Residues in the 1A Rod Domain Segment and the Linker L2 Are
Required for Stabilizing the A11 Molecular Alignment Mode
in Keratin Intermediate Filaments*
Taraneh
Mehrani
,
Kenneth C.
Wu
,
Maria I.
Morasso
,
Janine
T.
Bryan
,
Lyuben N.
Marekov
,
David A. D.
Parry§, and
Peter
M.
Steinert
¶
From the
Laboratory of Skin Biology, NIAMS, National
Institutes of Health, Bethesda, Maryland 20892-2752 and the
§ Institute of Fundamental Sciences, Massey University,
Palmerston North, New Zealand
Received for publication, August 10, 2000, and in revised form, September 26, 2000
 |
ABSTRACT |
Both analyses of x-ray diffraction patterns of
well oriented specimens of trichocyte keratin intermediate filaments
(IF) and in vitro cross-linking experiments on several
types of IF have documented that there are three modes of alignment of
pairs of antiparallel molecules in all IF: A11,
A22 and A12, based on which parts of the major
rod domain segments are overlapped. Here we have examined which
residues may be important for stabilizing the A11 mode.
Using the K5/K14 system, we have made point mutations of charged
residues along the chains and examined the propensities of equimolar
mixtures of wild type and mutant chains to reassemble using as
criteria: the formation (or not) of IF in vitro or in vivo; and stabilities of one- and two-molecule assemblies. We identified that the conserved residue Arg10 of the
1A rod domain, and the conserved residues Glu4 and
Glu6 of the linker L2, were essential for stability.
Additionally, conserved residues Lys31 of 1A and
Asp1 of 2A and non-conserved residues Asp/Asn9
of 1A, Asp/Asn3 of 2A, and Asp7 of L2 are
important for stability. Notably, these groups of residues lie close to
each other when two antiparallel molecules are aligned in the
A11 mode, and are located toward the ends of the overlap region. Although other sets of residues might theoretically also contribute, we conclude that these residues in particular engage in
favorable intermolecular ionic and/or H-bonding interactions and
thereby may play a role in stabilizing the A11 mode of
alignment in keratin IF.
 |
INTRODUCTION |
To date, approximately 50 different genes encoding intermediate
filament (IF)1 chains exist
in mammalian genomes. Based on differences in the organizations of
their primary structures and genes, six different types of IF are now
known (see Refs. 1-4 for reviews). The type I and type II keratins are
the most numerous. In human, for example, each contains approximately
20 members, which are differentially expressed in various epithelial
tissues. Each may be further divided into about 20 trichocyte keratin
chains expressed almost exclusively in "hard" keratinizing tissues
such as hair, and 20 cytokeratins. All keratin (as well as other) IF
chains consist of a central rod domain composed of four
-helical
segments (1A, 1B, 2A, and 2B) that possess a heptad repeat motif and
are separated from one another by non-
-helical linkers. The central
rod domain is flanked on the head and tail by domains of differing size
and chemical character. A large body of experimental evidence has now
documented that the fundamental building block of all keratin IF is the
heterodimer molecule, consisting of one type I and one type II chain
(1-7). Although a number of important details remain to be resolved,
this molecule is known to be stabilized in large part by the formation
of a segmented
-helical coiled-coil by the appropriate parallel
alignment of the central rod domain segments on the two chains. The
next step is the formation of a pair of such molecules. Typically, this
oligomer is the minimal IF structure that exists in solution,
especially below the critical protein concentration required for
assembly into macroscopic IF (~40 µg/ml). A number of biophysical,
electron microscopic, and biochemical experiments have documented that
the two molecules are aligned antiparallel and partly staggered in the
A11 or A22 alignment mode, depending on whether
the 1A+1B or 2A+2B rod domain segments overlap. Cross-linking data on
cytokeratins and trichocyte keratins have revealed that both of these
modes co-exist in solution, presumably in equilibrium with each other,
as various experimental manipulations allow realignments (8-10).
