Review
Structure and morphogenesis of bacteriophage T4
P. G. Leimana,
*, S. Kanamarua
,V.V. Mesyanzhinovb
, F.Arisakac
and M. G. Rossmanna
a
Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, Indiana 47907-2054
(USA), Fax +1 765 496 1189, e-mail: leiman@purdue.edu
b Laboratory of Molecular Bioengineering, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
16/10 Miklukho-Maklaya Street, 117997 Moscow (Russia)
c Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology,
4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)
Received 18 February 2003; received after revision 16 April 2003; accepted 9 May 2003
Abstract. Bacteriophage T4 is one of the most complex
viruses. More than 40 different proteins form the mature
virion, which consists of a protein shell encapsidating a
172-kbp double-stranded genomic DNA, a ‘tail,’ and
fibers, attached to the distal end of the tail. The fibers and
the tail carry the host cell recognition sensors and are required
for attachment of the phage to the cell surface. The
CMLS, Cell. Mol. Life Sci. 60 (2003) 2356–2370
1420-682X/03/112356-15
DOI 10.1007/s00018-003-3072-1
© Birkhäuser Verlag, Basel, 2003
CMLS Cellular and Molecular Life Sciences
tail also serves as a channel for delivery of the phage
DNA from the head into the host cell cytoplasm. The tail
is attached to the unique ‘portal’ vertex of the head
through which the phage DNA is packaged during head
assembly. Similar to other phages, and also herpes
viruses, the unique vertex is occupied by a dodecameric
portal protein, which is involved in DNA packaging.
Key words. Bacteriophage T4; baseplate; contractile tail; DNA packaging; fibrous protein; phage infection; prolate
head capsid.
Introduction
Viruses are ubiquitous and obligatory parasites of animals,
plants, and bacteria. Although all viruses require a
living host for reproduction, host range diversity is a consequence
of differences in virus structure and function. A
virus consists of a genome encapsidated into a protein or
proteolipid shell. The shell protects the genome when the
virus is transmitted between hosts. In addition, the shell
carries the host recognition sensors and, once a susceptible
host has been located, delivers the viral genome into
the host.
Bacterial viruses, or bacteriophages, range markedly in
their complexity from simple spherical viruses, whose
genome size is only about 5 kbp (e.g., bacteriophage
fX174) [1], to complex viruses with genomes of more than
* Corresponding author.
280 kbp (e.g. bacteriophage fKZ) [2]. Several families of
bacteriophage have a special tube-like organelle called a
‘tail,’absent in other bacterial, animal, and plant viral families,
attached to one of the vertices of the shell. The tail is
used during infection for penetration through the cell envelope
and subsequent DNA translocation from the shell
into the host cell. The host cell receptor recognition molecules
are attached to the tail, which helps to coordinate the
processes of host recognition and DNA delivery.
Bacteriophage T4 is a double-stranded DNA (dsDNA)
tailed virus that infects Escherichia coli (fig. 1). It is one of
the most complex viruses, with a genome that contains 274
open reading frames out of which more than 40 encode
structural proteins [3]. The mature virus, or ‘virion,’ consists
of a 1150 Å-long, 850 Å-wide prolate head with hemiicosahedral
ends [4] encapsidating the genomic DNA; a
1000 Å-long, 210 Å-diameter cocylindrical contractile tail
[5], terminated with a 460 Å-diameter baseplate [6]; and six
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 2357
1450 Å-long fibers attached to the baseplate [7]. The head,
tail, and fibers assemble via independent ordered pathways
and join together to form a mature virus particle.
Unlike animal viruses, infection of host cells by tailed
bacteriophages is highly efficient – only one bacteriophage
T4 particle is required, in general, to infect a host
cell [8]. Upon infection, the phage shuts down host-specific
nucleic acid and protein syntheses, thus ensuring
production of only its own components in amounts sufficient
to assemble up to 200 progeny virus particles per infected
cell.The efficiency of the infection process and the
large genome of bacteriophage T4, in which only half of
the genes are necessary for proliferation on E. coli, contribute
to the diversity of the phages from the T4-like
family, a subgroup of Myoviridae [9]. These phages propagate
on a wide range of bacterial hosts that grow in diverse
environments [9–12].
The structures of the bacteriophage T4 head, tail, and
fibers have been studied using various techniques [for reviews
see refs 13–15]. When combined with extensive
biological, genetic, and biochemical data, such as the results
from complementation assay studies and cross-linking
analysis, these studies provide a structural model of
bacteriophage T4 [16]. Recent advances in electron microscopy,
X-ray crystallography, and computing power
have extended the structural knowledge of T4 to higher
resolution. This review focuses on these advances.
