Structural basis for DNA recognition and
loading into a viral packaging motor
Carina R. Büttnera,1, Maria Chechika, Miguel Ortiz-Lombardíaa,2, Callum Smitsa, Ima-Obong Ebongb, Victor Chechikc,
Gunnar Jeschked, Eric Dykemane, Stefano Beninia,3, Carol V. Robinsonb, Juan C. Alonsof, and Alfred A. Antsona,4
aYork Structural Biology Laboratory, Department of Chemistry, University of York, York, YO10 5DD, United Kingdom;
University of Oxford, South Parks Road, Oxford, OX1 3QZ, United Kingdom;
Mathematics and Biology, University of York, York, YO10 5DD, United Kingdom; and
Investigaciones Cientificas, Darwin 3, 28049 Madrid, Spain
bDepartment of Chemistry,
cDepartment of Chemistry, University of York, York, YO10 5DD, United
eYork Centre for Complex Systems Analysis, Departments of
fCentro Nacional de Biotecnología, Consejo Superior de
dEidgenössische Technische Hochschule Zürich, 8093 Zürich, Switzerland;
Edited by Roger W. Hendrix, Pittsburgh Bacteriophage Institute, Pittsburgh, PA, and accepted by the Editorial Board November 20, 2011 (received for review
June 29, 2011)
Genome packaging into preformed viral procapsids is driven by
powerful molecular motors. The small terminase protein is essen-
tial for the initial recognition of viral DNA and regulates the
motor’s ATPase and nuclease activities during DNA translocation.
The crystal structure of a full-length small terminase protein from
the Siphoviridae bacteriophage SF6, comprising the N-terminal
DNA binding, the oligomerization core, and the C-terminal β-barrel
domains, reveals a nine-subunit circular assembly in which the
DNA-binding domains are arranged around the oligomerization
core in a highly flexible manner. Mass spectrometry analysis and
four further crystal structures show that, although the full-length
protein exclusively forms nine-subunit assemblies, protein con-
structs missing the C-terminal β-barrel form both nine-subunit
and ten-subunit assemblies, indicating the importance of the C
terminus for defining the oligomeric state. The mechanism by
which a ring-shaped small terminase oligomer binds viral DNA has
not previously been elucidated. Here, we probed binding in vitro
by using EPR and surface plasmon resonance experiments, which
indicated that interaction with DNA is mediated exclusively by the
DNA-binding domains and suggested a nucleosome-like model in
which DNA binds around the outside of the protein oligomer.
bacteriophage SPP1 ∣ DNA packaging ∣ virus assembly ∣
formed empty procapsid (1–3). A powerful ATP-fueled molecular
machine drives the DNA with a speed of up to 1;800 bp∕s
through the portal protein embedded in a unique vertex of the
icosahedral procapsid (2–4). The molecular motor usually con-
sists of the small and large terminase proteins. The small termi-
nase plays a dual role in virus particle assembly: It (i) recognizes
viral DNA during the initiation of packaging and (ii) modulates
the ATPase and nuclease activities of the large terminase during
DNA translocation (5, 6). After filling a procapsid, the rest of the
DNA is then docked to the portal entrance of another procapsid
where the process of DNA translocation is repeated (7).
X-ray structures have been determined for portal proteins
from bacteriophages φ29 (8), SPP1 (9), and P22 (10) and also for
large terminases from bacteriophages T4 (6), RB49 (6), and SPP1
(11). Three-dimensional information on small terminases is
limited to the cryo-EM structure of phage P22 small terminase
(12), the NMR structure of the DNA-binding domain of phage
λ gpNu1 (13), and the crystal structure of phage Sf6 small termi-
nase (14). In the absence of accurate three-dimensional data for
all three motor components of one particular phage, mapping of
functional information to the structure and modeling molecular
interactions between individual components is challenging. We
have addressed this issue by extending the structural information
on Bacillus subtilis bacteriophages SPP1 and SF6, two very closely
related viruses of the Siphoviridae family. Here we present five
he virus genome in tailed dsDNA bacteriophages and in the
evolutionarily related herpes viruses is packaged into a pre-
X-ray structures for several different constructs of the SF6 small
terminase. We also present mass spectrometry data on oligomeric
states of the small terminase. Structural observations on the full-
length protein containing the N-terminal DNA-binding domains,
together with DNA-binding data and normal mode analysis
calculations, suggest a model for packaging initiation in which
DNA-binding domains are adjusted to form a periodical nucleo-
Structure Determination. G1P, the small terminase of bacterioph-
age SF6, comprises 145 residues (Fig. 1A). We first determined
the crystal structure of the oligomerization core domain of
G1P, residues 53–120, in which the N-terminal DNA-binding and
the C-terminal β-barrel domains were truncated (G1P53–120). This
structure, determined at 1.85-Å resolution by using seleno-
methionine-substituted protein (SI Materials and Methods and
Table S1), revealed a ten-subunit assembly (Fig. 1 B and C) and
was used as a molecular replacement model to determine struc-
tures of a nine-subunit assembly (1.68-Å resolution, Fig. 1 D–F)
and a second crystal form of the ten-subunit assembly (2.19-Å
resolution) formed by the same truncated construct (G1P53–120).
