Structure of the coat protein in fd filamentous bacteriophage particles determined by solid-state NMR spectroscopy.

Abstract

The atomic resolution structure of fd coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles differs from that previously determined by x-ray fiber diffraction. Most notably, the 50-residue protein is not a single curved helix, but rather is a nearly ideal straight helix between residues 7 and 38, where there is a distinct kink, and then a straight helix with a different orientation between residues 39 and 49. Residues 1-5 have been shown to be mobile and unstructured, and proline 6 terminates the helix. The structure of the coat protein in virus particles, in combination with the structure of the membrane-bound form of the same protein in bilayers, also recently determined by solid-state NMR spectroscopy, provides insight into the viral assembly process. In addition to their roles in molecular biology and biotechnology, the filamentous bacteriophages continue to serve as model systems for the development of experimental methods for determining the structures of proteins in biological supramolecular assemblies. New NMR results include the complete sequential assignment of the two-dimensional polarization inversion spin-exchange at the magic angle spectrum of a uniformly 15N-labeled 50-residue protein in a 1.6 x 107 Da particle in solution, and the calculation of the three-dimensional structure of the protein from orientational restraints with an accuracy equivalent to an rms deviation of approximately 1A.

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Structure of the coat protein in fd filamentous
bacteriophage particles determined by
solid-state NMR spectroscopy
Ana Carolina Zeri, Michael F. Mesleh, Alexander A. Nevzorov, and Stanley J. Opella*
Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093
Communicated by John S. Waugh, Massachusetts Institute of Technology, Cambridge, MA, April 8, 2003 (received for review December 18, 2002)
The atomic resolution structure of fd coat protein determined by
solid-state NMR spectroscopy of magnetically aligned filamentous
bacteriophage particles differs from that previously determined by
x-ray fiber diffraction. Most notably, the 50-residue protein is not
a single curved helix, but rather is a nearly ideal straight helix
between residues 7 and 38, where there is a distinct kink, and then
a straight helix with a different orientation between residues 39
and 49. Residues 1–5 have been shown to be mobile and unstruc-
tured, and proline 6 terminates the helix. The structure of the coat
protein in virus particles, in combination with the structure of the
membrane-bound form of the same protein in bilayers, also re-
cently determined by solid-state NMR spectroscopy, provides in-
sight into the viral assembly process. In addition to their roles in
molecular biology and biotechnology, the filamentous bacterio-
phages continue to serve as model systems for the development of
experimentalmethodsfordeterminingthestructuresofproteinsin
biologicalsupramolecularassemblies.NewNMRresultsincludethe
complete sequential assignment of the two-dimensional polariza-
tion inversion spin-exchange at the magic angle spectrum of a
uniformly15N-labeled 50-residue protein in a 1.6 ? 107Da particle
in solution, and the calculation of the three-dimensional structure
of the protein from orientational restraints with an accuracy
equivalent to an rms deviation of ?1Å.
protein structure ? virus structure ? polarity index slant angle
wheel ? dipolar wave ? supramolecular assembly
F
essential roles in many laboratory procedures of molecular
biology and biotechnology, including the cloning and sequencing
of DNA, and the generation and screening of peptide libraries
(1, 4). fd, M13, and f1 are very similar inoviruses with their
single-stranded circular DNA packaged inside a cylinder that
consists almost entirely of ?2,700 copies of structurally identical
coat protein subunits. Although filamentous bacteriophages do
not have a membrane, the major coat protein (pVIII) is a
membrane protein in the course of the viral lifecycle because it
is stored in the bacterial membrane after synthesis and process-
ing before the assembly of new virus particles. Filamentous
bacteriophage coat proteins are popular systems for the devel-
opment of NMR methods applicable to helical membrane
proteins in micelles (5–10). The three-dimensional structure of
the membrane-bound form of fd coat protein, which has been
determined in micelles by solution NMR (11), and in bilayers by
solid-state NMR (12), is that of a typical membrane protein with
a hydrophobic transmembrane helix connected by a short loop
toanamphipathicin-planehelixthatinteractswiththelipidhead
groups. During the viral assembly process, the tertiary structure
of the protein changes dramatically as it undergoes the transition
from a membrane protein to the principal structural (and
DNA-binding) protein of bacteriophage particles. There have
been numerous x-ray fiber diffraction studies of filamentous
bacteriophages (3, 13–17), and although the resulting protein
structures have relatively low resolution, these studies provide
ilamentous bacteriophages have been studied in the context
of a wide range of prokaryotic biology (1–3), and they have
crucial information about the symmetry and packing of the coat
protein subunits (15, 18). Other spectroscopic investigations
(19–21), as well as our earlier solid-state NMR studies (22–26),
demonstrate that the coat protein is nearly all ?-helix, its
positively charged C-terminal residues are buried on the interior
of the particle where they interact with the DNA, and the
N-terminal residues are mobile (26) and exposed on the exterior
of the virus particles.
