THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 279, No. 45, Issue of November 5, pp. 46351–46354, 2004
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.
Minireview
Properties of Titin
Immunoglobulin and
Fibronectin-3 Domains*
Published, JBC Papers in Press, August 18, 2004,
DOI 10.1074/jbc.R400023200
Larissa Tskhovrebova and John Trinick‡
From the School of Biomedical Sciences, Leeds
University, Leeds LS2 9JT, United Kingdom
Immunoglobulin (Ig)1 and fibronectin-3 (Fn3) domains are common building blocks of many extracellular proteins involved in
ligand recognition and cell adhesion. Ig and Fn3 domains are also
the main components of a group of intracellular proteins associated
with the contractile apparatus of muscles. The largest of the intracellular group is titin (⬃3 MDa), which has key roles in the assembly and elasticity of vertebrate striated muscles. The titin molecule
spans one-half of the sarcomere, the repeating contractile unit that
gives striated muscle its characteristic striped appearance. Titin
interacts with a majority of sarcomere proteins and is also likely to
bind a variety of cytoplasmic signaling molecules. The Ig and Fn3
domains are important in titin interactions, whereas the levels of
interdomain mobility and structural stability relate directly to
mechanical functions. Titin and the other muscle Ig/Fn3 proteins
are likely to have a common ancestry. It is plausible that the
emergence and evolution of the muscle Ig/Fn3 proteins were closely
related to the origin and evolution of multicellularity and cell
differentiation. This review summarizes current knowledge regarding the properties of the Ig and Fn3 domains of titin. General
properties of titins are presented only briefly and are described in
detail in recent reviews (1–3).
Titin Structure
Titins are formed from the largest polypeptides so far found in
nature. The 363 exons estimated to comprise the human titin gene
contain 114,114 bp, corresponding to a polypeptide of up to 38,138
amino acid residues long (4, 5) (Swiss-Prot/TrEMBL accesion numbers Q10465, Q10466, and Q8WZ42). Isoforms sequenced from
cardiac and skeletal muscle cDNAs contain between 240 and 300 of
the Ig- and Fn-like domains. Assuming a linear arrangement of
domains each ⬃4 nm long, this indicates a molecule more than 1
␮m long, which compares well with electron microscope measurements of the purified protein. The domain arrangement is also
visible directly in a “string-of-beads” appearance in micrographs
(6).
In situ the titin molecule spans one-half of the sarcomere, which
is the repeating contractile unit of muscle, composed mainly of
ordered arrays of thin (actin) and thick (myosin) filaments (Fig. 1).
The N terminus is at the boundary of sarcomere, the Z-line, and the
C terminus is in the M-line at the center of the sarcomere. More
than one-half of the molecule (the A-band section) is an integral
part of the myosin filament. This region is formed mainly by Ig and
Fn3 domains.
The I-band part of titin forms an elastic connection between the
end of the thick filament and the Z-line, which changes its end-toend distance when muscle contracts or extends. This part of titin
does not contain Fn3 domains and has only Ig and unique se* This minireview will be reprinted in the 2004 Minireview Compendium,
which will be available in January, 2005. This work was supported by the
British Heart Foundation.
‡ To whom correspondence should be addressed. Tel./Fax: 44-113-3434350; E-mail: [email protected].
1
The abbreviations used are: Ig, immunoglobulin; Fn3, fibronectin-3; pN,
piconewtons.
This paper is available on line at http://www.jbc.org
quences. Most of the Ig domains are arranged in tandem in two
segments, “proximal” and “distal” to the Z-line, which flank the
N2-PEVK region containing mainly unique sequences.
On average, the titin Ig and Fn3 sequence motifs are less than
50% conserved within each family. However, each family has distinct subfamilies with sequence identity up to 90%. In the I-band
part, the Ig domains constitutively expressed and those differentially expressed in isoforms fall into different subfamilies (7). The
differentially expressed segment contains Ig domains from several
different subfamilies that are arranged in long range patterns or
“super-repeats” consisting of 6 and 10 domains (8).
In the A-band part, the Ig and Fn3 domains are arranged in 7and 11-domain super-repeats, which are repeated 6 and 11 times,
respectively. The domains at similar positions within different
super-repeats have higher sequence homology than the domains
from the same super-repeat, indicating that they belong to different subfamilies (9, 10).
