Structure and potential C-terminal dimerization of a recombinant mutant of surfactant-associated protein C in chloroform/methanol Burkhard Luy1, Alexander Diener2, Rolf-Peter Hummel3, Er
Trang 1Structure and potential C-terminal dimerization of a recombinant mutant of surfactant-associated protein C in chloroform/methanol
Burkhard Luy1, Alexander Diener2, Rolf-Peter Hummel3, Ernst Sturm3, Wolf-Ru¨diger Ulrich4
and Christian Griesinger5
1
Institut fu¨r Organische Chemie und Biochemie, Technische Universita¨t Mu¨nchen, Garching, Germany;2Institut fu¨r Organische Chemie, Johann Wolfgang Goethe-Universita¨t Frankfurt, Germany;3Department of Physical Organic Chemistry and4Department of Chemical Research, Altana Pharma AG, Konstanz, Germany;5Max Planck Institut fu¨r Biophysikalische Chemie, Go¨ttingen, Germany
The solution structure of a recombinant mutant
[rSP-C (FFI)] of the human surfactant-associatedprotein [rSP-C
(hSP-C) in a mixture of chloroform andmethanol was
determined by high-resolution NMR spectroscopy
rSP-C (FFI) contains a helix from Phe5 to the rSP-C-terminal Leu34
andis thus longer by two residues than the helix of porcine
SP-C (pSP-C), which is reportedto start at Val7 in the same
solvent Two sets of resonances at the C-terminus of the
peptide were observed, which are explained by low-order
oligomerization, probably dimerization of rSP-C (FFI) in
its a-helical form The dimerization may be induced by
hydrogen bonding of the C-terminal carboxylic groups or
by the strictly conservedC-terminal heptapeptide segment with a motif similar to the GxxxG dimerization motif of glycophorin A Dimerization at the heptapeptide segment wouldbe consistent with findings basedon electrospray ionization MS data, chemical cross-linking studies, and CNBr cleavage data
Keywords: dimerization; NMR spectroscopy; surfactant; surfactant protein C (SP-C)
Surfactant-associatedprotein C (SP-C) is a
34–35-amino-acidpeptide which is highly conservedamong species
(Table 1) It is part of the protein–phospholipidcomplex
that is secretedinto the alveolar space [1] andis
responsible for lowering of the alveolar surface tension
Recombinant (r)SP-C (FFI) surfactant (Venticute) has
provedto be highly effective in animal experiments [2,3]
as well as in pilot clinical trials [4,5] The structure of
porcine SP-C (pSP-C) has been solvedin CDCl3/CD3OH/
0.1M HCl (32 : 64 : 5, v/v/v), andit has been found
that the peptide forms an a-helix from residue 7 to the
C-terminal residue 34 [6] The N-terminal structure as well
as the hydrophobic a-helix seems to be conservedin the
micellar environment as shown for the N-terminal 17
residues of pSP-C in fully deuterated
dodecylphospho-choline micelles [7] A secondset of resonances was found
for the full-length pSP-C peptide in chloroform/methanol
at the C-terminus, which was explainedby partial
oxidation of the methionine residue M32 In general,
samples of the lipophilic pSP-C are not completely stable
in chloroform/methanol mixtures andform a gel-like
b-sheet aggregate after several days at 10C [8] A mutant
of the human SP-C (hSP-C) has been produced recom-binantly by omitting the residue [Phe() 1)] that is only partially present andperforming the following substitu-tions: C4F, C5F andM32I The rationale behindthe substitutions is that the two cysteine residues are naturally palmitoylated, which would have been difficult to achieve for a bacterially expressedprotein The mutation of residue 32 was to prevent the undesired putative oxidation
of methionine In this article, we present the structure of the rSP-C (FFI) mutant in CDCl3/CD3OH (1 : 1, v/v) with a comparison with the structure of pSP-C A second set of C-terminal signals is explainedby the coexistence
of monomeric andoligomeric (probably dimeric) rSP-C (FFI)
Materials and methods
Preparation of the sample For the studies on rSP-C (FFI) (Altana Pharma AG, Konstanz, Germany; WO patent no 95/32992), we usedthe solidsubstrate consisting of the peptide (90%), HCl (4%), propan-2-ol (3%), water (2%), andmethyl ester (1%) Samples of rSP-C (FFI) were preparedby dissolving 3–12 mg of the powder in 600 lL CDCl3/CD3OH (1 : 1, v/v) or CDCl3/CD3OD (1 : 1, v/v) The resulting
rSP-C (FFI) concentration was 1.1–4.4 mM, respectively The solidpeptide was storedat )20 C, andthe prepared samples were storedin liquidnitrogen between NMR measurements Dissolvedsamples hada lifetime of 72 h
at 10C Over time, the dissolved peptide maintained identical NMR chemical shifts, but strongly reduced intensity, indicating similar aggregation to b-sheet-like
Correspondence to C Griesinger, Max Planck Institut fu¨r
Biophysi-kalische Chemie, Abt NMR basedStructural Biology,
Am Fassberg 11, 37077 Go¨ttingen, Germany.