Bacteriophage T4 head
Head structure
The head of bacteriophage T4 is composed of more than
3000 polypeptide chains of at least 12 kinds of protein
(table 1) and a 172-kbp dsDNA chromosome, which
comprises 102% of the unique region of about 169 kbp.
The molecular weight (MW) of the head and the genomic
DNA are 194 MDa and 112 MDa, respectively [17]. The
shell has icosahedral ends and a cylindrical equatorial
midsection [4, 18] with a unique portal vertex where the
phage tail is attached. The icosahedral caps (T = 13 laevo
[19]) and the midsection (Q = 21 [4]) are formed by 160
Figure 1. Structure of bacteriophage T4 (modified from Eiserling & Black [16]). The proteins comprising the virion are labeled with their
corresponding gene number or name. [Provided by Fred Eiserling, University of California, Los Angeles.]
2358 P. G. Leiman et al. Structure and morphogenesis of bacteriophage T4
hexamers of gene product (gp)23*1
. The 12 pentagonal
vertices of the icosahedron are occupied by 11 pentamers
of gp24* [20] and one dodecamer of gp20 [21], which
substitutes the 12 pentamer of gp24* at the portal vertex.
The shell is decorated on the outside with gp hoc and
gp soc (highly antigenic outer capsid protein and small
outer capsid protein, respectively) [4, 22]. The latter two
proteins are non-essential for phage morphogenesis, although
biochemical data indicate that at least gp soc
helps to maintain head integrity in extreme environmental
conditions [23, 24].
A number of mutations in the capsid proteins (reviewed
in Black et al. [13]) lead to formation of heads with different
Q numbers [18]: isometrics (Q = T = 13), petites or
intermediates (Q = 17), wild type (Q = 21), and giants
(Q > 21). The semi-spherical ends in these mutants have
the same width as the T = 13 caps, but the lengths of their
midsection are different. The isometric heads are icosahedral
whereas the petites, the wild type, and the giants
are prolate. The isometric variants of heads have been
studied by cryo-electron microscopy (cryoEM). Threedimensional
(3D) maps of the empty capsids with and
without gp soc [25] and of DNA-filled capsids [20] have
been determined at 27-Å resolution and at 15-Å resolution,
respectively. The head diameter was found to vary
from ~973 Å along the fivefold axes to ~879 Å along the
threefold and twofold axes (fig. 2). As expected for a
T = 13 particle, the head is composed of 120 hexamers of
gp23* and 11 pentamers of gp24*. They form a shell that
is about 30 Å thick, encapsidating the genomic DNA
(fig. 3). Gp soc is a rod-like molecule (~39 ¥ 27 ¥ 14 Å),
which forms a continuous mesh on the surface of gp23*
hexamers (figs 2, 4). Every gp soc molecule binds between
two gp23* subunits, but not between gp23* and
gp24* (fig. 4) and, therefore, gp soc does not bind around
the gp24* pentamers. Gp soc forms trimers at the meeting
points of three gp23* hexamers, although when purified
from the phage or when expressed in E. coli, it is a
monomer in solution [25]. Since gp soc has been shown
to stabilize the head structure in extreme environmental
conditions, it might promote capsid stabilization by comTable
1. Protein compositions of the T4 prohead and mature head (modified from Black et al. [13]).
Gene Prohead Head Location
mass (kDa) copy number mass (kDa) copy number
23 56.0 960 48.7 960 shell, major capsid protein
20 61.0 12 61.0 12 shell, portal vertex
24 48.4 55 46.0 55 shell, fivefold vertices
soc – 0 9.7 840 shell, outer surface
hoc – 0 39.1 160 shell, outer surface
22 29.8 576 2.5 115 internal (scaffold)
21 23.2 72 18.5 3 internal (scaffold)
IPIII 21.7 370 20.4 370 internal (scaffold)
IPI 10.0 360 8.5 360 internal (scaffold)
IPII 11.1 360 9.9 360 internal (scaffold)
alt 76.8 40 75.9 40 internal (portal vertex)
68 17.0 240 – 0 internal
67 9.1 341 3.9 134 internal
Copy numbers of the shell proteins are derived from the EM studies [17, 20, 25]. Copy numbers of the internal proteins are based on the
SDS-PAGE analysis of Onorato et al. [95] and Isobe et al. [96].
Figure 2. Shaded-surface representation of the 3D cryoEM reconstruction
of the T4 isometric heads. The view is along a twofold axis
of the icosahedron. The prominent balloon-shaped projections are
gp hoc molecules. A total of 120 such molecules extend outward
from the center of each gp23* hexamer in the capsid. (Reprinted with
permission from Olson et al. [20]. Copyright 2001,Academic Press.)
