A critical motif for oligomerization and chaperone activityof bacterial a-heat shock proteins Sonja Studer, Markus Obrist, Nicolas Lentze and Franz Narberhaus Institute of Microbiology,
Trang 1A critical motif for oligomerization and chaperone activity
of bacterial a-heat shock proteins
Sonja Studer, Markus Obrist, Nicolas Lentze and Franz Narberhaus
Institute of Microbiology, Eidgeno¨ssische Technische Hochschule, Zu¨rich, Switzerland
Oligomerization into multimeric complexes is a prerequisite
for the chaperone function of almost all a-crystallin type
heat shock proteins (a-Hsp), but the molecular details of
complex assembly are poorly understood The a-Hsp
pro-teins from Bradyrhizobium japonicum are suitable bacterial
models for structure-function studies of these ubiquitous
stress proteins They fall into two distinct classes, A and B,
display chaperone activity in vitro and form oligomers of
24 subunits We constructed 19 derivatives containing
truncations or point mutations within the N- and C-terminal
regions and analyzed them by gel filtration, citrate synthase
assay and coaffinity purification Truncation of more than
the initial fewamino acids of the N-terminal region led to the
formation of distinct dimeric to octameric structures devoid
of chaperone activity In the C-terminal extension, integrity
of an isoleucine-X-isoleucine (I-X-I) motif was imperative for a-Hsp functionality This I-X-I motif is one of the characteristic consensus motifs of the a-Hsp family, and here
we provide experimental evidence of its structural and functional importance a-Hsp proteins lacking the C-termi-nal extension were inactive, but still able to form dimers Here, we demonstrate that the central a-crystallin domain alone is not sufficient for dimerization Additional residues
at the end of the N-terminal region were required for the assembly of two subunits
Keywords: a-crystallin; a-heat shock protein; small heat shock protein; chaperone; oligomerization
Heat shock or other forms of stress induce the expression of
a-heat shock proteins (a-Hsp proteins) in a broad range of
prokaryotic and eukaryotic organisms [1–5] a-Hsp proteins
take part in the cellular multichaperone protein-folding
network by binding to partially denatured proteins, thereby
creating a reservoir of unfolded proteins for subsequent
refolding by other chaperones such as DnaK and GroEL
[6–8] Virtually all a-Hsp proteins examined display
chap-erone activity in vitro, measured by their ability to protect
model substrates from thermally or chemically induced
aggregation [8–13]
a-Hsp proteins are named after their most prominent
representative, a-crystallin, which prevents protein
precipi-tation in the vertebrate eye lens They are mostly referred to
as small heat shock proteins (sHsp), because their
mono-meric molecular mass ranges between 12 and 43 kDa
However, the term sHsp is somewhat misleading, as several
other small heat-inducible proteins bear no resemblance to
a-Hsp proteins Moreover, native a-Hsp proteins are among
the largest protein particles in the cell, as they assemble into
complexes whose molecular mass often exceeds 500 kDa
Many of these complexes have been reported to consist of
24 subunits [13–16], but both larger and smaller structures
have also been described [11,12,17,18] The quaternary
structure of a-Hsp proteins is highly dynamic, which is often
reflected by pronounced size heterogeneity or rapid subunit
exchange [19] Mammalian members of the a-Hsp family are remarkably polydisperse [20,21], and some bacterial proteins such as Escherichia coli IbpB also display pro-nounced size heterogeneity [22] Particularly rigid structures are of prokaryotic origin, such as the 24-meric Hsp16.5 from Methanococcus jannaschii or nonameric Hsp16.3 from Mycobacterium tuberculosis[11,14]
a-Hsp proteins are widely distributed but poorly con-served One major characteristic of this protein family is the presence of a central a-crystallin domain, flanked by a N-terminal region and a C-terminal extension [23] The highest degree of amino-acid similarity is found within the a-crystallin domain, while the N-terminal region and the C-terminal extension are variable in length and sequence Naturally occurring a-Hsp proteins lacking these flanking regions acquire monomeric to tetrameric structures and are poor or inactive chaperones [24,25] In the last fewyears, systematic a-Hsp structure–function studies have mainly been focused on the mammalian representatives aA- and aB-crystallin These proteins proved remarkably resistant against mutational alterations For example, a wide variety
of truncations and point mutations within the N-terminal region had no consequence on protein structure and function [26–30] However, some modifications in the N-terminal region of eukaryotic a-Hsp proteins were reported to affect chaperone activity [31] or complex formation [26,32]
The C-terminal extension, being located on the outer surface of the a-Hsp oligomers, is generally assumed to contribute to complex solubility [33], but its further functional implication remains unclear The recently resolved crystal structures of a plant and an archaeal a-Hsp showthat in these proteins, the C-terminal extensions are involved in subunit–subunit interactions by strapping around the outer surface of the complex [14,34] The most
Correspondence to F Narberhaus, Institute of Microbiology,
ETH-Zentrum, Schmelzbergstrasse 7, CH-8092 Zu¨rich, Switzerland.