Fax: + 49 551201 2202, Tel.: + 49 551201 2201,
E-mail: cigr@nmr.mpibpc.mpg.de
Abbreviations: SP-C, surfactant-associatedprotein C; hSP-C, human
SP-C; pSP-C, porcine SP-C; rSP-C, recombinant human SP-C; rSP-C
(FFI), FFI variant of recombinant human SP-C; TACSY, taylored
correlation spectroscopy.
(Received17 December 2003, revised1 March 2004,
accepted23 March 2004)
Trang 2structures as observedfor natural pSP-C in the solvent used.
Because of the limitedlifetime, samples were prepared
immediately before NMR measurements
NMR measurements
2D1H-NMR spectra were recorded on Bruker DRX 800,
DMX 600, AMX 600 andAMX 400 spectrometers in the
pure-phase absorption mode using the States-TPPI method
[9] All spectra were recorded at 10C, andprocessing
andbaseline corrections were performedusing the standard
Bruker softwareXWINNMR The complete set of experiments
recorded is given in Table 2
The1H-NMR chemical shifts were calibratedrelative to
trimethylsilane The residual water signal and the signal
of the hydroxy proton of CD3OH are degenerate at
4.8 p.p.m andwere reducedusing presaturation [10]
Before Fourier transformation, the time domain data were
multipliedwith shiftedsquaredsinebell window functions
The vicinal scalar coupling constants 3JNHa were
deter-minedusing the SIAM-TACSY andKeeler–Titman
approaches [11,12] using macros written by T Prasch for
the programFELIX(Felix 95; MSI, San Diego, CA, USA)
Signal overlap in the 800-MHz NOESY made peak
integration unreliable So, instead, signal height of the
cross-peaks was usedfor a conservative estimation of the
maximum distances and classification of cross-peaks as
weak, medium and strong For the calibration of the
intensities of the NOE peaks, a statistical analysis of the
daN(i,i+3) signals of residues 11–30 was performed using
typical values for an ideal a-helix [13] The a-helical
structure of this part of the peptide is clearly evident from
Hachemical shifts [14,15]
Results
NMR assignment Sequence-specific 1H-NMR assignment was achievedby standard procedures for small proteins [13] using the computer program NDEE (Spin Up, Lu¨nen, Germany) Owing to the high abundance of the amino acids valine, leucine andisoleucine in the sequence of rSP-C (FFI), there was extensive overlap in the homonuclear1H-NMR spectra Nevertheless, almost all spin systems (vide infra) couldbe assignedfrom the TOCSY spectra (Fig 1A) andthe DQF-COSY spectra (not shown) collected under identical conditions (Table 3)
The unique spin systems His8, Lys10, Arg11 andAla29, andthe pairs of Phe andPro residues andGly28 andGly33 were unambiguously identified, as well as 10 of the 11 valines The N-terminal Gly1 shows a single very broad
HN/Hacross-peak Although all 34 amino acids were found, the spin systems of seven leucines, five isoleucines andthe residual valine could only be unambiguously identified using sequential NOE information
The high dispersion of the 800-MHz NOESY spectrum made it possible to obtain the complete assignment of
rSP-C (FFI) (Fig 1B,rSP-C) Starting from the unambiguously identified residues, we were able to carry out the sequential assignment for residues 1–17 and 24–34 by daNanddNN cross-peaks As an a-helical secondary structure was assumedfrom chemical-shift arguments, daN(i,i+3) and
daN(i,i+4) NOE cross-peaks were used, leading to the assignment of the residual amino acids 18–23
We encountered special difficulties in identifying the following connectivities: the chemical shifts of the amide
Table 2 NMR experiments.
