C-Terminal Sequence Determinants of T4
Bacteriophage Tail Fiber Adhesin for Specific
Ewa Brzozowska1*, Anna Pyra2, Malgorzata Miskow1, Sabina Gorska1 and Andrzej Gamian1
1Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wroclaw, Poland
2Faculty of Chemistry, Department of Crystallography, 14 F. Joliot-Curie, 50-383 Wroclaw, Poland
Ewa Brzozowska, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław,
Poland, Tel: +48-713-371-172; E-mail:
Received: April 14, 2015; Accepted: June 01, 2015; Published: June 15, 2015
Brzozowska E, Pyra A, Miskow M, Gorska S, Gamian A (2015) C-Terminal Sequence Determinants of T4 Bacteriophage Tail
Fiber Adhesin for Specific Lipopolysaccharide Recognition. SOJ Microbiol Infect Dis 3(1): 1-5. DOI: http://dx.doi.org/10.15226/sojmid/3/1/00130
Identification of short peptides that serve as specific ligands
to biological materials such as microbial cell surfaces has major
implications in better understanding the molecular recognition of
cell surfaces. The adsorption specificity of T4 phage is determined
by an adhesive protein (adhesin) localized on the tip of the phage tail
fiber. For the first time, we have shown that the specific interaction
between adhesin and Escherichia coli B Lipopolysaccharide (LPS)
occurs even if the phage protein is in denatured form. In this paper
the peptide sequences, that bind to the LPS were identified and are
characteristic only for T4 phage. Our results suggest that the primary
structure of the phage adhesin might be responsible for the receptor
recognition in the crucial role plays which the variable region of
the protein the sequences are localized in its C-terminal part of the
adhesin. This hypervariable area is flanked and interrupted by highly
conserved stretches of histidine residues and contains amino acids
which, favorably interact with terminal glucosyl-alpha-1,3-glucose of
the bacterial LPS. Moreover, in this paper the specific LPS recognition
and binding by short synthetic peptides, containing the variable
regions, as well as those with histidine boxes have been studied. Of
peptides of which amino acid sequences correspond to the variable
region of the adhesin and contain histidine boxes have been studied.
The peptides were synthesized on pins and analyzed in the
LPS binding assay. It was showed shown that these highly variable
peptides imitating T4 phage variable regions, specifically interacts
with the E. coli B LPS. The conserved regions, containing histidine
residues, and which occur also in other phage adhesins, don’t bind
to the LPS.
Keywords: Adhesin; Bacteriophage; Determinant; Recognition;
BSA: Bovine Serum Albumin; CBS: Carbohydrate
Binding Site; DMF: Dimethylformamide; HRP: Horseradish
Peroxidase; LPS: Lipopolysaccharide; SDS: Sodium Dodecyl
Sulfate; TBST: Tris Buffered Saline plus 0.1% Tween 20; TMP:
Bacteriophages (phages) are viruses specific towards bacteria strains. Their ability to a specific recognition of bacteria
is used in many fields of science and technology such as bacteria
identification by phage typing or as bacteria detection sensors
. They are also good model systems to study viral infection
and replication, gene transfer, proteins folding and assembly.
Bacteriophage T4 was isolated on Escherichia coli B (E. coli B) by
Demerec and Fanco . T4 phage binds to the Escherichia coli B
Lipopolysaccharide (LPS)or OmpC protein using C-terminal
region of phage adhesin, called as gene product 37 (gp37)
localized at the distal part of the long tail fibers [3,4]. A monomer
of gp37 consists of 1026 amino acid residues with receptorbinding
region encompasses C terminal amino acid residues.
These amino acids are involved in a specific interaction with two
terminal glucoses of E. coli B lipopolysaccharide .
In our previous paper, we have shown that the adhesin –
both in native and denatured form – recognizes and binds E.
coli B LPS with a high specificity . The next goal was to
establish which amino acid sequence (primary determinant)
of the phage protein is responsible for specific LPS recognition
and binding. We concerned with the C-terminal region of the
gp37 adhesin according to prior studies of Montag et al. [7,8]
or Miroshnikov et al. . They suggested that the specificity is
determined by repeating motifs: His-X-His-Y (His boxes) which
flank very variable sequences encompassing few to tens amino
acid residues (Figure 1). The authors have suggested, that the
His boxes participate in bacterial receptor recognition and their
relative positioning may affect host specificity .
Experiments with hybrid phages made by Montag et al.
