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Nayar 1
Structure and Function of Thymosin β4
Anita Nayar
Senior Comprehensive Paper
The Catholic University of America
Nayar 2
The thymosins are a group of proteins from the thymus gland. There are three
classes of thymosins based on their pI, α, β, and γ. One of the most prominent of the
thymosin family is thymosin β4. This protein contains 43 amino acids and is
approximately 5 kDa. Thymosin β4 is found predominately in platelets and has a higher
affinity for platelet actin. Thymosin β4 is known to be the main G-actin sequestering
peptide and to accelerate wound healing. Analysis of thymosin β4 gave insight into its
structure and how it sequesters G-actin. It was found that thymosin β4 has two α-helicies,
one at each terminal end of the protein and these two helicies link what is known as the
hexapeptide actin binding motif. A hydrophobic cluster is present in the N-terminal αhelix that helps in binding thymosin β4 to actin. Thymosin β4, prevents G-actin from
polyermizing into F-actin through these various binding sites to G-actin. It was also
discovered that thymosin β4 is unstructured in aqueous solution and upon binding to Gactin, thymosin β4 undergoes a conformational change into a folded structure.
Nayar 3
Up until the early 1960s the thymus gland was thought to be vestigial and
functionless. It was during that time that the thymus gland was discovered to be the
“master gland” of the immune system.1 The term “thymosin” was given to the group of
molecules which, heavily affected the immune system by increasing the number of
lymphocytes in the blood from the thymus gland. Research on the thymosins began in
1964 using calf thymus. The first thymosin fraction to be extracted was thymosin
fraction-3 (TF3). This fraction was only partially purified but it opened the doors to
research of the thymosins. TF3 was further purified through gel filtration, ultrafiltration,
and ammonium sulphate fractionation to give rise to a more pure preparation known as
thymosin fraction 5 (TF5) which was determined to be biologically active. TF5 has a
family of about forty small acidic ploypeptides. The isoelectric points were determined
for each of the forty polypeptides. The thymosins were then classified based on their
isoelectric points: α for isoelectric points below 5, β for isoelectric points between 5.0 –
7.0, and γ for isoelectric points above 7. The isoelectric points of the classes of the
thymosins can be seen in Figure 1. The β-thymosins are a family of highly conserved
water-soluble 5kDa polypeptides.2 Thymosin β4 is the most abundant of the β thymosins.1
Nayar 4
Figure 1. The isoelectric focusing for determining the three classes of the
thymosin family.1
Thymosin β4
Thymosin β4 is a 43 amino acid, 5kDa polypeptide. Its significant functions
include cell proliferation, promotion of angiogenesis (the formation of new blood vessels
from a pre-existing vasculature which is needed for both growth and wound repair),
acceleration of wound healing. It is also the main G-actin sequestering peptide. The motif
Nayar 5
which allows thymosin β4 to bind actin is a seven amino acid sequence found in each
thymosin: LKKTETQ.3
I. Structural Determination of Thymosin β4
Amino Acid Sequence of Thymosin β4
The amino acid sequence of thymosin β4 was obtained with subtractive Edman
procedures, proteolytic digestion, and a Beckman 890C sequencer.4 Upon reaction with
dansyl chloride or phenylisothiocyanate thymosin β4 did not yield an α-amino acid
derivative. This indicates that the NH2 terminus is blocked by an acetyl group. Tryptic
digestion gave two peptides which lacked a free α-amino group. These two peptides were
deduced to be a part of the N-terminus region. The tryptic digestion also produced a
peptide which lacks a lysine residue and was therefore placed in the C-terminus region.
The side chains were assigned their positions based on their charges determined through
high-voltage paper electrophoresis of the peptides derived from Edman degradation and
proteolytic digestion. Thymosin β4 contains nine lysine residues and eight glutamic acid
residues, however, it does not contain arginine, histidine, valine, tyrosine, and
tryptophan.4 The complete sequence of thymosin β4 and the enzymatic cleavage products
can be seen in Figure 2.