Structural modeling predicted that the subfamily-specific consensus sequences and residues would define the general surface
and interface differences between domain subfamilies. Thus, in the
I-band the constitutively and differentially expressed Ig domains
are likely to differ systematically in exposed polar and charged
residues and in the stiffness of domain interfaces (11). Likewise, in
the A-band the Fn3 domains are predicted to have subfamilyspecific clusters of conserved residues, mostly charged, on their
surfaces and interfaces (9, 10, 12).
Structure of Titin Domains
The structures of titin domains inferred from sequence were
confirmed by NMR and x-ray crystallography. The expressed domains that have been solved are representative of the M-line region, Ig-M5 (13), the constitutive distal segment, Ig-I27 (14), the
A-band, Fn3-A71 (9), and the constitutive proximal Ig segment,
Ig-I1 (16). In addition, atomic structures have been obtained of
polydomain constructs (17), the kinase domain (18), and a motif
repeat of the unique PEVK region (19).
The titin Ig structures have the ␤-sandwich fold characteristic of
the immunoglobulin superfamily (Fig. 2). The fold contains 8 –9
␤-strands arranged in two sheets, ABED and A⬘GFC(C⬘), packed
FIG. 1. Arrangement of titin in the sarcomere. Titin molecules span
one-half of the sarcomere, the structural and contractile unit of striated
muscle cells. The sarcomere is composed mainly of overlapping arrays of thin
(actin) and thick (myosin) filaments. The thick filaments are located in the
center of sarcomere in the A-band. The C-zone (shown striped) is a region of
constant protein packing. C-protein and its homologs are bound in this
region. The D-zone (distal from the center) is where the thick filament tapers
toward its end. On both sides thick filaments overlap with thin filaments
that extend through the I-band from the Z-lines at the ends of the sarcomere.
The N and C termini of the titin molecule are in the Z- and M-lines,
respectively. The A-band part of titin (red) is integral with the thick filament.
Estimates indicate a ratio of 6 titin molecules per half-thick filament. This
part of titin is formed mainly by Ig and Fn3 modules arranged in repeated
patterns, super-repeats, that correlate with the filament structure. The
kinase domain (green) is near the M-line region. I-band titin forms an elastic
connection between the tip of thick filament and the Z-line. This part consists
mainly of two tandem Ig segments (blue): the proximal (to the Z-line), and
the distal, which is near the thick filament end. The two segments are
connected by the N2-PEVK region (yellow). When the sarcomere contracts or
extends, the elastic I-band part of titin coils up or elongates.
46351
46352
Minireview: Properties of Titin Ig and Fn3 Domains
FIG. 2. Structures of titin Ig domains solved by the x-ray crystallography and NMR. These domains are representative for the constitutive
proximal Ig segment, I1 (Protein Data Bank entry 1GIC), the distal Ig
segment, I27 (Protein Data Bank entry 1TIT), and the M-line region, M5
(Protein Data Bank 1TNM). The N and C termini are on opposite sides of the
domains. The domains differ in size and conformation of the loops and overall
shape. Positions of the conserved residues specific for the domain subfamilies
are shown in red sticks (I1 and M5). These residues are likely to control
surface properties of the domains and thus to define their interactions at
interfaces and binding to other proteins. The network of interactions centered at the A⬘-G pair of strands (I27, green and red, respectively) defines
kinetic stability in both chemically and mechanically induced unfolding. The
four core residues essential for nucleation of the Ig-fold are shown as cyancolored spheres (I27). Within the molecule, the domains are arranged in a
linear array in a C to N terminus manner. The figure was created using
Pymol.
face-to-face. A disulfide bridge connecting the two sheets was observed only in one case, the I1 domain. This bridge, however, differs
from the classical Ig disulfide, which links strands B and F; instead
it connected strands C and E.
Close inspection of the structures and sequences placed the
domains in the intermediate I-set of the Ig superfamily (13). This
implied that, unlike domains of the constant C2-set to which they
were originally assigned, titin Ig domains are more similar to the
V-set (variable) domains. The conserved pattern of residues specific
to the I-set, the V-frame (20), had earlier been detected in other
muscle Ig proteins and was also found in all the titin Ig domains.