A further two structures, both revealing nine-subunit assemblies,
were determined for (i) a proteolytic fragment G1P65–141(3-Å re-
solution, Fig. 2A) containing the C-terminal β-barrel but missing
the N-terminal DNA-binding domains and (ii) the full-length
protein (approximately 4-Å resolution, Fig. 2 B and C and Fig. S1).
An overview of all constructs used in various experiments is in SI
Materials and Methods.
Author contributions: C.R.B., M.C., M.O.-L., C.S., I.-o.E., V.C., G.J., E.D., S.B., C.V.R., J.C.A.,
and A.A.A. designed research; C.R.B., M.C., M.O.-L., I.-o.E., V.C., G.J., E.D., S.B., C.V.R., and
A.A.A. performed research; J.C.A. contributed new reagents/analytic tools; C.R.B., M.C., M.
O.-L., C.S., I.-o.E., V.C., G.J., E.D., C.V.R., and A.A.A. analyzed data; and C.R.B., M.O.-L.,
C.S., I.-o.E., V.C., E.D., C.V.R., and A.A.A. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. R.W.H. is a guest editor invited by the Editorial
Freely available online through the PNAS open access option.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID codes 3ZQM, 3ZQN, 3ZQO, 3ZQP, and 3ZQQ).
1Present address: Department of Molecular Genetics, University of Toronto, 1 King’s
College Circle, Toronto, ON, M5S 1A8, Canada.
2Present address: Architecture et Fonction des Macromolécules Biologiques, Centre
National de la Recherche Scientifique, Universités d’Aix-Marseille I and II, 13288 Marseille
cedex 9, France.
3Present address: Bio-organic Chemistry Laboratory, Faculty of Science and Technology,
Free University of Bolzano, Piazza Università, 1, 39100 Bolzano, Italy.
4To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/
www.pnas.org/cgi/doi/10.1073/pnas.1110270109PNAS ∣ January 17, 2012 ∣ vol. 109 ∣ no. 3 ∣ 811–816
Oligomerization Core Domain. The conformation of individual sub-
units is essentially the same in the nine-subunit and ten-subunit
oligomeric states of the G1P53–120construct (Fig. 1 B–D). A seg-
ment of helix α4, present at the N terminus of this construct, and
the following peptide (residues 65–69) that link the N-terminal
DNA-binding domain with the oligomerization part are located
at the outer surface of the oligomer and do not contribute to sub-
unit interactions. The main body of the oligomerization core,
residues 70–120, is formed by two short antiparallel α-helices
(α5 and α6) connected by a β-hairpin (β1β2) (Figs. 1B and 2D).
This segment mediates multiple subunit-subunit interactions
comprising eleven hydrogen bonds, several hydrophobic contacts,
and two salt bridges formed between residues His102/Glu104
and Glu115/Lys112 (Fig. 1E and Table S2). Interactions are very
similar for both oligomeric states, resulting in similar intersubunit
buried surface areas of approximately 1;000 Å2. However, the
two oligomeric states differ in the dimensions of the central chan-
nel. The most constricted part of the oligomerization core do-
main has a van der Waals diameter of approximately 11 Å in the
9-mer and approximately 14.5 Å in the 10-mer (Fig. 1F). The
channel reaches its maximum dimension in its middle part with
a van der Waals diameter of approximately 29 Å in the 9-mer and
approximately 32 Å in the 10-mer.