Nayar 6
Figure 2. The complete amino acid sequence of thymosin β4. The F fragments are the
result of cyanogen bromide cleavage, the T fragments are the result of tryptic digestion,
and the P fragments are the result of partial acid hydrolysis.4
Determination of the Actin Binding Site
In order to determine the structure and binding motif of the actin binding site in
thymosin β4, mutational analysis was performed. The binding motif observed in virtually
all of the β thymosins is 17LKKTET22 and, furthermore, the first three residues are also
found in many other actin binding proteins.5 It is important to note that 23Q is considered
part of the actin binding motif in thymosin β4 the majority of the other thymosins,
however, at least three, do not contain 23Q which is why it is excluded from the conserved
motif discussed above.6 Therefore, the binding behavior of the motif was studied when
three residues of the six were mutated to L17A, K18E, and K19E. At high actin
concentrations, (e.g., 12 μM and 15 μM) wild-type thymosin β4 has 50% cross-links with
actin. However, at these high actin concentrations, K19E gave 30% cross-link with actin
and K18E only cross-links with actin slightly. Furthermore, when a double mutant of 18K
and 19K were exchanged to glutamates (KK18, 19EE), a total loss of inhibition was
Nayar 7
observed. Overall, mutant K19E produced an actin- thymosin β4 complex whereas mutant
K18E and L17A did not. This shows that these three residues make specific interactions
with actin and are essential for preserving the proper binding surface. Another mutant of
K was studied, K18A. This mutant did not affect the binding of thymosin β4 as severely
as K18E did. This suggests that 18K has electrostatic contact with a negatively charged
residue in actin. Moreover, when 18K was replaced with glutamate, a strong repulsive
effect was observed, which further confirms that 18K forms electrostatic contact with
The N-terminus of thymosin β4 (residues 1 through 16) is highly charged. To
study the significance of the N-terminus, many of the charged residues were substituted
with residues possessing the opposite charge. For example, a lysine was substituted with
glutamic acid. This charge reversal did not affect the binding as much as the mutations in
the binding site did. However, two mutations did have an unfavorable affect on actin
binding.5 These were the mutants of 16K and 14K. K16E had a 12-fold reduction of actin
affinity whereas K14E had essentially no affinity for actin. Further analysis of 14K with
another mutant K14A showed a similar very low actin binding affinity, and when
substituted with a glutamate gave a greater decrease in actin binding. This then suggests
that 14K is in an electrostatic interaction with a negatively charged actin residue. Then,
the nonpolar residues were studied and analysis showed that substitution of phenylalanine
to a smaller, less bulky side chain greatly residues thymosin β4’s affinity for actin.
Furthermore, when replaced by either tyrosine or tryptophan, the actin affinity is not
Nayar 8
affected as much. Therefore the bulky side chain of phenylalanine is of importance to the
binding of thymosin β4 to actin.5
An Intact N-terminal α-Helix is Required for Complete Thymosin β4 Activity5
To determine whether or not the N-terminus in thymosin β4 binds to actin, circular
dichroism (CD) measurements were taken.5 These measurements were carried out in both
water, and with 60% trifluoroethanol (TFE). It has been observed in NMR studies that
thymosin β4 is unstructured in aqueous solutions but the helices are observed in solutions
containing fluorinated alcohols6 such as TFE. Therefore the helices in thymosin β4 of
residues 4 – 16 and 30 – 40 were more pronounced in 60% TFE solution. The CD
measurements of wild type thymosin β4 showed a maximum at a wavelength of 190 nm
and double minima at wavelengths 207 nm and 222 nm, which correlate to the two αhelices shown in Figure 3. When CD analysis was performed on the mutants in the same
solutions, similar maxima and minima were observed. This shows that the loss of
thymosin β4 activity in these mutants is not due to an inability to form a secondary
structure. In K11P, a known helix-breaker, proline, replaced 11K as a control and the
affinity of thymosin β4 to actin severely diminished. CD analysis showed a decreased
intensity for K11P at peak 190nm, which is the marker for a helical conformation. The
minima which were associated with a helical conformation also shifted to a lower
wavelength for K11P as also shown in Figure 3. This mutation shows that overall loss of
activity in thymosin β4 is not entirely due to a change in conformation however, if the
integrity of the helix is altered, then thymosin β4 activity can be severely impaired.5 In
other words, this shows that thymosin β4 can still form an overall secondary helix, but the
Nayar 9
tertiary structure is impaired. If the helix is still present, thymosin β4 can still be
functional. However, once the helix is compromised, activity is lost. Therefore, if
thymosin β4 cannot form the secondary structures, it cannot function properly.