Thus I27, M5, and I1 all are similar to the typical I-set domain,
telokin (20). The deviations from this pattern include inconsistency
in structuring of the A, A⬘ and C⬘ strands and absence of some key
interactions involving loops A⬘B and EF and strand D. There are
also some variations between the domains in the length of the
␤-strands, in the size and conformation of loops, and in the overall
shape. These differences seem to reflect general structural differences between the domain subfamilies (7, 11).
Titin domain A71, the only structure of an intracellular Fn3
domain so far solved, was confirmed to have the characteristic Fn3
family fold (9). The strongest resemblance is to the first Fn3 domain of Drosophila neuroglian and to Fn3 domains in fibronectin
and tenascin. Sequence analysis indicated that all titin Fn3 domains contain key residues specific to the Fn3 family that form the
hydrophobic core of the domain. In addition to these residues, titin
domains were also found to contain titin-specific sets of conserved
residues.
Domains in the Molecule
Insights into the relative arrangement of domains and the overall conformation of the titin molecule have come from combined
experimental and modeling studies. A model of the constitutively
expressed I-band part, calculated from NMR data, supported the
expected beads-on-a-string arrangement (17). This also suggested
bending (⬃155°) and twisting (⬃90°) between successive domains,
resulting in 10 –17% shortening of the end-to-end distance from a
fully extended conformation. The interdomain mobility appeared to
be low, indicating a relatively well defined overall conformation of
the constitutively expressed segment. The differentially expressed
Ig segment was predicted to have an even more regular and stiff
conformation, because of the presence of super-repeats and higher
stiffness of interdomain interfaces suggested by sequence analysis.
A-band titin is also likely to have an extended and relatively stiff
conformation, based on modeling of the arrangement of the Fn3
domains, which are predominant in this part of the molecule (9, 10,
12). The super-repeat pattern of A-band titin and the predicted
high stiffness of the interdomain interfaces again suggest a regular
conformation.
Mobility between domains is likely to be the major source of the
flexibility in titin. Flexibility is especially important in the I-band
region that connects thick filaments to the Z-line. As described
above, this region has important mechanical roles and is the origin
of elasticity in relaxed sarcomeres. It also maintains the central
position of the thick filament in the sarcomere, which ensures
balance of forces between both its halves during the contractile
cycle. As the sarcomere changes length during muscle contraction
or passive extension, the I-band titin changes its conformation,
coiling up or elongating. Although mobility between domains is
predicted to be low throughout the molecule, its level appears to be
enough to provide the considerable overall flexibility. Studies of the
purified protein indicate a value of persistence length in the range
13–19 nm, implying substantial bending over molecule segments
containing 3– 4 domains (21–23).
Stability of Domains
Thermal Stability—Thermal unfolding of individual expressed
titin Ig domains has been shown to be reversible and to occur in a
simple two-state reaction. The relatively wide range of the transition temperatures, 35–70 °C (24), is similar to the range observed
for extracellular Ig domains. Studies of domain pairs indicate independent unfolding of each domain, as well as mutual stabilization (25). Confinement within narrow pores to mimic cellular environment also increases stability (26). With the exception of
M-line domains, the average domain stability appears to be higher
in the I-band than in the A-band part of the molecule.
Chemical Stability—Chemical denaturation of titin domains correlates with the thermal denaturation data. Unfolding again follows essentially a two-state process, although in some cases there
is evidence of low stability intermediates (24, 27, 28). The range of
stabilities is relatively wide, with estimates of the free energy of
unfolding between 2 and 10 kcal mol⫺1. The rates of folding and
unfolding also vary widely. Unfolding rate constants are in the
range 10⫺2 to 10⫺6 s⫺1, and folding rates are spread from 10⫺2 to
102 s⫺1 (28, 29). In multidomain constructs, stability increases
slightly, and unfolding and refolding rates are slowed.
I27 (from the distal Ig segment in the I-band) has been used
extensively to study folding and unfolding mechanisms of Ig domains (30 –32). Folding was shown to occur via a nucleation-condensation mechanism, in which the long range side-chain interactions of four key hydrophobic residues bring into close proximity
the central regions of strands B, C, E, and F of the two ␤-sheets.
These interactions define and stabilize the fold core, before the rest
of the polypeptide is arranged around it. This folding pathway
dominates in the physiological environment over alternative pathways controlled by local inter-residue interactions. Switching to the
alternative pathways may occur in the presence of denaturants.
