Solutionstructureofhumanproinsulin C-peptide
Claudia Elisabeth Munte
1
, Luciano Vilela
2
, Hans Robert Kalbitzer
3
and Richard Charles Garratt
1
1 Instituto de Fı
´
sica de Sa˜o Carlos, Universidade de Sa˜ o Paulo, Sa˜ o Carlos, Brazil
2 Biomm S.A., Montes Claros, Brazil
3 Institut fu
¨
r Biophysik und Physikalische Biochemie, Universita
¨
t Regensburg, Germany
C-peptide is an enzymatic cleavage product that arises
from proinsulin during maturation in the b cells of the
islets of Langerhans [1,2]. Two endopeptidases cleave
the proinsulin at two sites marked by pairs of dibasic
amino acids [2,3]. The type-I endopeptidase cleaves at
the junction of the B⁄ C chains ofproinsulin and the
type-II endopeptidase cleaves at the proinsulin C ⁄ A
junction. The basic amino acids at both sides are then
removed through the action of carboxypeptidase H.
After cleavage is complete, C-peptide and insulin are
produced and stored in mature secretory granules until
they are released in equimolar amounts from b cells
[4,5].
The 31-residue C-peptide has long been considered
to be merely auxiliary for the correct folding of insulin,
lacking any biological activity [6–8]. However, several
studies in diabetic patients and animal models during
the last 10 years have changed this view and it is now
considered to present biological activity by binding to
target cells, activating a G-protein-coupled signalling
response [9–12]. C-peptide elicits a number of cellular
responses, including Ca
2+
influx [9,13] and the activa-
tion of a series of enzymes including Na
+
⁄ K
+
-ATPase
[9,14], endothelial nitric oxide synthase [10,15], and
mitogen-activated protein kinases [16]. Administration
of C-peptide to insulin-dependent diabetic patients is
accompanied by a concentration-dependent rise in
blood flow to the kidneys, muscle, skin, and nerves in
the diabetic state [12,17,18].
Although the amino acid sequences of the C-peptide
from different species are quite variable, they do present
several relatively well conserved sequences, such as the
N-terminal acidic region, the glycine-rich central seg-
ment, and the highly conserved C-terminal pentapeptide
(Fig. 1). It has been recently demonstrated that muta-
tions in the N-terminal region have significant effects on
the in vitro refolding of proinsulin, probably due to
interactions with the A and B chains. It is therefore
believed to present an intramolecular chaperone-like
function important for proinsulin folding [19]. On the
Keywords
CA knuckle; NMR; proinsulin C-peptide;
protein secondary structure
Correspondence
H. R. Kalbitzer, Institut fu
¨
r Biophysik und
Physikalische Biochemie, Universita
¨
t
Regensburg, 93040 Regensburg, Germany
Tel: + 49 941 943 2595
E-mail: hans-robert.kalbitzer@biologie.
uni-regensburg.de
R. C. Garratt, Instituto de Fı
´
sica de Sa˜o
Carlos, Universidade de Sa˜o Paulo, Caixa
Postal 369, 13560–970 Sa˜o Carlos SP, Brazil
Tel: + 55 16 33739881
Fax: + 55 16 33739881
E-mail: richard@if.sc.usp.br
(Received 9 March 2005, revised 25 May
2005, accepted 4 July 2005)
doi:10.1111/j.1742-4658.2005.04843.x
The C-peptideofproinsulin is important for the biosynthesis of insulin,
but has been considered for a long time to be biologically inert. Recent
studies in diabetic patients have stimulated a new debate about its possible
regulatory role, suggesting that it is a hormonally active peptide. We des-
cribe structural studies of the C-peptide using 2D NMR spectroscopy. In
aqueous solution, the NOE patterns and chemical shifts indicate that the
ensemble is a nonrandom structure and contains substructures with defined
local conformations. These are more clearly visible in 50% H
2
O ⁄ 50%
2,2,2-trifluoroethanol. The N-terminal region (residues 2–5) forms a type I
b-turn, whereas the C-terminal region (residues 27–31) presents the most
well-defined structureof the whole molecule including a type III¢ b-turn.
The C-terminal pentapeptide (EGSLQ) has been suggested to be respon-
sible for chiral interactions with an as yet uncharacterized, probably a
G-protein-coupled, receptor. The three central regions of the molecule (resi-
dues 9–12, 15–18 and 22–25) show tendencies to form b-bends. We propose
that the structure described here for the C-terminal pentapeptide is consis-
tent with the previously postulated CA knuckle, believed to represent the
active site of the C-peptideofhuman proinsulin.