 Suggest the receptor-binding region of gp37 encompasses
residues 907–996, likewise structural analysis of the adhesin
made by Bartual et al.  also indicate that LPS binding is
initiated by the head domain comprising amino acid residues of
940-960. However, the LPS binding determinants have not been
precisely defined yet.
To determine the probability of carbohydrate binding by
single amino acid in the sequence, we made a sequence-based
prediction of the Carbohydrate Binding Site (CBS) using the CBS
Figure 1: Amino acid sequence of the C-terminal part of adhesin gp 37(
amino acid residues from 934 to 1026) containing His-X-His-Y motifs.
Pred web-server (http://tardis.nibio.go.jp/netasa/cbs-pred/) the
prediction methodology takes the predisposition of residues and
the sequential environments – solvent accessibility and neighbor
residue features as an input . According to this methodology,
a binary prediction was made for every residue – 1 for binding
and 0 for non-binding.
In the next step, we used pin method  to synthesize 8
different polypeptides of which amino acid sequences correspond
to the C-terminal end of T4 gp37within 930 and 1026 amino acid
residues. Two of them display a specific ability to bind LPS of E.
Materials and Methods
The prediction of carbohydrate binding sites in peptides
were performed using CBS-Pred (Carbohydrate Binding Side
Prediction) web-server (http://tardis.nibio.go.jp/netasa/cbspred/)
Synthetic peptides secondary structure predicted using PEPFOLD
1.5 . PEP-FOLD is a service for the de novo prediction of
peptide conformation from amino acid sequence. It is based on a
Hidden Markov Model derived Structural Alphabet that describes
a conformation as a series of local overlapping canonical
conformations. PEP-FOLD 3D generation is performed using a
discrete set of structural prototypes and using the OPEP coarsegrained
force field. PEP-FOLD will not show disulfide bonds. The
output files from PEP-FOLD are .pdb files which can be opened by
pymol - molecular visualization system .
Peptide synthesis on polyethylene pins
Peptide synthesis was performed using NCP Block of 96
Hydroxypropylmethacrylate pins (MIMOTOPES, Clayton,
Victoria, Australia) according to the standard protocol
described by Carter . F-moc amino acids* (60 mm in
Dimethylformamide DMF) were mixed with an equimolar
amount of diisopropylcarbodiimide and N-hydroxybenzotriazole
as coupling reagents for 4 h. Coupling reactions were tested using
10 mm bromophenol blue to check the presence of free amine
groups. After synthesis, the peptides were deprotected using
20% piperidine in DMF, washed with methanol, 0.5% acetic acid
in methanol and once again in methanol, drying and storage at
-20°C.Pins with bonded synthetic peptides were washed using
0.1 M phosphate buffer, pH 7.2, containing 1% sodium dodecyl
sulfate (SDS) and 0.1% 2-mercaptoethanol, heated to 60°C and
sonicated for 10 minutes.
Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Pro-OH, the
side group protected by Boc (tboc): removed using 95% TFA,
Fmoc-Trp(Boc)-OH or tboc, Fmoc-Lys(Boc)-OH or tboc, the side
group protected by tbu:Fmoc-Tyr(tbu)-OH, Fmoc-Thr(tbu)-OH,
Fmoc-Ser(tbu)-OH, the side group protected by otbu: removed
using 95% TFA: Fmoc-Asp(otbu)-OH, Fmoc-Glu(otbu)-OH, the
side group protected by Trt: Fmoc-His(Trt)-OH, Fmoc-Asn(Trt)-
OH, Fmoc-Gln(Trt)-OH, Fmoc-Cys(Trt)-OH
LPS binding assay
The E. coli B LPS binding assay was performed using
biotinylated LPS and synthetic peptides tethered to pins. The
LPS biotinylation was performed according to the method
described by Caccavo et al. . Pins with synthetic peptides
were transferred into wells in 96-well plate containing 1%
bovine serum albumin (BSA) in 50 mm Tris-Buffered Saline Plus
0.1% Tween 20 (TBST). The blocking reaction was performed for
1 h at room temperature (RT). After that, the biotinylated LPS
was added (100 ng/well) into the wells containing peptides and
incubated at 12°C overnight. The pins were washed three times
using TBST and incubated for 1 h, at RT with avidin conjugated
with Horseradish Peroxidase (HRP) (Thermoscientific) (avidin-
HRP dilution was 1:1000). TMP (3,3′,5,5′-tetramethylbenzidine)
(Promega) reagent was used to measure HRP activity according
to the protocol . The enzymatic reaction was stopped
by removal of pins from the plate and the amount of colored
product was measured at λ = 490 nm in a plate reader (biotek).