1
According to phage genetics usage, gpX* signifies the product of
maturation produced by the cleavage of gpX to gpX*. An explanation
of T and Q numbers that describe prolate head symmetry is
reviewed in reference 18.
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 2359
pensating for unfavorable interactions left on the gp23*
surface upon prohead maturation [23, 24]. Gp hoc is a
balloon-shaped molecule and extends to ~50 Å away
from the shell surface (figs 2, 4). Its protruding part is
composed of two domains: a rounded base (~19 Å high)
and a globular head (~20 Å wide, 24 Å high) connected
by a thin neck region. The overall mass of the protrusion
is about 12 kDa, suggesting that up to two-thirds of
gp hoc might be inserted in the center of each gp23*
hexamer. The function of gp hoc is unknown.
The gp20 dodecamer, which occupies one of the 12 fivefold
vertices, could not be detected in the cryoEM maps
reported by either Iwasaki et al. [25] or Olson et al. [20]
due to the 532-icosahedral symmetry imposed by the reconstruction,
causing the gp20 density to be equivalenced
with the 11 pentamers of gp24*. The density in the vicinity
of gp24* pentamers had about the same magnitude as
the rest of the gp23* shell, suggesting that the distortion
of the shell, introduced by the gp20 dodecamer around
the unique portal vertex, is minimal.
Head morphogenesis
Assembly of the T4 head (fig. 5) starts with the formation
of the gp20–gp40 membrane-spanning initiation complex
at the inner side of the cytoplasmic membrane [26].
The function of gp40 is unclear, since it is not present in
the assembled heads, but it renders gp20 competent to
start the assembly process [27]. Subsequently, gp21,
gp22, gp67, gp68, IPI, IPII, IPIII, and gp alt (table 1) atFigure
3. Central section of the reconstructed T4 head density map
viewed along a twofold axis. The DNA and protein density are dark.
The concentric dark layers beneath the outer capsid shell are attributed
to the densely packaged dsDNA. The inset plots the spherically averaged
density, computed from the 3D reconstruction, as a function of radius.The
highest-density peak at the far right of the plot corresponds to
the protein density in the outer capsid shell (~3.0 nm thick). Eight progressively
smaller peaks between radii of 38 and 22 nm arise from the
condensed layers of dsDNA. The spacing between successive rings is
~2.4 nm, which closely agrees with previous measurements of closepacked
DNA in bacteriophage T7 [56]. (Reprinted with permission
from Olson et al. [20]. Copyright 2001, Academic Press.)
Figure 4. (a) A view, approximately along a threefold axis of the T4 capsid icosahedron, showing the organization of the hexamers and
pentamers and the location of the two-, three-, and fivefold axes that limit an asymmetric unit (labeled 2, 3, and 5, respectively). (b)
Schematic representation of the view from a showing the distribution of the capsid protein subunits within the shell. One gp hoc molecule
associates with each hexamer of gp23*. The three gp23* hexamers nearest the icosahedral threefold axis are each surrounded by 12 gp soc
molecules. The gp23* hexamers adjacent to the gp24* pentamers are each surrounded by only 10 gp soc molecules. Gp soc molecules are
absent at the gp24*-gp23*interface. (Reprinted with permission from Olson et al. [20]. Copyright 2001, Academic Press.)
2360 P. G. Leiman et al. Structure and morphogenesis of bacteriophage T4
Figure 5. Morphogenesis of the bacteriophage T4 virion. The overall assembly pathway can be divided into three independent stages: head,
tail, and long tail fiber assembly. The chaperonines and catalytic proteins are indicated in brackets near the protein, or assembly step, that
requires the chaperonine. Known protein stoichiometries are given as subscripts. Crystal structures of structural proteins are shown as ribbon
drawings.
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 2361
Table 2. T4-encoded chaperonines necessary for phage morphogenesis
[13, 15, 59, 67–69, 94].
Gene Mass (kDa) Proteins that require the chaperonine
57A 5.7 gp12, gp34, gp37
26 23.9 activates gp29
28 17.3 activates gp29
51 29.3 activates gp29
31 12.0 gp23
38 22.3 gp37
40 13.2 activates gp20
wac 51.7 LTF to baseplate attachment
63 43.5 LTF to baseplate attachment
LTF, long tail fiber.
tach to the gp20-gp40 complex and form a scaffold,
which is then coated by a shell of gp23 and gp24. The dimensions
of this prohead are about 15–20% smaller than
those of the mature head. The proteins gp68, IPI, IPII,
IPIII, and gp alt are not required for assembly, but rather
help to eliminate incorrectly assembled intermediates
that form dead-end products of maturation.