4284 FEBS Journal 272 (2005) 4284–4293 ª 2005 FEBS
other hand, it is the C-terminal pentapeptide of
C-peptide that has been observed to elicit the full activ-
ity of intact C-peptide in stimulating Na
+
⁄ K
+
-ATPase
[20]. Furthermore it elicits an increase in intracellular
calcium [13], and causes phosphorylation of mitogen-
activated protein kinases in human renal tubular cells
[12]. In addition, the pentapeptide is capable of fully dis-
placing C-peptide bound to renal tubular cell mem-
branes [21,22], supporting the view that the C-terminal
segment may constitute an active site. The glycine-rich
central portion also exhibits some stimulatory effects on
Na
+
⁄ K
+
-ATPase activity [20], and it is reported to be
important for normalization of glucose-induced vascu-
lar dysfunction in a rat model [23]. Activities associated
with this central region appear not to be particularly
residue-specific, as fragments containing amino acid
substitutions or non-natural d-amino acid, are also
partly active. However, a peptide comprising amino acid
residues 11–19 derived from the central portion of the
C-peptide is unable to displace cell membrane-bound
human C-peptide, suggesting that the mechanisms asso-
ciated with this region are different from those of the
C-terminal segment [22].
Structural models for proinsulin and for the C-pep-
tide have been suggested in recent years, on the basis of
empirical analyses [23,24] and spectroscopic experi-
ments, such as NMR [25,26], photochemically induced
dynamic nuclear polarization [25], Fourier transformed
infrared [27] and CD [26]. Many of the resulting models
are consistent, at least in part, but there is remaining
conflict, principally about the probable C-terminal act-
ive site. With a view to determining structural motifs
within the humanproinsulinC-peptide that are consis-
tent with the clinical and physiological results thus far
reported for C-peptide fragments, we performed the
high-resolution 2D NMR studies presented here.
Results
Sequential assignments and secondary structure
The C-peptide was studied in different solvents, in
aqueous solution (95% H
2
O ⁄ 5% D
2
O) and in mix-
tures of trifluoroethanol with water (50% H
2
O ⁄ 50%
2,2,2-trifluoroethanol-d2, and 20% H
2
O ⁄ 80% 2,2,2-
trifluoroethanol-d2). We succeeded in obtaining com-
plete spin-system assignments under these conditions.
Data were deposited in the BioMagResBank (accession
code 6623). The NOE path for the sample in water is
shown in Fig. S1, and the same pattern is preserved in
the other two samples.
The chemical shifts in aqueous solution deviate
clearly from those observed for random-coil peptides.
This effect is strengthened when 2,2,2-trifluoroethanol
is added, which is known to stabilize secondary-struc-
ture formation in most cases [28]. In nonisotope
enriched samples especially, the chemical shifts of the
a-protons can be used to predict the secondary struc-
ture in well-folded proteins. In peptides existing in a
fast equilibrium between different partially folded con-
formations, it can be used to predict the secondary-
structure content of the time or ensemble average. The
differences DdH
a
of the measured chemical shifts from
random coil conformation values published by Wishart
et al. [29] have been depicted for the three samples in
Fig. 2A. In general, the tendencies visible in aqueous
solution are enhanced by the addition of 2,2,2-trifluoro-
ethanol, that is the content of the corresponding local
structures in the ensemble is increased by 2,2,2-trifluoro-
ethanol. In well-folded proteins, consecutive positive
DdH
a
values are indicative of b-strands, whereas con-
secutive negative values are characteristic of helices. In
our partially folded peptide, they indicate a helical
tendency (small negative DdH
a
) for the Glu1–Glu11
and Gln22–Glu27 sequences, suggesting that these resi-
dues belong to either short unstable helices or turns
coexisting in solution.
The NOE contact maps displayed in Fig. 2B,C show
a summary of sequential and intermediate-range NOEs
for the samples in water and in 50% H
2
O ⁄ 50% 2,2,2-
trifluoroethanol-d2. Together with the
3
J
NHa
coupling
constants and the secondary chemical shifts (Fig. 2A),
the NOE pattern characterizes the local structure of
the peptide. As already mentioned, the analysis of the
chemical shifts in aqueous solution clearly shows that
the peptide adopts nonrandom structures in the time
average. However, as even after the addition of 2,2,2-
trifluoroethanol no long-range NOEs could be
Fig. 1. Amino acid sequences ofproinsulinC-peptide from nine mammalian species. Conserved residues are marked in bold.
