Furthermore, resistance to CNBr cleavage and dual NMR resonances of porcine and human recombinant SP-C with Met32 replaced by isoleucine point to a dimerization site located at the C-ter
Trang 1Structural model for an AxxxG-mediated dimer of
surfactant-associated protein C
Visvaldas Kairys1, Michael K Gilson1and Burkhard Luy2
1
Center for Advanced Research in Biotechnology Rockville, MD, USA;2Institut fu¨r Organische Chemie und Biochemie der Technischen Universita¨t Mu¨nchen, Germany
The pulmonary surfactant prevents alveolar collapse and
is required for normal pulmonary function One of the
important components of the surfactant besides
phos-pholipids is surfactant-associated protein C (SP-C) SP-C
shows complex oligomerization behavior and a transition
to b-amyloid-like fibril structures, which are not yet fully
understood Besides this nonspecific oligomerization, MS
and chemical cross-linking data combined with CD spectra
provide evidence of a specific, mainly a-helical, dimer at
lowto neutral pH Furthermore, resistance to CNBr
cleavage and dual NMR resonances of porcine and human
recombinant SP-C with Met32 replaced by isoleucine point
to a dimerization site located at the C-terminus of the
hydrophobic a-helix of SP-C, where a strictly conserved
heptapeptide sequence is found Computational docking
of two SP-C helices, described here, reveals a dimer with
a helix–helix interface that strikingly resembles that of glycophorin A and is mediated by an AxxxG motif similar
to the experimentally determined GxxxG pattern of glycophorin A It is highly likely that mature SP-C adopts such a dimeric structure in the lamellar bilayer systems found in the surfactant Dimerization has been shown in previous studies to have a role in sorting and trafficking of SP-C and may also be important to the surfactant function
of this protein
Keywords: dimerization; docking; surfactant-associated protein C (SP-C)
The liquid–air interface in the alveoli of mammalian lungs
is coated with a surfactant monolayer that reduces surface
tension and thus opposes collapse of the alveoli [1] The
surfactant is composed of lipids ( 90%) and the
surfac-tant-associated proteins SP-A, SP-B, SP-C and SP-D
An abnormally lowlevel of surfactant, notably in preterm
infants, is associated with respiratory distress syndrome
[2,3], a condition that can be ameliorated by instillation of
surfactants into the lungs
The small hydrophobic proteins SP-B and SP-C are
important components of the pulmonary surfactant Thus,
SP-B deficiency causes fatal respiratory distress syndrome
[4], and SP-C deficiency in humans is associated with
childhood lung disease [5,6] and fatal respiratory distress
syndrome [7] It is also of interest that the therapeutic
surfactants nowon the market that include SP-B and SP-C
are regarded as more effective than those that are purely
lipidic [8] On the other hand, the animal-derived surfactant
proteins in current therapeutic preparations pose concerns about immunogenicity and transmission of disease, and efforts are under way to develop novel surfactant replace-ments with an improved protein component [9] As a consequence, there is considerable interest in the details
of howSP-B and SP-C stabilize the alveolar surface The mechanisms by which pulmonary surfactants act are still not fully understood Current thinking is that their actions are related to multilayer lipid–protein structures that underly the surface monolayer These subsurface structures growand contract as material leaves the monolayer during expiration and re-enters it during inspiration [10] The mechanism of removal presumably involves a process in which the lipidic surface of the monolayer, which is exposed
to air when the lungs are expanded, folds against itself and dips belowthe surface on expiration SP-B and SP-C appear
to play a role in the formation and stabilization of these dynamic, subsurface reservoirs of lipids [10–12] Experi-ments on peptides derived from SP-B and SP-C [13–15] and full-length proteins [16] also provide evidence that the two molecules promote formation of a fluid phase in the monolayer with a net-like topology that isolates patches of
a more rigid phase and inhibits alveolar collapse The molecular basis for these effects is still unclear, however, and development of structural information for SP-B and SP-C is
of central importance in establishing their mechanism SP-C is a 34–35-residue polypeptide the sequence of which (Table 1) can be viewed as consisting of four parts [17,18]: a palmitoylation motif with two surrounding pro-lines at residues 3–6; two highly conserved cationic residues
at positions 10 and 11; a rigid, totally hydrophobic a-helical segment with regularly stacked side chains spanning resi-dues 12–27; and a less hydrophobic, a-helical, C-terminal