As is the case for most proteins associated with biological
supramolecular assemblies, determinations of the structures of
fd coat protein in virus particles and in bilayers are problematic
for the x-ray crystallography and solution NMR methods that
work well with globular proteins. Fortunately, NMR studies of
proteins in large assemblies are formidable only because of the
correlation time problem, because crystals are not necessary,
and no other chemical or physical properties of the polypeptides
interfere with NMR experiments. The experimental methods,
instruments, and theories of solid-state NMR developed for
crystalline and amorphous solids are applicable to samples
where the molecules of interest are immobile on the millisecond
time scales of the operative chemical-shift and dipole–dipole
coupling spin interactions. With the exception of a few residues
at the N terminus, which have narrow isotropic resonances, there
is no evidence of motional averaging of backbone sites (27),
although many of the side chains undergo large amplitude
motions, in fd coat protein in bacteriophage particles (26, 28).
Because solid-state NMR procedures can be applied in ways
that lead to the selective averaging and temporal separation of
the evolution of the anisotropic spin interactions, spectra can
be obtained that provide directly interpretable orientational
restraints as input for structure determination (29). The
earliest NMR results on single crystals (30, 31) demonstrated
that high spectral resolution can be achieved through a com-
bination of radio frequency irradiations and sample alignment,
and that the observable spectral parameters reflect the ori-
entations of molecular sites with respect to the direction of the
applied magnetic field. Structure determination of proteins in
aligned samples by solid-state NMR (24) takes advantage of
these spectroscopic principles, as initially demonstrated with
uniaxially oriented polymer fibers (32), and subsequently with
magnetically aligned filamentous bacteriophage particles (25),
mechanically aligned membrane proteins in bilayers (33), and
magnetically aligned membrane proteins in bicelles (34).
The structures of several membrane-associated peptides (35)
and proteins (12, 36, 37) have been determined by solid-state
NMR of aligned bilayer samples. In this article, we describe the
structure determination of fd coat protein in magnetically
Abbreviations: PISEMA, polarization inversion spin-exchange at the magic angle; PISA,
polarity index slant angle.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,
www.rcsb.org (PDB ID code 1NH4).
*To whom correspondence should be addressed. E-mail: sopella@ucsd.edu.
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aligned filamentous bacteriophage particles in solution by
solid-state NMR spectroscopy.
Materials and Methods
Sample Preparation. Uniformly15N-labeled samples of Y21M fd
bacteriophage were obtained by infecting Escherichia coli cells
grown in M9 media containing15N-enriched ammonium sulfate
(Cambridge Isotope Laboratories, Cambridge, MA). Cells were
inoculated at mid-logarithmic phase (OD550? 0.3) with virus
stock at 10:1 multiplicity, based on infectivity determined by
plaque assay. Selectively labeled samples were obtained by using
M9 media supplemented with one
(Cambridge Isotopes Laboratories) and the other amino acids,
but not proline, in natural isotopic abundance (Sigma). The
isotopic labeling was verified with solution NMR spectra of
the coat proteins in micelles. Virus particles were isolated from
the growth media, after removal of bacteria by precipitation with
polyethylene glycol, and then purified by cesium chloride gra-
dient ultracentrifugation. The samples for the solid-state NMR
experiments were 5-mm o.d. thin-wall glass tubes containing 180
?l of 60 mg?ml solutions of bacteriophage in 5 mM sodium
borate buffer, pH 8.0, and 0.1 mM sodium azide. Under these
conditions, the bacteriophage particles align with their long axis
parallel to the direction of the applied magnetic field.
