(Received for publication, May 15, 1995; and in revised form, November 27, 1995)
From the
Analysis of deduced protein sequence and structural motifs of
5500 residues of human fetal skeletal muscle nebulin reveals the
design principles of this giant multifunctional protein in the
sarcomere. The bulk of the sequence is constructed of
150 tandem
copies of
35-residue modules that can be classified into seven
types. The majority of these modules form 20 super-repeats, with each
super-repeat containing a 7-module set (one of each type in the same
order). These super-repeats are further divided into eight segments:
with six segments containing adjacent, highly homologous super-repeats,
one single repeat segment consisting of 8 nebulin modules of the same
type, and a non-repeat segment terminating with a SH3 domain at the C
terminus.
The interactions of actin, tropomyosin, troponin, and calmodulin with nebulin fragments consisting of either repeating modules or the SH3 domain support its role as a giant actin-binding cofilament of the composite thin filament. Such affinity profiles also suggest that nebulin may bind to tropomyosin and troponin to form a composite calcium-linked regulatory complex on the thin filament. The modular construction, super-repeat structure, and segmental organization of nebulin sequence appear to encode thin filament length, periodicity, insertion, and sarcomere proportion in the resting muscle.
Nebulin comprises 2-3% of the myofibrillar protein of skeletal muscle and displays tissue- and development-specific isoforms (600 to 900 kDa)(1, 2, 3, 4) . It has been demonstrated by immunoelectron microscopy that a single nebulin molecule spans the whole length of the thin filaments with its C terminus anchored at the Z line and its N terminus extending toward the distal ends of thin filaments(2, 3, 5, 6, 7, 8) . Nebulin has been shown to accompany actin filaments during muscle contraction and extension(2, 5) . These data suggest that nebulin forms a composite filament with actin/tropomyosin/troponin, perhaps by binding laterally to actin in situ. Significantly, the size of nebulin isoforms is proportional to the length of thin filaments in many skeletal muscles(3, 4) . These correlations strongly suggest that nebulin may act as a protein ruler that regulates the length of thin filaments.
Although native nebulin is yet to be isolated and
characterized, evidence for nebulin's interactions with actin
have been demonstrated by analysis of partial cDNAs encoding nebulin
and protein interactions of expressed nebulin fragments. The deduced
amino acid sequence of nebulin shows an extensive tandem repeat of
35-residue modules that are organized into 7-module
super-repeats(4, 9, 10, 60) . It has
been proposed that the 35-residue module is the basic structural unit
of the actin binding domains in nebulin and that the super-repeats
reflect tropomyosin/troponin binding sites along the nebulin
polypeptides(3, 4, 7, 11) .
Recombinant nebulin fragments containing 2 to 15
modules(7, 12) , small native nebulin
fragments(13) , and 1-module synthetic peptides (11) all bind actin, consistent with this prediction. These
experiments indicate that nebulin may contain a string of about 200
actin binding domains along its length. If all sites are operative in situ, then nebulin would act as a zipper in its lateral
association with actin(12) . A one to one matching between
nebulin modules with actin protomers would allow nebulin to operate as
a protein ruler to determine or stabilize the length of actin
filaments(3, 4, 11) .
Recent studies on the effect of nebulin fragments on actin-myosin interaction and its regulation by calmodulin raise the intriguing possibility that nebulin might have regulatory functions on active contraction(14) . Nebulin fragments bind with high affinity to actin and the myosin head. Fragments from the N-terminal half of nebulin that are situated in the actomyosin overlap region of the sarcomere inhibit actomyosin ATPase activities as well as sliding velocities of actin over myosin during in vitro motility assays; while a nebulin fragment near the C terminus, which is localized to the Z line, does not prevent actin sliding. Significantly, calmodulin reverses the inhibition of ATPase and accelerates actin sliding in a calcium-dependent manner. Calmodulin with calcium greatly reduces the binding of nebulin fragments to both actin and myosin. Nebulin may hold the myosin heads close to actin in an orientation that prevents random interaction in resting muscles yet facilitates cross-bridge cycling upon activation by calcium and calmodulin. The data suggest that the nebulin-calmodulin system may function as a calcium-linked regulatory system.
