Organocatalysts of oxidative protein folding inspired by protein disulfide isomerase

Article abstract of DOI:10.1039/c4ob01738b

Organocatalysts derived from diethylenetriamine effect the rapid isomerization of non-native protein disulfide bonds to native ones. These catalysts contain a pendant hydrophobic moiety to encourage interaction with the non-native state, and two thiol gro

Full text of DOI:10.1039/c4ob01738b

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
COMMUNICATION
Organocatalysts of oxidative protein folding
inspired by protein disulfide isomerase†
Cite this: Org. Biomol. Chem., 2014,
2, 8598
1
a
b
c
a,c
John C. Lukesh III,‡ Kristen A. Andersen,‡ Kelly K. Wallin and Ronald T. Raines*
Received 14th August 2014,
Accepted 17th September 2014
DOI: 10.1039/c4ob01738b
www.rsc.org/obc
Organocatalysts derived from diethylenetriamine effect the rapid
isomerization of non-native protein disulfide bonds to native ones.
These catalysts contain a pendant hydrophobic moiety to encou-
rage interaction with the non-native state, and two thiol groups
a
with low pK values that form a disulfide bond with a high E°’
value.
The formation of native disulfide bonds is at the core of oxi-
1
–4
dative protein folding.
In oxidizing environments, reduced
proteins with multiple cysteine residues tend to oxidize rapidly
and nonspecifically. To attain a proper three-dimensional fold,
any non-native disulfide bonds must isomerize to the linkages
5
found in the native protein. In eukaryotic cells, this process is
mediated by the enzyme protein disulfide isomerase (PDI; EC
4
,6–14
5
.3.4.1).
Catalysis of disulfide-bond isomerization by PDI involves Fig. 1 Putative mechanism for catalysis of protein-disulfide isomeriza-
thiol–disulfide interchange chemistry. A putative mechanism tion by protein disulfide isomerase (PDI) and small-molecule dithiol
catalysts.
commences with the nucleophilic attack by a thiolate on a
non-native disulfide bond, generating a mixed-disulfide and a
1
5
new substrate thiolate (Fig. 1). This thiolate can then attack
another non-native disulfide bond, inducing further rearrange-
ments to achieve the stable native state. The ability of PDI
to catalyze disulfide-bond isomerization (rather than dithiol
oxidation) makes the enzyme essential to the viability of the
1
2,17,18
substrate binding.
The physicochemical properties of
its active-site make PDI an ideal catalyst for the reshuffling
of disulfide bonds in misfolded proteins. The deprotonated
thiolate of its N-terminal active-site cysteine residue (CGHC)
7
,16
yeast Saccharomyces cerevisiae.
1
9
initiates catalysis (Fig. 1). The amount of enzymic thiolate
PDI is abundant in the endoplasmic reticulum (ER) of
2
0,21
present is dependent on two factors.
active-site cysteine residue; the other is the reduction potential
E°′) of the disulfide bond formed between the two active-site
cysteine residues. In PDI, the cysteine pK is 6.7, and the
One is the pK of the
a
eukaryotic cells. The enzyme contains four domains: a, a′, b,
1
2
and b′. The a and a′ domains each contain one active-site
CGHC motif—a pattern analogous to that in many other oxido-
reductases, whereas the b and b′ domains appear to mediate
(
a
2
2,23
disulfide E°′ is −0.18 V.
Given the properties of the ER
(
pH 7.0; Esolution = –0.18 V), 1/3 of PDI active sites contain a
1
6,24
reactive thiolate.
Moreover, the high (less negative)
a
Department of Chemistry, University of Wisconsin–Madison, 1101 University
reduction potential of PDI renders the protein as a weak
disulfide-reducing agent, ensuring that ample time is available
for the catalyst to rearrange all of the disulfide bonds before
reducing its protein substrate to “escape” (Fig. 1). If necessary,
however, the second active-site cysteine residue can engage
to rescue the enzyme from non-productive mixed-disulfide
Avenue, Madison, WI 53706, USA
b
Molecular & Cellular Pharmacology Graduate Training Program, University of
Wisconsin–Madison, 1300 University Avenue, Madison, WI 53706, USA
c
Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive,
Madison, WI 53706, USA
†
Electronic supplementary information (ESI) available: Synthetic and analytical
procedures. See DOI: 10.1039/c4ob01738b
These authors contributed equally to this work.