Figure 3. CD measurements of wild-type thymosin β4 in TFE along with mutants
K18E, L17A, and double mutant KK18, 19EE (A) and mutated with K11P thymosin β4 in
TFE (B). The mutants in (A) only show a slight deviation from the WT and therefore the
loss in activity is not due to the fact that they cannot make wild type secondary structures
except L19A which shows more intense peaks indicating a stronger N-terminal α-helix or
that the helix extends over more residues. (B) shows that when the helix is severely
compromised, the activity is thymosin β4 is affected.5
The Proposed Structure of the Actin Binding Site
The analysis described above has shown that the sixteen residue N-terminus and
the conserved hexapeptide motif are both essential for thymosin β4 to bind to actin. Based
Nayar 10
on NMR studies, a final model of the structure was proposed. NMR measurements and
analysis suggested a α-helix for residues 4 – 16, an undefined conformation for the
hexapeptide binding site at residues 17 and 18, a loop which spans residues 24 through
28, and a less stable second α-helix for residues 30 through 40.5
It is likely that the N-terminus is an α-helix because it can support interaction with
actin and can position the hydrophobic cluster to also interact with actin in this
conformation. The hydrophobic cluster is formed by three residues: methionine 6,
isoleucine 9, and phenyalanine 12. Methionine occurs at position 6 and when methionine
is oxidized, thymosin β4 is known as thymosin β4 sulfoxide and the affinity for actin is
decreased 20-fold.6 The hydrophilic side of the N-terminal helix consists of 8E, 10E, 11K,
K, and 15S of which only 14K makes a salt bridge with actin. This proposed structure is
further confirmed with mutant K11P. K11P breaks the helix and renders thymosin β4
essentially inactive. This shows that an intact helix, along with the hydrophobic core, is
essential for its activity. Further analysis shows that if the N-terminal helix was made just
one residue longer, the placement of 18K and 19K would be shifted in a way that is
unfavorable to actin binding.5 This further confirms the necessity of the N-terminal helix
for the actin binding to occur and the exact length needed for the N-terminal residue. The
final proposed model for the thymosin β4 actin binding site can be seen in Figure 4. It is
important to note that the α-helical segment of thymosin β4 is the only completely
compacted, structured form of the protein where the hydrophobic segment of thymosin β4
forms an interface with the hydrophobic end of the barbed end (subdomains 1 and 3) of
Nayar 11
Figure 4. The N-terminal helix (residues 4 – 16) is in shown in white, the
hydrophobic cluster is shown in yellow surrounded by van der Waals forces, and the
important residues for actin binding are in red. The N-terminal helix is the underlined
sequence and the hexapeptide motif is the doubly underlined sequence.5
The Actin Binding Site is Conserved in Actin Binding Proteins
The entire β- thymosin family (including β- thymosins of various species) binds
to actin and therefore has an actin binding motif. It is important to note that thymosin β15
is the only member of the β- thymosin family to have an amino acid substitution in the Gactin binding motif. In thymosin β15, a glutamic acid is substituted with asparagine.6 The
motif and other residues such as 14K which are essential for actin binding are conserved
throughout the entire β- thymosin family as shown in Figure 5.6 Each lysine that is
essential in binding thymosin β4 to G-actin is conserved in the β- thymosin family.7
Nayar 12
Furthermore, other actin binding proteins such as actobindin have a similar, albeit not
exactly identical, motif for actin binding.5
Figure 5. The amino acid sequences of the β- thymosin family. The conserved
residues are bolded. thymosin β15 is the only member to have a substitution in the actin
binding motif.6
NMR Determination of the Structure of Thymosin β4
A NMR technique for the determination of structures of macro molecules is the
Nuclear Overhauser Effect (NOE). This technique was used to determine the structure of
thymosin 4.8 In order to obtain a basic insight into the structure, 1H – 15N NOE analysis
was performed on the protein at 2C and 25C. At both temperatures, thymosin 4
displayed incredibly low values of heteronuclear NOEs (under 0.5 for 2C and under 0
for 25C). Usually a fully structured protein has a heteronuclear NOE value around 0.7.