fragment, G1P65–141, comprising the C-terminal β-barrel and the
main body of the oligomerization core domain, forms a nine-
subunit assembly (Fig. 2A). In this structure, the N-terminal do-
mains which were shown to be responsible for DNA binding (15,
16) and are further on designated as DBDs (DNA-binding
domains), were missing because of degradation occurring during
the period required for crystal growth. However, the C-terminal
segment (residues 121–141), lacking in the previously crystallized
construct, is clearly visible in the electron density maps. The C-
terminal segments of all subunits assemble together in a parallel
β-barrel with each subunit contributing one β-strand (β3, residues
125–139). The internal van der Waals diameter of the β-barrel
varies from approximately 9.9 (measured for side-chain atoms
of Gln134) to 14.7 Å (side-chain atoms of Thr132). The slope
of the β-strands with respect to the barrel axis is approximately
55°, in good agreement with the theoretical value of 56.3° for this
type of β-barrel (17).
The N-Terminal DNA-Binding Domains Are Connected to the Oligomer
Via Flexible Linkers. The X-ray data for the full-length protein are
anisotropic and complete only to approximately 6-Å resolution,
although in directions perpendicular to the c axis they extend to
approximately 4 Å (Table S1). In this structure, shown in Fig. 2 B
and C, the C-terminal β-barrel as well as the N-terminal DBDs
of two out of three subunits in the asymmetric unit are defined in
the electron density maps (Fig. S1). The positions and orienta-
tions of the two defined DBDs with respect to the oligomeriza-
tion core domain differ markedly, with their centroids shifted
along the oligomer axis by approximately 33 Å. The DBD of the
third subunit of the asymmetric unit has no defined electron
density indicating variability in its position, which is permitted by
the lack of symmetry-related molecules in the vicinity of this do-
main. Taken together, the structural observations indicate signif-
icant variability in the positioning of DBDs.
To investigate further possible conformational changes in the
small terminase and, in particular, the potential adjustments in
the orientation of the DBDs, we performed normal mode analysis
calculations. These showed low-energy modes corresponding to
up-and-down movements of the DBDs (along the channel axis)
as well as rotational adjustments (Movies S1 and S2). These data
indicate a large degreeofflexibility intheorientationoftheDBDs,
consistent with the structural observations described above.
Full-Length Small Terminase Exclusively Forms Nine-Subunit Assem-
blies Stabilized by the C-Terminal β-Barrel. To understand whether
the nine-subunit state, observed for the full-length terminase,
predominates in solution or whether it was selected from a mix-
ture of different oligomeric states during crystallization, we per-
formed mass spectrometry analysis of the full-length protein
dues 53–120). (A) Domain organization showing location
of the oligomerization core within the full-length protein.
(B and C) Ribbon diagram of a ten-subunit assembly of
G1P53–120shown in two orthogonal views. The N and C termi-
ni as well as the secondary structure elements of a single sub-
unit are labeled. “Linker” refers to residues 61–65 connecting
α4 with the main body of the oligomerization core formed by
helices α5 and α6. (D) Ribbon diagram of a nine-subunit as-
sembly of the same construct, G1P53–120, shown in three alter-
nating colors. (E) Electrostatic surface potential at subunit
interface (ranging from −5, red, to þ5 kT∕e, blue) shown
for two opposing subunits with the central axis vertical as
in B. (F) Channel cross section indicating internal (van der
Waals) diameters. The red stars indicate the position of the
MTSSL in the S106C mutant used.
Structure of the oligomerization core domain (resi-
www.pnas.org/cgi/doi/10.1073/pnas.1110270109 Büttner et al.
under conditions where subunit interactions are maintained (18,
19). The only oligomer observed corresponded to the nine-sub-
unit assembly (Fig. 3A). Partial microheterogeneity was observed
at the C terminus of the protein, leading to peak splitting for the
9-mer species (Fig. S2). We also used mass spectrometry to probe
the oligomeric state of the protein in which the C-terminal seg-
ment was removed (G1P1–120). Mass spectra for this truncated
form indicated the presence of both nine-subunit and ten-subunit
species in an approximately 1∶1 ratio (Fig. 3B and Fig. S2).
