormancy assay. Details
are available in figs. S2 and S4.
htl-3
ShHTL10
ShHTL11
ShHTL2
ShHTL3
AtHTL
ShHTL1
ShHTL6
ShHTL7
ShHTL9
ShHTL8
ShHTL4
ShHTL5
GR24
Agonist
2
KAR
STR
5DS
Epi-DS
SOR
DMSO
1
3
4
5
6
7
0 Germination (%) 100
STR
5DS
SOR
Epi-DS
1
10
1
10
100
picoM
nanoM
htl-3 ShHTL7
Group 1
Group 2
Group 3
ShHTL6
ShHTL7
Group 4
0.180
100
35
64
68
66
44
100
100
42
100
42
ShHTL5
ShHTL10
ShHTL11
+++
++
+
++
++
++
++
+++
0
Striga
germination
The far right row represents agonist efficacy to germinate Striga seed. Details are
available in fig. S5. (B) Phylogenetic tree of ShHTLs in which the color of the gene
corresponds to the heat map value of germination on 5DS. Groups are based on
clustering by using chemical responsiveness. Bootstrap values of 100 replicates are
indicated by each node.
RESEARCH | REPORTS
encoded by the Arabidopsis genome. Thus, it
appears that different ShHTL hydrolases are
sufficient to confer the extreme sensitivity of
parasitic plants to strigolactones.
Although the introduction of different ShHTLs
into htl-3 resulted in variable GR24 seed re-
sponsiveness, this strigolactone is synthetic
and exists in two isomeric forms (GR24
rac
). To
test the contribution of each ShHTL more rigo-
rously, we analyzed the response of our transgen-
ics using a collection of naturally occurring
strigolactones, which are all ca p a b l e of ge rm i -
nating Striga.TransgenicsexpressingShHTL4
to ShHTL9 were the most sensitive to natural
strigolacto nes (Fig. 2A). This group of g en e s,
group 2, defines a subclade within the ShHTL
phylogeny that arose from a common ancestor
(Fig. 2B). ShHTL7 seed was the most responsive
because it ger mi n a t ed at pi co m o la r con c e n tr a -
tions for 5DS, 2-Epi-5DS, and sorgolactones but
responded to strigol in the nanomolar range (Fig.
2A). This suggests that the presence of a hydrox-
yl group on carbon 5 of the A-ring of strigol
decreases binding to this receptor and may ex-
plain why crop cultiva rs prod uc ing high levels
of 5DS that lack this hydroxyl group are so sus-
ceptible to infection (10). Last, two ShHTL lines
(ShHTL2 and ShHTL3)respondedtokarrikin,
although this butenolide does not germinate
Striga seed (Fig. 2A). This suggests that activation
of these recepto rs is not suffici ent for Striga ger-
mination. Overall, our functional results correlate
well with enzymatic activities of ShHTLson5DS
with the exception of ShHTL10 and ShHTL11
(13). Although ShHTL10 and ShHTL11 were bio-
chemically active, they could not suppress the
htl mutant phenotype, suggesting a different role
for these ShHTLsotherthangermination.
We then probed the promiscuity of ShHTL
receptors using a collection of synthetic strigo-
lactone agonists that show different potencies for
germinating Striga (Fig. 2A and fig. S4) (14). In
this experiment, we assayed germination of un-
derripened seed. This dormancy assay, similar
to thermoinhibition, is alleviated by strigolac-
tone and requires a functional HTL gene (11).
Within group 2, we found that ShHTL4 to
ShHTL7but not ShHTL8 and ShHTL9showed
the broadest range of sensitivity to various ag-
onists. Of the 11 strigolactone receptors in Striga,
it appears that ShHTL4 to ShHTL7 form a min-
imal set of receptors sufficient for Striga germi-
nation. Clustering analysis based on germination
efficiencies on natural strigolactones and syn-
thetic agonists confirmed the formation of the
ShHTL4 to ShHTL7 subclade and identified
ShHTL7 as the most functionally sensitive receptor
SCIENCE sciencemag.org 9OCTOBER2015 VOL 350 ISSUE 6257 205
Fig. 3. Structural analysis of ShHT L5. (A)StructuresofShHTL5and
AtHTL (PDB 4JYM). Spheres indicate solvent accessible volume comprising
activ e site cavities. (B)Comparisonofactivesiteresiduesofstrigolactone
receptors. Sticks are shown and labeled for active site residues showing notable
residue substitutions between ShHTL5 (yellow) and AtHTL (blue) plus the
catalytic serine and histidin e residues; equivalent ShHTL7 resid ues are labeled in
black. Karrikin1 (KAR, blue) bound to AtHTL (PDB 4JYM) and GR24 (red; A and D
rings of GR24 are labeled) in silico docked into the active sites of ShHTL5.