C. E. Munte et al. Solutionstructureof C-peptide
FEBS Journal 272 (2005) 4284–4293 ª 2005 FEBS 4285
observed, the peptide is not expected to occur in a
unique, compactly folded state but rather in an exten-
ded conformation or, more likely, in multiple conform-
ational states. As is to be expected, a significant
increase in the number of NOEs could be observed in
the sample in 50% H
2
O ⁄ 50% 2,2,2-trifluoroethanol-
d2. Despite some ambiguities, there are some NOEs
that are not observable in the sample in water, but
present in the sample in 50% H
2
O ⁄ 50% 2,2,2-trifluoro-
ethanol-d2, as can be seen in Fig. 3 for the H
N
-H
N
-
contact region. Addition of 2,2,2-trifluoroethanol shifts
the equilibrium to states with higher structural organ-
ization, particularly in the sequentially highly con-
served C-terminal region.
A
B
C
Fig. 2. Local structureof the C-peptide as obtained from the devia-
tions of
1
H
a
chemical shifts from random-coil values and from the
NOE contact map. (A) Values of the conformation-dependent sec-
ondary shifts DdH
a
are plotted with solid bars: in black for the
C-peptide in water, in dark grey for the C-peptide in 50%
H
2
O ⁄ 50% 2,2,2-trifluoroethanol-d2, and in pale grey for the C-pep-
tide in 20% H
2
O ⁄ 80% 2,2,2-trifluoroethanol-d2. (B,C) The intensi-
ties of the sequential proton–proton NOE connectivities d
NN
(i,i +1),
d
aN
(i,i +1),d
bN
(i,i +1)(d instead of N for proline residues) for the
peptide in water (B) and in 50% H
2
O ⁄ 50% 2,2,2-trifluoroethanol-d2
(C) are represented as strong, medium and weak by the height of
the bars; existing but ambiguous NOE cross-peaks are marked in
grey. The observed medium-range NOEs d
NN
(i,i +2), d
aN
(i,i +2),
d
bN
(i,i +2),d
aN
(i,i +3),d
ab
(i,i + 3) are indicated by lines connecting
the two residues that are related by the NOE. The absence of
some medium-range connectivities may be due to ambiguous or
nonexisting NOEs. J-coupling constants
3
J
NH-Ha
are displayed by
open circles for J > 8 Hz, filled circles for J < 6 Hz and crosses for
values between 6 and 8 Hz.
A
B
Fig. 3. Amide region of the 2D NOESY spectrum for the C-peptide.
The NOEs for the peptide in (A) water and (B) 50% H
2
O ⁄ 50%
2,2,2-trifluoroethanol-d2 are labelled on the spectrum. Some
sequential NOEs are unresolved because of resonance overlap.
Solution structureofC-peptide C. E. Munte et al.
4286 FEBS Journal 272 (2005) 4284–4293 ª 2005 FEBS
Three-dimensional structure
The chemical shifts of the peptide indicate that only a
small proportion of the peptide is locally folded in
aqueous solution. This is in line with the relatively
small number of intermediate range NOEs observable
under these conditions. The chemical shifts indicate
that with increasing 2,2,2-trifluoroethanol concentra-
tion the content of local secondary structures increases.
The best-resolved NOESY spectra were obtained for
50% H
2
O ⁄ 50% 2,2,2-trifluoroethanol-d2, therefore this
sample was used for the subsequent structure calcula-
tions. The NOESY spectrum for the sample in 20%
H
2
O ⁄ 80% 2,2,2-trifluoroethanol-d2 showed a lower
spectral quality with broad resonance lines (probably
owing to the increased viscosity of the solution and
exchange of amide protons with 2,2,2-trifluoroethanol
hydroxy protons).
For the 50% H
2
O ⁄ 50% 2,2,2-trifluoroethanol sam-
ple, a total of 268 NOE distance restraints were
obtained in the NOESY spectra and used in the final
structure calculation. In addition, 10
3
J
NHa
coupling
constant restraints were obtained from an analysis of
the COSY spectra. On the basis of both the total and
NOE energies, the 30 structures that presented the
lowest energies were selected for further analysis. The
structural statistics can be seen in Table 1. The relat-
ively large NOE energies probably reflect the overall
flexibility of the structure, which may cause conflicting
NOEs. However, the number of NOEs with violations
larger than 0.5 A
˚
(five) is rather small and concerns
mainly the ill-defined central region of the peptide.