15N-labeled amino acid
NMR Spectroscopy. Two-dimensional polarization inversion spin-
exchange at the magic angle (PISEMA) spectra (38, 39) were
obtainedonahome-builtspectrometerwithawide-boreMagnex
(Oxford) 550?89 magnet with a field strength corresponding to
a1H-resonance frequency of 550 MHz and a15N-resonance
frequency of 55.7 MHz. The sample tubes containing the
isotopically labeled bacteriophage particles were placed hori-
zontally within the solenoid coil of a home-built probe double-
tuned for the1H- and15N-resonance frequencies. The temper-
ature of the sample was controlled at 65°C. Typically, 256
transientswereacquiredfor64t1pointsintheindirectdimension
in these experiments. The initial spin-lock cross-polarization
time was 1 ms. Decoupling field strengths were between 60 and
70 kHz. The data were processed with the program FELIX
(Accelrys, San Diego) using sine bell apodization and zero filling
inthechemical-shiftdimensionandlinearpredictionfollowedby
sine bell apodization in the indirect heteronuclear dipolar-
coupling dimension.
Structure Determination. The resonances were assigned by using a
modified version of the shotgun NMR approach (12). The
two-dimensional PISEMA spectra from one uniformly
labeled and several selectively15N-labeled samples were ana-
lyzed with a combination of polarity index slant angle (PISA)
wheels (40, 41) and dipolar waves (42). A sinusoid with a period
of 3.6 residues was fit to the experimental
couplings as a function of residue number to characterize the
length and orientation of each helical segment relative to
the direction of the applied magnetic field (42). Based on the
assumption that the H–N bond vectors in an ideal ?-helix are
tilted 15.8° away from the long axis of the helix, the fit of the
properly parameterized expression yields values for the tilt and
rotation of the helix and serves as an index of the regularity of
the helix.
The atomic resolution structure was calculated through struc-
tural fitting (43) of the
chemical-shift frequencies measured from the spectrum in Fig.
1 and listed in Table 1, which is equivalent to the direct
calculation of the structure because all of the resonances are
assigned to specific residues (43). The accuracy of the structure
was evaluated with a statistical analysis that takes into account
both experimental errors in the measurement of the dipolar
coupling and chemical-shift frequencies and uncertainties in the
15N-
1H–15N dipolar
1H–15N dipolar coupling and
15N-
magnitudes of the principal values of the chemical-shift tensors.
For every calculation of the torsion angles between two residues
based on the pairs of frequencies associated with their reso-
nances, the chemical-shift tensor components were allowed to
vary within ?5 ppm relative to the canonical values (?11? 76
ppm, ?22 ? 89 ppm, and ?33 ? 229 ppm, relative to solid
[15N]ammonium sulfate at 26.8 ppm). The experimental accu-
racy for the determination of the frequencies was conservatively
estimated to be ?100 Hz in both frequency dimensions. To pick
up all torsion angles consistent with the resulting ranges in
frequencies and principal values, during the fitting the initial
values for the angles ? and ? were randomized within ?10°
relative to their ideal values, ?0? ?65o, ?0? ?40°. The mean
rms deviation of the 100 calculated structures was 0.98 Å relative
to their average structure.
Capsid Model. A model of the virus capsid was constructed by
using the structure of the coat protein obtained from the NMR
data and the known symmetry of the particles (16, 17). Because
the NMR data are ambiguous with respect to the rotation of the
protein about the alignment axis, initial backbone models of
the virus capsid were built with several different rotations of the
molecule about the z axis, consistent with the C terminus lysine
side chains on the inside pointing toward the DNA and the N
terminus pointing away from the particle. Side chains were
added to the backbone structure, using the program SCRWL
(side-chain placement with a rotamer library; ref. 44) and the
capsid model was energy minimized. The minimum energy
configuration obtained by using this algorithm was selected as
the best model because the vast majority of models produced
clashes that were not removable.
Results
The coat protein subunits are immobilized by, and aligned along
with, the filamentous bacteriophage particles parallel to the
direction of the applied magnetic field. Because all of the coat
protein subunits have the identical structure and are arranged
symmetrically in the virus particles, only by rotation and trans-
lation, each amide site in the polypeptide backbone contributes
one resonance to the NMR spectra of15N-labeled samples (25,
32). The two-dimensional PISEMA spectrum of a uniformly
15N-labeled sample of Y21M fd bacteriophage shown in Fig. 1
contains resonances from all of the structured amide sites of the
Fig.1.