Correspondence to B Luy, Institut fu¨r Organische Chemie und
Biochemie der Technischen Universita¨t Mu¨nchen, Lichtenbergstr 4,
D-85747 Garching, Germany Fax: + 49 89 28913210,
Tel.: + 49 89 28913275, E-mail: Burkhard.Luy@ch.tum.de
and M K Gilson, Center for Advanced Research in Biotechnology,
9600 Gudelsky Drive, Rockville, MD 20850, USA.
Fax: + 1 301 7386255, Tel.: + 1 301 7386217,
E-mail: Gilson@umbi.umd.edu
Abbreviations: SP-C, surfactant-associated protein C; rSP-C (FFI),
FFI variant of recombinant human SP-C; GpA, glycophorin A;
DPPC, dipalmitoylphosphatidylcholine.
(Received 17 December 2003, revised 29 February 2004,
accepted 23 March 2004)
Trang 2heptapeptide which is strictly conserved across species.
Although all four parts seem to be necessary for the function
of SP-C [18], the only component that has been assigned a
clear role is the hydrophobic a-helix, which is believed to
anchor SP-C in the lipidic surfactant layer Here we propose
that the role of the C-terminal segment of SP-C is to permit
dimerization of SP-C via an AxxxG helix interaction motif
similar to the GxxxG motif found in the
membrane-spanning helix dimer glycophorin A (GpA) [19,20]
This study was motivated by the similar observation of
two distinct sets of NMR resonances for the C-terminal
residues 28–34 of porcine SP-C [17] and recombinant
rSP-C (FFI) [21] (Table 1) in an organic solvent that mimics the
lipidic surfactant environment Although the second set of
resonances in porcine SP-C was originally thought to result
from a second oxidation state of the sulfur of Met32 [17],
this cannot explain the existence of dual resonances for
rSP-C (FFI) because it lacks Met32 (Table 1) As, in the
case of rSP-C (FFI), the relative intensities of the two sets of
resonances are clearly concentration dependent, it is
hypo-thesized that the dual resonances result instead from the
coexistence of a monomeric and dimeric form of SP-C with
its monomer–monomer junction near the C-termini of two
peptides Although such dimerization may be mediated by
hydrogen bonds between the C-terminal carboxy groups,
which are expected to be neutral under the experimental
conditions, the typical hydrogen exchange rates for carboxy
groups appear to be inconsistent with this model [21] It
therefore seems more likely that dimerization is mediated
by some other type of helix–helix interface However, this
hypothesis proved difficult to explore based on
experi-mentally obtained structural data because it was impossible
to assign intermolecular connectivities between the two
subunits of the putative dimer because of extensive overlap
in the homonuclear NMR spectra and to the weakness of
the NOE signal for molecules of this size We have therefore
used a computational method to explore the possible
dimeri-zation of SP-C The suitability of a computational approach
is supported by the success of previous computational
modeling of the GCN4 leucine zipper [22] and GpA [23]
Materials and methods
A fast code for conformational optimization [24,25] was
used to carry out extensive searches for the lowest energy
conformation of a homodimer of rSP-C (FFI) (Table 1) In
accordance with experimental data [17, 21, 26], the calcu-lations treated the peptide as a-helical Because the data suggested a dimer interface at the C-terminus, residues 1–15 were omitted, decreasing the number of residues to 19 in each a-helical monomer The artificial N-terminus of the shortened peptide was modeled as un-ionized, and the C-terminus was also treated as neutral, based on the acidic conditions of the NMR sample An extensive conforma-tional search was carried out with the Chemistry at Harvard Macromolecular Mechanics [27] force field and a simpli-fied but time-efficient distance-dependent dielectric model (e¼ 4rij) The calculations treat one monomer as fixed in space, and the energy of the system is minimized with respect to the position and orientation of the other mono-mer, along with selected bond rotations in both monomers (see below) The center of mass of the moving monomer was allowed to sample a large range of positions defined by a box of dimensions 36· 36 · 32 A˚, centered on the fixed monomer and with the fixed helix directed along the z-axis The backbone was kept rigid in this initial search, but all torsional angles in side chains Ile22 to the C-terminal Leu34 were treated as rotatable for both monomers because the NMR restraints of these side chains allowsome conform-ational flexibility During the search a total of 6· 106 conformations were generated and used to refine 500 low-energy candidates for the structure of the SP-C dimer The distribution of energies of the 500 low-energy structures is shown in Fig 1 The most stable structure is significantly separated from the rest by a marked energy step of 4.5 kcalÆmol)1