7
,25,26
‡
intermediates.
8598 | Org. Biomol. Chem., 2014, 12, 8598–8602
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Communication
Efficient oxidative protein folding requires a redox environ- disulfide E°′ values. Moreover, they did not mimic a hallmark
4
3
ment that supports both thiol oxidation and disulfide-bond of enzymic catalysts—binding to the substrate.
isomerization. In vitro and in cellulo, this environment can be The b and b′ domains of PDI have an exposed hydrophobic
provided by a redox buffer consisting of reduced and oxidized patch. The two patches unite to form a continuous hydro-
1
0,12,13,44,45
glutathione. For example, the oxidative folding of a favorite phobic surface between the two active sites.
model protein, bovine pancreatic ribonuclease (RNase A; EC hydrophobic surface could entice PDI to bind to unfolded or
.1.27.5), occurs readily in the presence of 1 mM glutathione misfolded proteins, which tend to expose more hydrophobic
This
3
2
7
46
(
GSH) and 0.2 mM oxidized glutathione (GSSG). Adding PDI residues than do proteins in their native state. Accordingly,
accelerates the process, but the large-scale use of PDI as a we set out to design organocatalysts that not only have low
catalyst for folding proteins in vitro is impractical due to its thiol pK and high disulfide E°′ values but also emulate sub-
a
high cost and conformational instability, and the complexity strate binding by PDI. We were inspired by the demonstrated
imposed by its separation from a substrate protein. Accord- ability of the hydrophobic effect to induce proximity in
ingly, the development of small-molecule PDI mimics has aqueous solution and thereby accelerate a variety of chemical
4
7,48
49,50
become a high priority.
reactions, such as O→N acyl transfer,
ester hydrolysis,
5
1,52
To date, most PDI mimics have been designed to replicate and dithiol oxidation.
We reasoned that analogous
the physicochemical properties of the CGHC active site—low induced proximity could enhance disulfide-bond isomeriza-
2
8
thiol pKa and high disulfide E°′. Previously, we reported tion in a misfolded protein, which is the key step in oxidative
7
,16
on (±)-trans-1,2-bis(mercaptoacetamido)cyclohexane (1; BMC) protein folding.
Fig. 2), a small molecule that catalyzes the formation of native We reasoned that dithiol 2 (Fig. 2) would provide an appro-
disulfide bonds in proteins, both in vitro and in cellulo. In priate scaffold for the development of useful catalysts. We were
(
2
9
2
005, other workers screened 14 reagents for their ability to drawn to dithiol 2 for three reasons. First, its mercaptoacet-
2
9,53
a
fold a variety of proteins, and concluded that BMC was the amido groups are known to have low thiol pK values.
3
0
best of known small-molecule catalysts. Though effective, Secondly, the disulfide bond of its oxidized form resides in a
BMC has shortcomings. For example, its low disulfide E°′ large, 13-membered ring containing two secondary amides,
renders the compound too reducing for optimal catalysis of which should lead to a high reduction potential. Finally,
disulfide-bond isomerization. Subsequently, various CXXC and dithiol 2 has an amino group that can be condensed with
CXC peptides, aromatic thiols, and selenium-based catalysts hydrophobic carboxylic acids to mimic the b and b′ domains
3
1–42
were developed and employed with some success.
Never- of PDI.
theless, these organocatalysts had non-optimal thiol pK and
Our experimental work commenced with the synthesis of
a
dithiol 2 from diethylenetriamine in a few high-yielding steps
(
see: ESI†). To determine its thiol pK
a
values, we monitored its
2
9,54
A238 nm as a function of pH.