Further, the cross-linking data have afforded quantitative estimates of
the degree of overlap of the molecules. Thus, for the A11
mode we have documented that the two molecules are displaced by
approximately 112 amino acid residues with respect to each other.
However, fundamental questions remain concerning the sequence features
that specify and stabilize these alignment modes. In this study we have
explored in K5/K14 IF which sequences may be involved in stabilizing
the A11 alignment mode. In this study, we have tested a
current hypothesis that charged residues located along the rod domain
segments may be important for molecular registration. Table I lists all
possible charged residues that theoretically could be involved. By use
of series of point substitutions of charged residues, we have
identified several conserved residue positions that are important for
stabilizing the A11 alignment mode.
 |
MATERIALS AND METHODS |
Expression and Purification of K5 and K14
Chains--
Full-length human K5 and K14 cDNAs were assembled into
a pET11a vector and expressed in bacteria as described (12). Several mutant forms of both chains were generated by use of the QuickChange site-directed mutagenesis kit (Stratagene) (Table II). DNA sequencing was performed to confirm the mutations. Following induction, inclusion bodies were recovered, dissolved in SDS-PAGE buffer, and resolved in
3-mm-thick slab gels. The desired keratin bands were cut out, eluted
into SDS gel buffer over night, and the solutions stored at
70 °C.
Protein concentrations were determined by amino acid analysis following
acid hydrolysis.
In Vitro IF Assembly--
Equimolar mixtures of either a wild
type and/or mutant K5 and K14 chain were made from the stored SDS gel
buffer solutions. The SDS was removed by ion-pair extraction (13) and
the pelleted acetone-wet proteins redissolved (0.05 or 0.5 mg/ml) in a
buffer of 9.5 M urea containing 50 mM Tris-HCl
(pH 7.6), 5 mM Tris(2-carboxyethyl)phosphine-HCl (TCEP)
(Pierce), and 1 mM EDTA. For electron microscopy studies, IF were assembled by 1-h dialyses through solutions of decreasing urea
solutions of 4, 2, and 1 M, and finally into assembly
buffer of 10 mM Tris-HCl (pH 7.6), 1 mM EDTA
and 5 mM TCEP (12). Final protein concentrations were
35-40 µg/ml, which is below the critical concentration
(Co) for IF assembly (14), wherein mostly
two-molecule assemblies formed, or 400 µg/ml for optimal IF assembly.
Particles were examined by electron microscopy following negative
staining with 0.2-0.7% uranyl acetate over holey carbon film grids.
Lengths of IF were measured (15) in fields of
400 µm2.
For IF assembly efficiency studies, protein mixtures in 9.5 M urea (40 µl of
500 µg/ml) were dialyzed directly
into assembly buffer for 4 h. Solutions were then pelleted at
100,000 × g for 30 min in an Airfuge (Beckman
Instruments). Yields of protein in pellet were estimated by measuring
the absorbance at 276 nm of the supernatant.
Transfection Experiments with K14-Green Fluorescent Protein (GFP)
Plasmids--
A construct encoding GFP coupled at the 5' end of the
full-length coding sequence of wild type K14 was a generous gift of Dr. R. D. Goldman (Northwestern University Medical School,
Chicago, IL). Point mutations were made in the plasmid as described above.
PtK2 (NBL-5) cells, epithelial-like rat kangaroo kidney cells, were
obtained from ATCC (no. CCL-56). The cells were grown in
25-cm2 tissue culture flasks and maintained in MEM
(Eagle's minimal essential medium with nonessential amino acids,
Earle's salts and reduced sodium bicarbonate at 0.85 g/liter) (Life
Technologies, Inc.) with 10% fetal bovine serum. For cell passage, the
cells were grown to near confluence, and the medium was aspirated,
washed once with phosphate-buffered saline, and trypsinized for 20 s (0.25% trypsin; Life Technologies, Inc.). The trypsin solution was
aspirated, and the cells were left at room temperature for 3 min. Five
milliliters of medium were pipetted over the cells to dislodge them
from the flask, and transferred to a 15-ml conical tube. Following
5 min of 1000 rpm centrifugation to pellet the cells, the medium was
aspirated, and the cells were resuspended in 2 ml of medium and counted.