The positive control in this test was reactivity of 30 ng of adhesin
with biotinylated LPS in the same conditions as described above.
Western blot assay
The reactivity of synthetic peptides was shown in a test
in which they were first pre-incubated with a biotinylated
LPS solution (1 μg/ml) and next the same solution of LPS was
used in the Western blot assay with adhesin immobilized on
Immobilon-P (Millipore) membrane. Adhesin was transferred
from SDS-PAGE . Blocking reaction was performed using 1%
BSA in Trisma Base Saline buffer containing 0.05% of Tween-20
(TBS-T). The membrane was washed three times with TBS-T.
Next, the solution of LPS was added for one-night incubation at
4°C. After subsequent washing with TBS-T, the avidin conjugated
with Horseradish Peroxidase (HRP) (Thermo Scientific) was
added for one hour incubation at RT. The membrane was
washed three times using TBS-T and installed on HRP substrate
- 3-amino-9-ehtylcabazole - solution (0.1 M sodium acetate, pH =
5.2 supplemented with H2O2).
The Positive control of the test was the immobilized adhesin
incubated an initial concentration of LPS (1μg/ml).
In our studies, we took under consideration the C-terminal
part of adhesin within 934 and 1026 residues because of its high
content of His-boxes (five repeats). The peptides containing at
least one His-box and peptides containing variable sequences
localized between His-boxes, but without histidine residues were
synthesized. The peptides are listed in Table 1.
The bioinformatic analysis showed that 72% of amino acid
building the C-end of gp37 can interact with carbohydrates. The
peptide analysis was performed using the CBS-prep software.
The binary values were obtained by choosing a cutoff and
marking residues with higher predicted probability as binding.
The Expected accuracy scores were: the best cutoff for binary
classification = 0.075793, the best binary classification, Net
Pred was 60.4%, Sensitivity was 51.87% and the Specificity
was 68.95% (Sensitivity S1 = TP/ (TP+FN), Specificity S2 = TN/
(TN+FP). TP - correctly identified binding sites, TN - correctly
identified non-binding sites, FP – number of non-binding
residues wrongly identified as binding by predictor, FN – number
of binding residues predicted as non-binding)
The LPS binding assay results showed that only peptide no
5 and no 8 bound to the biotinylated LPS of E. coli B (Figure
2). The Absorbance (A) at λ= 490 nm obtained in the test was
0.237 for peptide no 5 and 0.188 for peptide no 8, respectively,
with regard to the negative control (A at = 490 nm equal 0.062)
and the positive control – adhesin gp37 (λ = 490 equal 0.301).
These two peptides - 5 and 8 - were composed of 24 amino acid
residues and did not contain any His-boxes. Peptide number 5
Table 1: Synthetic peptides immobilized on pins.
had only 3 amino acid residues which did not bind carbohydrates
according to the CBS-Pred analysis. Peptide no 8 had 50% nonbinding
amino acid residues. Peptide number 7 contained three
His-boxes and according to bioinformatic analysis only 3 amino
acid residues (12%) did not interact with carbohydrates while in
the assay its activity was low. The binding levels of peptide 7 was
higher compared to the negative control, but over twice lower
than the binding level of peptide 8, the same peptide number
6. The peptides 6&7 bound to the LPS of E. coli B at a low level
(the A490< 0.1) were not analyzed further. The short peptides
containing one His-box did not bind the biotinylated LPS.
The LPS binding ability of the peptides 5 and 8 were
confirmed using the Western blot assay. In this test we showed,
that whole pool of LPS was bound to the peptides immobilized on
pins during the pre - incubation step. After pre-incubation, the
solution of biotinylated LPS was used in Western blot assay in
which the adhesin was transferred onto Immobilon-P membrane
from SDS-PAGE gel (Figure 3).
The 24 amino acid peptides can adopt a favorable
carbohydrate binding conformation in solution (in the ELISA
conditions). There is such a supposition because in the test the
peptides are not in denatured form. Thus, they could fold into
native form and their conformation could be responsible for
peptide-LPS interaction. The model structures of the peptides
5 and 8 according to Pep-fold server prediction (http://bioserv.
rpbs.univ-paris-diderot.fr/services/PEP-FOLD/) are α-helix with
a disordered loop of the N-terminal end and β-sheet with loop
respectively (Figure 4) .