We found pK values of 8.0 ±
a
0.2 and 9.2 ± 0.1 (Table 1). These values are slightly less than
those of BMC, presumably due to the additional electronega-
tive nitrogen atom. To determine the reduction potential of its
Table 1 Properties of PDI and mimics 1–8
Folding
yield (%)
a
b
Catalyst
pK
a
Disulfide E°′
log P
(
None)
— c
—
—
—
—
0.10
−0.74
0.66
1.67
0.90
1.82
2.06
45 ± 2
87 ± 2
42 ± 2
50 ± 2
45 ± 2
54 ± 4
57 ± 1
60 ± 2
66 ± 2
ND
PDI
6.7
−0.180 V
−0.232 V
−0.192 V
ND
ND
−0.203 V
ND
d
1
2
3
4
5
6
7
8
(BMC)
8.3; 9.9
8.0; 9.2
ND
ND
8.1; 9.3
ND
8.1; 9.4
ND
−0.206 V
ND
a
Values were calculated for dimethylamine in dithiol 2 and the tertiary
amide moiety in dithiols 3–8 (e.g., N,N-dimethylacetamide for dithiol
with software from Molinspiration (Slovenský Grob, Slovak
3
)
6
3 b
Republic), and are similar to known experimental values.
Values are
for the unscrambling of sRNase A to give native RNase A by 1 mM
c
catalyst in 5 h, as in Fig. 4. Value for the N-terminal cysteine residue
Fig. 2 Small-molecule PDI mimics synthesized and assessed in this in the active site of PDI.24 Values are from ref. 29. ND, not
d
study.
determined.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 8598–8602 | 8599

View Article Online
Communication
Organic & Biomolecular Chemistry
Fig. 3 Scheme showing the connectivity of the four disulfide bonds in
native RNase A. There are 104 other fully oxidized forms.
oxidized form, we equilibrated equimolar amounts of dithiol 2
and oxidized β-mercaptoethanol, and quantified the amount
of each reduced and oxidized species with analytical
2
9,55
HPLC.
We found a disulfide E°′ value of (−0.192 ± 0.003) V.
This value indicates that dithiol 2 is a weaker reducing
agent than is BMC, which is consistent with BMC being more
preorganized for disulfide-bond formation. Finally, to probe
the effect of increasing hydrophobicity on catalyzing the for-
mation of native disulfide bonds in proteins, we synthesized
dithiols 3–8. We isolated dithiols 3–6 as colorless oils, and
dithiols 7 and 8 as white solids. None had a strong odor.
Enzymatic catalysis provides an extremely sensitive measure
5
6
of native protein structure. RNase A contains eight cysteine
residues, which could form 105 (= 7 × 5 × 3 × 1) distinct fully
oxidized species, only one of which gives rises to enzymatic
5
7,58
activity (Fig. 3).
panel of compounds to catalyze the isomerization of
scrambled” RNase A (sRNase A), which is a random mixture
Accordingly, we tested the ability of this
“
of oxidized species, to its native state. The isomerization reac-
tion was monitored by measuring the gain of catalytic
5
9
activity. Dithiol 8 was excluded from the analysis due to its
low solubility in aqueous solution.
Some, but not all, of the PDI mimics led to a significant
increase in the yield of oxidative protein folding (Fig. 4A). Most
notably, the data with dithiols 2–7 revealed an overall trend
toward higher yield with increasing hydrophobicity of the
pendant carboxamide (Fig. 4B). This trend culminated with
dithiol 7, which increased the yield of folded RNase A by 47%
Fig. 4 Catalysis of disulfide-bond isomerization by PDI and PDI mimics
1
–7. (A) Graph of the time-course for the isomerization of sRNase A to
give native RNase A. All assays were performed in triplicate at 30 °C in
0 mM Tris–HCl buffer, pH 7.6, containing GSH (1.0 mM), GSSG
5
compared to that in the absence of a catalyst. These data con- (0.2 mM), and PDI or dithiol 1–7 (1.0 mM). (B) Graph of the yield of
trast markedly with those using monothiols (e.g., glutathione), native RNase A achieved by PDI mimics 2–7 after 5 h as a function of the
log P value of its side chain (Table 1).
which reduce the yield of properly folded protein by favoring
the accumulation of mixed-disulfide species.