For direct immunofluorescence studies, 3 × 105
cells/ml were plated in 35-mm sterile tissue culture dishes, each
containing a glass coverslip. After 24 h, the cells were
transfected with 1 µg of plasmid DNA and 3 µg of Lipofectin as
described by the manufacturer (Life Technologies, Inc.). After 4 h, the mix was aspirated and 1 ml of 15% glycerol in Keratinocyte-SFM
(Life Technologies, Inc.) was applied for 3.5 min. The glycerol
solution was replaced with 2 ml of fresh medium and the cells incubated
at 37 °C with 5% CO2 for at least 24 h. The
coverslips were washed in phosphate-buffered saline and mounted onto
glass slides with Gel/Mount (Biomeda Corp.). Intracellular localization
of GFP fusion proteins was determined by direct fluorescent microscopy.
Protein Chemistry Procedures--
To examine molecular
stabilities, equimolar mixtures of the desired K5/K14 chains (~40
µg/ml) were equilibrated by 2-h dialyses into urea solutions of the
desired concentration in a buffer of 10 mM triethanolamine
(pH 8.0). The proteins were cross-linked with 25 mM
disulfosuccinimidyl tartrate (DST) for 1 h at 23 °C, and
terminated with 0.1 M NH4HCO3
(final concentration) (16). Although significant random cross-linking
also occurs, these conditions were used because the near quantitative
modification of all lysines allows for less diffuse bands on
3.75-7.5% gradient PAGE gels.
To assess molecular alignments in the A11 and
A22 modes, cross-linking with DST was performed using 0.4 mM reagent as described before (8, 9). We used wild type
and mutant proteins that had been equilibrated into assembly buffer at
about 40 µg/ml for 1 h. In this case, <10% of the lysine
residues were chemically modified, except for several aligned residues
that formed cross-links with yields of up to about 0.3 mol/mol.
Following cleavage with CNBr and trypsin digestion, peptides were
resolved by HPLC as before, except that a non-linear gradient over a
120-min time period was used. The positions of elution of the peptides
cross-linked by DST corresponding to the A11 and
A22 molecular alignment modes were similar to those
published previously (9), although many were confirmed by sequencing
for five Edman degradation cycles on a Porton LF-3000 sequencer.
Semiquantitative estimates of molar yields of each were made based on
peak heights of the integrated HPLC profiles.
 |
RESULTS AND DISCUSSION |
In this paper we have made a systematic analysis of those charged
residues that, based on current structural information, are located in
rod domain positions that could influence the specificity and stability
of the A11 alignment mode of a pair of antiparallel heterodimer molecules in K5/K14 IF. These encompass the segments 1A,
1B, 2A, and beginning of the 2B, as well as the linkers L1, L12, and
L2. We found that 41 charged positions have been conserved in the type
II keratin 5 (K5) (Fig. 1,
upper row) and type I K14 (Fig. 1,
lower row) chains. Based on extant ideas (2-4),
we have hypothesized in this study that some of these may influence
molecular alignment stabilities. Indeed, using the known quantitative
estimate of molecular spacing of the A11 alignment mode for
keratin IF (about
112 residues) (9), we document in Table
I that most of the 41 conserved charged
residue positions lie opposite to each other and so are well sited to
theoretically form stabilizing ionic salt bond pairs and/or H-bonds.
Nevertheless, we discharged all conserved charged residue positions
(i.e. mutated them to a non-charged residue) (all mutations
are listed in Table II). In addition,
there are 59 residue positions in this set that are not conserved
between the K5 and K14 chains, and 4 others that are oppositely
charged; several of these are also theoretically good candidates to
form stabilizing salt bonds (Table I). Some of these residue positions
were discharged as well. We then examined the facility with which
equimolar mixtures of one mutant and one wild type chain could assemble
into one- and two-molecule oligomers, as well as IF in vitro
and in vivo.