Fax: + 41 1632 1148, Tel.: + 41 1632 2586,
E-mail: fnarber@micro.biol.ethz.ch
Abbreviations: a-Hsp, a-crystallin type heat shock protein; sHsp, small
heat shock protein; CS, citrate synthase.
(Received 1 March 2002, revised 25 April 2002, accepted 17 June 2002)
Trang 2striking feature of the poorly conserved C-terminal
exten-sion is the presence of an isoleucine-X-isoleucine (I-X-I)
motif in the majority of a-Hsp proteins This I-X-I (or, more
generally, I/V-X-I/V) motif was recognized as one of the
three main consensus regions of the a-Hsp family [5] There
is little experimental evidence to circumstantiate the role of
the C-terminal extension, and even less concerning the
conserved I-X-I motif Various modifications within the
C-terminal extension decreased chaperone activity of
a-crystallin and other vertebrate a-Hsp proteins
[27,31,33,35], but only in the case of one plant a-Hsp were
C-terminal truncations observed to reduce complex size [36]
Altogether, the oligomerization principles of a-Hsp
proteins, and especially of their bacterial representatives,
are still poorly understood We found in previous studies
that the soil bacterium Bradyrhizobium japonicum is a
suitable model organism for investigating prokaryotic
a-Hsp proteins [4,13,37] It contains at least 10 a-Hsp
proteins, which are highly induced upon heat shock [4] The
sequences of seven B japonicum a-Hsp genes are available
and can be assigned to two distinct classes, A and B These
two classes are not restricted to B japonicum, but also occur
in other rhizobia [38] Members of both classes have been
shown to prevent citrate synthase aggregation in vitro, to
form 400- to 500-kDa complexes and to interact with other
a-Hsp proteins of the same class [13]
The present study demonstrates that chaperone activity
of B japonicum a-Hsp proteins is stringently coupled to
multimerization, and that both the N- and C-terminal
regions are required for the formation of
chaperone-competent complexes In particular, we show that integrity
of the I-X-I motif is critical for assembly of functional a-Hsp
proteins, and that the isolated a-crystallin domain is unable
to dimerize
E X P E R I M E N T A L P R O C E D U R E S
Plasmid construction
Plasmids for the expression of B japonicum hspF (pRJ5306)
and hspH (pRJ5307) provided with a C-terminal His6tag
have been described previously [13] N- or C-terminal
truncations of hspF and hspH were constructed by PCR,
using pRJ5306 and pRJ5307 as templates For subsequent
cloning, we took advantage of two unique restriction sites,
namely BsaI on hspF and SfiI on hspH PCR products were
digested with either BsaI or SfiI and NdeI (N-terminal
truncations) or NotI (C-terminal truncations) The purified
fragments were used to replace the corresponding wild-type
fragments in pRJ5306 or pRJ5307 hspH variants encoding
the isolated a-crystallin domain or the a-crystallin domain
including either the N-terminal region or the C-terminal
extension were constructed in a similar manner Mutations
leading to single and double amino-acid exchanges were
introduced into hspH by means of the QuickChangeTM
Site-Directed Mutagenesis Kit (Stratagene) Expression vector
pRJ5307 was used as a template, and mutagenesis was
performed according to the manufacturer’s instructions All
resulting plasmids encoded a-Hsp versions carrying a His6
tag at the C-terminus A plasmid for the expression of an
untagged hspH variant lacking the C-terminal extension was
constructed by introducing a stop codon followed by an
XhoI site by PCR The amplification product was digested
with NdeI and XhoI and ligated into a pET24b vector digested with the same endonucleases The correct nucleo-tide sequence of all inserts was confirmed by automated DNA sequencing
Protein expression and purification Freshly transformed E coli BL21(DE3)pLysS strains were used for protein expression Overexpression cultures were grown at 30C to D600¼ 0.6, induced by addition of isopropyl thio-b-D-galactoside to a final concentration of 0.5 mM, and grown for a further 2–3 h After harvesting, cells were resuspended in binding buffer (500 mM KCl,
20 mM Tris/HCl, 5 mM imidazole, pH 7.9) containing
1 mM phenylmethanesulfonyl fluoride and 10 lgÆmL)1 DNaseI Lysis was performed in a French pressure cell at
1000 p.s.i, and soluble crude extracts were prepared by centrifugation at 12 000 g for 30 min at 4C
Proteins were purified by Ni-nitrilotriacetic acid affinity chromatography (Ni-nitrilotriacetic acid resin from Qiagen) under native conditions essentially as described previously [13] The column was pre-equilibrated with binding buffer and then washed with washing buffer (500 mMKCl, 20 mM Tris/HCl, pH 7.9) containing increasing imidazole concen-trations (5–50 mM) The imidazole concentration was finally raised to 250 mMin order to elute bound proteins If protein purity was not satisfactory, the eluate was diluted to an imidazole concentration below50 mM and applied to a second column, either Ni-nitrilotriacetic acid/agarose (Qia-gen) or Co-PDC/agarose (Acros Organics) Whenever possible, proteins were analyzed by gel filtration and citrate synthase assay immediately after purification Otherwise, eluates were supplemented with 20% glycerol and stored at )20 or )80 C Protein concentrations were determined by the Bradford assay All protein concentrations reported in this study are expressed in terms of protomers