These observations indicate that, although the C-terminal seg-
ment is not essential for G1P oligomerization, it appears to sta-
bilize the nine-subunit state, most probably through formation of
the intersubunit β-barrel observed in the crystal structure. This
hypothesis is supported by the electron paramagnetic resonance
(EPR) data (Table S3) showing that the construct G1P53–145con-
taining the C-terminal β-barrel forms predominantly oligomers.
Consistent with mass spectrometry analysis, variation of the sub-
unit number in the crystal structures was observed only when the
C-terminal β-barrel was truncated.
Comparison with Other Small Terminases. Sequence alignment of 33
small terminases from phages closely related to SPP1 indicated
several conserved residues that are exposed on the molecular
surface (SI Materials and Methods and Figs. S3 and S4 B and C).
One group of such residues (Lys5, Lys9, Arg12, and Gly32) is
found in the helix-turn-helix (HTH) motif involved in DNA
binding. Another group of conserved residues is present in the
central channel (Glu87, Lys112, and Lys119). These residues are
involved in electrostatic interactions that stabilize the oligomeric
assembly and may, potentially, also mediate interactions with
DNA. No conserved residues can be found at the outer surface
of the oligomerization core.
Structural comparisons with the small terminase of phage Sf6
(14) belonging to the Podoviridae family and a putative small ter-
minase from the Bacillus cereus prophage phBC6A51 [Protein
Data Bank (PDB) ID code 2ao9] reveal that, despite a lack of
sequence similarity, the folds are conserved (Fig. 2D). In all three
proteins the central oligomerization motif formed by α-helices
α5 and α6 in G1P and the C-terminal β-barrel are conserved fea-
tures. The fold of the DBD is also very similar (pairwise Cαrmsd
of 1.8–3.1 Å for approximately 45 DBD residues) although its
position with respect to the oligomerization core varies. G1P dif-
fers from the other two terminases by the presence of an extended
β-hairpin (β1∕β2in Fig. 2D) that connects the conserved α-helices
α5 and α6. Interestingly, the structure of the T4-like bacterioph-
age 44RR2 small terminase reported in the accompanying paper
(20) also reveals an oligomerization motif comprising two α-
helices, as indicated by earlier mutational analysis (21).
Previously, structural data were also obtained for the isolated
DNA-binding domain of the small terminase from bacteriophage
λ (13). Structural comparisons show that, although SF6 (PDB ID
code 2cmp) and Sf6 (14) small terminases share the same fold in
their DBDs, it differs markedly from the fold observed in bacter-
length small terminase. (A) Ribbon diagram of the proteolytic fragment
G1P65–141containing the C-terminal β-barrel in addition to the main body
of the oligomerization core domain shown with the central axis vertical, with
individual subunits in alternating colors. The indicated van der Waals dia-
meter of the β-barrel (9.8 Å) corresponds to the most constricted part of
the channel. (B) Ribbon diagram of the full-length G1P shown along the axis
with DBDs in red. Two DBDs of the asymmetric part (DBD-1 and DBD-2), that
were observed in the electron density maps, are shown as ribbons, and the
putative position of the third DBD (DBD-3) is depicted by dashed circles. (C)
Models offull-length G1P shown with all DBDs either in DBD-1 (left) or DBD-2
(right) orientation. For clarity, DBDs of only five subunits are shown, with a
10-bp dsDNA-DBD complex modeled only for one subunit. The rotational and
translational DBD movements derived from the normal mode analysis are
indicated on the left. (D) Stereo figure comparing superposed individual sub-
units of small terminases from Siphoviridae SF6 phage (two adjacent subu-
nits, red and pink), Podoviridae Sf6 phage (blue), and the putative small
terminase from prophage phBC6A51 (yellow). The structures were aligned
to fit the position of the channel axis, the first of the oligomerization α-he-
lices and the C-terminal β-strand. Although the position of the two helices is
conserved in the oligomer, the fold of the monomer differs in the Podovir-
idae Sf6 small terminase, where helix α6 occupies the same position as α6 of
an adjacent subunit in the small terminases from the other two phages.
Structure of N-terminal deletion construct (residues 65–141) and full-
length G1P reveals a 9-mer centered at 6;000 m∕z (green triangles). G1P has
some minor C-terminal truncations (see Fig. S2), which arise from partial clea-
vage of residues 128–145. To resolve these peaks, increased acceleration vol-
tageisapplied leadingtodissociation intomonomers (lowm∕z of1,000–2,000,
brown circles). (B) Mass spectrum recorded for the construct G1P1–120reveals
the formation of both 9-mers (green triangles) and 10-mers (purple hexagon)
in an approximately 1∶1 ratio. Gray circles correspond to monomers.