2
7
The apparent correlation of catalytic efficacy with hydropho-
bicity could be due to a physicochemical property other than itself. We note, however, that the molecular mass of PDI
2
hydrophobicity. Accordingly, we determined the thiol pK and (57 kDa) is >10 -fold greater than any of its mimics, enabling
a
disulfide E°′ values of the most efficacious dithiols containing optimization of substrate binding and turnover beyond that
an alkyl (5) and aryl (7) carboxamide. We found dithiol 5 to attainable with small-molecule catalysts. Also, each molecule
have thiol pK values of 8.1 and 9.3 and a disulfide E°′ value of of PDI has two active sites, and thus provides a higher concen-
a
−0.203 V (Table 1). We found dithiol 7 to have similar physico- tration of dithiol than do the organocatalysts.
chemical properties, with thiol pK values of 8.1 and 9.4 and a
a
Like the substrate-binding domains of PDI, the hydrophobi-
disulfide E°′ value of −0.206 V. Both of these compounds city of dithiols 4–7 likely encourages their interaction
possess thiol acidity and disulfide stability similar to those of with unfolded or misfolded proteins.10,12,13,44,45,60,61 Dithiols
parent dithiol 2, affirming that hydrophobicity is indeed corre- having moieties with higher log P values perform better, and
lative with catalytic efficacy.
aromatic moieties seem to be especially efficacious (Fig. 4B).
Our data are the first to indicate that adding a hydrophobic We note that a more hydrophobic catalyst could also increase
moiety to a small-molecule PDI mimic can have a profound the rate of the underlying thiol–disulfide interchange chemi-
effect on its ability to catalyze disulfide-bond isomerization. stry, as nonpolar environments are known to lower the free
6
2
Still, none of the organocatalysts were as efficacious as PDI energy of activation for this reaction.
8600 | Org. Biomol. Chem., 2014, 12, 8598–8602
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Communication
1
1 E. A. Kersteen and R. T. Raines, Antioxid. Redox Signaling,
Conclusions
2003, 5, 413–424.
We have designed, synthesized, and characterized novel 12 G. Tian, S. Xiang, R. Noiva, W. J. Lennarz and
organocatalysts that enhance the efficiency of oxidative protein H. Schindelin, Cell, 2006, 124, 61–73.
folding. Moreover, we have demonstrated that increasing the 13 C. W. Gruber, M. Cemazar, B. Heras, J. L. Martin and
hydrophobicity of the catalysts has a marked effect on their D. J. Craik, Trends Biochem. Sci., 2006, 31, 455–464.
catalytic efficacy. The production of proteins that contain 14 A. Y. Denisov, P. Maattanen, C. Dabrowski, G. Kozlov,
disulfide bonds by recombinant DNA technology often leads
D. Y. Thomas and K. Gehring, FEBS J., 2009, 276, 1440–
1449.
6
4,65
to the aggregation of misfolded proteins.
These aggregates
must be reduced, denatured, and solubilized to enable proper 15 E. A. Kersteen, S. R. Barrows and R. T. Raines, Biochemistry,
6
6
folding. Approximately 20% of all human proteins and many
proteins of high pharmaceutical relevance
2005, 44, 12168–12178.
6
7,68
contain at least 16 P. T. Chivers, M. C. A. Laboissière and R. T. Raines, EMBO
one disulfide bond between cysteine residues. For example,
J., 1996, 15, 2659–2667.
antibodies contain at least 12 intrachain and 4 interchain 17 A. Holmgren, J. Biol. Chem., 1979, 254, 9627–9632.
6
9
disulfide bonds, and there are >300 distinct antibodies in 18 J. C. Edman, L. Ellis, R. W. Blacher, R. A. Roth and
7
0
clinical development, including ∼30 antibody–drug conju-
W. J. Rutter, Nature, 1985, 317, 267–270.
19 N. J. Darby and T. E. Creighton, Biochemistry, 1995, 34,
3576–3587.
7
1
7,16
gates. The ability to mimic the essential function of PDI
in a small molecule could have a favorable impact on the
production of antibodies and other biologics, and usher in a 20 H. F. Gilbert, Adv. Enzymol., 1990, 63, 69–172.
new genre of organocatalysts for oxidative protein folding.
21 P. T. Chivers, K. E. Prehoda and R. T. Raines, Biochemistry,
997, 36, 4061–4066.