Single-letter abbr eviations for the amino acid residues are as follows: A, Ala; C,
Cys; D , Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Le u; M, Met; N, Asn; P,
Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V , V al; W, Trp; and Y ,Tyr . (C)Sequencealignment
of solven t-e xposed active site amino acid residues for ShHTL proteins. Amino
acids that differ fr om AtHTL are colored orange. AtHTL is blue, ShHTL5 is yellow ,
and ShHTL7 is black. A fully expanded alignment can be found in fig. S6. The far
right column is a heat map of germination of each transgenic in response to 5DS
from Fig. 2A. Color brackets represent groups based on Fig. 2B.
RESEARCH | REPORTS
(Fig. 2A). Although the phylogenetic tree based
on receptor sequence is mostly reflected by clust-
ering according to functional response, the use of
natural strigolactones and synthetic agonists
increases the functional resolution within a clade
of receptors (Fig. 2B). Thus, chemical probes
can be used to dissect functions of individual
Striga receptors not obvious through genetic
or evolutionary studies. Our result s indic a te that
this set of four receptors should be the focus of
chemical screens in the development of com-
pounds to combat Striga.
To establis h the molecularbasisforstrigolac-
tone sensitivity by ShHTL proteins, we determined
and characterized the crystal structure of ShHTL5
at a resolution of 2.48 Å because ShHTL5 be-
longs to the most sensitive group of receptors
(group 2) (table S1). Structural conservation
between ShHTL5 and AtHTL [Protein Data Bank
(PDB) 4JYM, PMID 23613584] structures (root
mean square deviation of 1.0 Å over 268 matching
Ca atoms) allowed comparative analysis of their
active site s (Fig. 3A). In both ShHTL5 and AtHTL ,
the active site is localized to a cavity formed by
respective a4, a5, and a7helices(Fig.3A),which
was more than twice as large for ShHTL5 as
compared with AtHTL (998 versus 403 Å
3
)(Fig.
3A). There is also substantial variation among
residues localized to the active site cavity, with
9outof18ShHTL5residuesshowingdeviation
from their counterpar ts in AtH T L (F i g. 3B ) . Pa r -
ticularly, the substitutions of Y124 and S196 in
AtHTL to V124 and Y196, respectively, in ShHTL5
deepened the active site cleft in the latter pro-
tein, making it more complementary to the D-
ring of the GR24 molecule (Fig. 3B, blue boxes).
In contrast, the Y124 in AtHTL would occlude
this region of GR24. Other differences in active
site composition included alteration at positions
(W153, F194, and A2 1 9 ) invol v e d in posi t i oning
the karrikin ligand in AtHTL but that are rep-
resented as M153, S194, and L219, respectively, in
ShHTL5 (Fig. 3B, pink boxe s) (9). These struc-
tural differences provide a s u bs t an t i al l y dif fe r e nt
chemical environment in the ShHTL5 active site,
suggesting tailoring of the active site to recognize
the strigolactone scaffold and discriminate against
karrikin.
Taking advantage of the structural character-
ization of ShHTL5, an analysis of the variation
among active-site residues across the ShHTL pro-
tein family rationalized the enhanced response
of ShHTL7- versus ShHTL5-harbouring transgenic
plants to strigolactones (Fig. 3B). Structural analy-
ses revealed that the active-site pocket of ShHTL1
was most similar to that of AtHTL (14 identical
and one chemically similar active-site residues);
the ShHTL7 active site was most distinct (two
identical and five chemically similar active-site
residues); and the ShHTL5 active site was inter-
mediary (four identical and five chemically sim-
ilar residues). The degree of similarity of ShHTL
active sites to those of AtHTL is consistent with
low, intermediate, and high response of ShHTL1-,
ShHTL5-, and ShHTL7-expressing transgenic plants
to GR24 (Fig. 3C). This observation prompted us to
(Fig. 4, A and B) hypothesize that ShHTLshave
evolved differential strigolactone-binding affin-
ities through modulation of their active site
architecture.
Success in controlling Striga infestations will
require integrated strategies involving both chem-
ical control and plant breeding (15, 16). Striga
itself is difficult to study and, as a noxious weed,
is generally inaccessible to most researchers.
Arabidopsis seed expressing ShHTL7 transgenic
seed could function as a bioassay for strigolac-
tone. Indeed, thermoinhibited ShHTL7 seed re-
sponded to GR24 concentrations as low as 2 pM.
High and low germination rates of ShHTL7 seeds
sprinkled on rice roots reflected cultivars emitting
high (IAC165) and low levels (TN1) of strigolac-
tone, respectively (Fig. 4C) (16).
Striga receptors possess larger and modified
active site architectures with high sensitivities
to strigolactone compared with that of Arabi-
dopsis HTL. We hypothesize that these changes
have contributed to the ability of Striga to ex-
pand its host range through recognition of a
wide range of strigolactones (12). Although broad
specificity and high sensitivity are not gener-
ally associated, biochemical studies involving
soluble multidrug transporters demonstrate that
the evolution of large binding pockets to ac-
commodate multiple ligands does not necessar-
ily result in a loss of sensitivity (17). Multidrug
transporters possess broad specificity because
of large hydrophobic binding pockets but also
form high-affinity interactions through hydro-
phobic and electrostatic interactions. It will be
interesting to determine whether Striga her-
monthica strigolactone receptors have similar
biochemical mechanisms.
