From the
Human parathyroid hormone (hPTH), amino acids Ser
Human parathyroid hormone (hPTH)
Contrasting these studies of
hPTH(1-34) in TFE-containing solution is NMR work on the same
fragment and on human parathyroid hormone-related protein in
H
TFE is commonly used as an additive to protein and peptide solutions
that stabilizes secondary structures, in particular
Thus, on one
hand, the structural results on hPTH(1-34) are at least partially
contradictory. On the other hand, determination of the
three-dimensional structure of the NH
hPTH(1-37) is not only the naturally occurring hPTH
fragment extractable from human blood, but also shows higher cAMP
generation activity in target cells than hPTH(1-34)(4) .
Compared to hPTH(1-34), hPTH(1-37) is extended by Val, Ala,
and Leu. Ala is a strong helix stabilizer(24) , and the
structure of hPTH in the region Asp
Data from the following 600 MHz NMR
spectra were employed for the sequence-specific assignment of spin
systems and the evaluation of NOESY distance constraints: DQF-COSY,
TOCSY with mixing times of 70 and 80 ms, respectively, NOESY with
mixing times of 100 and 200 ms, respectively. NOESY cross-peaks were
obtained from two sets of spectra at 283 and 298 K. For the present
calculations, only NOEs visible in the 298 K spectra with 200 ms mixing
time were taken into account, although identical calculations combining
information obtained at the two different temperatures resulted in
identical structures.
The structure calculations followed standard procedures employing a
hybrid DG-restrained MD approach with simulated annealing (SA)
refinement and subsequent energy minimization (dgsa;(29) ). For
the refinement, the dielectric constant was changed to
A cubic water box consisting of 8000
water molecules with a length of 6.22 nm in each dimension was set up,
based on an equilibrated box of 125 water molecules as supplied with
the X-PLOR program package. The overlay was achieved by placing the
protein in the center of the water box and by deleting all solvent
molecules closer than 0.26 nm to any heavy atom of the protein.
During the first 15 ps of the MD calculations, the
system was gradually heated to 300 K while coupled to an external water
bath(33) . The MD calculations were carried out using the Verlet
algorithm (34) with a time step of 2 fs. The SHAKE facility (35) was used to constrain the covalent bond length. A
dielectric constant of 1.0 was applied with a scaling factor of 0.4 for
one to four electrostatic interactions. All nonbonded interactions were
cut off at a distance of 0.85 nm.
The heating stage was followed by
200 ps of MD at 300 K using the parameters described above. During the
whole simulation minimum image periodic boundary conditions were used.
Coordinates, energies, and velocities were saved every 5 ps for further
analysis.
Simulations and analyses were performed on Cray YMP/EL and
Hewlett Packard HP 735 computers. A 1 ps simulation required about 2 h
of cpu time on a Cray YMP/EL computer.
In the current study,
only conservative semiquantitative NOE intensity based estimates of
interproton distances were used for the restrained molecular dynamics
calculations. Although this procedure weakens the danger of
overinterpretation of experimental data, it does not remove it
entirely.
The stability of the tertiary and secondary structure
presented here may be judged in various ways. First, relative NOE
intensities may be compared with results from other peptides and
proteins; second, for a purely helical peptide, the amount of helices
present at any given point in time may be estimated from the CD
spectra; third, local rmsd values may be calculated; fourth, the
structural stability may be probed by unrestrained molecular dynamics
calculations, and the time dependence of the structural elements may be
followed.