Mass spectrometry analysis. (A) Mass spectrum recorded for full-
Büttner et al.PNAS
January 17, 2012
iophage λ small terminase (Fig. S1). Although in all three cases,
the polypeptide chain folds into four α-helices, in the case of λ
small terminase the HTH motif is formed by the first and second
α-helices, whereas in the other two terminases it is formed by the
second and third α-helices. Unlike the SF6 and Sf6 terminases,
where the recognition helix (α3) is almost immediately followed
by another helix (α4), in bacteriophage λ small terminase the
recognition helix (α2) is followed by an extended hairpin, neces-
sitating classification of this fold as “winged HTH.” Fold differ-
ences result in a very different orientation of the HTH motif with
respect to helix α4, which connects the DBD with the C-terminal
oligomerization part of the protein (Fig. S1). So far, no structural
data have been obtained for the oligomerization part of the
bacteriophage λ small terminase, thereby preventing further
structural comparison, although analytical ultracentrifugation
data obtained for a complex of large and small λ terminases in-
dicated the formation of macromolecular assemblies comprising
eight subunits of the small terminase (22), thus suggesting a
potential similarity with the multisubunit assemblies found in
other small terminases.
Although all five small terminases (44RR2, P22, SF6,
phBC6A51, and Sf6), for which oligomerization domains have
been resolved by structural analysis, form ring-shaped oligomers,
they differ in their oligomeric state with subunit numbers ranging
from eight to eleven. Differences in oligomeric state result in dif-
ferent diameters of the internal channel.
DNA Binding by G1P is Exclusively Mediated by the DNA-Binding Do-
mains. The central channel as well as the presence of multiple,
peripheral DNA-binding domains per oligomer posed a question
about potential modes of interaction withDNA. We hypothesized
that, apart from interacting with the peripheral DBDs (Fig. 4A),
DNA could be accommodated in the central channel during
translocation (Fig. 4B). This assumption is supported by the
presence of Lys/Arg rings in the central channel (Fig. S4A) and
the intriguing finding that the geometry of the G1P β-barrel
matches the geometry of B-form DNA. Indeed, the strand angle
of the β-barrel matches the helical rise of double-stranded B-
DNA (Fig. 4B, Inset). We probed interaction with the two poten-
tial DNA-binding surfaces (the internal channel and the surface
around the outside of the oligomer formed by DBDs) by surface
plasmon resonance (SPR) and EPR.
SPR experiments were performed with a diverse range of
constructs for G1P and the SPP1 recognition pac site DNA, as
specified in Materials and Methods. The strongest interaction
was observed for the 428-bp pac DNA (pac1, comprising pacL,
pacC, and pacR) (Fig. 5A). Full-length G1P bound to this DNA
with a KDof 336 ? 4 nM and distinct association and dissociation
phases. The binding affinity decreased when fragments of the
pac site DNA were used (Table S4 and Fig. S5). Whereas the pre-
sence of the C-terminal β-barrel did not increase the affinity sig-
nificantly, a significant reduction was observed for constructs
lacking the DBDs. We hypothesized that the residual weak bind-
ing was due to nonspecific association of the DNA with basic
residues located at the outer surface of the oligomerization core,
in contrast tobinding in the central channel. Indeed, the mutation
of solvent-exposed positively charged Lys86 (Fig. S4) resulted in
a significant further drop in affinity (Table S4 and Fig. S5) ver-
ifying our assumption of nonspecific binding. Therefore, the in-
teraction with DNA as measured by SPR is predominantly
dependent on the presence of the DBDs.
To distinguish whether the weak residual binding after remov-
ing the DBDs was due to binding in the channel or whether it was
due to nonspecific interaction with the positively charged rings
present at the outer surface of the oligomerization domain, we
the outside of the oligomer mediated by the DNA-binding domains. Each
DBD in complex with a 10-bp oligonucleotide is reoriented so that DNA seg-
ments can form a continuous molecule. (B) DNA binding in the channel.
The Inset demonstrates the match between the geometry of the C-terminal
β-barrel and B-form DNA.