The optimum structure was refined via a more focused search with a comparatively realistic treatment of electro-statics This second search was intentionally guided toward the optimum from the first search by using the fact that the search algorithm stores a list of the low-energy conforma-tions found to date and can use subsets of their stored coordinates in generating newconformations to be tested during the optimization procedure The desired bias was thus established by inserting the optimum conformation from the first search into the list of found conformations
at the outset of the second search The monomers were afforded greater flexibility in this search: in addition to the flexible dihedrals of the previous optimization, the back-bone /,w angles of residues Leu31, Ile32, Gly33 and Leu34 were allowed to vary by ± 15 from the original structure because these residues are less well-defined by the NMR
Table 1 Sequences of SP-C variants, and partial sequences of human GpA, Neu and Ros-C Asterisks indicate residues defining the AxxxG or GxxxG motif in SP-C (A29, G33) and other transmembrane helices.
Trang 3data: they form the last turn of the a-helix where the amide
protons are not hydrogen-bonded and da,N(i,i+3) and
da,N(i,i+4) connectivities are absent However, to
some-what restrict the number of variables in the calculation,
valines 23, 24 and 27, which point away from the dimer
interface and which shifted little in the results of the initial
search, were locked into their optimized conformations The
total number of rotatable dihedrals in both monomers came
to 44 The Generalized Born electrostatics model [28–30]
was used with a solvent dielectric constant 4 and a molecular
dielectric constant of 1
In further studies, Ala29 was replaced by a Gly residue to
assess the effect of replacing AxxxG with the recognized
GxxxG dimerization motif Almost the same procedure
as described above was used for the docking study, now
allowing side chain flexibility only for residues pointing
towards the dimer interface side The resulting dimer, with
the artificial GxxxG motif, is basically identical to the
original calculations
Finally, to determine if wild-type SP-C, which has Met
instead of Ile at position 32, can adopt the dimer structure
found here for the FFI variant, the same technique was used
to optimize the structure of a wild-type SP-C dimer with
Generalized Born electrostatics The resulting geometry is
virtually the same as in rSP-C (FFI) (data not shown) In
addition, the Met32 side chains of each monomer do not
contact the other monomer and hence do not contribute to
the binding interface, so no special role could be identified
for this residue in the formation of the SP-C dimer
Results and Discussion
Structural model of the SP-C dimer
Figure 2A provides an overviewof the modeled structure of
the SP-C dimer that results from the extensive
conforma-tional search described in Materials and methods; for
comparison, the experimentally determined structure of GpA is also shown Interestingly, although no symmetry was imposed during the calculations, the modeled dimer is highly symmetrical The two helices are topologically parallel, with a right-handed crossing and a helix–helix angle of 44, as computed with the program INTERHLX (K L Yap, University of Toronto, Toronto, Canada) This
Fig 2 Comparison of the model-built SP-C dimer and the experi-mentally determined GpA dimer [20] (A) Each dimer (SP-C left, GpA right) is oriented to illustrate the splay between the helices Purple, backbone; green, side chains; orange, dimerization motifs (B) Sche-matic showing the dimers in their natural environment Whereas the transmembrane helix of GpA is situated in a membrane bilayer, SP-C
is found in the surfactant that forms lamellar bilayer systems on exhalation The C-terminus of SP-C still seems to be situated in the hydrophobic core of the lamellar bilayer and may be stabilized by the dimerization The interhelical angle of 44 for SP-C is consistent w ith experimental infrared reflection-absorption spectroscopy data [31] (C) Schematic showing the position of the dimerization motifs (A29 and G33 in SP-C, G79 and G83 in GpA, draw n in black) in the hydro-phobic core (green).