hPTH(1-34) in TFE-free
aqueous solution at pH 4.1 (10) shows a stable, extended
NH
The structure of
hPTH(1-37) showed a loop region from His
The Ser
Ser
E
The atomic
coordinates and structure factors (code 1HPH) have been deposited in
the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
The NDEE program was supplied by Franz Herrmann.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
to
Leu
, is biologically active with respect to both receptor
binding and activation of adenylate cyclase to influence the serum
calcium concentration. It induces DNA synthesis via an unknown signal
pathway. We investigated the structure of hPTH(1-37) in
H
O/buffer solution under near physiological conditions,
that is pH 6.0 and 270 mM salt, by circular dichroism,
ultracentrifugation, nuclear magnetic resonance spectroscopy, and
molecular dynamics calculations. Complete sequence specific assignments
of all
H resonances were performed by using
H
two-dimensional NMR measurements (double quantum-filtered correlated
spectroscopy, nuclear Overhauser effect spectroscopy (NOESY), and total
correlation spectroscopy with suppression of NOESY-type cross-peaks
spectra). hPTH(1-37) obtained helical structure and showed
hydrophobic interactions defining a tertiary structure. The
NH
-terminal four amino acids of hPTH(1-37) did not
show a stable conformation. Evidence for an
-helical region
between Ile
and Asn
was found. This region was
followed by a flexible link (Gly
, Lys
) and a
well defined turn region, His
to Ser
. The
latter was stabilized by hydrophobic interactions between Trp
and Leu
. Ser
through at least
Leu
formed an
-helix. Arg
and Lys
were involved in the core built by His
to
Ser
. Unrestrained molecular dynamics simulations indicated
that the structure was stable on the 200 ps time scale.
(
)contains three functionally distinct domains that are
responsible for receptor binding and activation of cAMP-cyclase to
maintain normocalcaemia and initiation of a cAMP-independent signal
transduction pathway for stimulation of DNA synthesis,
respectively(1, 2) . It is generally accepted that these
functionally active domains of the 84 amino acid hormone are located in
the NH
-terminal part of the protein(3) . Indeed,
hPTH(1-37) (for sequence, see ) is the naturally
occurring bioactive hormone extractable from human blood(4) .
The cAMP receptor-binding domain comprises His
to
Phe
(5, 6) , and the DNA synthesis
stimulating domain comprises Asp
to
Phe
(1) . The complete NH
-terminal
peptide hPTH(1-34) is required for stimulation of the
cAMP-dependent signal pathway(3) . Stimulatory potential is lost
on deletion of Ser
and Val
. cAMP-receptor
binding capacity is not influenced by this deletion, indicating that
the activation and receptor-binding sites are located in different
domains(7) . The intact cAMP-dependent signal pathway is
required to maintain normocalcaemia. Moreover, treatment of
osteoporotic patients with hPTH hormone stimulates an increase of axial
bone mass and bone formation (8). Thus, recent studies focused on
determination of the three-dimensional structure of
NH
-terminal peptides in solution by NMR spectroscopy. In
particular, hPTH(1-34) is an intensely studied hormone fragment.
It contains the functional domains and, in addition, is commercially
available in large quantities as necessary for solution structural
studies by NMR spectroscopy ((9-11); for a recent review, see
Ref. 12). The results of these studies are still under discussion, as
the majority concludes that hPTH(1-34) does not form secondary
structure elements under near physiological solvent
conditions(9, 11, 13) , but helix formation
under these conditions is also observed(10) . In
2,2,2-trifluoroethanol (TFE) containing solution, however, helical
structure is induced(9, 11, 13) . In particular,
in 40% TFE solution, hPTH(1-34) displays helical regions from
Ser
3 to Gly
and from Ser
to
Lys
(11) . In 70% TFE, hPTH(1-34) showed
similar behavior(13) .
O solution(14, 15) . These studies claim
that the NH
-terminal peptide of hPTH obtains stable
-helical structure in H
O solution from Glu
to Lys
and Val
to Gln
.
-helices
(16-21). It is also used to stabilize putative protein folding
intermediates(22) . Being mildly hydrophobic with a dielectric
constant
= 26.7, that is one-third that of
water(23) , TFE bears the risk of weakening hydrophobically
stabilized tertiary structure domains(19) .
-terminal part of hPTH
poses a problem of considerable medical and pharmaceutical importance,
as drugs mimicking this structure might eventually be extremely useful
as therapeutics against osteoporosis and parathyroid gland malfunction
or loss.