Potential models for the G1P-DNA complex. (A) DNA binding around
DNA recognition site comprising 428 bp (pac1). Equilibrium dissociation
constant KDwas estimated from a steady state analysis of three individual
experiments. (B) EPR experiments. Mims ENDOR spectra for G1P53–145
S106C labeled with MTSSL at the inner channel surface as shown in Fig. 1F.
The control experiment with 4-phosphonoxy-2,2,6,6-tetramethyl-piperidine-
1-oxyl (dashed line) clearly shows phosphorous coupling, but no coupling was
observed for the oligomer incubated with a 22-bp dsDNA (solid line).
DNA binding. (A) SPR experiments for full-length G1P and the pac
www.pnas.org/cgi/doi/10.1073/pnas.1110270109 Büttner et al.
performed EPR experiments with the spin label attached in the
internal groove of the channel, where there is sufficient space to
accommodate the label without affecting the diameter of the
channel (Fig. 1F). Pulsed EPR measurements were performed
by using the G1P53–145S106C mutant that lacks the DNA-binding
domains, labeled with the MTSSL (Materials and Methods). Our
modeling indicated that if DNA is present in the channel, the dis-
tance between the spin label and the nearest31P nucleus of the
DNA would be <1 nm. This distance is sufficiently short for the
hyperfine interactions between the electron spin of the spin label
in the protein and the nuclear spin of the31P nucleus in the DNA
to be observed by electron nuclear double resonance (ENDOR).
We used the Mims ENDOR pulse sequence at the Q band, which
was shown to be most sensitive to longer electron-31P distances
(23). Substoichiometrically labeled oligomers (containing approxi-
mately one spin label per oligomer) were used to prevent spin-
spin interactions between spin labels on adjacent subunits in the
oligomer. The control experiment using a phosphorylated spin
label (with ca. 0.6 nm nitroxide-31P distance) clearly showed phos-
phorous coupling in the Mims ENDOR spectra with an excellent
signal-to-noise ratio (Fig. 5B). However, ENDOR spectra of sub-
stoichiometrically labeled G1P oligomers incubated with a 22-bp
SPP1 dsDNA oligonucleotide showed no detectable phosphorous
The combination of SPR and EPR results demonstrates that
a complex of G1P with DNA in the inner channel could not be
assembled in vitro, at least under the conditions tested.
Small terminases play two crucial roles during DNA packaging.
Initially, the small terminase binds to a specific recognition site
within the viral chromosome and recruits the large terminase.
The assembled terminase-DNA complex then docks onto the
procapsid and the small terminase performs its second role, reg-
ulating the enzymatic activities of the large terminase.
Previous models describing the interaction between the small
terminase and the pac site DNA have suggested that the DNA
wraps around the circular oligomer. However, the presence of
the central channel suggested that it could accommodate DNA
(24). In this model of packaging initiation, the DNA would likely
interact first with the DNA-binding domains and then be trans-
located through the central channel of the small terminase. This
mode of DNA binding has been increasingly favored (2, 14, 24).
However, the observed inner diameters of the eight-subunit
assembly (14) and nine-subunit assembly (Figs. 1F and 2A) are
too narrow to accommodate the DNA double helix. Moreover,
small terminases from different phages differ in their oligomeric
states, containing either eight [Sf6 (14)], nine [SF6, P22 (24),
phBC6A51], or 11∕12 [44RR2 (20)] subunits, indicating that the
number of subunits and consequently the diameter of the inner
channel are not critical for the function of the small terminase.
Indeed, in the bacteriophage P22 small terminase, both nine- and
ten-subunit assemblies are functional for packaging in vivo (24).
In agreement with these observations, the combination of our
SPR and EPR data presented here strongly indicate that the
G1P-DNA interaction is mediated exclusively by DNA-binding
domains and not by the inner channel.