Here, we report the
determination and extensive sequence analysis of 5500 amino acids of
human fetal skeletal muscle nebulin based on DNA sequencing of five
partially overlapping cDNA clones in two open reading frames. Analysis
of these sequences, which represent at least 70% of the complete coding
sequence, shows that, with the exception of 163 residues at the C
terminus, the sequence is arranged as 150 tandem copies of
35-residue nebulin modules. These modules can be classified into
seven types, based on sequence homologies. These modules can be grouped
further into 20 super-repeats, with the 7 distinct types of modules in
each super-repeat, plus a single repeat region containing 8 modules of
the same type. Moreover, these super-repeats can be grouped further
into six segments, each containing a small number of adjacent, highly
homologous super-repeats. The C terminus of nebulin is distinct and
contains a Src homology domain 3 (SH3)(15, 16) .
The sequence analysis and protein binding studies of expressed nebulin fragments to be presented below suggest that the nebulin sequence encodes the blueprint for the structural and functional compartmentation of thin filaments in the sarcomere of skeletal muscles. The conclusions and implications of nebulin sequence analysis have been presented in a preliminary form(60) .
The initial screening
of gt11 library led to the identification of two independent
recombinants
HN1 and
HN2 containing nebulin cDNA fragments
HN1 and HN2. Both are used as probes to perform ``transcript
walks'' in the
gt10 libraries. Partial sequence analysis and
restriction maps established the order and extent of overlap. Although
the two walks have yet to overlap with each other, the outermost cDNA
fragments all localize to human chromosome 2 (9) , ruling out
ligation artifacts in the original cDNA cloning. Five large cDNA
clones: HNh20, HNd4 (from HN2 walks) and HNh19, HNe6, and HNb2 (from
HN1 walks) were selected for subcloning into pBluescript SK+
(Stratagene) and sequencing (Fig. 1).
Figure 1: Cloning, sequencing, and expression of human fetal nebulin. Five cDNA clones, HNh20 (4.5 kb), HNd4 (4.5 kb), HNh19 (1.7 kb), HNe6 (4.0 kb), and HNb2 (4.0 kb) are sequenced. Two open reading frames HNN (2468 residues) and HNC (3004 residues) are deduced, with HNN and HNC representing the N- and C-terminal sides of nebulin. Sequence numbers are assigned for each open reading frame, and the corresponding numbers for each cDNA clone are indicated. Seven nebulin fragments, NA4, NB5, NA3, NC17, ND66, ND8, and NSH3 are expressed, with their approximate positions within each open reading frame indicated (refer to Table 1for details).
which directs an in-frame ligation of the 5` end of the coding sequence to the ATG initiation codon in the vector sequence. Then a DdeI-BamHI adaptor
containing translation stop codons in all three reading frames
was added to join the remaining DdeI-cut end of the insert to
the BamHI-cut end of the vector. Transformation of BL21
(DE3)pLysS host cells (4 liters) with the resulting plasmid pHNb2D66
led to a high level of expression of soluble ND66 in the cytoplasm upon
IPTG induction (0.4 mM IPTG to an A = 0.6-0.8 culture for 3 h at 37 °C). The
bacteria were harvested at 5000 rpm in a Sorvall GSA rotor for 10 min
and lysed in a French Press (1,500 p.s.i., 3 times) in 50 ml of lysis
buffer (10 mM NaP
, 1 mM EDTA, 1 mM DTT, 2.5 µg/ml leupeptin, pH 7.0), followed by centrifugation
at 14,000 rpm for 20 min in a Sorvall SS-S4 rotor at 4 °C. The
supernatant was made 35% saturated in ammonium sulfate at 4 °C for
1 h and spun at 13,000
g for 30 min. The pellet was
dissolved in 50 ml of lysis buffer and dialyzed overnight, clarified,
and applied to a Whatman CM52 column (2
20 cm) equilibrated in
lysis buffer. Elution by a linear NaCl gradient (0-1 M NaCl, 150 ml each) yielded 95-99% pure ND66 between 0.35 and
0.40 M NaCl (40 mg per 4-liter culture).
To
facilitate quantitative analysis, the amount of adsorbed nebulin
fragments were kept sufficiently low (7-15 ng/well) so that the
total concentrations of the proteins in solution could be approximated
as the same as the free protein concentrations. The following
hyperbolic equation was derived to fit the data: A = AP/(K
+ P); where A is absorbance, A
is the maximal absorbance at
saturation, P is the total protein concentration, and K
is the microscopic dissociation constant. The stoichiometry of
interaction cannot be determined since this method determines only the
relative degree, not the absolute amount, of binding.
