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Communication
4S-Hydroxylation of insulin at ProB28 accelerates hexamer dissociation and delays fibrillation
Seth Lieblich, Katharine Fang, Jackson Kenai Blender Cahn, Jeffrey Rawson, Jeanne LeBon, H. Teresa Ku, and David A. Tirrell
J. Am. Chem. Soc.
,
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• Publication Date (Web): 09 Jun 2017
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0
100
200
300
400
500
600
0
60
120
180
Blood glucose concentration (mg/dL)
Time (min)
Vector
ProI
HzpI
HypI
AspI
C
RAI
Aspart
C
-
terminus
ProB28
ThrB27
TyrB26
Zn
2+
Zn
2+
Zn
2+
Aggregates
Ph
Ph
Ph
Ph
Ph
Ph
R
6
hexamer
(pharmaceutical
formulation)
T
6
hexamer
T
2
dimer
monomer
2
Ph
A
D
B
LysB29
N
H
OH
O
N
H
OH
O
HO
N
H
OH
O
HO
1
2
3
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3
B
C
A
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D
R
6
Insulin
PDB: 1ZNJ
Glu21 &
Gly20
Backbone
Glu21 &
Gly20
Backbone
Glu21 &
Gly20
Backbone
3.6 Å
3.8 Å
2.74 Å
4.5 Å
6.2
Å
4 Å
H
2
O
2.87 Å
3.3 Å
Glu21 &
Gly20
Backbone
3.3 Å
2.8 Å
Glu21 &
Gly20
Backbone
4.6 Å
3.5 Å
4.3 Å
A
T
2
Insulin
PDB: 3T2A
B
T
2
HzpI
PDB: 5HQI
C
T
2
HypI
PDB: 5HPR
E
R
6
HzpI
PDB: 5HRQ
F
R
6
HypI
PDB: 5HPU
4
Glu21 &
Gly20
Backbone
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4S
-Hydroxylation of insulin at ProB28 accelerates hex
amer
dissociation and delays fibrillation
Seth A. Lieblich
†,‡
, Katharine Y. Fang
†,‡
, Jackson K. B. Cahn
†
, Jeffrey Rawson
§,||
, Jeanne LeBon
§
, H.
Teresa Ku
§,||,¶
, David A. Tirrell
†,*
†Division of Chemistry and Chemical Engineering, Ca
lifornia Institute of Technology, Pasadena, CA 9112
5, USA.
§
Department of Translational Research and Cellular T
herapeutics, Diabetes and Metabolism Research Insti
tute, City of
Hope, Duarte, CA 91010, USA.
||
Beckman Research Institute of City of Hope, Duarte,
CA 91010, USA.
¶
Irell & Manella Graduate School of Biological Scien
ces, City of Hope, Duarte, CA 91010, USA.
‡
Authors contributed equally to this work.
*Corresponding author. Email:
tirrell@caltech.edu
Abstract.
Daily injections of insulin provide lifesaving ben
efits to
millions of diabetics. But currently available pran
dial insulins are
suboptimal: The onset of action is delayed by slow
dissociation of
the insulin hexamer in the subcutaneous space, and
insulin forms
amyloid fibrils upon storage in solution. Here we s
how, through
the use of non>canonical amino acid mutagenesis, th
at
replacement of the proline residue at position 28 o
f the insulin B>
chain (ProB28) by (4
S
)>hydroxyproline (Hzp) yields an active
form of insulin that dissociates more rapidly, and
fibrillates more
slowly, than the wild>type protein. Crystal structu
res of dimeric
and hexameric insulin preparations suggest that a h
ydrogen bond
between the hydroxyl group of Hzp and a backbone am
ide
carbonyl positioned across the dimer interface may
be responsible
for the altered behavior. The effects of hydroxylat
ion are
stereospecific; replacement of ProB28 by (4
R
)>hydroxyproline
(Hyp) causes little change in the rates of fibrilla
tion and hexamer
disassociation. These results demonstrate a new app
roach that
fuses the concepts of medicinal chemistry and prote
in design, and
paves the way to further engineering of insulin and
other
therapeutic proteins.
