Novel octavalent cross-linker displays efficient trapping of protein-protein interactions

Article abstract of DOI:10.1039/b701542a

A novel octavalent, resorcin[4]arene derived, cross-linker designed to overcome some of the limitations of commercially available reagents is significantly more efficient for covalent stabilisation of protein-protein interactions. The Royal Society of Che

Full text of DOI:10.1039/b701542a

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Novel octavalent cross-linker displays efficient trapping of protein–
protein interactions{
Simon R. Foster,a Alice Pearce,b Alexander J. Blake,a Melanie J. Welhamb and James Dowden*a
Received (in Cambridge, UK) 1st February 2007, Accepted 5th March 2007
First published as an Advance Article on the web 20th March 2007
DOI: 10.1039/b701542a
offering a greater number and span of reactive functionality than
contemporary cross-linkers, thus yielding greater efficiency. The
well defined geometry available from the resorcinarene architecture
offers potentially useful features for future developments, such as
analysis of its ‘footprint’ at the protein surface by mass spectro-
metry. Furthermore, various aldehydes incorporated during
resorcinarene synthesis could be easily varied to allow modular
enhancement of the design, for example by attaching functionality
that may assist in affinity purification.13–15
A
novel octavalent, resorcin[4]arene derived, cross-linker
designed to overcome some of the limitations of commercially
available reagents is significantly more efficient for covalent
stabilisation of protein–protein interactions.
Transient protein–protein interactions regulate a diversity of
cellular responses.1,2 Currently, it is difficult to predict such
interactions a priori from sequence information. Thus, methods to
characterise protein–protein interactions are of significant interest
for cell biology3 as well as the development of small molecule
modulators of such interfaces.4 A comparison of wide-scope
studies of the Saccharomyces cerevisiae protein interactions,
including yeast two-hybrid5,6 and tandem affinity purification,7,8
has highlighted poor overlap of data-sets and the need for an
intersection of data derived from diverse techniques.9
Chemical cross-linking10,11 provides covalent capture of tran-
sient protein–protein interactions and can facilitate topological
analysis using mass spectrometry.12 A limitation of cross-linking
reagents is that tether length and reactivity must be optimized for
individual protein–protein complexes. Furthermore, it is difficult
to discriminate between inter- vs. intra-molecular links or non-
productive modifications arising from protein modification at one
terminus of the cross-linker and, hydrolysis (for example) at the
other. Recently an isotope coding strategy has been used to
facilitate discrimination between some of these outcomes in the
mass spectrum.13 A modular synthetic route that offers rapid
access to a versatile arsenal of cross-linkers has also been reported,
but the optimum reagent for each protein system must be selected
by evaluation of each individual reagent.14
N-Hydroxysuccinimidylester octavalent cross-linker, SOXL 1,
was rapidly synthesised over four linear steps in y40% overall
yield on a gram scale. Reaction of 4-(2-(2-(2-methoxyethoxy)-
ethoxy)ethoxy)benzaldehyde16 with resorcinol in acidic absolute
ethanol at 80 uC furnished the resorcinarene precursor in 78% yield
after recrystallisation from hot ethanol (Scheme 1).17 Subsequent
alkylation of all eight resorcin[4]arene phenols proceeded in good
overall yield of the octaethyl ester 2 using a two molar excess of
ethyl 2-bromoacetate for each phenolate. Saponification of the
resulting ethyl esters provided the octa-acid 3, which was treated
with oxalyl chloride, then N-hydroxysuccinimide (NHS) to yield
the prototypical resorcin[4]arene derived succinimidyl octaester
cross-linker SOXL 1. We found that employing the polymer-
supported base piperidinomethyl polystyrene in this transforma-
tion provided high yields of essentially pure product 1.