Nayar 13
These data show that in aqueous solution, thymosin 4 is unfolded. When 1H – 15N NOE
analysis was performed on the thymosin 4 bound to actin, the value of the heteronuclear
NOE is around 0.7 which shows that when bound to actin, thymosin 4 is folded.8 This
NOE analysis can be seen in Figure 6.
Figure 6. The heteronuclear 1H – 15N NOE analysis of thymosin 4. The
represents the thymosin 4 – actin complex,  is thymosin 4 at 2C, and  is thymosin
4 at 25C.8
Analysis of the NOE peaks gives more insight into the structure of thymosin 4.
HN – HN peaks are indicative of helical folding and the most intense HN - H correlation
is considered intraresidual. Analysis of the peaks, HN, 15N, and Hα using density
functional theory method (CSI) calculations indicate that two α-helicies exist: one at the
N-terminus between residues 5D – 17L and one at the C-terminus between residues 31K –
A. These two helicies are responsible for linking the extended fragment, the actin
binding motif, which is residues 18K – 26N.8 The high correlation values of the NOE of
Nayar 14
the thymosin 4 actin complex shows that this region adopts an extended conformation
which binds to actin. This is known as the actin binding motif.8
One dimensional 1H-NMR analysis provides the same information about the
folding of thymosin β4 to actin.7 The shifts of thymosin β4 alone resemble that of an
unstructured protein and the chemical shifts of thymosin β4 bound to actin are in between
the shifts of thymosin β4 alone and G-actin alone which indicates that thymosin β4
becomes structured upon actin binding.7
To further confirm that thymosin 4 forms a complex with G-actin, the
heteronuclear single quantum coherence (HSQC) spectra was recorded at 25C with
thymosin 4 containing [U- 15N] thymosin 4 with an unlabeled actin. A large spreading
of amide protons was observed, which is expected of fully structured proteins, and can be
seen in Figure 7. Since thymosin β4 is unstructured in aqueous solution, these data show
that upon binding to G-actin, thymosin β4 undergoes a conformational change.
Furthermore, 26N was observed to undergo the largest shift, which indicates that this
amino acid lies on the interface of the thymosin β4 – G-actin complex. These data verify
that thymosin 4 forms a tight complex with G-actin.8 Therefore, NMR analysis confirms
the structure and binding pattern of thymosin 4 to G-actin, which coincides with the
ones proposed in the studies previously described.
Nayar 15
Figure 7. HSQC spectra of [U – 15N] thymosin 4. The red symbols indicate the
free thymosin 4 and the black dots indicate thymosin 4 bound to G-actin. The
additional black peaks indicate that the intact protein is structured when bound to actin.8
II. Function of Thymosin β4
Thymosin β4 and its Function in Wound Healing
It was first thought that thymosin β4 plays a role in wound healing because it is
involved in endothelial cell migration and because it is found in abundance in platelets.