15N-labeled Y21M fd bacteriophage particles aligned in the magnetic field of
the NMR spectrometer. The assignments of the amide resonances are indi-
cated. Each resonance is characterized by the orientationally dependent
frequencies associated with the1H–15N heteronuclear dipolar coupling and
15N-chemical-shift interactions that are listed in Table 1.
Experimentaltwo-dimensional1H?15NPISEMAspectrumofuniformly
Zeri et al.
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coat protein. Complementary data from selectively15N-labeled
samples (Fig. 2) provide assurances about the attributions of
partially overlapped resonances, and the information needed for
simultaneously assigning resonances and determining the pro-
tein structure through the shotgun NMR (12) and structural
fitting (43) approaches.
Samples of Y21M fd bacteriophage give better resolved solid-
state NMR spectra than those of wild-type fd (45). The associ-
ation of one resonance with each amide site is unambiguous in
the PISEMA spectra of the selectively15N-labeled samples in
Fig. 2. Each resonance is characterized by orientationally de-
pendent frequencies from the1H–15N heteronuclear dipole–
dipole coupling and15N-chemical-shift interactions, which, in
combination with the magnitudes and orientations of the spin-
interaction tensors in the molecular frame of the peptide bond
(38, 46), provide input for structure determination (24). In
aligned samples, the orientations of the spin-interaction tensors
effect the mapping of the protein structure onto the spectra.
Simple patterns are observed in the spectra for regular second-
ary structures of ?-helix (40, 41) and ?-sheet (47). The spectrum
in Fig. 1 has a distinct ‘‘wheel-like’’ pattern of resonances
because the helix axes are aligned approximately parallel to the
direction of the applied magnetic field.
PISA wheel patterns are sensitive indicators of the tilt (slant
angle) of the helix because the dipolar coupling and chemical-
shift frequencies reflect the orientations of the amide groups
relative to the direction of the magnetic field. However, spectra
for different rotations (polarities) of the helix vary only in their
resonance assignments, whose patterns mirror exactly the po-
larity of the corresponding helical wheel. When a wheel-like
pattern of resonances is observed in a PISEMA spectrum, no
resonance assignments are needed to determine the tilt of a
helix, and only a single resonance assignment or the patterns of
resonances from several labeled sites, as obtained from a selec-
tively15N-labeledsample,issufficienttodeterminehelixpolarity
because sequential resonance assignments are intimately linked
with the spectral features of PISA wheels (12, 40, 48, 49). Fig. 2
illustrates how PISA wheels simplify and accelerate the reso-
nance assignment and structure determination process. Fig. 2
A–C represent ideal PISA wheels for three segments of the
polypeptide. Ellipses that match the color and tilt angles of
the PISA wheels for the protein segments are superimposed on
theexperimentaldataanddemonstratethatalloftheresonances
fallon,orcloseto,thewheels.Theasymmetricdistributionofthe
four valine resonances on the wheel in Fig. 2E is sufficient to
index the wheel, determine the helix rotation, and assign reso-
nances from other residues in the helix, including those present
only in the spectrum of the uniformly15N-labeled sample. These
conclusions are reiterated in the spectra from other selectively
15N-labeled samples. Representative one-dimensional spectral
slices from the two-dimensional experimental PISEMA spectra
of uniformly and selectively15N-labeled Y21M fd bacteriophage
particles shown in Figs. 1 and 2 are shown in Fig. 6, which is
published as supporting information on the PNAS web site,
www.pnas.org.
Fig. 3 summarizes the structural analysis of the frequencies
measured from the spectrum in Fig. 1 and are listed in Table 1.
Although multiple samples are used in the qualitative assign-
ment process, all of the frequencies actually used for structure
determination are measured from a single spectrum, avoiding
potential problems due to variations among samples and con-
Table 1. Experimental frequencies measured from the PISEMA
spectrum in Fig. 1
Residue no.