-Phe
is
stabilized by flanking amino acids(1) . On these grounds we
decided to study the hPTH NH
-terminal fragment 1-37
in aqueous solution under conditions approaching the physiological
state with respect to salt concentration and pH, and compare the
results with the structures obtained earlier for hPTH(1-34) in
TFE-containing solution.
Peptide Synthesis
hPTH(1-37) was
synthesized on an automated peptide synthesizer (Millipore) 9050 using
standard Fmoc protocols with Fmoc-Arg(PMC) and
Fmoc-Trp(Boc)(25) . Peptide chains were assembled by
TBTU/DIPEA/HOBT activation on preloaded PEG-PS resin (Millipore).
Cleavage and deprotection reactions were performed with trifluoroacetic
acid/ethandithiole/water (94:3:3) for 90 min. The peptide was
precipitated by addition of cold tert-butylmethylether,
dissolved in 5% acetic acid for 15 h, and lyophilized. The crude
product was purified by reverse phase (RP) HPLC (Vydac C18, 10 µ,
25 mm 250 mm, 10 ml/min, 230 nm, buffer A: 0.1% HCl, buffer B:
0.1% HCl in PrOH/MeOH/water 50:30:20 with a constant gradient of
10-70% B in 60 min). Purity was checked with RP-HPLC (Vydac C18,
250
4.6 mm, 300 A, 10 m) and capillary zone electrophoresis
(Biofocus 3000, Bio-Rad). Electrospray mass spectrometry (Sciex API
III, Perkin-Elmer) showed correct relative molecular mass (calculated:
4401.01; measured: 4401 ± 1). Automated Edman degradation (473A
Protein sequencer, Applied Biosystems) and amino acid analysis
(Aminoquant 1090L, Hewlett Packard) confirmed amino acid composition as
well as amino acid sequence. The lyophilized product was stored at
-20 °C.
Biological Activity
Biological activity of the
synthetic hPTH(1-37) fragment was tested by observation of the
stimulation of cAMP generation in osteogenic cells (Rous sarcoma cells)
and in an established renal epithelial cell line (opossum kidney
cell).(
)
Sedimentation Analysis
Sedimentation
experiments were performed at room temperature in a Beckman model E
analytical ultracentrifuge, using UV scanning at 280, 290, 295 and 300
nm. Double sector cells with sapphire windows were applied in an AnH-Ti
rotor. The s value was
calculated from a lg(r) versus time plot, correcting
for temperature and water viscosity; high speed sedimentation
equilibria (26) were evaluated from ln(c) versusr
plots making use of a computer program
developed by G. Böhm, Regensburg. The partial specific volume of
the protein was calculated from the amino acid composition.
NMR Spectroscopy
NMR spectra were obtained on a
standard Bruker AMX600 spectrometer at 283 K and 298 K with standard
methods (27, 28). Advantages in lineshape and linewidth led us to use
3.4 mM protein in 50 mM potassium phosphate
buffer with
270 mM sodium chloride, pH 6.0. The
H
O resonance was presaturated by continuous coherent
irradiation at the resonance frequency prior to the reading pulse.
Differences in sample heating between TOCSY spectra and other spectra
were compensated for by temperature calibration of the probe for the
specific buffer system and accordingly corrected presettings for the
VT1000 temperature unit. The sweep widths in
and
were identical, 7042.3 Hz and 6024.1 Hz for the
spectra obtained in H
0/
H
O (9:1) and
H
O solution, respectively. Quadrature detection
was used in both dimensions, employing the time proportional phase
incrementation technique in
. 4 K data points were
collected in
and 512 data points were collected in
. Zero filling to 1 K and 2 K data points,
respectively, was used in
and
.
For determination of J-couplings, zero filling to 16 K in
was used. All two-dimensional spectra were multiplied by a
squared sinebell function phase shifted
/4 for NOESY spectra,
/6 for TOCSY spectra, and
/8 for COSY spectra. Base line and
phase correction to 6th order was used. Data were evaluated on X-window
workstations with the NDee program package (available to non-profit
organizations on request).