Blood glucose levels are tightly controlled in mamm
als through
a sensitive regulatory system mediated by insulin,
a 51>amino
acid endocrine hormone composed of two disulfide>li
nked
polypeptide chains (designated A and B). Upon bindi
ng to its
receptor, insulin initiates a signaling cascade tha
t accelerates
glucose uptake and glycogen production. In diabetic
patients, this
system malfunctions, and glucose levels must be con
trolled
through subcutaneous injections of insulin
1
. The
C
>terminus of
the B>chain is important in mediating dimerization
of the
hormone
2>3
, and the flexibility of the B>chain
C
>terminus is
believed to contribute to aggregation through forma
tion of
amyloid fibrils
4>6
. Pharmaceutical formulations of insulin are
stabilized with respect to fibrillation by addition
of zinc and
phenolic preservatives, which drive assembly of the
R
6
hexamer
(Fig. 1a)
7>9
. The R
6
form of insulin is inactive and dissociates
slowly to its active monomeric form after subcutane
ous injection;
the lag time for dissociation delays the onset of a
ction
10
. Mutation
of ProB28 yields rapid>acting insulins (RAIs) by di
srupting
contacts that are critical for dimer formation
8
, but replacement of
Pro through conventional mutagenesis also increases
the
flexibility and perturbs the trajectory of the prot
ein backbone
(Fig. 1B). We sought a means to disrupt the dimer i
nterface
without releasing the conformational constraints ch
aracteristic of
proline by using non>canonical amino acid (ncAA) mu
tagenesis
11>
13
. Specifically, we introduced hydroxyl groups at th
e 4>position
of ProB28 (Fig. 1B, C) by replacing Pro with Hzp or
Hyp. In
addition to introducing a polar functional group an
d the capacity
for hydrogen>bonding (including transannular hydrog
en bonding),
hydroxylation at the 4>position is known to alter t
he
endo
/
exo
preference of the pyrrolidine ring and the
cis
/
trans
equilibrium of
the backbone amide bond
14>16
.
We expressed modified proinsulins (PIs) in the prol
ine>
auxotrophic
E. coli
strain CAG18515 in M9 minimal media
supplemented with Hyp or Hzp. The extent of replace
ment of Pro
by either Hyp or Hzp was approximately 90%
17>18
as determined
by matrix>assisted laser desorption/ionization mass
spectrometry
(MALDI>MS; fig. S1). Denatured PIs were purified by
Ni>NTA
affinity chromatography in yields of 32 mg/L for Hz
p>PI and 29
mg/L for Hyp>PI (table S1) from the inclusion body
fraction. The
PIs were refolded and cleaved with trypsin and carb
oxypeptidase
B
19
. The resulting mature insulins were purified by r
eversed
phase HPLC
18>19
, and proper proteolytic processing of each
variant was verified by MALDI>MS (table S1). Wild>t
ype insulin
(ProI) and RAI Aspart (AspI, in which ProB28 is rep
laced by
aspartic acid) were produced similarly. All of the
variants caused
similar reductions in blood glucose upon subcutaneo
us injection
into diabetic mice (Fig. 1D)
2, 20>22
. RAIs cannot be distinguished
from ProI in rodent models
23
.
In the absence of Zn
2+
and phenolic preservatives, insulins
dimerize with a dissociation constant (K
D
) of approximately 10
μM. In contrast, K
D
for RAIs is typically >500 μM, and it is
believed that destabilization of the dimer interfac
e causes the
accelerated onset of insulin action after subcutane
ous injection
20,
24>25
. Monomeric forms of insulin give rise to character
istic
circular dichroism (CD) spectra with distinct minim
a at 208 and
222 nm
26>27
(e.g., AspI; Fig. 2A). Dimerization causes a loss
of
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Figure. 1
.
Hydroxyinsulins retain activity.
(
A
)
Schematic of hexamer disassembly (adapted from mech
anism previously described
8
).
Phenolic ligand (Ph), zinc ion (Zn
2+
), insulin monomer (triangle). Darker shading indic
ates the R>state of the hexamer. (
B
) Structures of
the B>chain
C
>termini of wild>type insulin (ProI) and RAI Aspart
(AspI). (
C
)
Chemical structures of L>proline (
1
), (
2S,4S
)>hydroxyproline
(Hzp;
2
), (
2S,4R
)>hydroxyproline (Hyp;
3
). (
D
)
Reduction of blood glucose following subcutaneous i
njection of 35 Pg/kg insulins into
streptozotocin>induced diabetic mice. Glucose level
s were measured post>injection via tail vein sampli
ng. ProI, AspI, HzpI, HypI or vector
were formulated as described
22
. Error bars denote one standard deviation (n = 3).
Figure. 2.
Hydroxylation at ProB28 modulates insulin dimerizat
ion, dissociation kinetics, and stability.