The scaffold and shell proteins in the fully assembled proheads
undergo proteolytic cleavage by gp21, which is
converted into its active form, called the prohead protease
(T4PPase), by slow self-cleavage [26, 28–31]. The portal
protein, gp20, similar to the portal vertex proteins of
other tailed phages, is not cleaved during this process.
The activatedT4PPase cleaves the amino termini of gp23,
gp24, IPI, IPII, IPIII, and gp alt and extensively digests
gp22, gp21, gp67, and gp68 (reviewed in Black et al.
[13]). All peptide fragments, except for peptide II and
peptide VII derived from gp67 and gp22, respectively, are
expelled from the prohead, thus providing free space to
accommodate the genomic DNA during packaging.
Gp21 performs self-cleavage only when incorporated
into the assembled prohead, suggesting that an exact 3D
arrangement of several gp21 subunits and, possibly, other
prohead proteins is required for the activation of gp21.
Post-assembly self-cleavage of the structural proteins is a
common feature of many viruses. For example, in picornaviruses,
VP0 is cleaved to produce VP2 and VP4, and
in retroviruses, the viral protease cleaves itself off the
Gag polyprotein [reviewed in ref. 32].
The folding of gp23 is controlled by the GroEL chaperone
working in concert with the phage-encoded chaperonine
gp31 (table 2), which substitutes GroES in the infected
cells [reviewed in ref. 33]. Despite low sequence
identity (about 14%), gp31 and GroES have similar folds,
form donut-shaped heptamers, and serve as a cap for the
GroEL protein-folding chamber [34]. The volume of the
GroEL/GroES chamber is insufficient to accommodate a
gp23 monomer and, hence, gp23 requires gp31, which
provides a folding chamber of a greater volume.
DNA packaging
The proteolytically processed proheads are released from
the cytoplasmic membrane for DNA packaging. The T4
DNA substrate is a long, branched concatemer created by
multiple rounds of homologous recombination between
the ends of several genomic DNA molecules synthesized
immediately after the infection has occurred. Upon recombination,
a branch in the concatemer can contain up
to 20 phage genomes along its length. The T4 endonuclease
VII, encoded by gene 49, cleaves the dsDNA concatemer
at the branch points [35], thus creating small regions
of single-stranded DNA (ssDNA). These regions
are recognized by gp17 [36], which is the large subunit of
the gp16-gp17 hetero-oligomeric complex called the terminase
[37]. Gp17 digests these ssDNA regions, but remains
associated with the rest of the DNA. The terminase
complex bound to DNA is then transported to the portal
vertex of the prohead where the gp17 ATPase interacts
with the gp20 portal protein [38]. The packaging proceeds
until the head is full, after which the terminase cuts
the capsid free from the rest of the DNA substrate. The
process of packaging requires ATPase activity of gp17
[39, 40], which is greatly facilitated by gp16 [41].
Tailed phages and herpes simplex viruses package DNA
into a preformed prohead in a similar fashion. The packaging
apparatus has been proposed to represent a rotary
motor powered by a virally encoded ATPase via ATP hydrolysis
[42, 43]. The DNA is a movable central spindle
of the motor, surrounded by a dodecameric portal protein
serving as a ball race between the DNA spindle and the
fivefold symmetric capsid vertex, to which the ATPase
complex is attached. During packaging, the ATPase powers
rotation of the portal protein, which causes translocation
of the helical DNA molecule into the capsid. Although
the sequence homology between the connector
proteins from different tailed phages is low (less than
20%), they all have a similar cone-shaped dodecameric
structure, and function similarly (fig. 6) [21, 43–46].
Furthermore, the connectors from phages f29 (19.3-kbp
genome) and l (48.5-kbp genome) are interchangeable
[47].
Similar to other dsDNA tailed phages, such as l, HK97,
T3, T7, and P22, initiation of DNA packaging in T4
causes prohead expansion [48, 49]. Upon initiation of
DNA packaging, the hexagonal lattice of the T4 prohead
expands from a 117-Å to a 140-Å repeat distance between
the gp23* hexamer centers, increasing the capsid
volume by 50% [19]. The expansion involves rotation of
the gp23* hexamers [50] and their partial refolding [51].
This transformation stabilizes the capsid structure and
creates binding sites for gp hoc and gp soc [24].