CD Spectroscopy
CD spectra were recorded at 25
°C in 0.1-mm cells from 250 to 190 nm at 20 nm/min on a Jasco J
600A CD spectropolarimeter with 310 µM protein in pH 6.0
solution containing 270 mM sodium chloride and 50 mM phosphate buffer in 30 µl volume. The reference sample
contained buffer without protein. Spectra were measured eight times and
averaged for sample and reference, respectively.
Structure Calculations with Restrained Molecular
Dynamics
Distance geometry (DG) and molecular dynamics (MD)
calculations were performed with the XPLOR 3.1 program package (29) on CRAY Y-MP/EL and HP735 computers. 520 NOESY cross-peaks
could be assigned, of which 183 were structure determining
interresidual cross-peaks (560 and 223 cross-peaks, respectively, when
combining the cross-peaks from the sets of spectra obtained at 283 K
and 293 K). We divided these cross-peaks into three groups according to
their relative intensities: strong intensity, 0.2-0.3 nm, medium
intensity, 0.2-0.4 nm, and weak intensity, 0.2-0.5 nm. 0.05
nm was added to the upper distance limit for distances involving
unresolved methyl or methylene proton resonances (pseudoatom approach).
=
4. Structure parameters were extracted from the standard parallhdg.pro
and topallhdg.pro parameter files(30) .
Unrestrained MD
Unrestrained MD calculations were
carried out using the parameters for the polypeptide chain and the
TIP3P water model (31) that were supplied with the standard
X-PLOR force field (29). A representative structure out of 30
structures calculated as above was chosen as starting structure for the
unrestrained MD calculations.
Close Nonbonded Solute-Solvent Interactions Were Removed
in Two Steps
First, 100 cycles of conjugate gradient energy
minimization (32) were carried out, keeping the positions of all
protein atoms fixed. Second, in 300 cycles of energy minimization, a
harmonic potential was used to restrain the peptide to its original
conformation.
Structural Analysis
The final structures were
analyzed with respect to stable idealized elements of regular secondary
structure using the Determination of Secondary Structure of Proteins
(DSSP) program package(36) . For visualization of structure
data, the SYBYL program package, version 6.0, (TRIPOS Association) was
used.
Molecular Mass
From sedimentation velocity
experiments at 68,000 revolutions/min, a sedimentation coefficient s = 0.51 ±
0.02 S was obtained, in accordance with a globular protein of less than
5 kDa molecular mass; boundary analysis provided clear evidence for
homogeneity. High speed sedimentation equilibria at 40,000 and 30,000
revolutions/min gave linear ln cversusr
plots from which a molecular mass of 4,105
± 175 was calculated (correlation coefficient 0.9994) (Fig. 1). In order to detect possible concentration-dependent
association, heterogeneity, and nonideality, the meniscus depletion
technique (26) was applied. Using scanning wavelengths between
280 and 300 nm, the maximum concentration at the bottom of the cell was
extended to 24 mg/ml. No tendency to form dimers or high molecular
weight aggregates could be detected. One-dimensional NMR spectra of
hPTH(1-37) were identical in a concentration range from 0.04 3.4
mM (data not shown), confirming that hPTH(1-37) did not
form aggregates at the concentrations used for further NMR experiments.
Figure 1:
Ultracentrifugal analysis of
parathyroid hormone in 50 mM potassium phosphate, pH 6.3, at
20 °C, initial peptide concentration 0.35 mg/ml. Meniscus depletion
high speed sedimentation equilibrium at 40,000 (left) and
68,000 revolutions/min (right). Upper panels, radial
distribution (cversusr), scanned at 280
nm. Lower panels: lncversusr linearization of the bottom region yield
the monomer molecular mass of 4,390 ± 228 and 4 105 ± 17
Da for 40,000 and 68,000 revolutions/min,
respectively.
Circular Dichroism Spectroscopy
The overall
content of helical structure elements was determined by far UV CD
spectroscopy (Fig. 2). The evaluation of the average helix
content of the peptide by two standard methods (37, 38) yielded an average helix content of
hPTH(1-37) of 28-30%.