Binding studies of biotinylated calmodulin to immobilized proteins were done by incubating 100 µl of biotinylated calmodulin (from 0.030-2.0 µM) in the binding buffer for 2 h at 37 °C. Plates were washed three times with TBS-T, followed by incubation with 100 µl/well streptavidin (Molecular Probes S-888) at 5 µg/ml in TBS-blocking solution for 15 min at 37 °C. The plates were washed three times with TBS-T and then incubated with 100 µl/well of biotinylated horseradish peroxidase (Molecular Probes P-917) at 5 µg/ml in TBS-blocking solution for 30 min at 37 °C. After washing, color development was done as described above.
A
total of 19 kb has been sequenced to give two open reading frames
designated as HNN and HNC with 2468 and 3004 amino acid residues,
respectively (Fig. 1). The HNb2 clone contains the 3` end of
nebulin transcript including the translation stop codon (TAG), a 422-bp
untranslated region and a 42-bp poly(A) tail (data not shown). The 5`
walking from HNh20 has yet to give clones that signify the 5` end
initiation codon and potential regulatory sequences. The protein
sequences encoded by the two open reading frames show extensive tandem
repeats of a sequence module that ranges from 31 to 40 residues, with
an average of
35 residues per module. These sequence repeats are
easily detected visually by a hexapeptide SXXXY(K/R) and a
single proline or a smaller cluster of 2-3 prolines that recur
every
35 residues. This module is repeated 69 times in the
N-terminal side open reading frame (HNN) and 81 times in the C-terminal
open reading frame (HNC), with no obvious linker sequences between
modules. We have arbitrarily defined each module as starting at serine
of the conserved hexapeptide. This repeating pattern however stops
short near the C terminus. The C-terminal segment of 163 residues
consists of a linker region of 105 residues enriched in acidic
residues, serine, threonines, and a 58-residue SH3 motif which is
highly homologous to those found in Src kinases and several
cytoskeletal proteins (see below).
The 7-module super-repeat
is best visualized by matrix plots with a window size of 8 to 15 with
PAM250 scoring matrix. As shown in Fig. 2(lower left
plot, with a window size of 12), numerous off-diagonal lines of
homologous modules are periodic with a spacing of 245 residues
between successive lines. For a given super-repeat, the intensity of
off-diagonal lines along the same vertical axis appears to diminish
gradually toward the C terminus. These patterns indicate that nebulin
modules are organized into 7-module super-repeats that extend nearly
the entire sequences of HNN and HNC. Additionally, the degrees of
similarity among nebulin modules are high near the N terminus side and
diminish gradually toward the C terminus. The 7 types of modules are
designated as type a to type g in Fig. 3. Each
module is designated sequentially from N to C termini as HNN1 to HNC81 (Fig. 3).
Figure 2:
Protein matrix plot of human fetal
nebulin. The combined HNN and HNC sequences are scored with PAM250
matrix with two sets of parameters. A 12-residue window (lower left
diagonal) is used to reveal super-repeats of nebulin modules. A
wider, 30-residue window is used to reveal segmental organization of
super-repeats (upper right diagonal). Segments B,
C, C
, D, I
, I
, N, and Z
are indicated (refer to Table 2for
details).
Figure 3: Human fetal nebulin sequence. The sequences of HNN and HNC are grouped into seven types of nebulin modules (types a to g on the top of each group) and arranged in 20 super-repeats (from HNN1 to HNC73) plus one single-repeat segment (from HNC74 to HNC81) and C-terminal linker and SH3. The highly conserved SXXXY residues at the beginning and prolines midway through the modules are highlighted with reverse-contrast. Charge residues are in red (DE) and green (HKR), respectively. Subsequences corresponding to protein kinase consensus sites are boxed in various colors. Gaps imposed by sequence alignment are indicated by hyphens(-). The consensus sequence of each type of module is based on 50% identity and is indicated on top of each group. X represents nonconserved residues (<50% identity). + and - represent conserved basic or acidic residues (50% conservation).