15N shift, ppm
1H–15N coupling, kHz
7
8
9
179.4
217.0
219.6
159.5
197.2
216.7
172.3
183.5
216.5
217.9
169.8
200.0
211.5
203.8
179.0
199.0
211.8
190.4
175.0
207.7
214.6
166.2
185.3
217.0
210.0
164.0
193.2
216.0
192.0
159.6
202.0
210.8
209.2
201.5
216.6
229.0
206.3
192.5
215.7
210.5
196.2
190.2
205.4
2.7
7.1
10.1
5.6
4.0
8.6
9.1
5.1
6.3
9.7
8.3
5.3
7.5
9.9
7.6
4.3
7.1
9.3
6.0
5.4
9.5
7.9
5.4
6.0
9.9
7.7
4.9
8.4
9.6
7.5
5.6
8.5
10.3
8.5
6.9
9.5
10.0
8.0
8.0
9.6
9.6
7.9
7.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
Fig. 2.
coat protein. The colored circles represent the resonances present in the
experimental spectra of selectively labeled fd virions in D–I with the labeled
amino acids identified by the single-letter code. Superimposed on the exper-
imental data are colored ellipses corresponding to the ideal wheels.
(A–C) Representations of ideal PISA wheels for three regions of the
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ditions. For clarity of presentation, and to focus on the principal
structural features of the protein, its sequence is divided into
four segments: residues 1–6 are not shown, because residues 1–5
are mobile (26) and proline 6 terminates the helix; residues 7–20
(red) constitute the amphipathic helix that lies on top of bilayers
in the membrane-bound form of the protein; residues 21–38
(blue) are hydrophobic and are part of the transmembrane helix
in the membrane-bound form of the protein; and the side chains
of residues 39–49 (green) provide the positive charges for DNA
interactions in the virus particles. Fig. 3A Left sorts the exper-
imental PISA wheels by segment for comparison to the corre-
sponding ideal PISA wheels in the adjacent column (Fig. 3B).
The residues at the ends of the helix, Ala-7 and Ser-50, are gray.
The dipolar coupling frequency measured for Ala-7 does not fit
well to a wave, indicating some distortion at the start of the helix,
and the resonance for the C-terminal Ser residue has not been
identified in the spectrum.
Dipolar waves for residues 8–49 are shown in Fig. 3C, where
sine waves with the 3.6-residues-per-turn periodicity character-
istic of a ?-helix are fit to the experimental dipolar-coupling
frequencies as a function of residue number (42). A break in the
helix was anticipated near residue 21, the site of the turn
connecting the hydrophobic and amphipathic helices in the
membrane-bound form of the protein (11, 12). Remarkably,
both the PISA wheels (Fig. 3B), and especially the dipolar waves
(Fig. 3C), indicate that residues 8–38 are in a single continuous
helix, with no evidence of a kink, curvature, or other distortion.
In contrast, there is an unexpected kink near residue 39. The
ideal and experimental PISA wheels corresponding to residues
21–38 and 39–49 reflect helices with different tilt angles, and the
dipolar waves demonstrate that the helix abruptly changes
direction near residue 39. Both the qualitative PISA wheels and
the quantitative dipolar waves indicate that all segments of the
protein form nearly ideal ?-helices. The discrepancies between
the experimental and ideal PISA wheels in Fig. 3 are typical of
those due to local variations of chemical-shift tensors. The R
factors for the fits of the experimental data to sine waves are less
than the experimental errors for the measurements of the
dipolar-coupling frequencies.
The backbone structure of the coat protein obtained by
structural fitting (43) to the data in Fig. 1 and Table 1 is shown
in Fig. 3D. The colored cylinders represent the orientations of
the three segments of the molecule obtained from the dipolar
waves. A conservative analysis of the accuracy of the structure
determination, as described in Materials and Methods, indicates
that it is equivalent to an rms deviation of ?1 Å. A model of the
virus capsid was constructed by placing the coat protein mono-
mers in a five-start helix with a two-fold screw axis with a 16-Å
pitch and ?33.23° rotation (3, 16, 17), and is presented in Fig. 4.
Sections of the capsid were built containing four interleaved
layers of the coat protein with rotations about the z axis varying
over a range of 180° (in 1° increments), which is consistent with
the C terminus on the inside of the capsid, and with a diameter
varying by a few angstroms about the literature value of 65 Å.