Fig 1 Energy distribution from initial helix–helix docking search The
500 lowest-energy structures were calculated for two associated
rSP-C (FFI) monomers as described in Materials and methods All helix–
helix interactions are attractive, but the most stable dimer used for
subsequent calculation is 4.5 kcalÆmol)1lower in energy than the next
most stable conformation.
Trang 4angle is consistent with the experimental observation of a
24 angle between the SP-C helix and the normal axis of
a membrane bilayer [31] if the membrane normal is
assumed to bisect the angle of the helix–helix dimer The
dimer interface appears to be stabilized by a combination
of factors First, as shown in Fig 3A, the two monomers
are linked by six Ca–HÆÆO hydrogen bonds [32–36] or
Ca–HÆÆO contacts, as previously defined [36]: Ala29(O)A–
Leu30(Ha)B, Leu30(O)A–Gly33(Ha)B and Gly33(O)A–
Leu34(Ha)B and their symmetric pairs The monomers also
pack intimately, forming a serpentine interface of close
van der Waals contacts, as highlighted in Fig 4 Finally,
two pairs of peptide groups in monomer A are positioned
with their carbonyl carbons directly across from the amide
nitrogens of the corresponding peptide groups in monomer
B, creating the possibility of attractive electrostatic
interactions [37], as follows: Gly33A–Leu34B, 3.53 A˚;
Gly33B–Leu34A, 3.59 A˚; Ala29A–Leu30B, 4.22 A˚;
Ala29B–Leu30A, 4.30 A˚ Interestingly, the side chain of
Ile32 in the dimer is significantly repositioned relative to the
monomer structure obtained by NMR studies [17, 21]; this
result is consistent with the experimental observation in NMR studies that Ile32 experiences significant changes
in side-chain chemical shifts in the two sets of resonances mentioned above [21]
Although no information on the structure of GpA was used in the calculations, the interface of the modeled SP-C homodimer strikingly resembles that of the GpA homo-dimer, which has been determined experimentally [19,20],
as illustrated in Fig 3B In particular, the GpA interface possesses six Ca–HÆÆO hydrogen bonds precisely analogous
to those in SP-C: Gly79(O)A–Val80(Ha)B, Val80(O)A– Gly83(Ha)B and Gly83(O)A–Val84(Ha)B and their sym-metric pairs As previously noted, the association of the GpA helices is mediated by a GxxxG motif in each helix (Gly79, Gly83) [19,20] In SP-C, the corresponding pattern
is AxxxG (Ala29, Gly33) Initially, we thought that the smaller helix–helix angle of GpAs (35 [20] vs 44 ) appeared to be attributable to the smaller size of Gly79 in GpA relative to Ala29 in SP-C However, docking studies
Fig 3 Electrostatic interactions of SP-C dimer (A) Dimerization site
of the computational model of rSP-C (FFI) Strong HaÆÆÆO¼C
inter-actions are labelled with their distances in Angstroms (B) Both dimers,
SP-C and GpA, have the backbone in close contact at the dimerization
motif Carbonyl groups are found directly opposite to amide groups of
the adjacent residue of the second monomeric unit The orientation
of the two polar groups reduces the usually observed electrostatic
repulsion and may even lead to an electrostatic attraction The
inter-actions shown in (A) and (B) seem to lead to a high specificity of the
helix–helix association.