Figure 2:
Far UV circular dichroism spectrum of
hPTH(1-37) as under ``Materials and Methods.'' The
presence of approximately 30% helical structure may be estimated from
this CD spectrum.
Two-dimensional NMR Spectroscopy
The
two-dimensional NMR spectra of hPTH(1-37) showed very little
resonance overlap (Fig. 3), so that the sequence-specific
resonance assignments could be performed with standard
techniques(28) . The complete NH-CH resonance
assignments are shown in the 200 ms NOESY spectrum (Fig. 3b), and the complete sequence-specific resonance
assignments are given in .
Figure 3:
200 ms NOESY spectrum of hPTH(1-37). a, amide-amide region; b, fingerprint region.
Experimental conditions: NMR: mixing time, 200 ms; data size: 4 k
1 k data points; filter: squared sine bell, phase shift
/4; ns, 96. Sample conditions: hPTH, 1.8 mM; pH, 6.0; T,
298 K; NaCl, 250 mM; potassium phosphate, 50
mM.
The high number of
cross-peaks in the backbone amide-amide region of the NOESY spectra (Fig. 3a) immediately suggested the presence of helical
structures. We used the chemical shift data available from our
experiments to perform a secondary structure estimate according to the
chemical shift index strategy(39) . The procedure depends on a
simple correlation between chemical shifts of C-proton
resonances of consecutive amino acids and local secondary structure:
C
proton resonances shifted to high field relative to
the corresponding random coil C
proton resonances
indicate local
-helical structure; C
proton
resonances shifted downfield compared to the corresponding resonances
in a random coil structure indicate local
-sheet structure. The
criterion for taking a particular shift into account in secondary
structure estimates is its deviation from the random coil value by more
than 0.1 parts/million. Also, to get a more reliable picture, it is
suggested that only resonances should be taken into account with the
same sense chemical shift deviation for a stretch of more than 3
sequential residues(39) . This procedure results in surprisingly
accurate estimates(40, 41, 42) . For
hPTH(1-37), chemical shift indexing indicated a helical region
extending from Met
to Lys
, and a shorter
helical region around Leu
(Fig. 4). No other elements
of regular secondary structure were suggested by this procedure.
Figure 4:
Chemical shift diagram according to (Ref.
39) as described in the text.
This preliminary estimate was corroborated by the detection of
CH-NH(i,i+3) and
C
H-C
H(i,i+3) NOESY cross-peaks (Fig. 5). From the (i,i+3) cross-peak pattern, it is
suggested that the peptide has a propensity to form helical regions
from Ile
to Asn
and from Met
to
at least Asp
. The C
H-NH (i,i+3)
cross-peak pattern may be interpreted to indicate that this region
extends to Phe
.
Figure 5:
NOEs versus sequence. The
thickness of the bars qualitatively indicates the relative strength of
the NOESY cross-peaks. An asterisk indicates that the NOE
could not be observed because of frequency
degeneration.
The dgsa protocol employed for
structure determination yielded the energy values shown in . Superposition of 10 backbone structures, His to Leu
, resulting from this procedure showed that
the structure was well defined in this sequence region (Fig. 6a). A single structure selected from this group
of structures clearly displayed the secondary structure elements and
the formation of tertiary structure around the His
to
Ser
turn region (Fig. 6b). From the
NH
terminus, the first regular secondary structure element
was a single helix turn, Ile
to His
. The second
-helix was experimentally well defined by helix type intermediate
range NOEs from Ser
to Leu
. This helix
extended to Asp
during the first 5 ps of an unrestrained
molecular dynamics calculation. A flexible region through the COOH
terminus followed. The very NH
and COOH termini were not
fixed by long range NOESY cross-peaks. A turn structure, His
to Ser
, was defined by 23 interresidual NOESY
cross-peaks in this region. This turn was probably stabilized by
hydrophobic interactions involving Leu
and
Trp
, among others. Experimentally, the relative position
of these two amino acids was fixed by five long range NOESY
cross-peaks. Furthermore, 13 NOESY cross-peaks between Trp
and Lys
, four NOESY cross-peaks between Trp
and Arg
, and six NOESY cross-peaks between
Leu
and Arg
defined the tertiary fold. This
observation of tertiary interactions was corroborated by the set of
spectra obtained at 283 K. Taking into account information at both
temperatures, the total number of interresidual NOEs defining the turn
region increased to 31, and the total number of interresidual NOEs
between Leu
and Trp
to six. These direct
observations of tertiary interactions clearly lent further support to
the results of the restrained molecular dynamics calculations that were
based on the 298 K data only.