(
A
)
Far UV CD spectra
collected on 60 PM insulins in 10 mM phosphate buff
er, pH 8.0 at 25°C. (
B
)
Insulin hexamer dissociation following sequestratio
n of Zn
2+
by terpyridine. Zn
2+
>(terpy) signal was monitored at 334 nm and fitted
to a mono>exponential decay. HzpI and HypI contain
10% ProI.
The curves for HypI and ProI are indistinguishable
in this plot.
(
C
) Representative fibrillation curves for 60 μM insu
lins (37°C, 960 RPM;
n=4). Insulin fibrils were detected by the rise in
Thioflavin T (ThT) fluorescence that accompanies bi
nding to fibrillar aggregates.
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Figure. 3. Crystal structures of HzpI and HypI.
In tan (left), wt insulins from PDB (3T2A, 1ZNJ) hi
ghlighting the distance between the
carbon atom at the 4
th
position of ProB28 and its closest neighbors, back
bone carbonyl oxygen atoms of GlyB20’ and GluB21 in
the T
2
dimer (
A
) and R
6
hexamer (
D
) forms. In grey (middle), HzpI in the T
2
dimer (
B
) and R
6
hexamer (
E
) forms. In blue (right), HypI in the T
2
dimer (
C
) and R
6
hexamer (
F
) forms.
negative ellipticity at 208 nm
27
(e.g., ProI; Fig. 2A). At a
concentration of 60 μM, HypI appears to be monomeri
c (with a
CD spectrum nearly identical to that of AspI; Fig.
2A) while the
spectrum of HzpI suggests a dimeric insulin (Fig. 2
A).
Sedimentation velocity (SV) and sedimentation equil
ibrium (SE)
experiments were consistent with the results of the
CD analysis
(fig. S3). SE data were fitted to a model of monome
r>dimer>
hexamer self>association (SEDPHAT)
28>29
, and yielded
monomer>dimer dissociation constants (K
D
) of >200 μM and 25
μM for HypI and HzpI, respectively.
Previous studies of RAIs have shown that destabiliz
ation of the
dimer interface correlates with accelerated dissoci
ation of the
hexamer and rapid onset of insulin action
8, 13
. Triggered
dissociation of Zn
2+
>hexamers by addition of terpyridine
30
revealed nearly identical rates of dissociation for
HypI and ProI,
(
1/2
= 87.0 ± 10 s and 90.4 ± 4.2 s, respectively; Fig.
2B and fig.
S4) while HzpI exhibited kinetics similar to those
of AspI (
1/2
=
53.6 ± 3.7 s and 42.7 ± 4.3 s, respectively; Fig. 2
B and fig. S4).
We found these results surprising – replacement of
Pro by Hyp
destabilizes the dimer but has essentially no effec
t on hexamer
dissociation, while introduction of Hzp causes litt
le change in
dimer stability but a substantial increase in the r
ate of hexamer
disassembly.
Each of the insulin variants was subjected to fibri
llation lag
time analysis (Fig. 2C)
31
. We found similar times to onset of
fibrillation for HypI, ProI and AspI; in contrast,
HzpI is markedly
more resistant to aggregation, with a mean time to
onset more
than three>fold longer than that observed for ProI.
The behavior of
HzpI is especially striking, in that it combines fa
st hexamer
dissociation with enhanced stability toward fibrill
ation.
Each subunit in the insulin hexamer adopts one of t
wo
conformational states (T or R), depending on the co
ncentration of
phenolic ligand (Fig. 1A)
13
. Pharmaceutical formulations are
prepared in the more stable R
6
form, whereas the T>state is
observed in the absence of phenolic ligands, most c
ommonly in
the form of T
2
>dimers
32
. To elucidate the molecular origins of the
dissociation and fibrillation behavior of HypI and
HzpI, we
examined crystal structures of both states.
Hydroxylation at ProB28 does not cause substantial
perturbation of the overall insulin structure (Fig.
3, fig. S5). In
comparison with ProI, the backbone RMSD values of H
ypI and
HzpI are 0.31 Å (T
2
> HypI), 0.44 Å (T
2
> HypI), 0.38 Å (R
6
> HypI)
and 0.69 Å (R
6
> HypI)
33
. The most notable feature of the HzpI
structures is the proximity of the hydroxyl group o
f Hzp to the
backbone carbonyl oxygen atom of GluB21′, which lie
s across the
dimer interface (denoted by prime; Fig. 3B, E). The
inter>oxygen
distances (2.8 Å in the T
2
structure, 2.7 Å in R
6
), are consistent
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