To test if thymosin β4 is involved in wound healing, six 8 mm punch biopsy wounds were
made in rats. These wounds punctured the epidermis and dermis and were in contact with
the fat and muscle.9 thymosin β4 was injected topically into 3 mice and intraperitonally in
3 other mice. Saline was injected in the same manner as a control. The injections were
made at the time the wound was made and 48 hours later. Figure 8 compares the size of
the wounds after four and seven days. Both topical and intraperitonal injections of
Nayar 16
thymosin β4 showed a greater decrease in wound size than saline injections. This proves
that thymosin β4 is involved in wound repair. It is thought that when the platelets arrive to
the injury, they release thymosin β4, among other factors, which attract endothelial cells
to being angiogenesis and then begins wound repair.9
Figure 8. a) Topical injection of thymosin β4 shows an 18% decrease in wound
size after 4 days and 62% after 7 days compared to the saline injection. b) Intraperitonal
injection shows a 42% decrease in wound size after day 4 and 61% after day 7 compared
to the saline injection.9
Thymosin β4 as the Main G-Actin Sequestering Peptide
Globular actin, also known as G-actin, is responsible for rapid generation of new
actin filaments. G-actin contains four subdomains which are all held together by bound
ATP or ADP as shown in Figure 9. When G-actin monomers come together they form
actin filaments known as F-actin. When G-actin polymerizes into F-actin, each monomer
is orientented in the same direction. This shows that the structure is polar with two
different ends, one known as the barded end (plus) and the other as the pointed end
(minus).10 However, nearly half the intracellular actin present is in its stable monomeric
Nayar 17
G-actin form. Monomeric actin is stabilized through its interaction with sequestering
factors. Thymosin β4 is known to form a 1:1 complex with G-actin which inhibits saltinduced polymerization into F-actin.2 Thymosin β4 binds to G-actin with a dissociation
constant (Kd) between 0.5 and 2.5 μM. This can be seen in Figure 10. Thymosin β4 has a
3 – 5 fold higher affinity for platelet actin over skeletal actin since the platelet actin, in β
form, has more amino acid residues on its surface that allows thymosin β4 to interact
with.7 A seven-amino-acid sequence has been proposed as the main actin binding motif in
thymosin β4. This sequence is: LKKTETQ.3 Furthermore, the first three residues of this
motif are essential for the interaction with G-actin as are the three hydrophobic residues
M, 9I, and 12F of thymosin β4. NMR analysis showed that the N-terminus of thymosin β4
must be in an α-helix for the interaction between G-actin and thymosin β4 to occur.6
Figure 9. The structure of G-actin showing the 4 subdomains bound to a central
Nayar 18
Figure 10. Thymosin β4 (red) interacts with subdomains 3, 1, and 2 of G-actin.
When G-actin is released from the complex, it is added to the (+) end of the actin
Zero-length cross linking was carried out to create isopeptide links between the
side chains of thymosin β4 and G-actin. The basis of cross-linking is taken under the
premise that certain noncovalent interactions between acidic and basic residues in the
structure can be converted into covalent bonds through cross-linking.7 Zero-cross linking
with 1-ethyl-3-[3-(dimethyamino)propyl]carbodiimide (EDC) showed that 3K of
thymosin β4 cross links with 167E of actin and 18K of thymosin β4 cross links with one of
the four N-terminal acidic amino acid residues of actin (1DEDE4). The two contact sites
on the barbed end of G-actin are approximately 25 – 30 Å apart. The 12 residue α-helix is
approximately 18 Å and binds to both these sites. Therefore the α-helix must be in a
stretched helix conformation to span both the sites.7 Also, 38K of thymosin β4 can be
cross-linked to 41Q of G-actin. Furthermore, the C-terminus of thymosin β4 can also block
Nayar 19
G-actin polymerization into F-actin by binding to the pointed end of actin. These three
points of contact are in subdomains 3, 1, and 2 of G-actin, respectively, as can be seen in
Figure 9 and 10. Subdomain 2 is known as the pointed end of G-actin and subdomains 1
and 3 are known as the barbed end.7 Thymosin β4 blocks G-actin from polymerizing into
F-actin by binding to the barbed and pointed ends of G-actin.7 Additionally, the
interaction of G-actin and thymosin β4 depends on either ADP or ATP bound to G-actin.