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Fig. 4. Strigolactone bioassay. (A)Germinationofthermoinhibitedhtl-3 seed containing different
ShHTL transgenes (ShHTL4 to ShHTL10). The center filter disc was impregnated with GR24
rac
,andblack
labels are the calculated final concentrations of GR24
rac
in the plate. (B)Germinationoftransgenichtl-3
seed containing ShHTL7 on varying concentrations of GR24
rac
.Thecenter filterdiscwasimpregnated
with GR24
rac
,andblacklabelsareasin(A).(C)Germinationofthermoinhibitedhtl-3 35S::ShHTL7 in the
presence of a high- (IAC165) or low- (TN1) strigolactoneemit ting rice cultivars. (Left) R epr esentativ e
picture of Arabidopsis htl-3 35S::ShHTL7 seeds sprinkled around rice roots. (Right) Quantification of
thre e independent experiments T SD. Scale bar , 1 mm.
RESEARCH | REPORTS
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ACK NOW LE DGM EN TS
We thank A. Babikier for providing the Striga seeds and T. Nakagawa
for the pGWB611 binary vector. Rice lines were provided by the National
Genetic Resources Program at the U.S. Department of Agriculture.
This work was supported by a grant from the National Science
and Engineering Research Council of Canada to P.M. We thank R. Di Leo
for technical assistance. Nucleotide and amino acid sequences
corresponding to ShD14 and ShHTLs have been deposited with
GenBank under accession KR013120 to KR013131. S.T., D.H.-S., P.M.,
and the University of Toronto have filed a U.S. provisional patent
application (US62/151,701) that relates to the specific topic
Strigolactone biosensors. We declare no financial conflicts
of interest in relation to the work. The supplementary
materials contain additional data.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/350/6257/203/suppl/DC1
Materials and Methods
Figs. S1 to S6
Table S1
References (1830)
2July2015;accepted9September2015
10.1126/science.aac9476
ONCOLOGY
Genomic correlates of response to CTLA-4
blockade in metastatic melanoma
Eliezer M. Van Allen,
1,2,3
* Diana Miao,
1,2
* Bastian Schilling,
4,5
* Sachet A. Shukla,
1,2
Christian Blank,
6
Lisa Zimmer,
4,5
Antje Sucker,
4,5
Uwe Hillen,
4,5
Marnix H. Geukes Foppen,
6
Simone M. Goldinger,
7
Jochen Utikal,
5,8,9
Jessica C. Hassel,
10
Benjamin Weide,
11
Katharina C. Kaehler,
12
Carmen Loquai,
13
Peter Mohr,
14
Ralf Gutzmer,
15
Reinhard Dummer,
7
Stacey Gabriel,
2
Catherine J. Wu,
1,2
Dirk Schadendorf,
4,5
Levi A. Garraway
1,2,3
Monoclonal antibodies directed against cytotoxic T lymphocyteassociated antigen-4
(CTLA-4), such as ipilimumab, yield considerable clinical benefit for patients with
metastatic melanoma by inhibiting immune checkp oint activity, but clinical predic tors of
response to these therapies remain incompl etely characterized. To inves tigate the roles
of tumor-specific neoantigens and alt erations in the tumor microenvironment in the
response to ipilimumab, we analyzed whole exomes from pretreatment melanoma tumor
biopsies and matchin g germline tissue samples from 110 patients. For 40 of these
patients, we also obtained and analyzed transcri ptome dat a from the pretreatment tumor
samples. Overall mutational load, neoantigen load, and express ion of cytolytic markers in
the immune mi croenvironment were significantly associa ted with clinical benefit.
However, no recurren t n eoantigen peptide sequences predicted responder patient
populations. Thus, detailed integrated mole cular characterization of large patient cohorts
may be needed to identify robust determinants of response and resistance to immune
checkpoint inhibitors.
B
lockade of cytotoxic T lymphocyte anti-
gen-4 (CTLA-4), an inhibitor of T cell
activation, with the monoclonal antibody
ipilimumab yields improvements in over-
all survival in patients with metastatic
melanoma as a monotherapy (1, 2)orincombi-
nation with other T cell immune checkpoint in-
hibitors (3, 4). Although overall single-agent
response rates are low, a long-term clinical ben-
efit is consistently observed for ~20% of treated
patients (5, 6). Preclinical and clinical studies
have suggeste d that tumor -specific missense muta-
tions may generate individual neoantigens that
mediate response to ipilimumab and other im-
mune checkpoint inhibitors (710). Clinical stud-
ies of exceptional responders (11)andofsmall
cohorts of melanoma patients have highlighted
NRAS mutation status, total neoantigen load,
and a neoantigen-derived tetrapeptide signature
as possible correlate s of res pons e to ipilimumab in
metastatic melanoma (12, 13). RNA-based studies
have also identified gene expression signatures
linked to immune infiltration within the tumor
microenvironment that correlate with overall sur-
vival, neoantigen load (14, 15), and resistance to
immunotherapy (16). To date, however, compre-
hensive genomic studies of tumor- and immune-
related factors in larger (i.e., >100 patients) clinical
cohorts have not been reported.