The backbone superimposition of the best structures
did not reveal a defined tertiary structure, so a search
for structured regions was undertaken by superimpo-
sing the peptide main chain in sections. RMSDs were
calculated within a sliding window of four, five and six
amino acids, as shown in Fig. 4A. The regions Ala2–
Leu5, Gln9–Leu12, Gly15–Ala18 and Gln22–Ala25 all
show a reasonable superimposition marked as local
minima in the red curve, indicating the presence of
more locally structured fragments in these portions of
the peptide. The best superimposition, however, occurs
in the C-terminal region, comprising the last five resi-
dues (Glu27–Gln31), which presents an RMSD for all
backbone atoms of 0.10 A
˚
. These five regions also all
exhibit a higher density of experimental restraints, as
can be seen in Fig. 4B. The C-peptide therefore
appears to be subdivided into a series of regions with
better-defined structures connected by regions with a
limited number of NOEs. The lack of observable con-
tacts between these regions may be due to a real spa-
tial separation between protons (greater than 4.5 A
˚
)
and ⁄ or ambiguous NOE cross-peaks that could not be
assigned. These five regions have been individually
analysed, and a superimposition of the main-chain
atoms of the 30 selected structures can be seen in
Fig. 4C.
Ala2–Leu5
In all structures the distances O(2)–H
N
(5) (between the
carbonyl oxygen of residue 2 and the amide hydrogen
of residue 5), as well as the angle defined by
O(2)–N
H
(5)–H
N
(5) are compatible with the presence
of a hydrogen bond (distance ¼ 1.9–2.3 A
˚
; angles ¼
30–38°). The /, w angles of residues 3 and 4 indicate a
type I b-turn. This appears to be the second most
highly structured part of the molecule after the C-ter-
minal pentapeptide (see below).
Gln9–Leu12, Gly15–Ala18 and Gln22–Ala25
In these three regions, the main-chain superimposition
does not indicate any well-defined structural element,
as can be seen for example in Fig. 4C for the region
Gly15–Ala18. The Ramachandran plot shows a large
Table 1. Structural statistics. C-peptide at pH 7.0 and 283 K, in
50% H
2
O ⁄ 50% 2,2,2-trifluoroethanol -d2.
Number of experimental restraints
NOEs 268
Intraresidual (i,i)134
Sequential (i,i+1) 90
Medium-range (i,i+j;1<j £ 4) 44
Long-range (i,i+j;4<j )0
3
J
NHa
10
Structural statistics for the 30 lowest energy structures (from 800
calculated)
Energy (kcalÆmol
-1
)
Total 549 ± 19
Bond 47.1 ± 3.1
Angle 115.7 ± 5.8
Improper 8.1 ± 1.0
van der Waals 85.9 ± 8.9
NOE 280 ± 12
Coupling 12.4 ± 3.3
RMSDs (A
˚
)
a
Whole peptide 4.731 (5.830)
Amino acid 2–5 0.130 (0.944)
Amino acid 9–12 0.490 (1.394)
Amino acid 15–18 0.309 (0.591)
Amino acid 22–25 0.350 (1.129)
Amino acid 27–31 0.103 (0.844)
a
All backbone atoms; values in parentheses all non-hydrogen
atoms.
C. E. Munte et al. Solutionstructureof C-peptide
FEBS Journal 272 (2005) 4284–4293 ª 2005 FEBS 4287
dispersion for the four residues in all three regions.
For these regions the number of distance restraints is
insufficient to define a unique conformation, and most
structures do not exhibit distances consistent with the
presence of hydrogen bonds. A slight tendency to
induce a turn in the peptide main chain could be clas-
sified as a bend, with distances between residues i and
i +3 below 7 A
˚
.
Glu27–Gln31
As shown in Fig. 4C, this region is in sharp contrast
with the remainder of the structure characterized by
the excellent main-chain superimposition of the 30
structures. The RMSD for this pentapeptide is signifi-
cantly smaller than that for the tetrapeptides described
above, showing that this region is by far the most
highly structured part of the C-peptide. The side
chains for this short segment also seem to be well
defined, especially that of Leu30. The O(27)–H
N
(30)
distance and the O(27)–N
H
(30)–H
N
(30) angle are
consistent with the presence of a hydrogen bond
(1.9–2.7 A
˚
and 21–29°, respectively). This bond seems
to be bifurcated in which the backbone carbonyl of
residue 27 is also hydrogen-bonded to the H
N
(31)
atom, with O(27)–H
N
(31) distance of 1.7–2.8 A
˚
and
O(27)–N
H
(31)–H
N
(31) angle of 12–21°. The two
predicted hydrogen bonds are indicated in Fig. 5A.