Spectrometer frequency (MHz)
Data matrix
Processed matrix
Mixing time (ms)
Total time (h) 1.1 m M rSP-C (FFI) in CDCl 3 /CD 3 OH (1 : 1) TOCSY 600 4096 · 768 4096 · 1024 70 11
DQF-COSY 600 4096 · 1024 4096 · 1024 – 12 4.4 m M rSP-C (FFI) in CDCl 3 /CD 3 OH (1 : 1) NOESY 800 8192 · 1024 8192 · 1024 50 24 1.1 m M rSP-C (FFI) in CDCl 3 /CD 3 OH (1 : 1) SIAM-TACSY 600 4096 · 400 4096 · 1024 70 12 1.1 m M rSP-C (FFI) in CDCl 3 /CD 3 OH (1 : 1) NOESY 400 4096 · 1024 4096 · 1024 50 12
Table 1 Amino-acid sequences of several SP-Cpolypeptides, including human, porcine and recombinant human SPCwith FFI substitution [rSP-C(FFI)].
Species Amino-acidsequence
Trang 3protons of Leu21, Val23 andLeu31 are degenerate so there
was a large overlap in the dNNcross-peaks As the amide
protons of Ile22 andLeu30 also overlapped, the assignment
was even more difficult The identical Hachemical shifts of
Leu12, Leu13 andLeu21 causedfurther problems in the
sequential assignment The same occurredfor the dNN connectivities to Val27 because Ile26 andGly28 have almost identical amide proton chemical shifts Except for some side chain protons of Ile14, Ile22 andIle26, all1H resonances of rSP-C (FFI) were assigned Stereochemical assignments for
Fig 1 NMR assignment (A) Assignment of the spin systems of 32 nonproline residues out of the 34 amino acids of rSP-C (FFI) illustrated in the TOCSY experiment with a mixing time of 70 ms Shown is the so-calledfingerprint region where the well-dispersedHNprotons are correlatedto the H a andside chain protons The spin systems of Lys10 andArg11 are indicatedby rectangles as both contain a secondH N in the side chain The N-terminal Gly1 appears as a weak andvery broadpeak All H a chemical shifts of residues 5–31 show an upfieldshift comparedwith random-coil data indicating an a-helical structure in an empirical pattern-recognition approach [13,16] (B) HN-HNregion of the 800-MHz NOESY experiment Sequential d NN (i,i+1) connectivities can be found for all nonproline amino acids For the C-terminal residues 31–34, a second set of resonances can
be sequentially assignedindicatedby the prime in the annotation of the corresponding NOE connectivity (C) H N -H a region of the 800-MHz NOESY experiment All resolved interresidual NOE connectivities are annotated In particular, the d Na (i,i+3) andd Na (i,i+4) connectivities are indicators of an a-helical secondary structure Intraresidual signals are not annotated (D) Summation of the experimental NMR data Shown are all resolvedNOE connectivities, where thin bars indicate distances > 4.0 A˚, medium bars distances of 3.0–4.0 A˚, andthick bars distances < 3.0 A˚ The d Na (i,i+3), d Na (i,i+4) as well as the d NN (i,i+2) andthe strong d NN (i,i+1) connectivities clearly show the a-helical structure of rSP-C (FFI) In addition,3J NHa coupling constants are summarized, with small circles indicating couplings < 5.0 Hz and large circles for constants > 6.0 Hz Pentagons classify the exchange properties of amide protons in weak exchange (filled pentagons), medium exchange (open pentagons) and strong exchange (no pentagon) as described in the text.