Fig 4 Van der Waals contacts between the helices of the computed
SP-C dimer in a view along the helix axes (A) and from the bottom of the dimer (B) ‘‘Hotter’’ colors indicate closer contacts Figures were gen-erated using the programs PROBE [57] and MAGE [58].
Trang 5using monomers with Ala29 replaced by a Gly residue
resulted in an interhelical angle of 43.2, which is virtually
identical with the original angle determined for the SP-C
dimer The helix–helix interface must therefore be mainly
determined by surrounding residues at the dimerization site,
as is the case with Thr87 in GpA, which probably forms
a hydrogen bond to the backbone carboxy group of
Val84 [38]
Another difference between SP-C and GpA is that the
helix–helix interface of SP-C is very near the C-termini of
the helices, whereas the interface of GpA is relatively central
(Fig 2) The stability of the two dimers therefore seems
quite different Owing to its position at the C-terminus, the
contact surface of the SP-C dimer is considerably smaller
than the GpA dimer and also lacks the equivalent to the
Thr87–Val84 hydrogen bond Furthermore, the dimer of
GpA is stabilized in a membranous bilayer by its
hydro-philic ends, whereas the C-termini of SP-C are just buried in
the hydrophobic core All this is reflected by SDS/PAGE
experiments in which the GpA dimer is clearly visible, and
SP-C without additional chemical cross-linking appears to
be monomeric (Fig 8C in [53]) Besides the reduced
stability, the C-terminal position of the interface in SP-C
along with the relatively large interhelical angle causes SP-C
to have a much more V-shaped appearance than the more
compact X-shaped GpA, as seen in Fig 2A,C
The GxxxG and AxxxG motifs belong to one of the two
basic types of helix–helix contacts recently identified in
membrane proteins [37] Here, small residues, especially
Gly, Ser, Ala and Thr, create smooth surfaces which allow
the close approach of the helices’ backbones The
import-ance of such contacts is supported by statistical analyses
showing enrichment of small residues at helix–helix
inter-faces [39,40], with an especially high occurrence of G–G
contacts [40] Moreover, the GxxxG motif emerged
spon-taneously in an experimental system where dimerization was
applied as a selection criterion for randomized
trans-membrane helices [41], and GxxxG was also found to be
overrepresented in protein segments identified as
trans-membrane helices by sequence analysis [42] The GxxxG
motif, as well as variants in which the first G is replaced by
other small amino acids, appear to play a role in the
dimerization of epidermal growth factor receptors [43] and
the receptor tyrosine kinases Neu and Ros-C (chicken) [44–
46], and also in the function of the aIIbb3 integrins [47]
Interestingly, GxxxG, AxxxA and GxxxA sequences have
recently been implicated in helix–helix contacts in
water-soluble proteins [48,49]
Experimental correlations and functional implications
The natural environment of SP-C is the surfactant, a
complicated structure of monolayer and lamellar bilayer
systems consisting mainly of the phopholipids
dipalmi-toylphosphatidylcholine (DPPC) and
dipalmitoylphos-phatidylglycerol This environment favors the formation
of a dimeric structure in several ways For one thing, the
bulk concentration of SP-C in the surfactant appears to
be of the same order of magnitude as the 1 mM
concentration used in the NMR studies [21, 26] [Given
the weight percentage of SP-C in surfactant (1% [16]),
and the specific volume of DPPC in a membrane (about
1000 A˚3), the bulk concentration of SP-C in the mem-brane is about 2 mM.] The concentration of SP-C is even higher in surfactant prepared by lung lavage and in several therapeutic preparations [50] Furthermore, it is likely that SP-C molecules in the surfactant are oriented with their positively charged residues (10 and 11) close to the lipids’ polar head groups and their hydrophobic a-helices pointing into the lipidic part of the layer This orientation positions the AxxxG dimerization motifs near each other, increasing their local concentration relative to bulk and thereby increasing their tendency to dimerize The palmitoylated residues Cys4 and Cys5 of wild-type SP-C are probably situated near the polar head groups as well, in which case the relatively short fatty acid chains cannot interfere with the proposed dimerization site but may even support the dimer formation by filling in the gap between the two monomeric units