Comparison of NMR data with closely related polyether
derivatives initially suggested a flattened cone structure,17 but we
subsequently obtained crystals of the ethyl ester 2 in which the
asymmetric unit contains two independent half molecules each
lying about an inversion centre.18 This structure confirmed our
assignment of the C2h isomer in which adjacent pairs of aldehyde
There remains an unmet need to augment the ratio of cross-
linked compared to surface-labeled protein species.11 A reagent
that does not require optimisation for individual protein systems
would be of general utility. Increasing the efficiency of cross-
derived groups sit on opposite faces of the central macrocycle. The
…
terminal esters occupy a distorted rhomboid geometry, with C
C
˚
distances for the carbonyls ranging between 4.5 and 13.5 A (Fig. 1).
We then set out to compare the octaester 1 with a commercial
homobivalent cross-linker disuccinimidyl suberate, DSS 4, for their
efficiency to cross-link a known protein dimer. Glutathione
S-transferases (GST) [E.C.2.5.1.18]19 exist as homodimers,
although higher order oligomers have also been reported.20
Experiments were performed on the Schistosoma japonicum form
of GST purified from E. coli transformed with the pGEX2T
expression vector.21
linking reagents is therefore
a key design criterion. This
communication describes a novel cross-linker architecture that is
a more efficient cross-linker than a representative panel of
commercial reagents in preliminary comparative experiments.
We envisaged that a resorcinarene scaffold could be elaborated
to display multiple functional groups over a large surface area. In
turn, this should bias the reaction toward inter-protein links by
aSchool of Chemistry, University of Nottingham, University Park,
Nottingham, UK NG7 2RD. E-mail: james.dowden@nottingham.ac.uk;
Fax: 0115 95135654; Tel: 0115 9513566
Aliquots of between 1 and 8 molar equivalents of SOXL 1 were
added to GST solution (2.7 mM), quenched after 1 h and separated
through 10% poly-acrylamide SDS gel, with immunoblotting
providing the principal means for visual inspection of the results. In
the absence of cross-linker, only monomeric GST is apparent
(Fig. 2(a), lane 13). At the same stoichiometry DSS was
bDepartment of Pharmacy and Pharmacology, University of Bath,
Calverton Down, Bath, UK BA2 7AY
{ Electronic supplementary information (ESI) available: General experi-
mental details and characterisation data for compounds 1–3. See DOI:
10.1039/b701542a
2512 | Chem. Commun., 2007, 2512–2514
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Scheme 1 Reagents and conditions: (a) KOH, H2O/THF, rt; (b) (COCl)2, then N-hydroxysuccinimide, piperidinomethyl polystyrene, THF 210 uC.
Fig. 1 One centrosymmetric dimer from octaester 2 crystal structure.18
significantly less effective at achieving measurable cross-linking of
GST. At 2, 4, 6 and 8 molar equivalents of cross-linker to GST,
increased levels of dimer (y56 kDa) are apparent in samples
treated with SOXL 1 (Fig. 2(a), lanes 3, 5, 7 and 9) compared to
DSS 4 (Fig. 2(a), lanes 4, 6, 8 and 10). For SOXL1 treated lanes, an
additional band consistent with addition of at least one copy of
SOXL 1 (y1.6 kDa) to GST monomer (y27 kDa) is apparent.
SOXL 1 treated GST-dimer has a slightly higher molecular weight
Fig. 2 Cross-linking of GST was performed with molar equivalents of
either SOXL 1 (lanes 1, 3, 5, 7, 9, 11) or DSS 5 (lanes 2, 4, 6, 8, 10, 12) as
than that treated with DSS 4, most likely due to the additional
indicated. In lanes 11 and 12 an equimolar mixture of GST and BSA was
molecular weight contributed by SOXL 1. The nature of the higher
molecular weight band in the DSS 4 treated lanes is currently not
clear.
used and a 4-fold excess of cross-linker added. Lanes 13 and 14 represent
GST and BSA alone, respectively, in the absence of added cross-linkers.
(a) Immunoblotting with anti-GST antibody. (b) Immunoblotting
performed on the same gel using anti-BSA antibody.
The improved efficiency is presumably due to presentation of
multiple reactive groups that increase the effective molarity of the
reactive NHS esters at the protein surface. Lanes 3 and 10
(Fig. 2(a)) offer comparative concentrations of NHS ester and
apparently provide similar levels of GST dimer after 1 h. Kinetic
measurements of protein cross linking at relevant concentrations
of buffer are generally difficult,10,22 thus we were unable to
compare the rate of reaction for DSS 4 and SOXL 1 with GST.