Figure 6:
a, best fit superposition of the peptide
backbone atoms, amino acids His to Leu
, of
the 10 structures calculated and selected as described under
``Material and Methods.'' b, stereo picture of a
single structure selected from the set in a, with key residues
labeled.
Stability of Structure
Small linear peptide
fragments usually display a higher amount of flexibility than globular
proteins, that is they populate a whole range of
conformations(43) . A general problem of structural NMR studies
of flexible molecules is the very nature of the observed NOE data as a
population weighted average. Thus, in particular in peptide studies,
additional information from other sources, spectroscopic and
computational, is needed to further characterize the dynamic state of
the system(43) . Suggestions have been made to resolve this
situation with restrained molecular dynamics methods(44) , but
these procedures are not yet applied widely.
Relative NOE Intensities
The geometry of an
idealized -helix suggests that the backbone atom distances dC
(i,i+1)
and dC
(i,i+3)
are very similar to each other, whereas d
(i,i+1)
distances are appreciably shorter (approximately 0.35 versus 0.28 nm;(45, 46) ). Distances involving side chain
C
protons may occupy a wider range of distances in
-helices (dC
(i,i+3)
= 0.25 - 0.44 nm, dC
(i,i+1)
= 0.25 - 0.41 nm(28) ). Consequently, the ratio of
these intensities may be used as a qualitative estimate of the
fractional helix content of specific sequence regions. In the
dimerization stabilized
-helix of the GCN4 leuzine zipper, the
intensity of several classes of these NOESY cross-peaks is of
comparable magnitude in some
studies(47, 48, 49) . The same is true for
typical zinc-finger
-helices that are stabilized by the divalent
metal ion and related
motifs(50, 51, 52, 53) . Nascent helix
structures (54) do not show i,i+3 NOESY
cross-peaks, and the basic domain-leucine zipper motif basic
DNA-binding domain of GCN4 (55) may be considered an example of
this type of nascent helix structure. Absence or very low intensity of i,i+3 cross-peaks is also observed in studies of
some leucine zipper motifs, for example the Jun oncoprotein homodimer
(56). From this comparison of NOESY cross- peaks, the helical regions
of hPTH(1-37) in TFE-free solution showed a fractional helix
content similar to the zinc-finger and the more stable category of
leucine zipper
-helices. Appearance of strong
N(i,i+1) cross-peaks in the
helices of hPTH(1-37) indicated contribution of some extended
conformation even in these structured regions. Direct quantitative
estimate, however, of fractional helix content for PTH(1-37)
based on the present NMR data is not possible.
CD Spectroscopy
Estimates of the secondary
structure content of hPTH(1-37) based on published procedures (37, 38) indicated 28-30% helical structure. This
corresponds to 11 amino acids employed in perfectly stable regular
secondary structure or, alternatively, to more than 11 amino acids
involved in weaker, transient helix formation. Secondary structure
determination by NMR and NOE-based restrained molecular dynamics
calculations suggested that Gln to His
and
Ser
to Leu
formed helical structures
according to structure analysis with the DSSP program
package(36) . Assuming that the length of the helices is
reflected correctly by this procedure, the CD measurements showed that
the helical sequences in hPTH were in helical conformation in 70% of
the time on average. Judging this result, one has to take into account
the low sensitivity of CD spectroscopy with respect to the
determination of helices less than 6 amino acids in length. It is
believed that 4 amino acid helices result in CD spectra that clearly
deviate from the ideal helix type spectra, but these matters are still
under discussion ((57, 58) and literature cited therein). Thus, a time
average of 70% for the presence of helices in hPTH(1-37) clearly
is a conservative lower limit of the time averaged formation of helices
in the two hPTH regions Gln
to His
and
Ser
to Leu
.