Mutants with altered lengths of the capsid cylindrical
midsection are capable of DNA packaging [52–54]. The
length of the packaged DNA is proportional to the volume
of the head: the isometric heads contain only about
2362 P. G. Leiman et al. Structure and morphogenesis of bacteriophage T4
70% of the genome, whereas the giant heads can contain
more than a dozen of the genomic sequences repeated
along a single dsDNA molecule. The density of packaged
DNA is similar in all head types [13] and, therefore, the
termination of packaging is regulated by the stress applied
during DNA packaging on the portal complex
and/or the entire shell. The packaged DNA is probably
wound in a tight toroidal bundle [55] whose axis is perpendicular
to the head length. In phage T7, however, the
axis of the bundle is along the phage length [56]. The
toroid consists of DNA layers separated by 24 Å in both
T4 and T7 [20, 56].
Upon completion of DNA packaging, the gp16-gp17 terminase
complex dissociates from the head.The head maturation
is finalized by attachment of gp13, gp14, gp2, and
gp4 to the portal vertex. These proteins are necessary for
subsequent attachment of the independently assembled
tail and fibers to produce the infectious virus particles.
Since the tails do not attach to the empty but otherwise
mature heads, a DNA-mediated interaction is involved in
the joining process. In T4, as is the case for phages l and
T5 [57], one end of the packaged DNA might descend
into the tail.
The tail, fibers, and infection process
Tail structure and morphogenesis
Phages from the Myoviridae family have exceptionally
complex, contractile tails. Bacteriophage T4 devotes
25 kbp of its genome to tail assembly, which is comparaFigure
6. Structure of the dsDNA tailed phage f29 portal vertex.
(a) Cross-section of the cryoEM prohead density (gray mesh) fitted
with the Ca backbone of the f29 portal protein, called the head-tail
connector (solid lighter gray), and the difference cryoEM map of
the structural prohead RNA (pRNA) surrounding the portal vertex
(light-gray mesh). Shown also is a DNA molecule passing through
the central channel of the connector. The viral ATPase, which powers
the DNA-packaging process, binds to the pRNA during packaging.
(Reprinted with permission from Simpson et al. [43]. Copyright
2000, Nature Publishing Group.) (b) Structure of the f29 connector,
which is homologous to the T4 portal protein, gp20. Helices
are shown as cylinders. The 12 monomers and the DNA, passing
through the central pore, are colored in different shades of gray.
(Reprinted with permission from Simpson et al. [97]. Copyright
2001, International Union of Crystallographers.)
Table 3. T4 baseplate and tail tube proteins in order of appearance
in the T4 genetic map (modified from Coombs and Arisaka [14]).
Gene Mass (kDa) Copy number Location
3 19.7 6 tail tube terminator
53 23.0 6 wedge
5 63.7 3 hub
6 74.4 12 wedge
7 119.2 6 wedge-vertex
8 38.0 12 wedge
9 31.0 18 wedge-vertex
10 66.2 18 wedge-pin
11 23.7 18 wedge-pin
12 55.3 18 STF
15 31.4 6 tail terminator
18 71.2 144 tail sheath
19 18.5 144 tail tube
25 15.1 6 wedge
27 44.4 3 hub
29 64.4 6 tail tube
48 39.7 6 baseplate
54 35.0 6 baseplate
td 33.1 3 hub?
frd 21.7 6 wedge?
STF, short tail fiber.
ble with the size of the entire adenovirus genome
(36 kbp). Products of at least 22 genes are involved in tail
assembly (table 3), which include a phage-encoded chaperone
that participates in folding of the long and short tail
fibers (table 2) [58–60].
The bacteriophage T4 tail is composed of two concentric
protein cylinders, at one end of which is the baseplate and
fibers (fig. 1) [6]. The inner cylinder, called the tail tube,
is built of 144 copies of gp19 [61, 62]. The tail tube has a
40 Å-diameter channel for DNA passage from the head to
the infected cell [5]. The outer cylinder, called the tail
sheath, tightly envelopes the 90 Å-diameter tail tube and
has a width of about 210 Å. It is composed of 144 copies
of gp18 [5, 61, 63]. The subunits comprising each cylinder
form a six-start helix with a pitch of 41 Å and a righthanded
twist angle of 17°.The helix has a length of 984 Å
and contains 24 repeats [5, 63].
During infection, the phage recognizes an E. coli bacterium
using its long tail fibers (LTFs) connected to the
baseplate. The phage then anchors the baseplate to the
lipopolysaccharide cell surface receptors using the short
tail fibers (STF), which are initially assembled under the
baseplate. This event triggers a hexagon-to-star conformational
change in the baseplate [6] and causes an irreversible
contraction of the tail sheath [64], releasing about 25 kcal/
mol of energy per gp18 monomer [65]. During this
process, the gp18 hexamers flatten, rotate, and expand radially,
resulting in a decrease of their thickness by 26 Å and
an increase of the twist angle by 15°. The contracted tail
sheath has a length of only 360 Å and a width of 270 Å
[66]. The tail tube does not change its length during sheath
contraction. As a result, almost half of the tube protrudes
out of the contracted tail sheath and the baseplate.