Trang 4the diastereotopic groups were inferred from NOEs
through floating chirality calculations
Second set of resonances
Closer inspection of the spectra revealedtwo sets of
resonances for Gly28, Leu30, Ile32, Gly33 andLeu34,
which differ mainly in the chemical shifts of the amide
protons andthe c protons of Ile32 andLeu34 A
compar-ison of the spectra showeddifferent relative intensities of the
two sets of resonances with respect to the concentration of
rSP-C (FFI) in CDCl3/CD3OH (1 : 1, v/v) andthe age of
the sample For a systematic analysis, freshly prepared
samples with concentrations of 0.7–3.5 mM were usedin
NOESY experiments with a mixing time of 50 ms At low
concentration, the two sets of signals were almost equally strong, whereas at higher concentrations of rSP-C, one of the signal sets was more predominant Attempts to fit the relative intensities of the two sets of resonances to a quantitative monomer–dimer equilibrium model failed (data not shown) However, the concentration dependence shown in Fig 5 can be considered an indication of intermolecular interaction The comparable linewidths of the signals of the two sets of resonances still suggest that monomeric and dimeric units are involved
Amide proton exchange The exchange properties of the amide protons were
rSP-C (FFI) in CDCl3/CD3OD (1 : 1, v/v) with the sample freshly preparedabout 1 h before the experiment All measurable Ha-HNcross-peaks were integratedandcom-paredwith the integrals of the 800-MHz NOESY spectrum The most intense signals were taken as 100% relative intensity, making the assumption that no significant exchange occurredin the given time frame within the center
of the well-ordered a-helix The relative intensities of the
Ha-HNcross-peaks of residues His8, Ala29 and Leu31 were about 50% of those recorded in the 800-MHz NOESY spectrum in CDCl3/CD3OH (1 : 1, v/v), andthe intensities
of residues 9–28 and 30 were 80% or higher From these estimates of the relative intensities, hydrogen bonds for the structure calculations were assumedfor His8 to Leu31 The amide protons of residues 1–7 and 32–34 could not be detected in the fully deuterated solvent
Structure of rSP-C (FFI) Using the empirical pattern-recognition approach [16], the combination of strong sequential dNNconnectivities, obser-vation of a significant number of daN(i,i+3), dab(i,i+3), anddaN(i,i+4) connectivities,3JNHacoupling constants of less than 5 Hz for all non-Gly residues in the polypeptide segment Phe5, Val7–Leu30, andretardedamide proton exchange for residues 8–31 indicate that rSP-C (FFI) forms
a long a-helix comprising approximately residues 5–34 For a more precise definition of the structure of
rSP-C (FFI), a set of 203 intraresidual, 201 interresidual and seven ambiguous NOE-derived upper distances were used together with 23 / angles derived from3J(HN,Ha) coupling constants as input data for a structure calculation using the programXPLOR[17] In addition, we introduced 24 hydro-gen bonds derived from the slow exchange rate of the amide protons No stereospecific assignments were usedin the floating chirality simulatedannealing protocol For residues 28–34, we usedonly the set of resonances with the stronger intensities because identical relative NOEs were observed for the two species
For the structure calculations, we useda standard simulatedannealing protocol designedfor proteins [18] After an initial energy minimization involving 50 optimiza-tion steps with conjugatedgradients, a high temperature phase with 2000 K was simulatedfor 32.5 ps in which all upper limits built the active constraints The following step was the first cooling phase from 2000 K to 1000 K in 25 ps with the dihedral angles as additional constraints After the
Table 3 Chemical shifts of rSP-C (FFI).
Residue H N H a H b Others
Gly1 8.23 3.73
Ile2 8.61 4.45 1.90 c1.66, 1.00; d1.23,0.95
Pro3 4.38 2.15, 1.99 c2.10; d3.95, 3.72