We hypothesized that both tumor-specific
neoantigens and the tumor immune micro-
environment might influence clinical benefit from
ipilimumab. To test this, we performed whole-
exome sequencing (WES) on a cohort of 110 pa-
tients with metastatic melanoma from whom
pretrea tm ent tumor biopsies were availab le for
study (Fig. 1A). Tumor whole-transcriptome se-
quencing was performed in 42 of these patients,
of whom 40 had matched WES. This cohort in-
cluded 92 cutaneous, 4 mucosal, and 14 occult
melanomas. After WES of matched tumor and
germline samples (17), quality-control metrics were
applied to ensure sensitive mutation detection
(18). Average exome-wide target coverage was
183.7-fold for tumor samples and 157.2-fold for
germline samples. We performed somatic muta-
tion identification (table S1) and germline human
lymphocyte antigen (HLA) typing (table S2) using
established methods (14, 19). The median non-
synonymous mutational load was 197 per sample
(range: 7 to 5854), which is consistent with the
known high mutational loads in cutaneous mel-
anoma (13, 20).
To stra tify our cohort, clinical benefit was de-
fined using a composite end point of complete
response or partial response to ipilimumab by
RECIST criteria (21)orstablediseasebyRECIST
criteria with overall survival greater than 1 year
(n = 27). No clinical benefit was defined as
progressive disease by RECIST criteria or stable
disease with overall survival less than 1 year (n =
73). The basis for these designations stems from
clinical trials demonstrating that ipilimumab sig-
nificantly improves median overall survival, with
asubsetofpatientssurvivingbeyond2years
(~20%), but does not affect progression-free survival
SCIENCE sciencemag.org 9OCTOBER2015 VOL 350 ISSUE 6257 207
1
Department of Medical Oncology, Dana-Farber Cancer
Institute, Boston, MA 02215, USA.
2
Broad Institute of MIT
and Harvard, Cambridge, MA 02142, USA.
3
Center for Cancer
Precision Medicine, Dana-Farber Cancer Institute, Boston,
MA 02215, USA.
4
Department of Dermatology, University
Hospital, University DuisburgEssen, 45147 Essen, Germany.
5
German Cancer Consortium (DKTK), 69121 Heidelberg,
Germany.
6
Department of Medical Oncology, Netherlands
Cancer Institute, 1066 CX Amsterdam, Netherlands.
7
Department of Dermatology, University Hospital Zurich,
8091 Zurich, Switzerland.
8
Skin Cancer Unit, German Cancer
Research Center (DKFZ), 69121 Heidelberg, Germany.
9
Department of Dermatology, Venerology, and Allergology,
University Medical Center, Ruprecht-Karls University of
Heidelberg, 68167 Mannheim, Germany.
10
Department of
Dermatology, University Hospital, Ruprecht-Karls University
of Heidelberg, 69120 Heidelberg, Germany.
11
Department of
Dermatology, University Hospital Tübinge n, 72076 Tübingen,
Germany.
12
Department of Dermatology, University Hospital
Kiel, 24105 Kiel, Germany.
13
Department of Dermatology,
University Medical Center Mainz, 55131 Mainz, Germany.
14
Department of Dermatology, Elbe-Kliniken, 21614
Buxtehude, Germany.
15
Department of Dermatology and
Allergy, Skin Cancer Center Hannover, Hannover Medical
School, 30625 Hannover, Germany.
*These authors contributed equally to this work. Corresponding
author. E-mail: levi_garraway@dfci.harvard.edu (L.A.G.);
dirk.schadendorf@uk-essen.de (D.S.)
RESEARCH | REPORTS
... F-box protein MAX2 is pre-requisite for their downward signal transmission, though distinct growth responses are produced through regulation of distinct members of the SMAX1-LIKE/D53 family like Lotus japonicus (Carbonnel et al. 2020a). Unlike D14 or SL-deficient mutants, kai2 and max2 mutants share and display different phenotypes (seed germination, seedling growth, petiole orientation, and leaf shape) indicating that KAI2 identifies an unidentified endogenous KAI2 ligand (KL) (Conn and Nelson 2016;Toh et al. 2015). ...
... Parasitic plants belonging to Orobanchaceae have multiple copies of KAI2 in their genome. Among which some are evolved for SLsperception, others perceive KARs and some others fail to perceive any stimulus in Arabidopsis (Conn et al. 2015;Toh et al. 2015). ...