Local rmsd Values
The superposition of the helical
structure of hPTH(1-37) resulted in a comparatively low rmsd
value. This indication of helix stability is confirmed by calculating
local rmsd values(59) . The latter showed a very marked decrease
in the helical regions (Fig. 7).
Figure 7:
Plot of
local rmsd values (59) versus amino acid sequence. Backbone
atoms of 5 amino acid segments of two structures were aligned and the
pairwise rmsd values calculated. The procedure was performed for the 10
structures in Fig. 6a. The average backbone rmsd value is
represented as a function of the sequence number of the residue in the
window center.
Unrestrained Molecular Dynamics
Unrestrained MD
calculations of the hPTH(1-37) peptide in HO were
performed to probe the structural stability of the two helical regions
determined by NMR and restrained MD calculations, that is Gln
to His
and Ser
to Leu
. The
200 ps simulation calculations clearly showed that the extent of the
helical conformation was slightly increasing, and the helical regions
were from Ile
to Asn
and Ser
to
Asp
/Val
for most of the time. The local
formation of
-helices proved to be stable as shown by a diagram of
helix length versus time (Fig. 8b). The rmsd
value of the backbone and side chain heavy atoms of the structures in
the unrestrained molecular dynamics calculation as compared to the
starting structure increased only slightly at the very beginning of the
calculation, as expected, and remained stable for the remaining period (Fig. 8a). Earlier 200 ps simulations of this type
performed on
-helix H8-HC5 of myoglobin (residues 132-153)
showed that this helix is stable in TFE, whereas it is unstable in
H
O on this time scale(60) , validating the MD
approach to peptide stability characterization. Comparison of the
results of our unrestrained MD calculations on hPTH(1-37) with
those on
-helix H8-HC5 of myoglobin clearly show that the latter
is much less stable in H
O on this time scale(60) .
Furthermore, the unrestrained molecular dynamics simulations on
hPTH(1-37) also showed that the small hydrophobic core formed by
Leu
and Trp
is stable during the simulation
time of 200 ps.
Figure 8:
a, backbone rmsd values of current
structure versus starting structure during the 200-ps
unrestrained molecular dynamics calculation. Lower trace,
backbone heavy atoms; upper trace, all heavy atoms. b, time dependence of helix length during the 200-ps
unrestrained molecular dynamics calculation. Helix structure content
was evaluated by use of the DSSP program every 5 ps. The two helices
are symbolized as bars in this
figure.
Using the longer helices obtained during the
unrestrained MD calculations as the basis for an estimate of the time
averaged helix content by CD spectroscopy reduces the lower bound for
the existence of full-length helices to 55%.
Comparison with Other Peptides
Secondary structure
formation is reported for a number of small peptides (43, 61 and
literature therein). Many of these small peptides are preferentially in
-helical conformation, although a few
-sheet-containing
peptides are known(43) . Observation of tertiary interactions,
however, in monomeric, linear peptides as small as hPTH(1-37) or
hPTH(1-34)
(
)is unusual. In particular,
conformations as rigid as the one we suggest for hPTH(1-37) on
the basis of NMR spectroscopy, CD spectroscopy, and restrained as well
as unrestrained molecular dynamics calculations, are rarely reported.
Although it is difficult to point out any single reason for structure
formation, it seems that the helix from Ser
to Leu
plays a crucial role in positioning the central Trp
residue so that it may establish stable hydrophobic interactions
with Leu
. A similar example of a peptide showing tertiary
interactions is the chimeric 25 amino acid peptide made of the core
domain of equine infectious anemia virus transactivator (Tat) protein
and the basic domain of human immunodeficiency virus Tat
protein(42) . This peptide forms a hydrophobic core that is
structurally highly similar to the identical sequence region in
full-length Tat proteins(63, 64) . In the peptide, this
hydrophobic core is also flanked by an
-helix, but no long range
interactions between core and helical region are observed.