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Fig. 1.
Distribution of charged residues in the K5
(upper row of each pair) and K14
(lower row) chains in the 1A, L1, 1B, L12,
2A, L2, and first part of 2B rod domain segments. Conserved
charged positions are colored red (acidics) or
blue (basics). Non-conserved (black) or
oppositely charged (green) positions are also shown.
a-g denote the heptad position. Numbers on
right denote the domain residue position number, not protein
sequence residue number.
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Table I
Charged residues that are theoretically well sited to form stabilizing
ionic salt bond pairs in the A11 alignment mode
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Assembly of IF in Vitro and in Vivo--
The initial criterion of
assembly competence was formation of pelletable IF particles by use of
a sedimentation assay in the Airfuge. Experience has shown that
particles must be
750 kDa in size in order to pellet with high
efficiency.2 This corresponds
to an oligomer of as many as 16 chains (8 molecules), i.e.
it consists of a full-length half-width entity characteristic of an
early stage of IF assembly (17). In almost all cases, however, we found
empirically that assembly of mixtures of mutant and/or wild type chains
either resulted in macroscopic IF (
0.5 µm long), which were readily
pelletable in the Airfuge and clearly visible by electron microscopy
after negative staining, or no large IF particles were formed at all
(<0.1 µm long and <4 nm wide), which did not pellet in the Airfuge
and required examination over holey carbon film grids to be visible.
Sixty-six combinations of K5/14 chains were examined in in
vitro assays (Fig. 2, Table II).
These included every case of a conserved charged residue position in
the K5 and K14 chains documented in Fig. 1, as well as several other
positions that had not been conserved. Almost all formed pelletable
particles in high yield and appeared as native-type IF >1 µm in
length. However, several combinations did not, including three
positions in the 1A rod domain segment (K14 Asp9
Ala,
Arg10
Leu, K5 Lys10
Met, K14
Lys31
Glu or Lys31
Met), three
positions in the L2 linker (several combinations of K5 and/or K14 in
which positions 4, 6, and 7 (K14 chain only) of L2 had been
discharged), and three positions in the 2B rod domain segment (residues
100, 104, and 106). In addition, the 2A rod domain residue
Asp1
Ala (K5) produced particles that only partially
pelleted in the Airfuge and visibly were only 0.1-0.3 µm in length
(Table III).

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Fig. 2.
Electron microscopy of wild type and IF
assembled from wild type and/or mutant K5/K14 chains. These and
all data are summarized in Table III. A, wild type;
B, K5 wild type and K14 1A Arg10 Leu;
C, K5 wild type and K14 1A Arg10 Lys;
D, K5 1A Lys10 Arg and K14 wild type;
E, K5 wild type and K14 1A Lys31 Met;
F, K5 wild type and K14 2A Asp1 Ala;
G, K5 L2 Arg2 Leu and K14 wild type;
H, K5 L2 Glu4 Ala/Glu6 Ala
and K14 wild type. Scale bar, 200 nm.
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In a related second set of experiments, nine of these mutations were
introduced into the GFP-K14 construct and their propensities for
assembly into keratin IF in vivo were examined after
transfection into PtK2 cells (Fig. 3).
These cells express predominantly the K6, K7, K16, and K17 keratin
chains but have been shown previously to accommodate incorporation of
transfected wild type or mutant K14 chains (19). Additionally, the
efficacy of incorporation of transfected GFP-K14 constructs into
cultured cells to explore keratin IF cytoskeletons is now established
(20).3 Four mutants (1A
Arg10
Leu (Fig. 3B), 1A Lys31
Met (Fig. 3D), L2 Glu6
Ala (Fig.
3G), and 2B Glu106
Ala (Fig.
3H)), resulted in severely disrupted cytoskeletons in which
most of the keratin IF had withdrawn to a perinuclear location, and
there were bright spots of unassembled GFP-labeled protein. In five
other cases, the keratin IF cytoskeletons were either unchanged (1A
Lys17
Met (Fig. 3C), 1B Lys71
Ile (data not shown)), or mildly abnormal due to some apparent clumping and/or elongation of the keratin IF (1A Glu22
Ala (data not shown), 1B Glu56
Ala (Fig.