Phe4 8.08 4.49 3.18, 3.09 d7.17; e7.27; f7.19
Phe5 8.46 4.68 3.29 d7.28; e7.42; f7.36
Pro6 4.25 2.34, 2.00 c2.14; d3.65
Val7 7.62 3.69 2.28 c1.13, 1.01
His8 8.05 4.47 3.35, 3.29 d7.22; e8.74
Leu9 8.13 3.97 1.70, 1.60 c1.65; d1.03, 0.98
Lys10 7.95 3.91 2.03 c1.64, 1.50; d1.79;
e2.93; f2.92 Arg11 7.89 3.94 2.02, 1.99 c1.70; d3.30, 3.24; e7.50;
1g7.18; 2g6.68 Leu12 7.82 4.01 1.69 c1.81; d0.94
Leu13 8.04 4.01 1.89 c1.71; d0.95
Ile14 7.77 3.64 2.08 c1.94, 1.20; d0.97, 0.93
Val15 7.67 3.52 2.40 c1.17, 1.01
Val16 8.01 3.51 2.33 c1.15, 1.00
Val17 8.03 3.52 2.33 c1.16, 1.02
Val18 8.15 3.57 2.32 c1.14, 1.03
Val19 8.36 3.57 2.32 c1.15, 1.00
Val20 8.35 3.49 2.31 c1.15, 1.00
Leu21 8.25 4.01 1.99, 1.93 c1.75; d1.02, 0.94
Ile22 8.30 3.60 2.16 c1.17; d0.99
Val23 8.25 3.53 2.40 c1.16, 0.99
Val24 8.59 3.57 2.42 c1.17, 1.02
Val25 8.28 3.71 2.39 c1.17, 1.03
Ile26 8.45 3.70 2.09 c1.95, 0.98; d1.17
Val27 8.93 3.59 2.23 c1.14, 1.02
Gly28 8.45 3.88, 3.77
8.42 3.86, 3.77
Ala29 8.21 4.09 1.62
8.19 4.08 1.62
Leu30 8.25 4.18 2.12, 2.03 c1.60; d0.99
Leu31 8.30 4.14 2.07 c1.60; d0.99, 0.96
8.28 4.13 2.07 g1.60; d0.99,0.96
Ile32 7.70 4.35 2.18 c1.64,1.51; d1.02
7.64 4.38 2.18 g1.61,1.55; d1.02
Gly33 7.95 4.09, 3.87
7.91 4.09, 3,85
Leu34 8.02 4.51 1.73 c1.78; d1.00
8.08 4.55 1.64 g1.74; d1.00
Trang 5secondcooling phase from 1000 K to 100 K in 10 ps, a
secondenergy minimization was performedwith 200 steps
of conjugatedgradients for each structure The rmsdvalues
and the distance and dihedral angle violations for the best 10
out of 60 structures are given in Table 4 The final structures
shown in Figs 3 and 4 were determined by an additional
refinement in vacuo including the experimental restraints,
full charges, anda dielectric constant set to e¼ 4rijusing a
heating andcooling protocol
Figure 2 showsMOLMOLstereographic projections [19] of
the heavy atoms of C (FFI) The structure of
rSP-C (FFI) is a well-defined a-helix ranging from Phe5 to
Leu34 Note that the distribution of the / and w angles
indicates an a-helical structure up to Phe5, although residue
6 is a proline Strong evidence for this comes from the
unambiguously identified daN(i,i+3) anddaN(i,i+4)
cross-signals for Phe5 andPro6 (cf Fig 1D)
Discussion
Comparison of rSP-C (FFI) with pSP-C
The 34-residue peptide rSP-C (FFI) contains mainly
apolar amino acids, i.e 11 valines, seven leucines and
five isoleucines, andforms a well-defineda-helix along
residues 5–34 dissolved in CDCl3/CD3OH (1 : 1, v/v) The
solution structure of pSP-C with 76% sequence identity
(Table 1) in CDCl3/CD3OH/0.1MHCl (32 : 64 : 5, v/v/v)
was investigatedby Johansson et al [6] To compare the
structure of pSP-C with rSP-C (FFI), we show in Fig 3
the differences in chemical shifts of the HNandHasignals
of the corresponding residues It can be seen that the
chemical shifts for residues 10–29 are almost identical,
with slightly greater variations at nonidentical amino
acids Only the N-terminal nine residues show significant
chemical-shift differences mainly introduced by the
sequence deviations at residues 4, 5 and 8 This difference
at the N-terminus can also be seen when the two resulting
structures shown in Fig 4 are compared Whereas the
backbone of the central a-helix is very well defined in
both structures, the N-terminal variability for the pSP-C is
greater than that of rSP-C (FFI) This reflects the
NOE-data-based fact that rSP-C (FFI) has a defined a-helix
comprising residues 5–34, whereas for pSP-C an a-helical region at residues 7–34 has been reported [6]
However, the slow deuterium exchange for Leu9 and small distances dNa(i,i+3) anddab(i,i+3) for Pro6 andVal7 suggest that even pSP-C adopts an a-helix starting with capping at residue Cys5 [8] Substitution of acylated Cys with Phe in the polypeptide seems to influence the N-terminal a-helix formation including Pro6 in rSP-C (FFI) A possible explanation is the occurrence of aromatic interactions between Phe5 andHis8 which may leadto stabilization of the extendeda-helix The structures