The /,w angles of Gly28 and Ser29 characterize a type
III¢ b-turn, as can be confirmed in the Ramachandran
plot (Fig. 5B). Leu30 exhibits a poorly favoured, but
not forbidden main-chain conformation for a leucine.
The type III¢ b-turn is extremely well defined, showing
/,w angles for Gly28–Leu30, which consistently reside
in the same regions of the Ramachandran plot for all
800 structures initially generated by the simulated
A
B
C
Fig. 4. Structured regions of the C-peptide
in 50% H
2
O ⁄ 50% 2,2,2-trifluoroethanol-d2.
(A) RMSD calculated from the peptide main-
chain superimposition within a sliding win-
dow of four (red), five (pale blue) and six
(dark blue) amino acids. (B) Density of
experimental distance restraints (blue lines).
(C) Superimposition of the main-chain atoms
of the 30 selected structures for the C-pep-
tide in 50% H
2
O ⁄ 50% 2,2,2-trifluoroethanol-
d2, for the N-terminal region Ala2–Leu5, the
central region Gly15–Ala18, and the C-ter-
minal region Glu27–Gln31 (the main chains
are indicated in black and the side chains in
grey).
Solution structureofC-peptide C. E. Munte et al.
4288 FEBS Journal 272 (2005) 4284–4293 ª 2005 FEBS
annealing protocol. To eliminate the possibility that the
type III¢ b-turn may result from the force field only and
not the experimental NOEs, the structures obtained
were submitted to a new refinement at low temperature
in the absence of experimental restraints. In none of the
models so produced did the type III¢ b-turn persist,
showing it to be a consequence of the experimental
NOEs. In larger protein structures, type III¢ b-turns are
relatively rare, and its appearance in these simulations
suggested the possibility that the number of experimen-
tal restraints may not be sufficient to distinguish it from
its more common mirror image, the type III b-turn. To
eliminate this possibility, the /,w angles of Ser29–Leu30
for one of the structures were changed in such a way as
to convert the type III¢ b-turn into a type III b-turn.
However, the resulting interatom distances found in the
new model were inconsistent with the NOESY spectra
measured for the C-peptide.
Discussion
The analysis of the chemical shifts and NOEs of the
C-peptide dissolved in solution shows that it is neither
well folded nor has a random structure. The data are
typical for a structural ensemble in fast equilibrium on
the NMR time scale, favouring some local structures.
The addition of 2,2,2-trifluoroethanol shifts the equi-
librium in the accessible conformational space towards
specific local structures. However, as is often found for
peptides in aqueous solution, some typical NOE con-
tacts that are present in 2,2,2-trifluoroethanol are still
observed with reduced intensity in water, and the
chemical shift deviations from the random-coil values
are qualitatively still in agreement with the structure
found in the presence of 2,2,2-trifluoroethanol. This is
usually interpreted as the existence of a small popula-
tion of the local and global structural states stabilized
by 2,2,2-trifluoroethanol, which are mixed with other
‘random’ structures. An indication of such an avera-
ging would be a concentration dependence on the
cosolvent, the extrapolation of which to zero would
lead to nonrandom, qualitatively still correct values for
the chemical shift changes and interatomic distances.
Such behaviour is found for the C-peptide in our stud-
ies. The local structures determined in the presence of
2,2,2-trifluoroethanol, especially the extremely well-
defined structure found for the C-terminal region,
would thus also exist in low populations in aqueous
solution and would be stabilized in a less polar envi-
ronment, as is to be expected during the interaction
with its receptor or with cell membranes. Water is in
general excluded from these interactions favouring the
formation of this structure. Therefore the human pro-
insulin C-peptidestructure presented in this work is
expected to be physiologically relevant, despite the
nonphysiological conditions used for the structure
determination itself (presence of 2,2,2-trifluoroethanol).
Human proinsulinC-peptide dissolved in 50%
H
2
O ⁄ 50% 2,2,2-trifluoroethanol-d2 does not present a
well-defined global tertiary structure. No long-range
inter-residual NOEs could be assigned in the NOESY
A
B
Fig. 5. Structureof the C-terminal pentapeptide of the C-peptide in
50% H
2
O ⁄ 50% 2,2,2-trifluoroethanol-d2. (A) One selected model
showing the two predicted hydrogen-bonds. (B) Ramachandran
plot, indicating the main-chain conformation of Glu27 (green dots),
Gly28 (red dots), Ser 29 (blue dots) and Leu30 (black dots).