The sheath can be caused to contract by exposing the
phage to 3 M urea. Nevertheless, the DNA is not released
until the tail tube tip binds to a cytoplasmic membrane receptor
common to enteric bacteria, suggesting that tail
contraction does not cause the release of DNA [8]. The interaction
of the tail tube tip with the cytoplasmic membrane
involves creation of a channel for DNA passage.
During DNA transfer from the capsid into the cell, the
membrane remains virtually undamaged since the transfer
requires a proton motive force across the membrane [8].
The assembly pathway of the bacteriophage T4 tail
(fig. 5) is regulated by ordered sequential interactions of
proteins rather than sequential gene expression [67–75].
The baseplate, a remarkably complex multiprotein structure,
is assembled first. It is composed of about 150 subunits
of at least 16 different gene products, many of which
are oligomeric (table 3) (reviewed in Coombs and Arisaka
[14]). These proteins form six independently assembled
wedges that join together around the central hub with the
help of the trimeric proteins (gp9)3 [76] and (gp12)3 [77].
Each wedge is assembled by sequential interactions of the
seven protein oligomers: (gp11)3 [78], (gp10)3 [75], gp7,
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 2363
(gp8)2 [79], (gp6)2, gp53, and gp25. The baseplate hub is
formed by (gp5)3, (gp27)3 [80], gp29 [81] and, probably,
gp28 [82].The assembly of the baseplate is completed with
the attachment of six copies of gp48 and six copies of gp54
[72] to the external interface between the wedges and the
hub. The latter proteins serve as a starting point for polymerization
of gp19 to form the tail tube [83], which is terminated
with gp3 [84, 85].The tail tube serves as a scaffold
for polymerization of the tail sheath around it. During this
process, gp18 stores energy in its conformation (possibly
byATP hydrolysis [86]), making the non-contractedT4 tail
a stretched spring. The length of the tail tube is controlled
by the ruler protein, gp29 [81], which also participates in
assembly of the central part of the baseplate [69]. The
length of the tail sheath is determined by the length of the
tube. The assembly of the tail is completed by attachment
of a gp15 hexamer to the last ring of the tail sheath [87].
CryoEM reconstruction of the baseplate-tail tube complex
[80] shows that the baseplate is a dome-shaped
object which is 270 Å high and 520 Å in diameter at its
widest part (fig. 7). The hollow tail tube stems from the
center of the baseplate. One of the most remarkable feaFigure
7. CryoEM reconstruction of the T4 baseplate-tail tube assembly.
(a) Stereo view of the surface-shaded representation.The top
quarter of the baseplate has been removed to visualize the internal
features. (b) Cross-sectioned density fitted with the atomic structure
of the gp5-gp27 complex. For clarity, the contour level of the surfaceshaded
figure in a is higher than the contour level of the cross-sectioned
density in b. Thus, some of the density representing gp27 in a
is missing compared to b. (Reprinted with permission from Kanamaru
et al. [80]. Copyright 2002, Nature Publishing Group.)
2364 P. G. Leiman et al. Structure and morphogenesis of bacteriophage T4
Figure 8. Structure of the gp5-gp27 complex. (a) Ribbon stereo diagram. The three gp5 monomers are colored red, green, and blue. The
three gp27 monomers are colored yellow, gray, and purple. The K+
ion within gp5C is shown in pink. The (PO4)–
is hidden behind the
lysozyme domain. (b)The structure of the gp27 monomer with its four domains colored cyan, pink, light green, and gold along the polypeptide
chain. (c) Top view of the gp27 cylinder shows that the cyan and green domains form a hexagonal torus. (Reprinted with permission
from Kanamaru et al. [81]. Copyright 2002, Nature Publishing Group.)
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 2365
tures of the baseplate is a 30-Å wide, 110-Å long spike,
or needle, along the axis of the dome. The crystal structure
of the gp5-gp27 complex (fig. 8) can be fitted into
the baseplate map so that the needle density is occupied
by the C-terminal domain of gp5 (fig. 7). The gp27 trimer
forms a channel suitable for passage of a dsDNA and
serves as an extension of the tail tube (figs 7, 8).