of both pSP-C andrSP-C (FFI) were determinedin chloroform/methanol, an environment in which hydropho-bic elements can move freely Membranous environments such as the surfactant, however, have a directional effect on the hydrophobic palmitoylatedCys andPhe residues andon the chargedLys andArg residues at positions 10 and11, which probably results in slightly different N-terminal structures for the SP-C variants in their biologically active form
The central helix of pSP-C has a slightly lower rmsdvalue than that of rSP-C, probably because of the longer stretch of Val residues, leading to extremely stable stacking In
rSP-C (FFI) this homogeneous stacking is interruptedby Ile14 andIle22, which may introduce slight mobility into the hydrophobic a-helix However, this increasedmobility still leaves the central helix quite rigidanddoes not seem to be important, as it was shown in mutation studies that SP-C retains its function even after the replacement of all valines
by leucines or other a-helical amino-acidsequences [20,21] Two sets of resonances
Two sets of resonances were foundfor rSP-C (FFI) at the C-terminal residues Gly28, Leu30, Ile32, Gly33 and Leu34 Similar duplication of resonances has been reported for pSP-C, affecting residues Val27, Ala29, Leu30, Leu31 and Met32 [6] In the case of pSP-C, the additional signals were explainedby partial oxidation of Met32 to methionine sulfoxide In the case of rSP-C (FFI), a different explan-ation must be foundfor the secondset of resonances because Met32 is substitutedby Ile32 The careful studies on pSP-C show a variation of 20–50% of the minor populated
Table 4 Analysis of the 10 best calculated structures before and after the refinement.
E tot (kcalÆmol)1) 165.9 ± 12.7 (142.4.181.6) ) 265.4 ± 7.5 ()266.9 … )246.1) Distance violations
Torsion-angle violations
Rmsds (A˚)
Heavy atoms (8–33) 1.05 ± 0.18 (0.90.1.44) 0.82 ± 0.13 (0.67.1.00) Backbone (18–28) 0.23 ± 0.08 (0.14.0.41) 0.07 ± 0.02 (0.04.0.10) Heavy atoms (18–28) 0.61 ± 0.08 (0.51.0.77) 0.45 ± 0.12 (0.35.0.68)
Trang 6set of resonances among samples preparedfrom different
batches We observedthe same variation even in samples
preparedfrom the same batch A closer look at the acquired
spectra indicates a dependence of the relative population
of the signals on the overall SP-C concentration As a
consequence, we acquireda set of 2D NOESY spectra with
identical mixing times but different concentrations of
rSP-C (FFI) in rSP-CDrSP-Cl3/CD3OH (1 : 1, v/v) The relative
popu-lations of the two sets of resonances in these spectra with
respect to the overall SP-C concentration are shown in
Fig 5 The dependence observed is a clear indication of
intermolecular interaction The relatively narrow linewidths
of the observedsignals ledto the conclusion that oligomers
of low order are present, probably monomeric and dimeric
units, but trimeric or tetrameric units may also be possible; larger oligomers can be excluded because the linewidths wouldhave to be significantly broader than observed The linewidths of the two sets of resonances do not differ significantly, therefore the two oligomers must be of comparable size, anda monomer/tetramer equilibrium, for example, cannot explain the observedsignals The absence of further resonances implies that we are observing specific oligomers Finally, chemical shifts of the Ha resonances are a clear indication that both oligomers are mainly a-helical andthat their structures differ only slightly The NMR data therefore point to the coexistence of
a monomeric andhomodimeric a-helical form of rSP-C (FFI)
Fig 2 Stereographic projection of the best 10
out of 60 structures of rSP-C(FFI) (A) Side
view of the heavy atoms of the full-length
peptide (B) View from the bottom along
residues 15–27 of the tightly packed a-helix.