Article
Full-text available
Plant-derived smoke is a source of several butenolide compounds collectively termed, karrikins (KARs). These have been recognized as a recent class of plant growth regulators associated with diverse phenomena especially seed germination, seedling vigor and other photo-morphogenetic responses. Role of KARs in improving the performance of plants exposed to different environmental constraints has been recognized recently. KARs mitigate oxidative stress in plants caused by salinity, drought, shade and heavy metals by modulating antioxidative metabolism (SOD, POX, GR, APX) and up-regulating the expression of various stress related genes in plants. Such a modulation involves an interplay with different transcription factors and endogenous plant growth regulators predominantly auxins, gibberellins, ethylene and abscissic acid. The present article is an attempt to gain a comprehensive understanding regarding the KARs-induced plant responses. Moreover, role of KARs in plant abiotic stress tolerance, perception of KARs in plants, expression of different enzymes and genes involved in abiotic stress mitigation, their cross-talk with other endogenous hormones under normal and chellanging environmental circumstances.
... (8). These weeds are obligate parasites that developed the ability to sense SLs as a seed germination signal enabling the coordination of their life cycle with the presence of an available host in the close vicinity (9). Infestation by root parasitic plants, such as Striga hermonthica, is a severe problem for agriculture and a major threat for global food security, in particular in Africa where it causes more than U.S. $7 billion annual losses in cereal production (10,11). ...
Article
Full-text available
Strigolactones (SLs) are a plant hormone inhibiting shoot branching/tillering and a rhizospheric, chemical signal that triggers seed germination of the noxious root parasitic plant Striga and mediates symbiosis with beneficial arbuscular mycorrhizal fungi. Identifying specific roles of canonical and noncanonical SLs, the two SL subfamilies, is important for developing Striga-resistant cereals and for engineering plant architecture. Here, we report that rice mutants lacking canonical SLs do not show the shoot phenotypes known for SL-deficient plants, exhibiting only a delay in establishing arbuscular mycorrhizal symbiosis, but release exudates with a significantly decreased Striga seed-germinating activity. Blocking the biosynthesis of canonical SLs by TIS108, a specific enzyme inhibi-tor, significantly lowered Striga infestation without affecting rice growth. These results indicate that canonical SLs are not the determinant of shoot architecture and pave the way for increasing crop resistance by gene editing or chemical treatment.
... Here, we present the crystal structure of A. thaliana DLK2, which, as a major difference to its paralogs AtD14 and AtKAI2, features a significantly smaller substrate binding pocket. Previous structures of strigolactone receptors or KAI2 clade proteins have shown that not only the substitution of amino acids inside the substrate binding pocket determines its size or ligand specificity (Guercio et al., 2022;Toh et al., 2015) but that also interactions between residues located in different secondary structure elements can influence the volume and shape of the pocket. This has been observed in a hydrogen bond between helices αD1 and αD3 in hyposensitive to light proteins from Striga hermonthica (ShHTL) (Xu et al., 2018) and in a loop region between helices αE and αF in KAI2-like proteins from Physcomitrium patens (Bürger et al., 2019). ...
Article
Full-text available
In Arabidopsis thaliana, the Sigma factor B regulator RsbQ‐like family of α/β hydrolases contains the strigolactone (SL) receptor DWARF14 (AtD14), the karrikin receptor KARRIKIN INSENSITIVE2 (AtKAI2), and DWARF14‐LIKE2 (AtDLK2), a protein of unknown function. Despite very similar protein folds, AtD14 and AtKAI2 differ in size and architecture of their ligand binding pockets, influencing their substrate specificity. We present the 1.5 Å crystal structure of AtDLK2, revealing the smallest ligand binding pocket in the protein family, bordered by two unique glycine residues. We identified a gatekeeper residue in the protein's lid domain and present a pyrrolo‐quinoline‐dione compound that inhibits AtDLK2's enzymatic activity.
... In petunia, DAD2 encoded α/β-hydrolase, hydrolyses GR24, and binds to the MAX2 proteins leading to the signalling of SCF-mediated pathway and phenotypic responses of plants 46 . About 11 SLs receptors have been characterized by Striga hermonthica 47 . Bioinformatical and other analyses have also shown that DAD2, DWARF14 may also work as SLs receptors 46,48 . ...
Article
The strigolactones (SLs) are plants hormones that have multiple functions in architecture and development. The roles of SLs in shoot branching and stem secondary growth of autotrophic plants are established. SL is also involved in the interaction between root parasitic plants and their host plants. SLs are exudates by the root of the host plant in search of a fungal partner for symbiotic association, while parasitic plants utilize this facility to detect the host root. The first formed tubercle of Philapanhche, whose germinations are driven by host-derived SLs, exudates parasitic derived SLs (PSLs) and could encourages germination of the adjacent parasitic seeds, resulting in parasite cluster formation. The existence of aboveground spikes in clusters suggests an intriguing approach for increasing parasite population by amplifying PSLs, which result in massive parasitic seed germination. PSLs probably have a role in the increased branching of Broomrapes opposing the host plant, resulting in the parasites' clustered appearance aboveground. This review highlights the distinct roles of SLs and PSLs, and their potential role in host-parasitic interaction.