In our studies, we showed that amino acid
sequences: AWNGTGVGGNKMSSYAISYRAGGS (no 5) and
TVNSTGNTENTVKNIAFNYIVRLA (no 8) bind to the E. coli B
Figure 2: The results of E. coli B LPS binding by the synthetic peptides (1 – 8) immobilized on pins in the LPS binding assay – ELISA – compared to
the positive control- adhesin.
LPS in a specific manner. Synthetic peptides containing His-box
motifs, the long and the short ones, are not capable to bind to
the lipopolysaccharide molecule. The studies included the part
of adhesin gp37, which was previously indicated by Montag et
al. ; as a part responsible for phage-bacteria interaction. The
sequence no 5, that determines the specificity of LPS binding
belongs to the variable region of T4 adhesin and our studies
showed that the region of the adhesin is responsible for LPS
specific recognition and binding. As was previously noted, the
His-X-His-Y boxes (which X and Y are predominantly Ser or Thr)
flank the variable regions and are present at the sites almost
exactly corresponding to the sites in adhesive proteins of phages:
T4, tuia, tuib, Stf and λ. However, the only T4 phage can infect E.
coli B using LPS as receptor[8,14,17]. The other phages use
bacterial outer membrane proteins (porins) to infect their host
bacteria (but not the E. coli B). It is known, that the His-box motifs
are metal-binding sites which interact with divalent ions such as
Fe2+ or Zn2+ . The divalent cations as Zn2+ enhance phage T4
infectivity and contribute to cell lysis in environmental systems
. The amino acid sequence of peptide no 5, which showed
the highest ability of the E. coli B LPS binding is present only in
T4 phage adhesin, while the sequence of peptide no 8 is highly
homologous to those present in all adhesive proteins of phages
listed above. The amino acid sequence of the peptide no 5 is identical to the sequence belonging to the head domain of the
adhesin located at the extreme distal end of the long tail fiber.
Previously, it was suspected to play a primary role in receptor
binding . The peptide no 8 can also bind the LPS of E. coli B,
so the presence of the homological sequence should also allow
to bind the LPS by the other phages (tuia, tuib, Stf and λ). The
adhesive protein of tuib phage contains exactly the same amino
acid sequence like peptide no5 and the T4 adhesin - gp37.The
experiments presented by Yu et al.  confirmed that tuib
phage also uses LPS as receptor initiating the infection process.
It is very likely, that the other phages can also bind to the same
lipopolysaccharide receptor. The LPS binding is an intermediate
stage of bacterial infection, but it is not enough to infect the
bacteria. It should be emphasized, that if the infection does not
occur, it does not mean the lack of LPS binding. It is known, that
the adhesin gp37 interacts with the terminal glucosyl-alpha-
1,3-glucose of lipopolysaccharides  and protein–saccharide
interactions almost always involve stacking of sugar residues
onto aromatic amino acid side chains . So the side chain of
aromatic amino acids of the peptide no 5 (Trp-936, Tyr-949,
and Tyr-953) are the attractive candidates for receptor binding.
Moreover, Lys-945 (near Trp-936) and Arg-954 present in
the peptide 5 may also interact with the lipopolysaccharides
phosphate groups. Peptide no 8 also contains aromatic amino
Figure 3: Western blot assay of biotinylated LPS binding by the adhesin using LPS solutions which were first pre-incubated with the synthetic
peptides on pins. Numbers 5 and 8 show the absence (no pink band) of the LPS that was previously bound to the peptides. (1-8 peptide numbers,
C- positive control).
Figure 4: Model structures of peptide 5 (A) and 8 (B) according to the PEP-FOLD 1.5. .
acid side chains (Phe-1018 and Tyr-1020) capable to bind the
carbohydrate receptor as well as charged amino acid (Lys-1014
and Arg-1024) involved in the LPS phosphate group interaction.
E. coli B infection by T4 phage has been studied for a long
time and it is considered as a model system of bacteria – phage
interaction. In this paper, the amino acid sequence responsible
for the interaction has been described. However, identification
of short peptides those serve as specific ligands to biological
materials such as microbial cell surfaces become more and more
popular. Short peptides recognizing bacterial cells in specific
manner have major implications in better understanding the
molecular recognition of cell surfaces and for bacteria detection.
The authors gratefully acknowledge support for this work
from the Polish National Science Centre under grant No.
- Brzozowska E, Smietana M, Koba M, Górska S, Pawlik K, Gamian A, et
al. Recognition of bacterial lipopolysaccharide using bacteriophageadhesin-
coated long-period gratings. Biosensors & Bioelectronic.