Using the program SCRWL (44), side chains were added to the
models with the backbone atoms of the capsid-held fix. This
method begins by placing side chains into the model in their most
favorable rotamer and then relieving sterical clashes by varying
the preferred rotameric configurations of those side chains to
lower the overall energy of the model. The algorithm then
performs a brief energy minimization by using a hard repulsive
potential to remove steric clashes. Using the parameters of the
AMBER 4.1 potential, the final energy of the models are compared
and the lowest energy configuration was chosen as the final
model of the capsid. This calculation converges to a single
orientation of the protein molecule. A section of the capsid is
shown in Fig. 4. In Fig. 4 A and B, the molecular surface of the
capsid is colored according to the electrostatic potential calcu-
lated with the program GRASP (50). Fig. 4A is a view from the
Fig. 3.
formly and selectively15N-labeled Y21M fd bacteriophage particles. The
midpoint of each circle represents the pair of frequencies associated with a
resonance in the spectrum shown in Fig. 1 and the top of column, and listed
in Table 1. (B) Ideal PISA wheels corresponding to the experimental data in A.
(C) Dipolar waves fit to the experimental dipolar coupling frequencies as a
function of residue number. The frequencies are measured from the reso-
nancesinthespectruminFig.1,listedinTable1,andrepresentedbythecircles
in A. (D) Three-dimensional structure of Y21M fd coat protein in bacterio-
phage particles, whose coordinates are deposited as PDB ID code 1NH4,
superimposed on colored tubes with orientations determined from the fits to
the dipolar waves in C.
(A) Representations of the experimental PISEMA spectrum of uni-
Fig. 4.
built from the coat protein subunit structure, which was determined by
solid-state NMR spectroscopy. The symmetry was derived from fiber diffrac-
tion studies. (A and B) Representations of the electrostatic potential on the
molecularsurfaceofthevirus(AisabottomviewandBisasideviewalongthe
virus axis) obtained by using the program GRASP (57). (C and D) Views of the
capsid structure showing the arrangement of the coat proteins in pentamers
and further assembly of the 2-fold helical structure obtained by using the
program INSIGHT II (Accelrys).
Model of a section of the Y21M fd filamentous bacteriophage capsid
Zeri et al.
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top along the virion axis and Fig. 4B is a side view. Figs. 4 C and
D present the same views of the capsid as rendered by the
program INSIGHT II (Accelrys).
Discussion
The structure of fd coat protein shown in Figs. 3D and 5A results
from a direct fit to the experimental data. Protein structures
based on ideal ?-helices represented by tubes (Fig. 3D) and
ribbons are also consistent with the experimental PISA wheels
and well fit dipolar waves. In contrast, the fiber-diffraction
structure results from model fitting of data with a resolution of
?7 Å (16, 17). However, the differences between the protein
structures determined by NMR spectroscopy and x-ray fiber
diffraction cannot be explained on the basis of differences in
resolution alone. The structure of the coat protein determined
by x-ray fiber diffraction has been described as a single gently
curving ?-helix (16), a gently curved ?-helix (17) oriented
roughly parallel to the virion axis (14, 15), and as having a 20°
tilt with respect to the same axis (16). Raman spectroscopy data
suggest an average tilt of the helix between 13° and 20° (51). The
solid-state NMR data give very accurate values based on dipolar
couplings for the tilt of the helical regions of the coat protein,
with residues 8–38 having a 23° tilt and the C-terminal region,
starting at residue 40, having a somewhat smaller tilt, of 18° with
respect to the filament (and magnetic field) axis. The difference
is patent in the dipolar waves in Fig. 3D, from which the tilt
angles are calculated. The PISA wheel analysis and the structural
fitting of the spectra confirm these findings.
In comparisons between the solid-state NMR structure (PDB
IDcode1NH4)andthefiber-diffractionstructureoffd(PDBID
code 1IFI), a small difference in the length of the individual coat
protein subunits is apparent. Ideal ?-helices have a periodicity
very close to 3.6 residues per turn (52). The fiber diffraction
structure has a periodicity of 3.55 residues per turn, whereas the
solid-state NMR data have 3.68 residues per turn. These small
variations in periodicity might reflect slight differences in the
peptide plane geometries and bond lengths used to calculate the
helical structures, and result in the overall differences in length
of the protein. A simulated dipolar wave fit to 1IFI is shown in
Fig. 5D, and compared with dipolar waves corresponding to the
NMR structure in Fig. 5B, and to ideal helices: in Fig. 5F, a
straight, 45-residue helix tilted 20° with respect to the z axis; and
in Fig. 5H, a slightly curved 45-residue helix. The ribbon
representation for each structure is shown in Fig. 5 A, C, E, and
G. The dipolar waves used to determine the structure by NMR
(Fig. 5B) are clearly different from that derived from the fiber
diffraction structure [Fig. 5D, because the coordinates from 1IFI
give rise to a dipolar wave that appears to correspond to a
straight helix with a tilt of ?23o, and neither is consistent with
the dipolar wave for a curved helix (Fig. 5F) or a straight helix
with a 20° tilt (Fig. 5H)].