3E), and 1B Glu84
Ala (Fig.
3F)).

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Fig. 3.
Direct immunofluorescence of wild type and
mutant GFP-K14 constructs transfected into PtK2 cells. These and
all other data are summarized in Table III. A, wild type;
B, 1A Arg10 Leu; C, 1A
Lys17 Met; D, 1A Lys31 Met;
E, 1B Glu56 Ala; F, 1B
Glu84 Ala; G, L2 Glu6 Ala;
H, 2B Glu106 Ala.
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Some of these data were expected and thus serve as controls. The 1A
positions 9 and 10 have been shown previously to be sites for mutation
in various keratinopathy diseases (18); in vivo and/or
in vitro expression of proteins containing these mutations revealed limited or no IF assembly (21). Similarly, we have recently
documented that the 2B residue positions 100, 104, and 106 are required
to form stable molecules because they participate in coiled-coil
trigger formation in IF (11).
Cross-linking Studies with DST in Urea Solutions to Assess One- and
Two-molecule Stabilities--
The second criterion of assembly
competence used in this study was the formation of stable one- and
two-molecule assemblies. We have previously established (16) a method
to assess the stabilities of single coiled-coil molecules and pairs of
them by use of a graduated urea concentration titration assay coupled
with cross-linking by DST. At protein concentrations below the critical
concentration for IF assembly (~40 µg/ml) in assembly buffer in the
absence of urea, the K5 and K14 chains form mostly two-molecule (and
traces of one-, three-, and four-molecule) oligomers (8, 9). These dissociate into single molecules at about 6.5 M urea
(approximate concentration of half loss), and then the molecules
dissociate to individual chains by about 9.5 M urea, as
reported earlier by Wawersik et al. (22) for K5/14 keratin
IF and for vimentin and
-internexin (16) (see Fig.
4A).

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Fig. 4.
Stabilities of dimer (one-molecule) and
tetramer (two-molecule) assemblies of wild type and/or mutant chains in
concentrated urea solutions (as shown) following cross-linking.
These and all other data are summarized in Table IV. The compositions
of the assembly reactions are as shown. T, D, and
M, respectively, mark the position of migration of the
tetramer (two-molecule), dimer (one-molecule), and single-chain
species.
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Mutants representing every single conserved charged residue position in
either the K5 or K14 chain (from Fig. 1), and some nonconserved ones,
were tested in this assay. Several observations are apparent (Table
IV). First, for only the 2B rod domain
positions 100, 104, and 106 were both the two-molecule and
single-molecule entities unstable in even 1 M urea. This is
expected from our earlier data, as these residues participate in the
formation of a stabilizing coiled-coil trigger motif for IF (11).
Second, in all other cases, the one-molecule species was essentially as stable as the wild type. However, third, there were several conserved charged residue positions that resulted in significantly destabilized two-molecule entities (~4 M urea), including 1A positions
9 and 10; 2A position 1; and L2 positions 4, 6, and 7. Finally, in a few other positions, the two-molecule entity was somewhat less stable
(
5 M): 1A position 31 and 2A position 3.
Taken together with the in vivo and in vitro IF
assembly experiments, these data reveal that 1A positions 9, 10, and
31; 2A positions 1 and 3; and linker L2 positions 4, 6, and 7 are
important for the stability of the two-molecule entity of K5/14.