Segmental organization of these super-repeats is
detected by matrix analysis with a much wider window size that is
comparable to the length of nebulin modules. As shown in Fig. 2(upper right plot, with a window size of 30), the
off-diagonal lines, 245 residues apart, display staircase steps of
various lengths or heights. These patterns indicate a higher degree of
homology for super-repeats within each step or segment. Closer
examination of local similarity scores by MACAW allows the
identification of six segments of homologous super-repeats which are
designed as B, C, C
, D, I
, and
I
(see ``Discussion'' for the choice of
terminology). Within a given segment, each module type of super-repeats
shows a higher similarity score with one another than with those of the
same type from other segments. Modules from HNC74 to -81 are all of the
same type (type d) and, as such, are designated as a
single-repeat segment (N segment). Additionally, the C-terminal 163
residues are designated as Z segment, ( Table 2and Fig. 2).
Nebulin sequence, as presented in Fig. 3, is organized to highlight the 20 seven-module super-repeats that are evident from HNN1 to HNC73. The consensus sequence of each module type, based on a minimum of 50% identity or higher, is annotated on the top of each module and summarized in Fig. 4.
Figure 4: The consensus sequences of human fetal nebulin modules. The consensus of consensus sequences of seven types of modules (Cons Cons) is derived by selecting residues with 40% identity and allowing gaps(-) in order to align conserved SXXXY residues and prolines. X represents nonconserved residues.
The regularity of conserved sequences as well as charge
profiles becomes less prominent in HNC, especially for the modules
after HNC35. The number and location of prolines and charge group
distribution in segments I and I
are fairly
variable and deviate frequently from the consensus. However, the
conserved Tyr and some of the major grouping of basic and acidic groups
are still conserved within each type despite sequence variability.
The modules bordering HNC74 and HNC79 are somewhat difficult to
classify. HNC70 to -73 are tentatively classified as part of segment
I, but appear to be sufficiently homologous to be
considered as three single repeats. HNC80 is strikingly similar to a type d module HNN1. HNC81 starts with SXXXY, yet
without the conserved Pro midway or other conserved residues of type d module.
Figure 5:
Nebulin homologs. A, homologs of
nebulin modules are found in a hypothetical protein (25.7 kDa) in C. elegans (GenBank P34417, 221 amino acids). B, SH3 domain homologs. The sequence of nebulin SH3 (HNC) is
compared with similar domains from chicken cortactin (GenBank
G01406, 563 amino acids), human HS1 (GenBank
P14317,
486 amino acids), yeast ABP1 (GenBank
A48096, 550 amino
acids), Dictyostelium discoideum myosin 1D (GenBank
P34109, 1111 amino acids), and c-Src kinase (GenBank
P12931, 536 amino acids). Key residues of c-Src SH3 domain are
numbered below. The consensus sequence of these SH3s is based on 60%
identity and labeled on the top.
The 105 residues spanning HNC81 and the SH3 domain is designed as a linker. This linker begins with the SXXXY sequence characteristic of the first half of the nebulin module, but otherwise shares no homology or charge profile with the remaining portion of any of the 7 types of modules. It is highly enriched in acidic residues, serine and threonine totaling 39 mol %.
As shown in Fig. 6,
the calculated pI values of these modules fall into three classes:
basic (8.5 to 10), neutral (6.0 to 7.3), and acidic (4.5 to 5.9). A
plot of pI along the sequence revealed striking periodicity throughout
most of the HNN region. For example, within HNN modules 3 to 58, a
7-module super-repeat consisting of 5 basic, 1 neutral, and 1 acidic
modules is repeated eight times. This pI distribution pattern became
less regular from HNN59 to HNC23, with 2-3 basic, 2-3
neutral, and 2-3 acidic modules per super-repeat. From HNC38 to
-73, most modules are basic, with at most 1 acidic module per
super-repeat. The single repeat segment (HNC74 to -81) is mostly basic
and neutral with no acidic ones. The linker is nearly neutral (pI
= 6.04) and the C-terminal SH3 is acidic (pI = 4.10). It
is striking that this segmental variation in regularity of pI profiles
corresponds closely to the staircase-like diagonal matrix plot based on
sequence homology (Fig. 2). Taking these independent criteria
into consideration, we group these super-repeats into segments that
display higher degrees of sequence homology as well as similar pI
profiles among the contiguous super-repeats. This segmental
organization is illustrated in Fig. 6. Our earlier
immunolocalization studies were useful to estimate the distance between
HNN and HNC. It is known that HNN41-47 (as an expressed fragment
NB5) is localized at 0.88 µm away from the Z line in adult human
quadriceps muscle by site-specific anti-nebulin monoclonal antibody
N101(8) . Assuming that fetal and adult nebulin have similar
sequences and that each module spans 5.5 nm (i.e. span of
actin subunit), then HNN and HNC is roughly 45 modules apart,
corresponding to 1600 residues or 4.7 kb of cDNA.