C. E. Munte et al. Solutionstructureof C-peptide
FEBS Journal 272 (2005) 4284–4293 ª 2005 FEBS 4289
spectra, which would be essential for the convergence
of the models to a well-defined, compact tertiary struc-
ture. However, a detailed analysis of the models
obtained shows the existence of five regions with rather
well-defined local structures.
The N-terminal region (Ala2–Leu5) possesses the
basic features of a type-I b-turn. The potential helical
structure initiated by this turn is consistent with the
lower frequency chemical-shift deviations in this region
and with previous results, including theoretical predic-
tions [23,24] and NMR spectroscopy [26]. However,
there is a discrepancy about the size of the structured
region. Unlike in previous studies, the nascent helix
encountered here is broken by Gln6, which is evi-
denced by its random-coil H
a
chemical shift and by
the random /,w-angle distribution in the Ramachan-
dran plot of the experimental structures. The unambig-
uous absence of an NOE between the H
a
proton of
Asp4 and the H
N
proton of Val7 (expected always to
exist in a helix because of separation within the range
3.3–3.5 A
˚
between these atoms) endorses our conjec-
ture that the helix is short. Despite the fact that Gln6
is highly conserved in C-peptides from different spe-
cies, Leu5 is found to be replaced in all species but
humans by a proline, a known helix breaker. Recent
experiments performed with proinsulin reveal that
deletions or alanine mutations of the N-terminal acidic
amino acids of the C-peptide result in the formation of
large aggregates during in vitro refolding [19]. It is sug-
gested that these results indicate that the highly con-
served acidic N-terminal part of the C-peptide may
have some intramolecular chaperone-like function in
the folding of the insulin precursor. The presence of a
highly conserved N-terminal tetrapeptide also suggests
the existence of a functionally active site in the B ⁄ C
junction of proinsulin, and has been proposed to con-
stitute part of the type I endopeptidase recognition site
[19,30]. The b-turn structure that has been found for
the Ala2–Leu5 region in this study is coherent with
these previous findings.
The superimposition of the three central regions
(Gln9–Leu12, Gly15–Ala18 and Gln22–Ala25) is not
as good as that of the N-terminal region, with little
indication of any well-defined, typical secondary struc-
ture. The number of experimental restraints in these
regions is low, probably resulting from ambiguities in
the NOESY spectra, making it impossible to conclude
if the three regions are really unordered or not. Previ-
ous results with the use of spectroscopic methods, such
as Fourier transformed infrared [27], NMR and
CD [25,26], confirm that these regions tend to be dis-
ordered. Published studies of Na
+
⁄ K
+
-ATPase activ-
ity in rat renal tubule segments (the stimulation of
which by C-peptide has been previously described [18])
revealed the existence of peptide fragments derived
from part of the central portion of the molecule that
exhibited some stimulatory activity [20]. In human
C-peptide, the sequence from residues 13–19 (GGGP-
GAG) is unusual in that it is nearly symmetrical with
respect to the central proline, possesses solely nonpolar
residues, and has a high content of the nonchiral
amino acid glycine. These residues have been proposed
to form a turn-like structure, which is relevant to non-
chiral interactions with membranes [23]. The slight ten-
dency to a bend found in the three central regions is
consistent with these results.
The C-terminal region (Glu27–Gln31) contrasts
sharply with the remainder of the structure in that it
presents an excellent superimposition for the 30 mod-
els. The Glu27–Leu30 tetrapeptide forms a type-III¢
b-turn stabilized by a hydrogen bond between the
Glu27 carbonyl and the Leu30 amide. This hydrogen
bond is bifurcated and also involves the amide from
Gln31 (the highly conserved C-terminal residue of
the C-peptide). These results are consistent with the
existence of a well-defined structure for the EGSLQ
C-terminal pentapeptide. Although a type-III¢ b-turn is
a secondary-structure element not commonly found in
polypeptides, it is favoured by the presence of glycine
in position (i + 1) and serine in (i + 2), both of which
are able to adopt a left-handed helical conformation.
Residue Leu30 possesses an excellent side-chain super-
imposition among the structures and seems to be sta-
bilized by van der Waals interactions.