Trang 7The literature on SP-C describes many oligomerization
processes, most of which are either aggregates with mainly
b-sheet-like or undetermined structure Specific
oligomeri-zation, i.e dimerioligomeri-zation, is only reported in a few cases:
MS data provide evidence for dimeric SP-C [22,23], and
chemical cross-linking studies also show mainly a specific
dimer of mature SP-C (Fig 8C in [24]) Yet unpublished
high-resolution Fourier-transform ion-cyclotron-resonance
MS, light-scattering andCD experiments reveal the
exist-ence of an a-helical dimer at acidic pH ([25]; A Seidl,
G Maccarone, N Youhnovski, K P Schaefer and
M Przybylski, unpublished data) CNBr cleavage data
even put the dimerization site near Met32 at the C-terminus,
i.e at the site at which the dual resonances are observed [23]
The coexistence of monomeric andhomodimeric
rSP-C(FFI) as derived from the NMR data therefore
corres-ponds well to other reported experimental observations
Fibril formation
The data from Fig 5 couldnot be fittedto a simple
monomer–dimer equilibrium model, but this is not
surpri-sing considering that rSP-C (FFI), like pSP-C, shows a
complete transition to b-sheet fibrils over time [8,26,27]
Immediately after rSP-C (FFI) is dissolved in chloroform/
methanol, short, fiber-like impurities of up to 1 mm length
are observedin solution andon the glass walls of the NMR
tube on visual inspection This indication of already formed
fibrils makes it necessary to describe rSP-C (FFI) by at least
a three-state model with two a-helical states, probably
monomer anddimer, andb-sheet fibrils that cannot be
observedby high-resolution NMR because of their high
molecular mass A three-state model with monomeric, nonhelical and b-fibril states has already been presented [8] Interestingly, the existence of an a-helical transition state (SPC#in [8]) was proposedin that publication, which would
Fig 4 Comparison of the 10 best structures of rSP-C (FFI) (left) and pSP-C(right) The backbone of the a-helix is shown Clearly visible
is the better defined secondary structure of rSP-C (FFI) near the N-terminus.
Fig 5 Concentration dependence of the relative integrals of the two sets
of resonances observed at the C-terminus Ratios are given for well-resolvedresidues Ile32, Gly33 andLeu34.
Fig 3 Differences in the chemical shifts of rSP-C(FFI) compared with
pSP-C[6] for the H N (A) and the H a protons (B) Whereas residues
10–29 show almost identical chemical shifts, residues at the N-terminus
andC-terminus differ more strongly.
Trang 8match the potential a-helical dimer found here The
interpretation of the potential dimeric state as the transition
state to b-fibril formation wouldalso match recent
solid-state andliquid-solid-state NMR results, which suggest that the
smallest fibril diameter in b-amyloidfibrils is due to a
parallel b-sheet dimer [28,29] and also that the minimum
unit needed for fibril growth of a-synuclein is a dimer [30]
The disappearance of high-resolution NMR signals after
several days at 10C in chloroform/methanol shows that
the equilibrium state of rSP-C (FFI) is the b-sheet-like
multimer The a-helical states are therefore not equilibrated,
and, in addition to the observed concentration dependence,
a dependence on the age of the prepared samples can be
predictedin the given solvent It shouldbe notedthat
neither rSP-C (FFI) nor pSP-C [8] show any transition to
b-fibrils in dodecylphosphocholine micelles even after
several weeks at room temperature
Sample handling and the situationin vivo
SP-C is very difficult to handle In general, basic conditions
shouldbe avoidedandproperties of the molecule depend
strongly on the conditions for synthesis, the kind of
purification used, and the aggregation states it was
trans-ferred to In this study, we relied on the elaborate procedure
developedby Altana Pharma andonly suspendedthe
powder provided directly in chloroform/methanol The
NMR spectra yieldedgoodresults andtherefore there
appearedto be no needto change the method Whether
oligomerization can be avoided by different sample
treat-ment remains to be proven
The local environment of the molecule also has a large
impact on its behavior Wild-type SP-C, like rSP-C (FFI),
is solely monomeric at micromolar concentrations in pure
organic solvents but has a strong tendency to aggregate in
more hydrophilic environments Relatively high
concentra-tions can be obtained in dodecylphosphocholine micelles in
which SP-C is stable for months in its a-helical form [8] The
surfactant consists of 1% by weight of SP-C [31] The
concentration of rSP-C (FFI) andextractedpSP-C in
the NMR studies is therefore similar to the concentration
of SP-C in its natural environment, although it shows a slow
transition to b-sheet fibrils However, whether the
homo-dimer in chloroform/methanol is representative of the