Gp5 consists of three domains: an N-terminal oligosaccharide
binding-fold domain, the middle lysozyme domain,
and the C-terminal triple b-helix domain (figs 8, 9)
[80]. The gp5 lysozyme domain has 43% sequence identity
[88] and a closely similar structure to the T4
lysozyme encoded by gene e (T4L) [89]. The active center
residues in both proteins are the same, suggesting a
common evolutionary origin. Gp5 undergoes a maturation
cleavage between residues Ser351 and Ala352
(fig. 9), but both the resultant parts, the N-terminal part
(gp5*) and the C-terminal part (gp5C), remain associated
and are found in the phage particle [90]. Dissociation of
the gp5C trimer from gp5* frees the gp5* substrate-binding
sites [81] and increases the lysozyme activity of gp5*
by at least tenfold [90] (fig. 9).
The T4 STF (table 4) is a gp12 trimer, which is attached
to the baseplate via gp11 [78]. The STF is a 340 Å-long,
club-shaped molecule, which has a narrowing in the middle
of the shaft where it can bend by up to 90° [91]. This
feature might be necessary to accommodate the folded
fiber into the baseplate. Three polypeptide chains of gp12
run in register through the length of the club starting from
the shaft end towards the club head end [77, 91]. During
infection, the club heads (the C termini of gp12 molecules)
bind to the core region of the lipopolysaccharide
cell surface receptors [91], while the club shaft remains
associated with the baseplate [91].
Both gp12 and gp5 contain remarkable right-handed,
triple-stranded, b-helix domains, which are composed
of three intertwined, in register protein chains. The
triple-stranded b-helices in gp5 and gp12 are somewhat
different to each other. The gp5 b-helix has a welldefined,
eight-residue repeat with a common motif
(VXGXXXXX) [88, 90], whereas the gp12 helix does
not have a repeating sequence motif. Moreover, the
strands in the gp12 helix vary slightly in length (five to
seven residues per strand), although a repeat consensus
of six residues can be established. The inside of the
gp12 helix is hydrophobic, whereas the inside of the gp5
helix has hydrophobic, polar, and charged patches. In addition,
on the inside of the gp5 helix there are more than
40 water molecules and two ions situated on the threefold
axis. The gp5 helix probably represents a mechanically
firmer structure than the gp12 helix, making gp5
especially suitable as a needle for puncturing the host
membrane.
Figure 9. Assembly of the gp5-gp27 complex. (a) Domain organization
within the gp5 monomer. The maturation cleavage is indicated
by the dotted line. The three gp5 domains are shown as rectangles
with the initial and last residue number indicated. (b)
Alignment of the octapeptide units composing the intertwined part
of the c-terminal b-helix domain of gp5. Conserved residues are in
bold print; residues facing the inside are underlined.The main chain
dihedral angle configuration of each residue in the octapeptide is
indicated at the top by k (kink), b (sheet), or a (helix). (c) Assembly
of gp5 and gp27 into the hub and needle of the baseplate.
(Reprinted with permission from Kanamaru et al. [80]. Copyright
2002, Nature Publishing Group.)
Long tail fibers
The LTFs (table 4) of bacteriophageT4 are adsorption devices
and environmental sensors [8]. They are connected
to the baseplate via gp9 and gp7 [76, 84]. The fibers are
about 1450 Å long and only 40 Å in diameter. Each fiber
consists of the rigid proximal and distal parts connected
by a hinge region [7, 15].The gp34 trimer forms the proximal
part, whereas the distal part is composed of the
trimeric gp36 and gp37 and the monomeric gp35 (fig. 10)
[7, 15]. The N termini of the gp34 trimer form the baseplate-binding
bulge, whereas its C termini, gp35, and a
part of the gp36 trimer participate in formation of the
hinge. The gp36 trimer comprises about 13 nm of the distal
part of the LTF and interacts with the gp34 trimer,
which forms the rest of the fiber (fig. 10). The fiber is
composed of a repeating motif, common to gp34, gp37,
and gp12 (STF), with intervening sequences of different
lengths (fig. 10). The C-terminal regions of gp37 and
gp12 are homologous, presumably because both function
to bind lipopolysaccharides [7, 93]. Two phage-encoded
chaperones, gp57A and gp38, are required for assembly
2366 P. G. Leiman et al. Structure and morphogenesis of bacteriophage T4
Figure 10. Domain organization of the long and short tail fibers. (a)The phageT4 LTF.The proteins comprising the fiber and their oligomerization
states are indicated. The baseplate-binding bulge of the fiber is on the left and the receptor recognition tip is on the right. The bar represents
10 nm. (b) Repeat segments and homologous regions shared among theT4 tail fiber proteins. Linear representations of the amino acid
sequences (total number of amino acid residues in each protein is indicated in parentheses) of gp37, gp34, and gp12 are shown. The regions