... To test the SL-responding ability of PjKAI2d2, PjKAI2d3 and PjKAI2d3.2, we adopted a modified cross-species complementation method, which had been successful in previous studies 27,46,47 . We used the A. thaliana d14 kai2 double mutant in the Col-0 background 48 and evaluated responses to rac-strigol by seed germination rates (Fig. 4c). ...
Article
Full-text available
Parasitic plants are worldwide threats that damage major agricultural crops. To initiate infection, parasitic plants have developed the ability to locate hosts and grow towards them. This ability, called host tropism, is critical for parasite survival, but its underlying mechanism remains mostly unresolved. To characterise host tropism, we used the model facultative root parasite Phtheirospermum japonicum, a member of the Orobanchaceae. Here, we show that strigolactones (SLs) function as host-derived chemoattractants. Chemotropism to SLs is also found in Striga hermonthica, a parasitic member of the Orobanchaceae, but not in non-parasites. Intriguingly, chemotropism to SLs in P. japonicum is attenuated in ammonium ion-rich conditions, where SLs are perceived, but the resulting asymmetrical accumulation of the auxin transporter PIN2 is diminished. P. japonicum encodes putative receptors that sense exogenous SLs, whereas expression of a dominant-negative form reduces its chemotropic ability. We propose a function for SLs as navigators for parasite roots. Parasitic plants are able to grow towards potential hosts. Here the authors show that strigolactones produced by the host plants can act as chemoattractants for the root parasites Phtheirospermum japonicum and Striga hermonthica.
... 44) One of the factors responsible for the strong biological activity of SPL7 is its role as an agonist that selectively acts on ShHTL7, which has a high affinity for SL among the paralogs of the SL receptor D14, which has been shown to be present in at least 11 in S. hermonthica. 45,46) SPL7 is a structural analog of a hit compound obtained by a chemical screen conducted to discover agonists of ShHTLs, using seed germination stimulation of S. hermonthica as an indicator. Since the minimum effective concentration is 1/100 million of that of GR24, it can overcome the chemical instability caused by the D-ring. ...
Article
Full-text available
Parasitic plants in the Orobanchaceae family include devastating weed species, such as Striga, Orobanche, and Phelipanche, which parasitize major crops, drastically reduces crop yields and cause economic losses of over a billion US dollars worldwide. Advances in basic research on molecular and cellular processes responsible for parasitic relationships has now achieved steady progress through advances in genome analysis, biochemical analysis and structural biology. On the basis of these advances it is now possible to develop chemicals that control parasitism and reduce agricultural damage. In this review we summarized the recent development of chemicals that can control each step of parasitism from strigolactone biosynthesis in host plants to haustorium formation.
... For example, SLs do not promote germination of Arabidopsis seeds, while KARs cannot rescue the excess branching phenotype of SL-deficient mutants . It is worth mentioning that KAI2 family members have undergone numerous duplication events, and D14 was found to be an ancient duplication in the KAI2 receptor in the seed plant lineage followed by subfunctionalization of the receptor, enabling SL perception (Toh et al. 2015;Xu et al. 2016;Swarbreck et al. 2019;Guercio et al. 2022). ...
Article
In this study, we investigated the potential role of the karrikin receptor KARRIKIN INSENSITIVE2 (KAI2) in the response of Arabidopsis seedlings to high temperature stress. We performed phenotypic, physiological and transcriptome analyses of Arabidopsis kai2 mutants and wild-type (WT) plants under control (kai2_C and WT_C, respectively), and 6 h and 24 h heat stress conditions (kai2_H6, kai2_H24, WT_H6, and WT_H24, respectively) to understand the basis for KAI2-regulated heat stress tolerance. We discovered that kai2 mutants exhibited hypersensitivity to high temperature stress relative to WT plants, which might be associated with more highly increased leaf surface temperature and cell membrane damage of kai2 mutant plants. Next, we performed comparative transcriptome analysis of kai2_C, kai2_H6, kai2_H24, WT_C, WT_H6 and WT_H24 to identify transcriptome differences between WT and kai2 mutant in response to heat stress. K-mean clustering of normalized gene expression separated the investigated genotypes into three clusters based on heat-treated and non-treated control conditions. Within each cluster, kai2 mutants were separated from WT plants, implying that kai2 exhibited distinct transcriptome profiles relative to WT plants. Gene ontology and KEGG enrichment analyses showed a repression in ‘misfolded protein binding’, ‘heat shock protein binding’, ‘unfolded protein binding’ and ‘protein processing in endoplasmic reticulum’ pathways, which was consistent with the down-regulation of several genes encoding heat shock proteins and heat shock transcription factors in kai2 mutant versus WT plants under control and heat stress conditions. Our findings suggest that chemical or genetic manipulation of KAI2 signaling may provide a novel way to improve heat tolerance in plants.