Mechanical Stability—Titin and its expressed fragments were
among the first macromolecules, the mechanical stabilities of
which were explored at the single molecule level. Early availability
of the atomic structure of I27 also led to it becoming popular for
experimental and theoretical studies of mechanically induced unfolding. Studies of titin domains have provided powerful insights
into the relationship between protein internal architecture and the
levels of force and deformation it can withstand.
Application of mechanical force deforms the domains and initiates unfolding by breaking the inter- and intrasheet bonds and
stretching the polypeptide. Unfolding of multidomain constructs
generates a series of steps or peaks in force-distance curves, reflecting sequential unfolding of the domains. Values of peak unfolding force and the pattern of unfolding depend on various factors, including not only the rate of extension, as was shown first,
but also structural variations between domains, the pulling geometry (33) and overall compliance (34).
One of the major differences observed between the unfolding
patterns of different Ig domains relates to unfolding intermediates.
Domains from the distal Ig segment of the I-band titin, such as I27,
unfold in a three-state process (35), whereas domains from the
proximal Ig segment, such as I1, unfold by a two-state process (36).
Differences in unfolding behavior were predicted and explained by
theoretical modeling as resulting from differences in the relative
Minireview: Properties of Titin Ig and Fn3 Domains
46353
FIG. 3. Scheme for the structure of titin. The shortest cardiac isoform is shown. There are several distinct regions with differing domain arrangements.
The Z- and M-line parts contain Ig domains alternating with unique sequences. The I-band part contains two tandem Ig segments, proximal and distal to
the Z-line. These are separated by the N2-PEVK region formed mainly by unique sequences. The A-band part contains Ig and Fn3 domains arranged into
super-repeat patterns. The 11-domain super-repeats are located in the C-zone in situ, and the 7-domain super-repeats are located in the D-zone (see Fig. 1).
Potential ligands, indicated by in vitro studies, are shown. The Z-line and M-line domains can bind to structural proteins of the respective sarcomere regions,
as well as to cytoplasmic signaling molecules. A-band modules bind to major thick filament proteins. The unique sequences of the I-band part may also bind
signaling molecules. The letter P indicates location of the phosphorylation sites.
density of hydrogen bonding networks connecting pairs of the
strands A and B and A⬘ and G in the N- and C-terminal parts of the
domains (37). Stronger bonding network between strands A⬘ and G,
compared with strands A and B in I27, acts as a “mechanical
clamp” that stabilizes the intermediate state after early detachment of the A strand. Indeed, a mutant I27 lacking the A strand
was found to be an adequate model of this intermediate, maintaining the architecture and integrity of the wild type protein with only
a little loss of stability (38).
An important question raised by the stability studies concerns
the similarity of unfolding mechanisms probed by different methods (27). It was realized that the same C-terminal region of the
domain, stabilized by a network of interactions centered on the A⬘
and G strands, defines the level of its kinetic stability in resisting
both mechanically and chemically induced unfolding (39). However, the interactions in the core important for thermodynamic
stability of the domain do not contribute to its mechanical properties (30, 41). Also, the routes of unfolding induced by different
methods are different. This was first suggested by the apparent
absence of the mechanical unfolding intermediate of I27 in chemical denaturation data. Theoretical modeling also indicated that
the earliest events are either different or differ in the proportions of
conformational changes occurring in parallel (42). Further experiments illustrated that I27 unfolds differently with these two methods (38, 41).
Forces that unfold titin Fn3 domains were in the region 100 –200
pN, suggesting that these domains are weaker than the Ig domains, which needed 150 –300 pN at similar force-loading rates
(43). Studies of homologous domains in tenascin and fibronectin
also showed generally lower mechanical strength. Overall, the experiments agree with conclusions drawn from comparative modeling of Ig and Fn3 domains, which emphasize how the details of
domain structure influence unfolding (42).
Domain Stability and Mechanical Functions of Titin—Domain
stability, especially mechanical stability in the I-band region, directly relates to the mechanical function of titin in the sarcomere.
Recent reports show how the molecule structure has adapted to
functional demands.