We then sought to model the interaction between DNA and
the small terminase oligomer comprising multiple DNA-binding
domains, as observed in SF6, Sf6, and phBC6A51. We did not use
DNA-interaction data available for the bacteriophage λ small
terminase (13, 25)because of significant differences in DBD folds
and oligomeric states (dimeric species reported for λ are not
observed in the SF6, Sf6, and phBC6A51 small terminases) and
because of DNA bending in λ involving an additional host factor
(26). Interestingly, when modeling the DNA, guided by HTH-
DNA complexes of closest structural homologues (27), the DNA
orientation is roughly parallel to the oligomer axis. This mode of
interaction suggests that one DNA molecule could connect the
DBDs of several small terminase oligomers. However, from the
mass difference accumulating to the chip surface in our SPR
experiments, we calculated that there was only about one G1P
oligomer per approximately 100 bp DNA. Moreover, previous
DNase footprinting experiments with G1P bound to pac DNA
revealed nine protected sites within an approximately 100-nu-
cleotide segment, one site every 10 ? 1 bp (every turn of the
DNA helix); these sites were separated by DNase-sensitive re-
gions (16). Taken together, these data indicate that in the com-
plex an approximately 100-bp DNA fragment wraps around the
protein oligomer interacting with multiple HTH motifs separated
by approximately 34 Å (16). The approximately 34-Å separations
would be in good agreement with the DBD separation observed
in crystal structures and with their flexibility. This model suggests
that small terminases with varying numbers of subunits would still
be functional as long as the DBDs were appropriately spaced.
Our structure of full-length G1P has two out of three DNA-
binding domains in the asymmetric unit defined, because no elec-
tron density was observed for the third domain. The two DBDs
for which electron density was observed are in different positions
relative to the oligomerization domain, which indicates signifi-
cant flexibility in their position. The observed flexibility is further
supported by normal mode analysis calculations. This flexibility
may be essential for binding DNA because to bind DNA in the
expected circular/helicoidal orientation, each DBD must reorient
in order to match its HTH motif with the corresponding segment
of DNA. On the basis of the observed flexibility of the DBD
positions, we generated a model starting with the two extreme
DBD positions defined in our crystal structure. We interpolated
the positions of the other DBDs around the oligomer, adjusted
them to be equidistant from the oligomer and each other, and
aligned the HTH motifs. Intriguingly, the two starting positions
provide enough spatial separation to allow a helicoidal assembly
where the DNA wraps completely around the oligomer (Fig. 6).
In contrast, in a model with all DBDs adopting the same constant
position relative to the oligomerization domain, the DNA mole-
cule cannot encircle the G1P oligomer without unrealistic dis-
The small terminase performs at least two distinct tasks during
DNA packaging: First, it assures the specific recognition of the
pac DNA, and second, it regulates the enzymatic activities of the
large terminase during DNA translocation, which requires that
the small terminase is engaged in the molecular motor. We show
that the DNA recognition task involves the small terminase DBDs
and that the DBDs are the dominant interaction site for the for-
mation of a nucleoprotein complex. Our structure of full-length
G1P, in which the DBD exists in two very different positions and
orientations, together with normal mode analysis, DNA-binding
data, and the DNase footprinting results (16) allowed us to model
Positionsof DBDs were remodeled so that the vertical (channel) coordinate of
the two extreme positions corresponds to coordinates observed in the crystal
structure with all other DBDs occupying intermediate positions; orientation
of each DBD was adjusted so that helix-turn-helix (HTH) motifs fit the heli-
coidal DNA, with approximately 34-Å spacing between adjacent HTH motifs.
Model of the small terminase-DNA complex during DNA recognition.
Büttner et al.PNAS
January 17, 2012
the interaction with DNA prior to recruiting the large terminase to
perform the initial cut. We argue that the interaction must involve
remodeling at the level of the tertiary structure and aligning the
DBDs with the DNA to form a helicoidal, nucleosome-like assem-
bly. After directing the DNA cleavage, the small terminase up-
regulates ATP hydrolysis and thereby DNA translocation while
inhibiting nuclease activity (5, 28, 29). Despite the conserved over-
all structure of the small terminase, no conserved residues were
located on the exterior surface of G1P, indicating that interaction
with the large terminase or portal protein is mediated by the over-
all architecture ofthemolecular motor andbythe distinctive shape
of the small terminase.