The N-terminal amphipathic helix (residues 7–20), which lies
on the surface of lipid bilayers (16, 11, 12), undergoes a large
rearrangement during the assembly process, which is equivalent
to a rigid body rotation of ?90°, after which it is seamlessly
integrated, along with the residues that act as a hinge, into a helix
that starts immediately after Pro 6 and continues through the C
terminus in the bacteriophage particles. The helix is straight,
except for a kink near residue 39, which enables the C-terminal
residues (39–49) to have a somewhat different orientation than
residues 8–38. The change in orientation at residue 39 may be
necessary to optimize interactions of the positive charges of the
side chains of the C-terminal residues with the DNA packed
inside. Alternatively, the length of the C-terminal region is
compatible with the rise of ?16 Å per turn of the 2-fold screw
axis symmetry observed for the virus capsid, considering the
?-helicalriseof5.4Åperturn,or16.5Åfor11residues(52).The
same kink is present in the structures of the membrane-bound
form of the coat protein determined in micelles (11) and bilayers
(12), therefore it may satisfy structural requirements of the
membrane-boundformoftheproteinunrelatedtoitsrolesinthe
bacteriophage particles.
Depending on the extent of hydration, the diameter of the
capsid as determined by x-ray fiber diffraction varies between 55
Å and 65 Å (53), and most studies report external diameters of
60 Å (17) or 65 Å (16). In our model-building approach to the
virus particles based on the atomic resolution structure of the
coat protein subunits, an external diameter of 67 Å was the most
energetically favorable after the side chains were added. The
model of the virus capsid shown in Fig. 4, as well as those derived
from the x-ray fiber diffraction results, have similar side-chain
interactions among the coat protein subunits and between the
C-terminal residues and the DNA.
Recent mutational analysis (54) has shown that only a small
group of residues is absolutely required for virus viability,
including residues Ile-39, Leu-41, Phe-42, and Phe-45. This
finding is supported by hydrogen-exchange experiments (data
not shown) where two-dimensional PISEMA spectra were ob-
tained after resuspension of uniformly15N-labeled bacterio-
phage particles in D2O at pH 8.0. The resonances corresponding
to these four residues are present in spectra obtained on samples
exposed to D2O for as long as 6 months, whereas nearly all other
resonances disappear within a few hours of resuspension of the
sample in D2O. This result suggests that the inter-subunit
interactions involving these residues are very stable, and reflect
the resistance of the virus particles to extremes of temperature,
pH, and ionic strength (55). Three of the C-terminal lysine
residues (40, 43, and 44) are also highly conserved, and their side
chains point toward the center of the capsid and interact with the
DNA. The electrostatic surface of the capsid in Fig. 4 shows
the prevalence of positively charged residues on the interior and
the location of acidic residues on the exterior.
Solid-state NMR spectroscopy of aligned samples yields
atomicresolutionstructuresofproteinsinassembliesthatcannot
be crystallized and are too large to reorient rapidly in solution.
Previous examples were membrane proteins in bilayers aligned
between glass plates (12, 35–37), and here we present the
structure of the coat protein in virus particles. The structure of
the membrane-bound form of fd coat protein was determined in
Fig. 5.
by NMR spectroscopy and shown in Fig. 3D. (C) Ribbon rendering of the x-ray
fiberdiffractionstructureoffdcoatprotein(PDBIDcode1IFI).(E)Idealgently
curved helix. (G) Ideal straight tilted helix. (B, D, F, and H) The corresponding
dipolar waves. (B) The fit to the experimental NMR data in Fig. 4C. (D)
Calculated from the x-ray fiber diffraction structure. (F and H) Simulations for
the corresponding ideal helices.
(A) Ribbon rendering of the structure of fd coat protein determined
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www.pnas.org?cgi?doi?10.1073?pnas.1132059100 Zeri et al.