Time-course experiments suggest that cross-linking with SOXL 1
but not DSS 4, is faintly detectable after 1 min of reaction,
apparently reaching completion in 30–60 min (data not shown).
The protein concentration and reagent stoichiometry are at the
lower limit typically used for cross-linking experiments.10,22 Cross-
linking with SOXL 1, however, can be observed in experiments
run at lower protein concentrations (0.27 and 0.027 mM), although
detection is rather faint, particularly at 0.027 mM GST (data not
shown). Consistent observation of GST monomer modified with
SOXL 1 suggests that much reagent is consumed by protein rather
than hydrolysis, thus linking efficiency could be further improved.
Multiple reactive groups will increase the chance of reaction
between any protein in addition to the target protein complex.
Ideally, this outcome will be mitigated by the short lifetime of the
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Notes and references
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Fig. 3 Cross-linking of GST was performed with 1, 2 and 4 molar
equivalents of either SOXL 1 (lanes 2, 6 and 10), DSS 4 (lanes 3, 7 and 11),
DSG 5 (lanes 4, 8 and 12), or SAB 6 (lanes 5, 9 and 13), as indicated. Lane
1 contains molecular weight markers only.
reactive ester in water,10 which is the dominant reaction for NHS
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cross-linked by including bovine serum albumin (BSA), which
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then examined by immunoblotting did not display altered GST-
dimer formation (Fig. 2(a), lanes 11 and 12) and importantly, anti-
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linking the GST homodimer than DSS 4. Neither five-carbon
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˚
disuccinimidyl glutarate (DSG, 5 y8 A) or ten-carbon sebacic acid
˚
bis(N-succinimidyl) ester (SAB, 6 y13 A) performed any better
˚
than DSS 4 (y11 A, Fig. 3) and were significantly less efficient
than SOXL 1, which discounts a simple distance effect and
18 Compound 2: (crystallizes with two independent half molecules in the
asymmetric unit), colourless tablet, 1.12 6 1.12 6 0.58 mm, triclinic,
¯
˚
P1, a = 11.7830(8), b = 15.3349(11), c = 31.054(2) A, a = 91.283(2), b =
reinforces the benefit of the multivalent architecture.
3
23
˚
91.879(2), c = 102.979(2)u, V = 5462.3(6) A , Z = 2, Dc = 1.295 g cm
,
Evaluation of SOXL 1 for cross-linking other protein–protein
interactions is underway. Recently, collaborators have used higher
concentrations of SOXL 1 in potentially reactive Tris buffer to
define oligomerisation activity of N-terminal and C-terminal
domains of the Bacillus subtilis DnaD protein.23 Meanwhile, we
are actively exploiting the rapid, adaptable synthesis to achieve
modular improvements to prototype 1, such as other protein
reactive groups, modulation of solubility and incorporation of
biotin for affinity purification. We anticipate that the defined
geometry of SOXL 1 may be useful for topological analysis of
protein complexes by mass spectrometry and are pursuing further
improvement of its architecture to this end, the progress of which
will be described in subsequent publications.
˚
2hmax = 55u, Mo-Ka, l = 0.710 73 A, v scans, T = 150(2) K, 50 321
reflections measured, all 24 449 unique used in the refinement, no
absorption or extinction corrections applied, structure solution by direct
and difference Fourier methods using SHELXS97, structure refinement
used SHELXL97, 1374 parameters, H atoms geometrically placed and
refined using a riding model, R = 0.0758, wR = 0.235, full-matrix least
2
23
.
˚
squares on F , final residual electron density 1.19 and 20.75 e A
CCDC 297190. For crystallographic data in CIF or other electronic
format see DOI: 10.1039/b701542a.
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This work was supported by grants from BBSRC (E18957) and
Wellcome Trust (069955MA). We acknowledge the use of the
EPSRC Chemical Database Service at Daresbury.
2514 | Chem. Commun., 2007, 2512–2514
This journal is ß The Royal Society of Chemistry 2007