Materials and Methods
Protein Production, Crystallization, and X-Ray Structure Determination. The
gene encoding full-length small terminase (G1Pfl, residues 1–145) of bacter-
iophage SF6 was cloned, overexpressed, and purified as described in SI
Materials and Methods. Crystals of G1Pflgrew within 1 wk by vapor diffusion
at 293 K from solution containing 30% Jeffamine 600 M and 0.1 M sodium
cacodylate, pH 6.0. The 4-Å–resolution data were collected at ID-14 beamline
at European Synchrotron Radiation Facility (ESRF). A single better-diffracting
crystal from the G1Pflsample grew after 6 mo from solution containing 0.2 M
MgCl2, 8% (wt∕vol) PEG 20,000, 8% (wt∕vol) PEG 550 monomethyl ether, buf-
fered with Tris·HCl, pH 8.0. The 3-Å–resolution data from this crystal were
collected in house by using a Rigaku RU200 X-ray generator with a rotating
anode. Crystals of G1P53–120-SeMet were obtained under several crystalliza-
tion conditions: 0.1 M malonic acid, imidazole, and boric acid buffer, pH 7.0,
25% (wt∕vol), PEG 1500 (crystal form #1); 0.2 M KCl, 50 mM Hepes, pH 7.5,
35% (vol∕vol) pentaerythritol propoxylate (5/4 PO/OH) (crystal form #2) and
2.7 M ðNH4Þ2SO4, 0.1 M Tris·HCl, pH 8.5 (crystal form #3). Single-wavelength
anomalous dispersion X-ray diffraction data at the selenium absorption edge
(λ ¼ 0.9716 Å) for crystal forms #1 and #2 were collected at the I04 beam line
(Diamond). Data for crystal form #3 were collected at BM14 (ESRF). Most crys-
tallographic calculations were performed by using the CCP4 program pack-
age (30). G1Pfland G1P65–141datasets were processed with MOSFLM/SCALA
(Table S1). All other data were processed by using HKL2000 (31). A detailed
description of structure determination is given in SI Materials and Methods.
Coordinates have been deposited with the Protein Data Bank under acces-
sion ID codes 3ZQM (G1P53–120, #1), 3ZQN (G1P53–120, #2), 3ZQO (G1P53–120,
#3), 3ZQP (G1P65–141), and 3ZQQ (G1Pfl).
Mass Spectrometry. Thirty microliters of a solution containing G1Pfl or
G1P1–120were buffer-exchanged into 1 M ammonium acetate (pH 7.5) by
using Bio-Spin columns (Bio-Rad). MS analysis was conducted on a Q-ToF2
mass spectrometer (Waters) modified for high mass detection and conserva-
tion of noncovalent interactions between protein subunits (18, 19). Then 2 μL
of this solution was introduced into the mass spectrometer from a gold-pla-
ted capillary needle. Instrument parameters used during experiments were
capillary voltage, 1.7 kV; cone voltages, up to 110 V; extractor cone voltage,
5 V; and collision energy, ranging from 30 to 100 V. The mass spectra recorded
were calibrated by using cesium iodide. All data were acquired and processed
by using MassLynx v4.1 (Waters).
Surface Plasmon Resonance. On the basis of DNA-interaction regions identi-
fied by DNAse footprinting of the G1P-pac DNA complex (16) with G1P
found to bind to pacL and pacR but not to pacC, three different SPP1 pac
site variants were generated by PCR using the SPP1 genome as template
(pac1: complete pac site—i.e., including all three functional sites pacL-
pacC-pacR—and spans nucleotides −326 to þ108 relative to the cleavage site
in pacC, length 428 bp; pac2: ends after pacL, spans nucleotides −326 to −82,
length 253 bp; pac3: starts after pacL and covers pacC and pacR, nucleotides
−82 to þ108, length 190 bp). Further details are given in SI Materials and
EPR Measurements. The protein was spin labeled with 1-oxyl-2,2,5,5-tetra-
methylpyrroline-3-methyl)-methanethiosulfonate (MTSSL) using a standard
protocol. Continuous wave-EPR measurementswere carried out at room tem-
perature by using an X-band spectrometer, and Mims ENDOR was carried
out by using a pulsed Q-band spectrometer at 50 K. Full details can be found
in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Bernie Strongitharm and Andrew Leech
(University of York) and also John Butler and Tim Fagge (Biacore) for help
with SPR measurements. Ralf Flaig and Martin Walsh (Diamond, Oxford) are
thanked for help during data collection. We are grateful to Andrey Lebedev,
Eleonor Dodson, Johan Turkenburg, and Guy Dodson (University of York) for
useful suggestions during data analysis. Ma Yun (University of York), Emma
Carter, and Damien Murphy (University of Cardiff) are thanked for help with
EPR spectroscopy. This work was supported by the Welcome Trust (fellowship
081916 to A.A.A.).
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