2015; 67: 93-99. doi: 10.1016/j.bios.2014.07.027.
- Demwrec M, Fano U. Bacteriophage resistant mutants in Eschericha
coli. Genetics. 1945; 30(2): 119–136.
- Hashemolhosseini S, Montag D, Kramer L, Henning U. Determinants of
receptor specificity of coliphages of the T4 family. A chaperone alters
the host range. J Mol Biol. 1994; 241(4): 524-33.
- Riede I, Degan M, Henning U. The receptor specificity of bacteriophages
can be determined by a tail fiber modifying protein. EMBO J. 1985;
- Bartual SG, Otero JM, Garcia-Doval C, Llamas-Saiz AL, Kahn R, Fox GC,
et al. Structure of the bacteriophage T4 long tail fiber receptor-binding
tip. Proc Natl Acad Sci USA. 2010; 107(47): 20287-92. doi: 10.1073/
- Prehm P, Jann B, Schmidt G, Strim S. On a bacteriophage T3 and T4
receptor region within the cell wall Lipopolysaccharide of Escherichia
coli B. J Mol Biol. 1976;101(2):277-81.
- Montag D, Riede I, Eschbach M.L, Degen M, Hening U. Receptorrecognizing
proteins of T-even type bacteriophages. Constant and
hyper variable regions and an unusual case of evolution. J Mol Biol.
1987; 196(1): 165-74.
- Montag D, Hashemolhosseini S, Henning U. Receptor-recogniizing
proteins of T-even type bacteriophages. The receptor-recognizig area of proteins 37 of phages T4, TuIa and TuIb. J Mol Biol. 1990; 216(2):
- Miroshnikov KA, Marusich EI, Cerritelli ME, Cheng N, Hyde CC, Steven
AC, et al. Engineering trimeric fibrous protein based on bacteriophage
T4 adhesins. Protein Eng. 1998; 11(4): 329-32.
- Malik A, Firoz A, Jha V, Ahmad S. PROCARB: A Database of Known and
Modelled Carbohydrate-Binding Protein Structures with Sequence-
Based Prediction Tools. Adv Bioinformatics. 2010; 436036. doi:
- Malik A, Ahmad S. Sequence and structural features of carbohydrate
binding in proteins and assessment of predictability using a neural
network. BMC Struct Biol. 2007; 7: 1.
- Thévenet P, Shen Y, Maupetit J, Guyon F, Derreumaux P, Tufféry
P. PEP-FOLD: an updated de novo structure prediction server for
both linear and disulfide bonded cyclic peptides. Nucleic Acids
Research.2012; 40(Web Server issue): W288-W293. doi:10.1093/
- The PyMOL Molecular Graphics System, Version 1.2r3pre. Schrödinger
LLC. Available from https://www.pymol.org/pymol.
- Michael CJ, Jacq B, Argues G, Bickle TA. A remarkable amino acid
sequence homology between a phage T4 tail fibre protein and ORF314
of phage lambda located in the tail operon. Gene. 1986; 44(1): 147-50.
- Caccavo D, Afeltra A, Pece S, Giuliani G, Freudenberg M, Galanos C,
et al. Lactoferrin-lipid A-lipopolysaccharide interaction: inhibition
by anti-human lactoferrin monoclonal antibody AGM 10.14. Infect
Immun. 1999; 67(9): 4668-72.
- Gosling JP. Immunoassays: A Practical Approach. Oxford: Oxford
University Press. 2000.
- Laemmli UK. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature. 1970; 227(5259):680-5.
- Montag D, Schwarz H, Henninh U. A component of the side tail fiber
of Escherichia coli bacteriophage lambda can functionally replace the
receptor-recognizing part of a long tail fiber protein of the unrelated
bacteriophage T4. J Bacteriol. 1989; 171(8): 4378-84.
- Adams MH. Bacteriophages. New York: Interscience publishers. 1959.
- Yu F, Yamada H, Mizushima S. Role of lipopolysaccharide in the
receptor function for bacteriophage TuIb in Eschericha coli. J Bacteriol.
1981; 148(2): 712-5.
- Yu F, Mizushima S. Roles of lipopolysaccharide and outer membrane
protein OmpC of Escherichia coli K-12 in the receptor function for
bacteriophage T4. J Bacteriol. 1982; 151: 718–722.
- Vyas NK. Atomic features of protein-carbohydrate interactions. Curr
Opin Struct Biol. 1991; 1(5): 732–740.