In 2D NMR experiments comparing proinsulin and
insulin [25], the authors described perturbations to the
2D NMR resonances assigned to the hydrophobic core
of the insulin moiety of proinsulin. These perturba-
tions were reversed by site-specific cleavage at the C ⁄ A
junction but not the B ⁄ C junction. The authors suggest
the existence of a stable local structure at the C ⁄ A
junction, which has been designated the ‘CA knuckle’,
involving a nonstandard secondary structure, accessible
to solvent and not involving distant regions of the
C-peptide [25]. These results are wholly consistent with
the structure presented in this work for the C-terminal
region of the humanproinsulin C-peptide. In the same
C-peptide fragment activity experiments described
above for the central region, a second fragment invol-
ving residues 27–31 was found to elicit stimulatory
effects on the Na
+
⁄ K
+
-ATPase [20]. In addition, the
C-terminal pentapeptide was also effective in the dis-
placement binding studies in which C-peptide had been
previously bound to membranes of several human cell
types (renal tubular cells, skin, fibroblasts, and saph-
enous vein endothelial cells) [21]. The results, which
Solution structureofC-peptide C. E. Munte et al.
4290 FEBS Journal 272 (2005) 4284–4293 ª 2005 FEBS
are sequence-specific, point to the existence of a C-pep-
tide-specific receptor that recognizes the C-terminal
pentapeptide [20,21]. Thus a well-defined secondary
structure is to be expected for the EGSLQ pentapep-
tide, perhaps the previously foreseen ‘CA knuckle’,
which may well represent the elusive active site of the
C-peptide itself [25]. Our results showing an extremely
well-defined structure for the Glu27–Gln31 region are
compatible with these concepts.
Proinsulin is processed to produce insulin by the
action of two distinct endopeptidases followed by the
loss of two basic amino acids via carboxypeptidase H
cleavage acting on both the B-peptide and C-peptide. It
is well known that the removal of the C-terminal argi-
nines from the B-chain of insulin is essential for the
interaction of mature insulin with its receptor [2]. How-
ever, the processing of the C-peptide is rather curious if
it does not have any useful physiological role. The loss
of the two C-terminal basic amino acids from the proin-
sulin C-peptide has been shown to be fundamental to its
biological activities [21], conceivably because of the
exposure of the highly conserved Gln31, suggesting an
important functional role for this residue. The conclu-
sion drawn here that the C-terminal region of the proc-
essed C-peptide is its most highly structured portion,
consistent with a physiological role, provides an explan-
ation for C-peptide C-terminal processing during insulin
maturation.
Gly28–Ser29, which are expected to be structurally
important for the maintenance of the type III¢ b-turn,
are not absolutely conserved in different species, being
notably absent in both rat C-peptides (Fig. 1). This
is probably connected with either the existence of
species-specific C-peptide receptors or a nonconserved
C-peptide activity across species. It is relevant to note
therefore that rat C-peptide failed to bind to human
cells [11,22], suggesting that the structure found in the
present work for the human C-terminal pentapeptide is
not expected to exist in the rat homologues. Indeed in
the rat C-terminal pentapeptides, the Gly28–Ser29
sequence is replaced by Val–Ala, which would not be
expected to form a type III¢ b-turn principally because
of the bifurcation of the valine b-carbon which makes
the left-handed a-helical region of the Ramachandran
space inaccessible.
Finally, and most importantly, our results support
the idea of structured N-terminal and C-terminal
regions for the peptide. The latter has been previously
suggested to form the active site of the human proinsu-
lin C-peptide, and has been baptised the CA knuckle.
The results described here have gone further than
previous studies in terms of the structural characteriza-
tion of this region. We suggest that the knuckle, as
described here, represents the most structurally ordered
segment of an otherwise flexible peptide. However,
whether this structure is assumed by the peptide under
physiological conditions remains to be demonstrated.
It is hoped that our observation that the most highly
structured region of the molecule appears also to
correspond to the predicted active site will stimulate
further investigation.
Experimental procedures
Sample preparation
Human C-peptide, a byproduct of the industrial prepar-
ation ofhuman recombinant insulin, was a gift from
Biomm S.A. A stock solutionof 6 mm unlabelled human
C-peptide in distilled water was used to prepare three dif-
ferent samples: C-peptide in 95% H
2
O ⁄ 5% D
2
O(v⁄ v), in
50% H
2
O ⁄ 50% 2,2,2-trifluoroethanol-d2 (v ⁄ v), and in 20%
H
2
O ⁄ 80% 2,2,2-trifluoroethanol-d2 (v ⁄ v). The pH of each
was adjusted to 7.0 by the addition of appropriate quanti-
ties of NaOH. As internal reference 2,2-dimethyl-2-silapen-
tane-5-sulfonate was added to a final concentration of
0.05 mm.