Figure 6:
Profile of isoelectric point of nebulin
modules and segmental organization of nebulin super-repeats. The
calculated isoelectric points of nebulin modules (as numbered in Fig. 3) are plotted along the HNN and HNC sequences. The
gap between HNN and HNC is estimated by immunolocalization of modules
HNN41 to -47 with monoclonal anti-nebulin N101 to 0.88 µm from the
Z line(8) . The segments B, C, C
, D,
I
, and I
are presented as boxes of
super-repeats. The single repeat region (segment N) and C terminus SH3
and linker (segment Z) are found near the end of HNC. The approximate
location of nebulin segments in a rest length (2.3 µm) sarcomere is
illustrated on the lower panel. The division of the
half-A-band into the M region, P zone, C zone, and D zone is from
Sjöström and
Squire(35) . Two landmarks (NB5 and ND8) were localized by
site-specific monoclonal antibodies (N101 and N113)(8) . The numbers refer to the individual modules, and the boxes are for the 7-module super-repeats.
It is worth noting that 13 of the 34 tyrosine kinase
consensus sites are found in the SXXXY motif of type b modules in HNN and HNC, with 2 to 6 sites in the same region of
each of the other types of modules. None is found in the single repeat
region. Interestingly, 14 of the 15 cAMP-dependent kinase sites are
found in segment I, a small adjacent region of segment
I
(starting at HNC34) and Z segment of HNC, with only 1 in
HNN. In the short HNC linker region are localized 3 cAMP kinase sites
and 1 Ca
-calmodulin kinase site, perhaps reflecting
its enrichment in serines and threonines. Of the 11 protein kinase C
sites, 6 are in type e and f modules, 0 in type b module, and 1 is found in the NSH3 domain at RTGR (HNC residues
2989-2992). It should be noted that actual phosphorylation sites
are unknown and may occur at only a small proportion of the possible
sites identified in this manner.
Two cortactin homologs have been reported. Amplaxin, a gene product
of EMS1 gene that is located within the amplified chromosome
11q13 region in human carcinomas(31) , exhibits 6 tandem
repeats of cortactin-like modules and an SH3 domain at its C terminus.
HS1, a hematopoeitic lineage cell-specific protein contains 3
tandem repeats and a C-terminal SH3 domain(32) . None of these
repeats, however, contain the SXXDYK motif of cortactin or
nebulin modules. These proteins, however, do share the same domain
architecture, with tandem repeats of 37-residue modules joined to a
C-terminal SH3 by a linker enriched in serine, threonine, and acidic
residues (Fig. 7).
Figure 7: Domain structure of SH3-containing nebulin homologs. The tandem repeats of 35-residue nebulin modules (HNC) and 37-residue cortactin modules (C. Cortactin, H.HS1, and H.Amplaxin) are represented by filled squares. The cortactin half-module is represented by a half-square. C-terminal SH3s and linkers are represented as shaded spheres and lines, respectively. Both nebulin and cortactin modules bind F-actin.
Nebulin-like modules are also detected in
a 25.7-kDa hypothetical protein encoded by F42H104 of chromosome III of C. elegans (GenBank P34417)(33) . This
sequence (c25.7, residue 64-141) is homologous to HNC80 and -81
(HNC residues 2747-2826) with a 40% identity. The possibility
exists that this sequence may be part of a larger nebulin-like protein,
such as the giant thin filament-associated protein in the body wall
muscle of C. elegans(34) .
As a first step, the binding of six nebulin fragments to three major thin filament proteins (actin, tropomyosin, troponin) and calmodulin was studied by solid phase binding assays at physiological ionic strength.