Part of the surfactant is believed to exist as a lipid monolayer, and in vitro studies of SP-C in lipid monolayers reveal that the main a-helix of SP-C adopts a tilt angle of
70 relative to the membrane normal It seems possible that SP-C still exists as a dimer under these conditions, as tilting the entire structure may just bury the hydrophobic helix in the lipid’s acyl chains, but it is also likely that the dimer is disrupted
SP-C is known to oligomerize under various conditions Higher-order aggregates, however, are usually of b-amy-loidogenic structure and should not be confused with the specific a-helical dimer presented in this article A number of experimental studies provide evidence for a dimeric form of SP-C Indeed, a decade ago direct evidence for dimerization
of human wild-type SP-C was provided by electrospray ionization MS data based on the detection of specific odd-charged molecular ions ([M + 5H]5+) [51,52] In addition, cross-linking studies on mature SP-C using bismaleimido-hexane showa distinct dimer at pH 7.4 (Fig 8C of [53]) Finally, in very recent studies a specific dimer could be unambiguously identified by high-resolution Fourier-trans-form ion cyclotron resonance MS and light-scattering methods ([54], A Seidl, G Maccarone, N Youhnovski,
K P Schaefer and M Przybylski, unpublished data) It was also found that the dimer appears only at lowto neutral pH and is mainly a-helical (verified by CD spectra), whereas tetramers and higher-order oligomers of nonhelical confor-mation were only detected at higher pH Evidence that the binding site of the a-helical dimer is positioned close to the C-terminus can be found in the resistance of Met32 to CNBr cleavage [51] and the concentration-dependent dual set of NMR resonances of the C-terminal heptapeptide segment already mentioned [21] All experimental results fit well with the computational homodimeric model described
in this work
It is interesting to speculate on the possible significance of SP-C dimerization for its role in the pulmonary surfactant,
in addition to the apparent role of homomeric association in trafficking [53] One possibility is that the V shape of the dimer allows it to stabilize the membrane curvature required
to form the multilamellar structures that underly the surfactant monolayer (e.g [55] and references therein) Also, the specific shape of the SP-C dimer may be important
in promoting the selective squeeze-out of non-DPPC lipids during surface film compression [10], or in the formation
Trang 6and 2D patterning of the expanded and
liquid-condensed phases observed in surfactant preparations
(e.g [56] and references therein) Finally, it is conceivable
that compression and expansion of the surface film shifts the
monomer–dimer equilibrium of SP-C by mass action and
that this shift buffers and stabilizes the physical
character-istics of the surface
Conclusions
The computational analysis of SP-C described here reveals a
dimer with a helix–helix interface that strikingly resembles
that of GpA, except that it is based on an AxxxG motif
rather than GxxxG, and that the dimerization takes place
near the C-terminus rather than in the center of a
membrane-spanning helix This result is consistent with
the existence of dual chemical shifts at the C-terminus of
rSP-C (FFI), which cannot be explained by alternative
oxidation states of a methionine residue, and with a growing
body of biophysical and biological data In particular,
recent experimental evidence strongly suggests that
dimeri-zation is important in the trafficking of SP-C The potential
that it is also important for surfactant function should be
borne in mind in developing therapeutic pulmonary
surf-actants
Acknowledgements
We thank S O Smith (State University of NewYork at Stony Brook)
for kindly providing coordinates of glycophorin A and M Przybylski
(University of Konstanz, Germany) for providing his results on SP-C
dimerization in advance of publication This work was supported by a
grant from the National Institutes of Health (GM61300) B L thanks
the Fonds der Chemischen Industrie, the Alexander von Humboldt
Foundation, and the Deutsche Forschungsgemeinschaft (Emmy Noether
LU 835/1-1) for financial support.
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