2 H. C. Hawkins and R. B. Freedman, Biochem. J., 1991, 275,
35–339.
3 J. Lundstrom and A. Holmgren, Biochemistry, 1993, 32,
6649–6655.
1
2
2
3
Acknowledgements
K.A.A. was supported by a predoctoral fellowship from the
PhRMA Foundation and by Molecular and Cellular Pharma- 24 C. Hwang, A. J. Sinskey and H. F. Lodish, Science, 1992,
cology Training Grant T32 GM008688 (NIH). This work was 257, 1496–1502.
supported by grant R01 GM044783 (NIH). NMR spectra were 25 K. W. Walker, M. M. Lyles and H. F. Gilbert, Biochemistry,
obtained at NMRFAM, which is supported by grant P41
GM103399 (NIH).
1996, 35, 1972–1980.
26 K. W. Walker and H. F. Gilbert, J. Biol. Chem., 1997, 272,
8
845–8848.
7 M. M. Lyles and H. F. Gilbert, Biochemistry, 1991, 30, 619–
25.
2
2
6
Notes and references
8 W. J. Lees, Curr. Opin. Chem. Biol., 2008, 12, 740–
1
Biochemistry of the SH Group: The Occurence, Chemical
745.
Properties, Metabolism and Biological Function of Thiols and 29 K. J. Woycechowsky, K. D. Wittrup and R. T. Raines, Chem.
Disulfides, ed. P. C. Jocelyn, London, U.K., 1972. Biol., 1999, 6, 871–879.
Oxidative Folding of Peptides and Proteins, ed. J. Buchner 30 M. S. Willis, J. K. Hogan, P. Prabhakar, X. Liu,
2
and L. Moroder, The Royal Society of Chemistry,
Cambridge, UK, 2009.
K. Tsai, Y. Wei and T. Fox, Protein Sci., 2005, 14, 1818–
1826.
3
4
M. Lindahl, A. Mata-Cabana and T. Kieselbach, Antioxid. 31 K. J. Woycechowsky, B. A. Hook and R. T. Raines, Biotech-
Redox Signaling, 2011, 14, 2581–2642. nol. Prog., 2003, 19, 1307–1314.
O. B. Oka and N. J. Bulleid, Biochim. Biophys. Acta, 2013, 32 K. J. Woycechowsky and R. T. Raines, Biochemistry, 2003,
1
833, 2425–2429.
42, 5387–5394.
5
6
C. B. Anfinsen, Science, 1973, 181, 223–230.
A. S. Robinson, V. Hines and K. D. Wittrup, Biotechnology,
33 J. D. Gough, J. M. Gargano, A. E. Donofrio and W. J. Lees,
Biochemistry, 2003, 42, 11787–11797.
34 J. D. Gough and W. J. Lees, J. Biotechnol., 2005, 115, 279–
290.
35 J. D. Gough and W. J. Lees, Bioorg. Med. Chem., 2005, 15,
777–781.
1
994, 12, 381–384.
7
8
M. C. A. Laboissière, S. L. Sturley and R. T. Raines, J. Biol.
Chem., 1995, 270, 28006–28009.
Prolyl Hydroxylase, Protein Disulfide Isomerase, and Other
Structurally Related Proteins, ed. N. A. Guzman, Marcel 36 J. D. Gough, E. J. Barrett, Y. Silva and W. J. Lees, J. Biotech-
Dekker, New York, NY, 1998. nol., 2006, 125, 39–47.
K. J. Woycechowsky and R. T. Raines, Curr. Opin. Chem. 37 J. Beld, K. J. Woycechowsky and D. Hilvert, Biochemistry,
Biol., 2000, 4, 533–539. 2008, 47, 6985–6987.
0 R. B. Freedman, P. Klappa and L. W. Ruddock, EMBO Rep., 38 J. Beld, K. J. Woycechowsky and D. Hilvert, Biochemistry,
002, 3, 136–140. 2009, 48, 4662–4662.