As shown in Fig. 8A, all tested nebulin fragments bind actin. Since
phalloidin is included in the buffer to lower the critical
concentration of actin polymerization, the binding curves reflect
mainly F-actin interaction. The relative affinity of actin binding
follows the order: NSH3 (K
0.1
µM) > ND66, NA3, NA4 (K
0.5
µM) > NC17, ND8 (nonsaturated at 2 µM actin). This trend is consistent with our previous estimates by
cosedimentation studies (NA3, NA4 >
ND8)(7, 12, 14) . The tight binding of NSH3
to actin is unexpected (see ``Discussion'') and provides the
first evidence that a SH3 domain binds directly to actin.
Figure 8:
Affinity profiles of nebulin fragments
toward thin filament proteins and calmodulin. Nebulin fragments NA3,
NA4, NC17, ND66, ND8, and NSH3 (5 nM in 10 mM Tris
Cl, 150 mM NaCl, pH 7.4) were adsorbed onto microtiter plates
overnight at 4 °C, washed, blocked, and incubated with actin (A), tropomyosin (B), troponin (C), and
biotinylated calmodulin (b-CaM, D) in a binding
buffer (10 mM imidazole, 4 mM MgCl, 1
mM CaCl
, 150 mM NaCl, 0.05% bovine serum
albumin, pH 7.0) for 2 h at 37 °C. Phalloidin (5 µM)
was present in actin solutions. The bound proteins were detected with
peroxidase-conjugated antibodies (A, B, and C) or streptavidin (D). All solid curves were calculated using a one-class binding model. Dotted lines indicate binding isotherms that have not reached
saturation.
The
binding of tropomyosin to nebulin fragment is generally weaker than
actin binding (Fig. 8B). It binds moderately strongly
to ND66, NC17, and NSH3 (K
0.5-1.0
µM). Its binding to NA3, NA4, and ND8 is not saturated
even at 5 µM tropomyosin. Interestingly, the binding of
troponin to nebulin fragments is much stronger than tropomyosin-nebulin
interaction (Fig. 8C). NA4, NSH3, and ND8 are among the
stronger ones (K
0.1 µM) and
NC17, ND66, and NA3 display relative weaker binding (K
0.2 µM).
Calmodulin (as biotinylated
calmodulin), a calcium-mediator of the inhibitory effect of nebulin
fragments on actomyosin interaction(14) , binds to NA4, NSH3,
ND8, and ND66 with higher affinity (K
0.1
µM) than NC17 and NA3 (unsaturated up to 2 µM calmodulin). This profile thus follows a similar trend as
troponin/nebulin affinity, except that the ND66-calmodulin interaction
is somewhat stronger. We have previously determined that NA3 binds to
calmodulin with a K
0.6 µM in a
low ionic strength buffer(14) . Additional binding studies have
shown that nebulin-protein interactions are fairly sensitive to ionic
strength and conditions. Details of these studies will be reported
elsewhere.
The bulk of the sequence is constructed of more than 150
copies of nebulin modules. Homology analysis of these modules reveal
that most of these modules can be classified into seven types and that
one of each type forms a 7-module set, to yield 20 super-repeats.
Further analysis indicates that the similarity among modules diminishes
toward the C terminus. This gradient of diminishing similarity is
consistent with the idea that nebulin has evolved from tandem
duplication of nebulin modules initiated from its C-terminal end, with
the most recently duplicated and conserved ones near the N terminus. We
speculate that the total number of duplicated nebulin modules would be
determined by the length of thin filaments and the number of actin
subunits per helical strand. Further evolution might have led to the
formation of the 7-module super-repeats to provide appropriately spaced
sites for tropomyosin and troponin binding, thereby satisfying the
spatial constraint of the actin-tropomyosin-troponin complex. The
segmentation of super-repeats might be evolved to accommodate its
interaction with A-band or I-band components which are themselves
segmented in the sarcomere. This speculative idea arose from the
following observations. i) The order and span of these segments appear
to correlate with the morphological zones within the sarcomere. Close
examination of the segmental organization suggests that, provided each
super-repeat spans 40 nm, the highly homologous segments of
C
, C
, and D are expected to overlap with the C
zone and D zone of the A-band in a resting muscle sarcomere of 2.3
µm(35) . The binding to both actin and myosin by several
nebulin fragments in this region (NA3, NA4, ND5, and NC17) supports
this notion(14, 19) . Interestingly, the combined span
of I
and I
segments near the C terminus would
be between 0.30 and 0.50 µm, corresponding to half the width of an
I band of a 2.3- to 2.6-µm resting length sarcomere. (ii) The
charge profile of modules in HNC display an abrupt transition between
the D and I
segments (demarcated at HNC24 module, Fig. 5). Most modules in segments I
, I
,
and N lack the highly regular repeating pattern observed in segments B,
C
, C
, and D (Fig. 5). This transition is
also evident when primary sequences of nebulin modules on either side
of this transition are compared (Fig. 3). Modules in the
I
, I, and N segments display significant variations,
especially in the second half of each module. This is in great contrast
to the highly homologous super-repeats in the C
,
C
, and D segments. We speculate that these sharp
transitions between D and I segments might signal the entry of thin
filaments into the A band environment of a rest length sarcomere.