The Arg10 Residue of the 1A Rod Domain Segment and the
Glu4 and Glu6 Residues of the L2 Linker Are
Especially Required for the Stability of the A11 Mode of
Alignment of Two Molecules--
We have documented that there are
three possible modes of alignment of a pair antiparallel molecules to
form the two-molecule oligomer, known as A11,
A22, and A12. In our hands, the A12
mode exists only at high pH values and is not assembly-competent (14). However, a variety of chromatographic, ultracentrifugation, electron microscopic, solution birefringence, and cross-linking data have documented that two-molecule oligomer of a variety of mammalian IF
exist in assembly-competent solutions as 60-70-nm-long particles in
which the two molecules are aligned in the A11 and/or
A22 mode. Our previous cross-linking experiments have shown
that the two must co-exist in solution, since we have been able to
recover DST cross-linked peptides arising from links between
antiparallel molecules aligned in both modes (8-10). Therefore, we
reasoned in the present experiments that the destabilization of the
two-molecule oligomer in the several mutations identified above should
be due to loss of one or both of these alignment modes. To check this, we performed additional larger scale cross-linking experiments with 0.4 mM DST. The proteins were then cleaved with CNBr and trypsin, and the resulting peptides were resolved by HPLC (Fig. 5), but using a broader and flatter
gradient extending over 120 min versus 70 min previously
(8). We found six common peaks in the one- and two-molecule species of
wild type K5/14 arising from intramolecular links (Fig. 5B).
In the wild type two-molecule oligomer, there were an additional 15 peaks due to intermolecular links, of which 5 could be assigned to
linkages between molecules aligned in the A22 mode (Fig.
5C, closed circles), and 10 to
linkages denoting the A11 alignment mode (Fig.
5C, open circles). Semiquantitative data on the amounts of each were determined on the basis of peak areas
(all data summarized in Table V). These
experiments were repeated for seven mutant mixtures. As found
previously (11), the Glu106
Ala substitution in the 2B
rod domain segment resulted in loss of the A22 alignment
mode, and resultant substantial loss of the A11 mode.
However, the 1A Arg10
Leu (Fig. 5D) and
Lys31
Met, and L2 Glu6
Ala single (Fig.
5E) or Glu4
Ala/Glu6
Ala
double substitutions resulted in almost complete loss of the
A11 mode. Further, the yields of the cross-links denoting to the A22 mode were generally increased over the wild type
amounts (Table V). These data confirm that the A11 and
A22 modes of molecular alignment in fact exist in
equilibrium in solution and suggest that loss of the former by
destabilization results in a net reduction of the stability of all
tetramers, together with an accumulation of molecules into the latter.
The Arg10
Lys substitution in 1A retained near wild
type yields of both modes, consistent with the data of Tables III and
IV. In summary, these data document that discharging of
Arg10 of 1A, or one or both of the acidic residues
Glu4 and Glu6 of the L2 linker singly or
together, results in loss of the A11 mode of alignment.
Discharging of Lys31 in 1A results in substantial loss of
the A11 mode.

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Fig. 5.
HPLC profiles of DST-cross-linked peptides
reveal loss of the A11 alignment mode in certain conserved
charged residue positions of wild type and/or mutant K5/14 chains.
Oligomer sizes and chain compositions are indicated. Small
dots represent common intrachain cross-links; open
circles indicate cross-links denoting the A11 alignment mode; closed circles indicate those denoting
the A22 mode. Semiquantitative information of each peak is
listed in Table V.
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Model for the Stabilization of the A11 Alignment Mode
in Keratin IF: A Molecular Explanation for the Role of
Arg10 Substitutions in Keratinopathy Diseases--
Thus,
we have presented three sets of data, which document that certain
residues along the keratin IF chains are especially important for:
successful IF formation in vitro and in vivo; the stability of the two-molecule hierarchical stage of IF assembly; and,
in particular, for specifying and stabilizing the A11 mode of alignment of two antiparallel molecules. Indeed, several of the
residues identified here correspond to residue pairs documented in
Table I that are theoretically good candidates to form stabilizing ionic salt bonds. Based on the known alignment parameters of two antiparallel molecules in the A11 mode, these residues are
likely to lie very close to each other in the A11 mode
(Fig. 6). Thus, the conserved
Arg10 position of the 1A rod domain segment is closely
adjacent to the conserved set of two (and in type I IF chains, three)
acidic residues in positions 4, 6, and 7 in the linker L2. Notably,
discharging of any one of these residues severely compromised the
A11 alignment of the two-molecule hierarchical stage of IF
structure. Asp9 (often an isosteric Asn in many IF chains)
is likewise adjacent to these residues in L2. In addition, we note from
Fig. 6 that the conserved 1A residue Lys31 lies near the
conserved 2A residue Asp1 and Asp3 (K5 chain
only); likewise, discharging of these residues resulted in impaired
stability of the A11 alignment mode.