NMR spectroscopy
NMR experiments were performed on a Bruker DRX-600
spectrometer (proton frequency of 600 MHz). All spectra
were recorded at 283 K. The water signal was suppressed
by selective presaturation. 2D data sets were recorded
with 4096 complex t
2
points and 1024 t
1
increments; phase-
sensitive detection in the t
1
direction was obtained with time-
proportional phase incrementation [31]. NOESY [32] spectra
were recorded with a mixing time of 80, 100, 150 and 200 ms
to check for possible spin diffusion effects and to allow nor-
mally weak NOEs to become more apparent. TOCSY [33]
spectra were recorded with spin-lock times of 80 ms using a
MLEV-17 [34] sequence. DQF-COSY spectra were obtained
as described by Rance et al. [35]. The time-domain data were
processed using the xwinnmr package (Bruker) and evalu-
ated with the program aurelia [36].
Experimental restraints
Assignment of resonance lines was performed according to
the standard strategy for homonuclear spectroscopy [37]
using DQF-COSY and TOCSY spectra for the identification
of the spin systems and NOESY spectra for the sequence-
specific and NOE assignment. Amide–Ha coupling constants
3
J
NHa
were determined from a slightly exponentially filtered
DQF-COSY spectrum by fitting the antiphase signals to a
pair of Lorentzians using the corresponding routine from
aurelia. Upper and lower boundaries for coupling constant
C. E. Munte et al. Solutionstructureof C-peptide
FEBS Journal 272 (2005) 4284–4293 ª 2005 FEBS 4291
restraints were set to 0.5 Hz. The NOESY cross-peaks were
integrated by the automated segmentation procedure of the
program aurelia, and distances were calculated applying
the initial slope approximation. A set of well-resolved methy-
lene resonances (assumed interproton distance of 1.76 A
˚
)
were taken as reference distances. The upper and lower dis-
tance bounds were taken as described in [38].
Structure calculations and analysis
Structures of the C-peptide in 50% H
2
O ⁄ 50% 2,2,2-trifluoro-
ethanol-d2 were calculated by simulated annealing using the
program CNS 1.0 [39], starting from extended structures.
High-temperature torsion-angle dynamics were run for 15 ps
at an initial temperature of 3000 K. The system was then
slowly cooled to a temperature of 0 K in 25 K steps over a
period of 5 ps. At 0 K, a final stage of 150 steps of Powell
minimization was performed to yield the final structures. The
final values of the force constants used in the simulated
annealing calculations are as follows: 1 kcalÆmol
)1
ÆA
˚
)2
for
bond lengths, 1 kcal Æ mol
)1
Ærad
)2
for angles and improper
torsions, 1 kcalÆmol
)1
ÆA
˚
)4
for the quadratic van der Waals
repulsion term, 300 kcalÆmol
)1
ÆA
˚
)2
for NOE-derived distance
restraints and 1 kcalÆmol
)1
Æ Hz
)2
for the
3
J
NHa
coupling con-
stant restraints. Analysis of secondary-structure elements
and calculation of RMSD values were performed using the
program molmol 2.6 [40]. Co-ordinates for the 30 lowest
energy structures have been deposited in the Protein Data
Bank (accession code 1T0C).
Acknowledgements
This work was supported by Fundac¸ a
˜
o de Amparo a
`
Pesquisa do Estado de Sa
˜
o Paulo (FAPESP), Brazil,
grant 96 ⁄ 12386-3, the DFG, and the Bayerische Fors-
chungsstiftung.
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Supplementary material
The following material is available for this article
online:
Fig. S1. NOE path of the C-peptide in water.
C. E. Munte et al. Solutionstructureof C-peptide
FEBS Journal 272 (2005) 4284–4293 ª 2005 FEBS 4293
. the
active site of the C-peptide of human proinsulin.
4284 FEBS Journal 272 (2005) 4284–4293 ª 2005 FEBS
other hand, it is the C-terminal pentapeptide of
C-peptide. sequences of proinsulin C-peptide from nine mammalian species. Conserved residues are marked in bold.
C. E. Munte et al. Solution structure of C-peptide
FEBS