Article
The discovery of strigolactones has resulted in a confluence of various research topics like parasitic plants, arbuscular mycorrhizal fungi and phytohormones, which all play a big role in current global agricultural production. Over the past few decades, strigolactone research swiftly gained a spotlight, as to reveal their possible functions within plants and also the surrounding organisms in the rhizosphere. In this review, we explore the discovered functions of strigolactones with the main focus on the chemical structure of strigolactones and how it relates to the various biological responses they cause. We highlight their involvement in plant responses to abiotic stressors, like lack of available nutrients, high salinity, drought, extreme temperatures and presence of potentially toxic elements of environmental importance, while reflecting upon the strigolactone-mediated plant associations with arbuscular mycorrhizal fungi and nodule-forming, N-fixing bacteria. Furthermore, we elaborate on the current state of applied strigolactone research in agriculture and the probable bright future these compounds have in commercial use and what hurdles need to be overcome before they can be fully utilized.
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Arbuscular mycorrhizal fungi (AMF) play important roles in some key steps of nitrogen cycling in agroecosystems, such as the transformation of soil nitrogen and plant nitrogen uptake. However, whether AMF-mediated nitrogen cycling can be affected by other soil organisms, such as earthworms, remains to be elucidated, particularly under straw incorporation. A factorial microcosm experiment was conducted to test the effects of AMF-earthworm interactions on nitrogen cycling by including two earthworm species with different feeding behaviors (Eisenia fetida, epigeic, EP and Metaphire guillelmi, endogeic, EN) under different straw management strategies (no straw, NS; straw mixing with soil in 0–10 cm depth, SM; and the deep burial of straw at 20 cm deep, DB). Our results showed that AMF significantly increased the aboveground plant nitrogen uptake under NS (+27.01 %), SM (+7.63 %) and DB (+14.42 %) treatments. However, interactions between earthworms with different feeding types and straw management could affect AMF-mediated nitrification and denitrification in soils. In addition, earthworms were found to promote the uptake of mycorrhizal ¹⁵N uptake in the SM treatment but reduced it under the DB treatment. However, the interaction between AMF and EN earthworms promoted the total plant N uptake in the DB treatment. Our study suggests that AMF can affect the transformation of soil nitrogen and plant nitrogen uptake, but their impacts could be altered by agronomic management, such as straw incorporation, which induced changes in the activities of soil fauna, such as earthworms.
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Orobanchaceae is the largest family of parasitic plants, containing autotrophic and parasitic plants with all degrees of parasitism. This makes it by far the best family for studying the origin and evolution of plant parasitism. Here we provide three high-quality genomes of orobanchaceous plants, the autotrophic Lindenbergia luchunensis and the holoparasitic plants Phelipanche aegyptiaca and Orobanche cumana. Phylogenomic analysis of these three genomes together with those previously published and the transcriptomes of other orobanchaceous species, created a robust phylogenetic framework for Orobanchaceae. We found that an ancient whole-genome duplication (WGD; about 73.48 Mya), which occurred earlier than the origin of Orobanchaceae, might have contributed to the emergence of parasitism. However, no WGD events occurred in any lineage of orobanchaceous parasites except for Striga after divergence from their autotrophic common ancestor, suggesting that, in contrast to previous speculations, WGD is not associated with the emergence of holoparasitism. We detected evident convergent gene loss in all parasites within Orobanchaceae and between Orobanchaceae and dodder Cuscuta australis. The gene families in the orobanchaceous parasites showed a clear pattern of recent gains and expansions. The expanded gene families are enriched in functions related to the development of the haustorium, suggesting that recent gene family expansions may have facilitated the adaptation of orobanchaceous parasites to different hosts. This study illustrates a stepwise pattern in the evolution of parasitism in the orobanchaceous parasites, and will facilitate future studies on parasitism and the control of parasitic plants in agriculture.
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Elucidating the signaling mechanism of strigolactones has been the key to controlling the devastating problem caused by the parasitic plant Striga hermonthica. To overcome the genetic intractability that has previously interfered with identification of the strigolactone receptor, we developed a fluorescence turn-on probe, Yoshimulactone Green (YLG), which activates strigolactone signaling and illuminates signal perception by the strigolactone receptors. Here we describe how strigolactones bind to and act via ShHTLs, the diverged family of α/β hydrolase-fold proteins in Striga. Live imaging using YLGs revealed that a dynamic wavelike propagation of strigolactone perception wakes up Striga seeds. We conclude that ShHTLs function as the strigolactone receptors mediating seed germination in Striga. Our findings enable access to strigolactone receptors and observation of the regulatory dynamics for strigolactone signal transduction in Striga.
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Strigolactones (SLs), a newly discovered class of carotenoid-derived phytohormones, are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signalling mechanisms of SL remain poorly understood. Here we show that DWARF 53 (D53) acts as a repressor of SL signalling and that SLs induce its degradation. We find that the rice (Oryza sativa) d53 mutant, which produces an exaggerated number of tillers compared to wild-type plants, is caused by a gain-of-function mutation and is insensitive to exogenous SL treatment. The D53 gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with the α/β hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL perception. We demonstrate that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth. Our combined genetic and biochemical data reveal that D53 acts as a repressor of the SL signalling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses.