hPTH Structure in TFE Containing versus TFE-free
Solution
Comparing the results of the present structure
determination close to physiological conditions with the secondary
structures determined in TFE-containing solution (see, for
example,(9, 13) ), the following picture emerges:
hPTH(1-34) does not display a well-defined tertiary structure in
TFE-containing solution, a fact that is made obvious by the complete
lack of long range NOEs in these studies. In contrast, in TFE-free
solution the tertiary structure of the peptide is experimentally well
defined by five long range NOEs between Leu and
Trp
. The turn region from His
to Ser
is stabilized by hydrophobic interactions. TFE with its lower
polarity,
= 26.7(23) , has a strong tendency to
overcome the hydrophobic stabilization energy. This adverse effect of
TFE on hydrophobically stabilized protein structures was repeatedly
observed (see, for example, Ref. 19).
-terminal
-helical region from Glu
to
Lys
, well defined by NOEs typical for stable
-helical
structure. In contrast to these studies, we found a shorter helix from
Gln
to His
by NOE-based restrained MD
calculations (Ile
to Asn
on the basis of
subsequent unrestrained MD calculations as above), also indicated by
the chemical shift index plot (Fig. 5) and the backbone NOE
pattern (Fig. 6). In this respect, our results were in agreement
with earlier NMR studies of hPTH(1-34) in TFE containing aqueous
solution, where the NH
-terminal helix ends with
His
(9) . We found a flexible hinge around Gly
and a Gly
J
coupling constant of
approximately 12 Hz, as observed earlier by others(9) . The
flexible region around Gly
and Lys
is
necessary for the hormonal activity in an in vitro adenylate
cyclase assay(65) . This fact contrasts the notion of a long
NH
-terminal helix suggested earlier for hPTH(1-34) in
TFE-free aqueous solution(10) .
to
Ser
followed by an
-helix from Ser
to
Leu
as determined by NOE-based restrained MD calculations
(Ser
to Asp
on the basis of subsequent
unrestrained MD calculations as above). This region of the molecule
forms a major part of the receptor binding
domain(5, 6) . The extent of the COOH-terminal helix is
in good agreement with previous work in TFE containing as well as in
TFE-free aqueous solution. Not only did the length of this helix in
hPTH(1-37) vary in the unrestrained MD calculation, but also
strong
N(i,i+1) NOEs were observed
in this region. This helix thus seems to be in a less compact, more
stretched conformation compared to the corresponding helix in
hPTH(1-34) in TFE-containing aqueous solution.
to Leu
helix observed in TFE-free solution is nearly
an ideal helix when compared with the propensity of amino acids for
certain positions in an
-helix(62) . The N-cap is
Asn
or Ser
, depending on the definition of
the helix start. The positively charged side chains in the last turn
are Arg
, Lys
, and Lys
. Glu has a
high probability to be at the second position COOH-terminal from the
C-cap. These residues are represented by Glu
and
Asp
in the case of hPTH(1-37). Hydrophobic amino
acids are suggested to occupy positions +4 from the NH
terminus and -4 from the COOH terminus of the helix.
Indeed, Val
and Leu
are at just these
positions.
, Val
, Ser
3, and
Glu
of hPTH(1-37) are flexible and not included in
any obvious secondary structural element. They also do not take part in
hydrophobic interactions. Independent experiments, however, show that
cleavage of hPTH between Val
and Ser
abolishes
stimulation of cAMP-related activity(3) . This indicates that
the role of the NH
-terminal amino acids cannot be explained
on the basis of their local structure in the absence of receptor
interaction.
Table: H chemical shifts and assignments
for hPTH(1-37) at pH 6.0, 298 K, 250 mM NaCl relative to
DSS as an external standard, accuracy +/- 0.01 parts/m
Table: Energy contributions to the structure
and deviations from standard geometry
,
total energy; E
, van der Waals energy; E
, effective NOE energy term resulting from a
soft square-well potential function as described in the text. All
calculations were carried out using the standard X-PLOR force field and
energy terms. The values are mean values over 10 refined structures.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.