First, as already outlined, Ig domains are strongest. The weaker
Fn3 domains are present only in the A-band part of the molecule,
which is stabilized within the thick filament structure. Second,
within the I-band part, Ig domains differing in stability and unfolding properties are arranged in distinct patterns. Adjacent to the
Z-line, the constitutive proximal Ig segment contains most of the
weak Ig domains, with average unfolding forces of ⬃150 pN (36,
44). The differentially expressed domains, which are centrally positioned in the I-band, have intermediate stability and unfolding
forces of ⬃200 pN (43, 45). Near the I/A junction, the constitutive
distal segment contains the strongest domains, with an unfolding
force of ⬃250 pN (43– 45). Finally, the average unfolding rate
constant of the proximal Ig domains is higher (⬃10⫺3 s⫺1) than that
of the distal Ig domains (⬃10⫺4 s⫺1) (44). This implies that, if
muscle stretch initiates multiple unfolding of I-band Ig domains,
the process is likely to start near the Z-line region and then spread
toward the A-band part of the molecule.
For refolding to begin, the polypeptide has to collapse to almost
the size of the intact domain to form the early native contacts (27,
30, 46). Refolding times, estimated from mechanical experiments,
are on the order of seconds, with faster rates for the distal than the
proximal domains (44). This indicates refolding may occur in situ,
provided that after stretch the muscle is allowed to relax to its
resting length and the time interval before the next extension is not
too short. The relatively low probability of simultaneous unfolding
of contiguous domains will minimize misfolding (28).
The probability of domain unfolding during normal sarcomere
function is relatively low. Domains are protected by the limits of
normal muscle extension and by the high compliance N2-PEVK
region of titin. However, if muscle is overextended, reversible unfolding of domains may protect titin and the sarcomere structure
from permanent damage.
Interactions of Titin Domains
Titin, with its giant size and hundreds of Ig and Fn3 domains,
illustrates a natural “molecular platform,” capable of binding diverse ligands according to a strict spatial pattern (Fig. 3). Among
the first titin ligands identified were the thick filament proteins,
myosin and C-protein. Since then, many other potential binding
partners have been found, mostly by yeast two-hybrid assay. Overall, the data suggest that titin provides scaffolding not only for
most structural proteins in the sarcomere, but also for many cytoplasmic proteins.
Interactions of titin in the Z- and M-line regions have two roles:
they contribute to the structural integrity of these parts of the
sarcomere and they provide site-specific links with regulatory systems. The regulatory links are likely to differ in cardiac and skeletal muscles, in accordance with the structural and biochemical
differences of these muscle types.
Within the elastic I-band part of titin, which spans between the
Z-line and the tip of thick filament, most of the interactions appear
to be concentrated in the N2-PEVK region. Interactions here are
mainly with cytosolic proteins of various signaling systems. The
A-band part of titin is integral with the thick filament. Two major
partners here, myosin and C-protein, are bound in a regular manner correlating with the periodic structure of the thick filament.
46354
Minireview: Properties of Titin Ig and Fn3 Domains
Evolutionary Origin and Relationships of Titin Domains
Attempts have been made to trace evolutionary relationships
between titin domains and other members of the muscle Ig family
(47, 48). Major conclusions of this analysis are as follows. Firstly,
the different regions of titin, the A-band part and the constitutively
and differentially expressed I-band parts, evolved independently
from common ancestral domains. Evolution of these regions involved a series of gene duplication events, including duplications at
the level of groups of domains, giving rise to the domain superrepeats. Secondly, titin and the other vertebrate and invertebrate
members of the muscle Ig family have a common ancestry, and a
primordial protein must have existed before the divergence of vertebrates and nematodes.
The evolutionary origin and expansion of multimodular proteins
are thought to be closely related to the emergence and expansion of
multicellularity and the animal world (15). The Ig-like domains
apparently considerably predated multicellular organisms and
were some of the first to be recruited to produce adhesion molecules, which were needed for cell-cell and cell-substrate communications. It seems plausible that, later in evolution, increased complexity of multicellular organisms and cell differentiation
necessitated new generations of intracellular “adhesive” proteins.
Evolution of striated muscle cells, in particular, would have needed
such proteins to order and stabilize the growing network of the
contractile apparatus. The ancient adhesive Ig fold, with its low
requirements for sequence conservation, and thus high ability to
maintain structural framework while changing ligand-binding
properties, was well suited to these tasks in muscle.
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