Book
Witchweeds (Striga species) decimate agriculture in much of Africa and parts of Asia, attacking the major cereal grains and legumes, and halving the already very low yields of subsistence farmers. Several years of research have provided promising technologies, based on the fundamental biology of the Parasite-Host associations, for dealing with this scourge. However, there is an apparent realization that these technologies will fail because highly successful weeds such as Striga evolve resistance to all types of controls unless proven methods are integrated with each other for a more sustainable solution. Integration is often an anathema to basic scientists who typically deal with single variables and solutions. However, key leaders in the development of the new Knowledge-Based control strategies, already in the field and under development, recently joined forces to develop strategies and projects in order to integrate the technologies in a symposium in Ethiopia in November 2006. The encouraging results are described in this Peer-Reviewed book, authored by leaders in the field who have been supplying the basic biology, genetics, biochemistry, and molecular information that have offered insights and generated technologies in how to deal with Striga. © 2007 by World Scientific Publishing Co. Pte. Ltd. All rights reserved.
Article
Obligate parasitic plants in the Orobanchaceae germinate after sensing plant hormones, strigolactones, exuded from host roots. In Arabidopsis thaliana, the α/β-hydrolase D14 acts as a strigolactone receptor that controls shoot branching, whereas its ancestral paralog, KAI2, mediates karrikin-specific germination responses. We observed that KAI2, but not D14, is present at higher copy numbers in parasitic species than in nonparasitic relatives. KAI2 paralogs in parasites are distributed into three phylogenetic clades. The fastest-evolving clade, KAI2d, contains the majority of KAI2 paralogs. Homology models predict that the ligand-binding pockets of KAI2d resemble D14. KAI2d transgenes confer strigolactone-specific germination responses to Arabidopsis thaliana. Thus, the KAI2 paralogs D14 and KAI2d underwent convergent evolution of strigolactone recognition, respectively enabling developmental responses to strigolactones in angiosperms and host detection in parasites. Copyright © 2015, American Association for the Advancement of Science.
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The latest version of the CATH-Gene3D protein structure classification database (4.0, http://www.cathdb.info) provides annotations for over 235 000 protein domain structures and includes 25 million domain predictions. This article provides an update on the major developments in the 2 years since the last publication in this journal including: significant improvements to the predictive power of our functional families (FunFams); the release of our ‘current’ putative domain assignments (CATH-B); a new, strictly non-redundant data set of CATH domains suitable for homology benchmarking experiments (CATH-40) and a number of improvements to the web pages.
Article
Strigolactones are terpenoid-based plant hormones that act as communication signals within a plant, between plants and fungi, and between parasitic plants and their hosts. Here we show that an active enantiomer form of the strigolactone GR24, the germination stimulant karrikin, and a number of structurally related small molecules called cotylimides all bind the HTL/KAI2 α/β hydrolase in Arabidopsis. Strigolactones and cotylimides also promoted an interaction between HTL/KAI2 and the F-box protein MAX2 in yeast. Identification of this chemically dependent protein-protein interaction prompted the development of a yeast-based, high-throughput chemical screen for potential strigolactone mimics. Of the 40 lead compounds identified, three were found to have in planta strigolactone activity using Arabidopsis-based assays. More importantly, these three compounds were all found to stimulate suicide germination of the obligate parasitic plant Striga hermonthica. These results suggest that screening strategies involving yeast/Arabidopsis models may be useful in combating parasitic plant infestations.
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Protein structure determination by X-ray crystallography is dependent on obtaining a single protein crystal suitable for diffraction data collection. Due to this requirement, protein crystallization represents a key step in protein structure determination. The conditions for protein crystallization have to be determined empirically for each protein, making this step also a bottleneck in the structure determination process. Typical protein crystallization practice involves parallel setup and monitoring of a considerable number of individual protein crystallization experiments (also called crystallization trials). In these trials the aliquots of purified protein are mixed with a range of solutions composed of a precipitating agent, buffer, and sometimes an additive that have been previously successful in prompting protein crystallization. The individual chemical conditions in which a particular protein shows signs of crystallization are used as a starting point for further crystallization experiments. The goal is optimizing the formation of individual protein crystals of sufficient size and quality to make them suitable for diffraction data collection. Thus the composition of the primary crystallization screen is critical for successful crystallization. Systematic analysis of crystallization experiments carried out on several hundred proteins as part of large-scale structural genomics efforts allowed the optimization of the protein crystallization protocol and identification of a minimal set of 96 crystallization solutions (the “TRAP” screen) that, in our experience, led to crystallization of the maximum number of proteins.
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Genome sequencing projects have resulted in a rapid increase in the number of known protein sequences. In contrast, only about one-hundredth of these sequences have been characterized at atomic resolution using experimental structure determination methods. Computational protein structure modeling techniques have the potential to bridge this sequence-structure gap. In this chapter, we present an example that illustrates the use of MODELLER to construct a comparative model for a protein with unknown structure. Automation of a similar protocol has resulted in models of useful accuracy for domains in more than half of all known protein sequences.