of homology are boxed and labeled with Greek letters. p15B indicates regions of homology shared with the plasmid p15B of E. coli [98]. The
numbered boxes correspond to occurrences of the repeat motifs called ‘A’ and ‘B,’ the sequences of which are listed below. Each repeat is labeled
with the gene product number followed by the corresponding repeat number within that protein (i.e. the sequence labeled ‘34-1’ corresponds
to the first boxed repeat within gp34). The positions of the start and stop residues of the A and B motifs are indicated. Intervening
sequences, if present, are included. Highly conserved residues are boxed, and the residue positions detected as key sites by the iterative sampling
algorithm of Lawrence et al. [99] are indicated by an asterisk. The secondary structure elements derived from the gp12 fragment crystal
structure are shown in register with the sequence below the alignment. The open box labeled ‘A’ is the a helix, the arrows ‘I’ and ‘II’ are
the two b strands, and the dashed line represents the region of the polypeptide chain that was disordered in the crystal structure. The bar represents
100 Å. (Reprinted with permission from Cerritelli et al. [7]. Copyright 1996, Elsevier Science.) (c) Crystal structure of the gp12 STF
fragment fitted into a cryoEM reconstruction of the whole fiber. The conserved domain, consisting of the sequence labeled as ‘12-6’ in b is
highlighted with the gray box. (d) A ribbon representation of the conserved domain. The first and last residues are numbered. The secondarystructure
elements are named as in b. (Reprinted with permission from van Raaij et al. [77]. Copyright 2001, Elsevier Science.)
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review Article 2367
Figure 11. Infection process. (a) T4 phage recognizes the E. coli LPS molecules using the LTFs. (b) The phage attaches the baseplate to
the cell surface initiating contraction of the tail sheath. (c) Tail contraction causes the gp5 needle to puncture the outer cell membrane. (d)
Gp5C dissociates from the tail tube, thus activating the three lysozyme domains. (e) The lysozyme domains create an opening in the peptidoglycan
layer. ( f ) Gp27 associates with a receptor on the inner membrane, which initiates release of DNA into the cytoplasm.
Table 4. T4 fibers.
Gene Mass (kDa) Copy number Fiber Location
12 55.3 3 STF baseplate
34 140.0 3 LTF proximal part, connected to the baseplate
35 30.0 1 LTF hinge region
36 23.0 3 LTF distal part, hinge connection
37 109.0 3 LTF distal part, receptor recognition tip
wac 51.9 3 head whisker head-tail joining region
The copy number is given per fiber. There are six fibers of each type per virion [7, 15, 91, 94].
of the LTFs (fig. 5) and another two, gp63 and gp wac
[94], for attachment of the fibers to the baseplate
(table 2). Gp57A is also required for solubility and
trimerization of gp12 in vitro and in vivo [59].
Infection process
The structural and biochemical results lead to a probable
mechanism of infection [8], which is likely related to the
infection processes in many other viruses (fig. 11).TheT4
phage initiates infection of an E. coli bacterium by recognizing
the lipopolysaccharide cell surface receptors with
the distal ends of its LTFs. The recognition signal is then
transmitted through the LTFs to the baseplate attachment
protein, gp9, and then to the baseplate itself. Subsequently,
the STFs unravel from underneath the baseplate
and bind irreversibly to the lipopolysaccharide cell surface
receptors, thus securely anchoring the baseplate to the cell
membrane. The baseplate changes its conformation from
hexagonal to star-shaped, causing contraction of the tail
sheath. The contracted tail sheath drives the head closer to
the cell surface and, therefore, exerts a force onto the tail
tube directed toward the cell membrane. This force is
transmitted through the gp27 cylinder and the N-terminal
domain of gp5 to the b-helix needle, causing the latter to
puncture the outer membrane of the cell [80]. As the tail
sheath contraction progresses, the b-helix needle spans
the entire 40 Å width of the outer membrane, thereby enlarging
the pore in the membrane. Subsequently, when the
b-helix needle comes into contact with the periplasmic
peptidoglycan layer, it dissociates from the tip of the tube,
thus activating the lysozyme domain of gp5 [90]. The latter
digests the cell wall, allowing penetration of the tail
tube to the inner membrane [92]. The gp27 trimer, forming
the tip of the tail tube, probably interacts with a specific
receptor molecule on the cytoplasmic membrane to
initiate release of DNA from the phage head through the
tail tube into the host cell.
Acknowledgements. We are grateful to V. Kostyuchenko for many
helpful discussions and to S. Wilder and C.Towell for help in preparation
of the manuscript. This work was supported by a National
Science Foundation grant to M. G. R. and a Howard Hughes Medical
Institute grant to V. V. M.
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