biologically active SP-C in the surfactant cannot be judged
from the experiments presented A hint may be gained from
chemical cross-linking data on mature SP-C in cytosolic
vesicles of A549 cells (Fig 8C in [24]), which provide
evidence of dimer formation during trafficking
Potential dimerization site
The evidence suggests dimerization of SP-C, and it might be
allowedto speculate on the potential dimerization site The
C-terminus of rSP-C (FFI) only contains apolar side chains
andit can be assumedthat it is situatedat the hydrophobic
palmitoyl chains of the surfactant phospholipids In this
environment, hydrogen-bonding interactions and strong
hydrophobic associations are most likely to be the source of
intermolecular attraction A minor dimerization motif can
be foundin the C-terminal carboxylic group Similar to the
dimer formation of acetic acid, SP-C may form a dimer via
hydrogen bonding (Fig 6A) The acidic conditions of the NMR sample as well as the natural environment of SP-C would allow such a dimer formation However, in the acidic NMR sample, relatively fast hydrogen exchange rates are expectedwhich do not match the slow exchange regime observedfor the two sets of resonances Therefore, hydro-gen bonding of the carboxylic group is unlikely to be the cause of the observeddimerization, but we cannot exclude it
An alternative dimerization motif can be found in the strictly conservedC-terminal heptapeptide segment-span-ning residues Gly28 to Leu34: the heptapeptide segment
of rSP-C (FFI), as well as all other SP-C variants, has an AxxxG pattern that perfectly matches the requirements for helix–helix association as described in [32] Interestingly, the residues for which double resonances are observed are all within the strictly conservedheptapeptide segment with the AxxxG motif (Fig 6B) Attempts to model two distinct structures for the two sets of resonances failedbecause of massive overlap of the side chain resonances in the region
of interest in particular However, as mentionedabove, we can conclude from chemical-shift arguments that the two structures shouldbe very similar andare a-helical in character For the same reasons, it was impossible to obtain
a structure of the potential dimer based on intermonomeric NOEs A theoretical model based on the monomeric structure presentedin this paper andcomputational dock-ing studies is derived in the followdock-ing paper [33]
Conclusion
We have derived by NMR spectroscopy the high-resolution 3D structure of rSP-C (FFI) dissolved in CDCl3/CD3OH (1 : 1, v/v) The lipophilic peptide forms a tight a-helix for residues 5–34 which is two residues longer than the a-helix
Fig 6 Potential dimerization motifs for rSP-C(FFI) (A) Hydrogen bonding at the C-terminal carboxy group may lead to dimerization (B) Comparison of the amino-acidsequences of glycophorin A and rSP-C (FFI) shows a potential AxxxG dimerization motif similar to the van der Waals dimer of glycophorin A [33–35] at the strictly conservedheptapeptid e segment where two sets of resonances are observed.
Trang 9observedin pSP-C, with 76% sequence identity in the same
solvent Both peptides show two sets of resonances for a
number of C-terminal residues Because of the lack of Met
we can exclude oxidation to methionine sulfoxide as the
cause of the secondset of resonances for rSP-C (FFI),
which was previously assumedin the case of pSP-C [6]
Studies on the concentration dependence of the dual
resonances together with the narrow linewidth of the
NMR signals suggest the coexistence of a monomeric and
dimeric a-helical structure in the given solvent There are
two potential dimerization sites in SP-C: the C-terminal
carboxylic group may form a dimer via hydrogen bonding;
the C-terminal heptapeptide segment, which is conserved in
all known SP-C species, contains an AxxxG motif that
closely resembles the GxxxG helix–helix dimer motif of
glycophorin A Even though the latter dimerization motif is
consistent with other experimental results andtherefore
highly likely, additional studies such as point mutations at
the potential dimerization site are necessary to
unambigu-ously determine the origin of the intermolecular interaction
that leads to the second set of resonances
Acknowledgements
C.G gratefully acknowledges support from the DFG, the MPG, and
the Fonds der Chemischen Industrie B.L andA.D were supportedby
the Fonds der Chemischen Industrie B.L is also supportedby the DFG
(Emmy Noether LU 835/1–1) We thank Bettina Elshorst for help with
NDEE , Michael Nilges for help with the XPLOR protocols, andMichael
Przybylski (University of Konstanz) for providing his results before
publication Special thanks go to Michael K Gilson (CARB, Rockville,
MD, USA) for many detailed scientific discussions.
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