9
1
2
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 8598–8602 | 8601

View Article Online
Communication
Organic & Biomolecular Chemistry
3
4
4
9 J. Beld, K. J. Woycechowsky and D. Hilvert, J. Biotechnol., 54 J. C. Lukesh, M. J. Palte and R. T. Raines, J. Am. Chem. Soc.,
010, 150, 481–489. 2012, 134, 4057–4059.
0 G. Z. Wang, X. Y. Dong and Y. Sun, Biochem. Eng. J., 2011, 55 G. V. Lamoureux and G. M. Whitesides, J. Org. Chem., 1993,
5, 169–175. 58, 633–641.
1 A. S. Patel and W. J. Lees, Bioorg. Med. Chem., 2012, 20, 56 J. R. Knowles, Science, 1987, 236, 1252–1258.
2
5
1
020–1028.
57 R. T. Raines, Chem. Rev., 1998, 98, 1045–1066.
58 T. A. Klink, K. J. Woycechowsky, K. M. Taylor and
R. T. Raines, Eur. J. Biochem., 2000, 267, 566–572.
59 B. R. Kelemen, T. A. Klink, M. A. Behlke, S. R. Eubanks,
P. A. Leland and R. T. Raines, Nucleic Acids Res., 1999, 27,
3696–3701.
4
4
2 W. J. Lees, ChemBioChem, 2012, 13, 1725–1727.
3 W. R. Jencks, Catalysis in Chemistry and Enzymology,
McGraw–Hill, New York, NY, 1969.
4 S. R. Shouldice, B. Heras, P. M. Walden, M. Totsika,
M. A. Schembri and J. L. Martin, Antioxid. Redox Signaling,
4
4
2
011, 14, 1729–1760.
60 L. W. Guddat, J. C. Bardwell, T. Zander and J. L. Martin,
Protein Sci., 1997, 6, 1148–1156.
5 F. X. Kober, W. Koelmel, J. Kuper, J. Drechsler, C. Mais,
H. M. Hermanns and H. Schindelin, J. Biol. Chem., 2013, 61 H. F. Gilbert, J. Biol. Chem., 1997, 272, 29399–29402.
2
88, 2029–2039.
62 P. A. Fernandes and M. J. Ramos, Chem. – Eur. J., 2004, 10,
257–266.
4
4
6 L. Lins and R. Brasseur, FASEB J., 1995, 9, 535–540.
7 J. R. Knowles and C. A. Parsons, Chem. Commun., 1967, 63 J. Sangster, J. Phys. Chem. Ref. Data, 1989, 18, 1111–1227.
55–757. 64 F. A. O. Marston, Biochem. J., 1986, 240, 1–12.
8 C. A. Blyth and J. R. Knowles, J. Am. Chem. Soc., 1971, 93, 65 E. De Bernardez Clark, Curr. Opin. Biotechnol., 1998, 9,
017–3021. 157–163.
9 J. R. Knowles and C. A. Parsons, Nature, 1969, 221, 5553– 66 P. L. Martelli, P. Fariselli and R. Casadio, Proteomics, 2004,
554. 4, 1665–1671.
0 C. A. Blyth and J. R. Knowles, J. Am. Chem. Soc., 1971, 93, 67 R. C. Fahey, J. S. Hunt and G. C. Windham, J. Mol. Evol.,
021–3027. 1977, 10, 155–160.
1 G. Z. Wang, X. Y. Dong and Y. Sun, Biotechnol. Prog., 2011, 68 J. M. Thorton, J. Mol. Biol., 1981, 151, 261–287.
7, 377–385. 69 H. Liu and K. May, mAbs, 2012, 4, 17–23.
2 H. Liu, X. Y. Dong and Y. Sun, Biochem. Eng. J., 2013, 79, 70 Pharmaceutical Research and Manufacturers of America
7
4
4
5
5
5
5
3
5
3
2
2
9–32.
3 W. J. Lees, R. Singh and G. M. Whitesides, J. Org. Chem.,
991, 56, 7328–7331.
report “Medicines in Development—Biologics, 2013
Report”.
1
71 A. Mullard, Nat. Rev. Drug Discov., 2013, 12, 329–332.
8602 | Org. Biomol. Chem., 2014, 12, 8598–8602
This journal is © The Royal Society of Chemistry 2014