Near the C terminus, a short segment of eight tandem repeats of type d modules has been immunolocalized to the edge of the Z
line (8) and probably corresponds to the N line to
the Z region of the thin filaments which usually appears thicker and
stiffer (36) . Two nebulin fragments (ND66 and ND8) from this
region bind actin but display no affinity toward myosin(14) ,
reflecting a functional distinction from the super-repeat region.
The C-terminal segment of nebulin includes the linker and an SH3 domain, both of which are distinct from nebulin modules. Significantly, the SH3 domain also binds actin (Fig. 8). This interaction may contribute to the anchoring of nebulin to the Z line(8) .
If the basic premises of the foregoing speculative analysis are correct, then the nebulin sequence encodes not only a blueprint for the length and architecture of thin filaments, but also instructions for the degree of overlap of thin and thick filaments and sarcomere length in the resting muscle. Nebulin thus may impart a functional and structural compartmentation along the otherwise uniform actin/tropomyosin/troponin filaments. Remarkably, this task is accomplished mostly by modifying and duplicating a short yet versatile 35-residue building block.
Whether the N terminus of nebulin also possesses a unique segment that binds the capping protein for the pointed end of actin filaments such as tropomodulin (46) is yet unclear. The similarity of module HNN1 with the C-terminal end module HNC80 hints that the 5`-most sequence may be nearby the N terminus of HNN.
Figure 9:
A hypothetical model of a composite
regulatory complex containing nebulin and tropomyosin/troponin on the
thin filaments of skeletal muscle sarcomere. In this working model,
each seven-module nebulin super-repeat (squares with graded
shading) binds one tropomyosin, possibly through the seven charge
clusters along the length of each tropomyosin (partitioned rod), and
one troponin complex (shaded spheres with a tail), consisting
of TnT, TnI, and TnC in orientations as specified by the N and C
termini(49) . Each nebulin super-repeat binds to seven actin
monomers (open circles) along the thin filament. Two strands
of this composite regulatory complex are depicted as winding around the
outer actin domains (subdomains 1 and 3) along the double helical
polymer. Tropomodulin is thought to bind to the pointed end of actin
filaments and/or the N terminus of nebulin. Cap Z, -actinin may
bind to the barbed end of actin filaments and/or the C terminus of
nebulin.
In non-muscle cells, cortactin and its homologs share the same modular architecture as the C-terminal end of human nebulin (see Fig. 5) and may be considered as functional or structural analogs of a family of SH3-containing proteins which bind actin polymers to SH3 target sites in the cortical cytoskeleton or in the Z line. It would be of great interest to know if the short cortactin tandem repeats regulate the length of the attached actin oligomers or polymers.
Other proteins that contain nebulin modules are also being detected. The presence of nebulin-like modules in a hypothetical protein (25.7 kDa) in C. elegans demonstrates that nebulin modules are ancient and conserved. The similarity between these modules with HNC81, found near the C-terminal of human nebulin also point to the importance of nebulin modules in this anchor region. In this connection, it is significant that at least 11 nebulin modules that are highly homologous to (HNC69 to -79) human nebulin are present in cardiac nebulette, a 107-kDa nebulin-like protein found in cardiomyocytes of human and chicken(58) . This anchor region structure may be a theme upon which structural or functional variants evolve in non-muscle and cardiac muscle cells.