When G-actin is bound to ATP, there is a 50-fold higher affinity to thymosin β4 than the
G-actin ADP complex.6
The discussion above states that thymosin β4 is the main G-actin sequestering
peptide in the cytoplasm. Thymosin β4 is also the main G-actin sequestering peptide in
the nucleus. To first determine the presence of G-actin in the nucleus, DNase I, another
actin binding protein, was labeled with Oregon Green in MCF-7 cells. All these cells
displayed the Oregon Green-labeled DNase I in the nucleus. Thymosin β4 was
fluorescently labeled with Oregon Green cadaverine (OGC) to determine its presence in
the nucleus. Some other thymosin β4s were bis-labeled with OGC at 23Q and 36Q. Both of
these labels appeared in the analysis of thymosin β4 in the nucleus. When the Kd was
determined for the bis-labeled thymosin β4, it did not differ significantly from the
unlabeled thymosin β4. In order to further test the distribution of thymosin β4
fluorescently labeled thymosin β4 was also injected into Vero cells. Both MCF-7 and
Vero cells showed distribution of the peptide in the nucleus. Then the bis-labeled
thymosin β4 was digested using AsnC-endoproteinase. Thymosin β4 contains only one
asparagine residue at position 26. Therefore this endoproteinase produced two fragments,
the N-terminal fragment containing residues 1 – 26 and the C-terminal fragment
Nayar 20
containing residues 27 – 43. Each of the fragments contained a fluorescent label. HPLC
analysis showed that the N-terminal fragment containing the actin binding domain was
predominant in the nucleus while the C-terminal fragment remained in the cytoplasm as
shown in Figure 11. Residues 14 – 16, KSKLKK, is rich in lysine residues and is
believed that be a part of thymosin β4’s translocation into the nucleus.2
Figure 11. A and B show the N-terminal in the nucleus and C and D show the Cterminal fragment of thymosin β4 in the cytoplasm. A and C display the fragment of
thymosin β4 also injected with rabbit immunoglobin G and B and D display the fragment
of thymosin β4 alone.2
Many proteins involved in cell cycle regulation are naturally unstructured and
fold when they bind to their biological target.8 Thymosin β4 falls into this category. This
protein is involved in both wound healing and is the main G-actin sequestering peptide.
G-actin is responsible for rapid generation of new actin filaments.2 Thymosin β4 binds to
Nayar 21
G-actin in order to prevent G-actin from polymerizing into F-actin. The α-helix of the Nterminus of thymosin β4 binds to subdomains 1 and 3 of G-actin while the α-helix of the
C-terminus binds to subdomain 2 of G-actin. In between these two helicies, an actin
binding motif of thymosin β4 binds to actin also. Each of these contacts prevents G- actin
from polymerization. Thymosin β4 is found predominately in the platelets and even has a
higher affinity for platelet actin. Therefore, it is highly possible, especially since
thymosin β4 has a role in wound healing, that future drug research can make certain drugs
focusing on thymosin β4 to aid in the wound healing process. These types of drugs for
example might be able to help people deficient in clotting or shortening the wound
healing duration of a serious wound.
Nayar 22
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Mannherz, H.G.; Journal of Cell Science 2004, 117, 5333 – 5343.
(3) Philip, D.; Huff, T.; Gho, Y.S.; Hannappel, E.; Kleinman, H.K.; The FASEB Journal
2003, 17, 2103 – 2105.
(4) Low, T.L.K.; Hu. Shu-Kuang; Goldstein, A.; Proc. Natl. Acad. Sci. USA 1981, 78,
1162 – 1166.
(5) Troys, M.V.; Dewitte, D.; Goethals, M.; Carlier, M.F.; Vandekerckhove, J.; Ampe,
C.; The EMBO Journal 1996, 15, 201 – 210.
(6) Huff, T.; Müller, C.S.G.; Otto, A.M.; Netzker, R.; Hannappel, E.; The International
Journal of Biochemistry & Cell Biology 2001, 33, 205 – 220.
(7) Safer, D.; Sosnick, T.R.; Elzinga, M.; Biochemistry 1997, 36, 5806 – 5816.
(8) Domanski, M.; Hertzhog, M.; Coutant, J.; Gutsche-Perelroizen, I.; Bontems, F.;
Carlier, M.F.; Guittet, E.; van Heijenoort, C.; The Journal of Biological Chemistry
2004, 279, 23637 – 23645.
(9) Malinda, K.M.; Sidhu, G.S.; Mani, H.; Banaudha, K.; Maheshwari, R.K.; Goldstein,
A.; Kleinman, H.K.; The Society for Investigative Dermatology 1999, 113, 364 –
(10) Berg, J.; Tymoczko, J.; Stryer, L. Molecular Motors; In Biochemistry; WH Freeman
and Company: New York, 2007; pp. 985 – 986.