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Fig. 6.
A model of two heterodimer keratin molecules
(the two chain types are shown in white and
green) aligned in the A11 mode using the
statistically averaged displacement values determined from earlier
cross-linking studies (2, 3, 10). This displays the close
proximity of the two conserved and Arg/Lys10 residues of
one molecule (blue lines, large
blue dots) with the conserved acidic residues
Glu4 and Glu6 in the L2 linker (red
lines, large red dots) of
the other molecule. In addition, large dots
delineate the possible interactions between 1A Lys31 and 2A
Asp1. Smaller dots delineate possible
interactions involving 2A Asp3 and L2 Glu7. We
hypothesize that these may form several intermolecular salt and/or
H-bonds and thereby contribute essential specificity and stability to
the A11 alignment mode. The segments of the molecules are
marked.
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The simplest explanation of these data is that the key residues
identified in this study interact to afford essential stability. One
possibility is that this stability is provided by the formation of a
complex intermolecular network of salt bonds and/or H-bonds. However,
we cannot formally exclude the possibility that head and/or tail domain
sequences also cooperate in these stabilizing phenomena. In addition,
it is to be expected that many other charged residues, in addition to
the key ones identified here, may also contribute in important ways to
the alignment of the A11 mode. It is also possible that
these residues may participate in higher orders of IF structure, in
particular the lateral association of molecules in the A12
alignment mode, and elongation of molecules by overlapping of the
ACN alignment mode. The availability of the complete atomic
structure of a single IF molecule should provide the opportunity to
further explore these possibilities in model building studies. Finally,
it is interesting to note that the key potential interactions
identified here do not involve the 1B segment, which corresponds to the
central region of the A11 overlap. Instead, both ends
appear to be crucial in making favorable intermolecular interactions.
Nevertheless, we speculate that apolar interactions between residues in
the antiparallel 1B segments could also play a role in stabilizing the
A11 alignment mode.
Interestingly, substitution of the Arg10 residue in the 1A
rod domain segment of especially type I keratins often results in a
very serious phenotype in a variety of keratinopathy diseases (recently
reviewed in Ref. 18). The molecular basis of the consequence of this
substitution on keratin IF structure has not heretofore been
determined, although one report (23) suggested the problem occurred at
a structural hierarchical level above the stability of a single
molecule. Our present data indicate in a straightforward way that this
substitution causes a serious problem at the level of the two molecule
stage of IF assembly, in particular by destabilizing the
A11 alignment mode.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom all correspondence should be addressed: NIAMS, Bldg.
6, Rm. 425, National Institutes of Health, Bethesda, MD 20892-2752. Tel.: 301-496-1578; Fax: 301-402-2886; E-mail:
pemast@helix.nih.gov.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M007260200
2
L. N. Marekov, D. A. D. Parry,
and P. M. Steinert, unpublished observations.
3
R. D. Goldman, personal communication.
 |
ABBREVIATIONS |
The abbreviations used are:
IF, intermediate
filament;
DST, disulfosuccinimidyl tartrate;
K, keratin, as in K5
(keratin 5);
TCEP, tris(2-carboxyethyl)phosphine-HCl;
PAGE, polyacrylamide gel electrophoresis;
HPLC, high performance liquid
chromatography;
GFP, green fluorescent protein.
 |
REFERENCES |
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|
Fuchs, E.,
and Weber, K.
(1994)
Annu. Rev. Biochem.
63,
345-382
|
2.
|
Parry, D. A. D.,
and Steinert, P. M.
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