Chaperone activity assay Chaperone activity was determined by the citrate synthase (CS) assay a-Hsp proteins were preincubated at 43C in
1 mL of 50 mM sodium phosphate, pH 6.8, for at least
15 min The assay was started by addition of CS to a final concentration of 600 nM CS aggregation was measured by monitoring light scattering at 360 nm for 30 min in an Ultrospec 3000 spectrophotometer (Amersham Pharmacia Biotech) Prior to use, CS (Sigma) was dialyzed against Tris/ EDTA buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0) For each protein, chaperone activity assays were performed
at least twice with preparations from independent purifica-tions
Gel filtration Analytical size exclusion chromatography of purified pro-teins was performed at room temperature on a Superdex
200 HR 30/10 column (Amersham Pharmacia Biotech) using a BioCAD perfusion chromatography system (PerSeptive Biosystems) After equilibrating the column with elution buffer (500 mMKCl, 20 mMTris/HCl, 250 mM imidazole, pH 7.9), 200-lL protein samples were injected and separated at a flowrate of 0.6 mLÆmin)1 Absorbance was recorded at 280 nm The following standards were used
Trang 3for calibration: thyroglobulin (669 kDa), ferritin (440 kDa),
aldolase (158 kDa), albumin (67 kDa), and ribonuclease A
(13.7 kDa), all from Amersham Pharmacia Biotech Gel
filtrations were performed at least twice with protein
obtained from independent preparations
Co-affinity purification
Copurification of untagged a-Hsp proteins with His6-tagged
variants by Ni-nitrilotriacetic acid affinity chromatography
was used to investigate protein–protein interactions of certain
truncated a-Hsp proteins The applied
denaturation–renat-uration protocol has been described previously [13]
R E S U L T S
Construction of HspH and HspF derivatives
for structure-function studies
In order to study the principles of a-Hsp assembly and the
relation between complex formation and chaperone activity,
we constructed a series of truncated and point-mutated
a-Hsp variants A total of 19 a-Hsp derivatives were
analyzed in the course of this study An overviewof these
constructs is given in Fig 1 The alignment presents the
seven known a-Hsp sequences from B japonicum, i.e five
class A (HspA, HspB, HspD, HspE and HspH) and two
class B proteins (HspC and HspF), as well as the class A
E coliproteins IbpA and IbpB A representative example
from each class, A (HspH) and B (HspF), was chosen for further analysis
The N-terminal region of HspH and HspF was gradually shortened by eliminating an increasing number of amino-acid residues The resulting proteins were named HspH (D3N), HspH(D9N), HspH(D15N), HspH(D20N), and HspF(D5N), HspF(D30N), HspF(D40N) In HspH(D20N) and HspF(D40N), approximately half of the N-terminal region was eliminated, including a proline and an arginine residue conserved in all seven B japonicum a-Hsp proteins (Fig 1) To examine the importance of these two particular residues, we constructed two HspH derivatives in which the proline (P8A) or the arginine (R18A) was replaced by an alanine
The characteristic I-X-I motif in the C-terminal extension
is present in all B japonicum and E coli a-Hsp proteins To investigate its role in oligomerization, we constructed two a-Hsp derivatives lacking one [HspF(D5C)] or both [HspH(D20C)] isoleucine residues Two further constructs, HspH(D5C) and HspH(D15C), contained the entire I-X-I motif To assess the role of the conserved motif in the context of the full-length protein, three HspH variants were constructed, in which the isoleucines were replaced by alanine, either individually, resulting in HspH(I133A) and
HspH(II133,135AA)
Finally, we searched for the minimal fragment required for dimer formation For this purpose HspH variants lacking entire subregions were constructed Three proteins,
Fig 1 Amino-acid alignment of B japonicum and E coli a-Hsp proteins The alignment includes the seven known sequences of B japonicum a-Hsp proteins, belonging to class A [HspH (accession number O86110), HspA (P70917), HspB (P70918), HspD (O69241) and HspE (O69242)] and B [HspC (AAC44757) and HspF (CAA05837)] IbpA (P29209) and IbpB (G65170) from E coli (both class A) were added for comparison The alignment was created with CLUSTAL W [48] Amino acids that are identical in all analyzed proteins are shown in white letters shaded in black White letters and black letters shaded in grey indicate amino acids that are identical in at least 80 or 60% of all proteins, respectively Arrows mark truncations introduced to HspH and HspF, and asterisks indicate alanine exchange mutations in HspH Note that four N-terminal residues were included in HspH(a) and HspH(aC) to avoid disturbance of potential secondary structures.
Trang 4consisting of the a-crystallin domain alone [HspH(a)], the
a-crystallin domain plus the N-terminal region [HspH(Na)],
or the a-crystallin domain followed by the C-terminal
extension [HspH(aC)], were analyzed
Alterations in the N-terminal region affect chaperone
activity and oligomerization
As demonstrated previously [13], full-length class A and
class B a-Hsp proteins of B japonicum act as efficient
chaperones in vitro (Fig 2H,I) They form oligomers with
an apparent molecular mass of 400–500 kDa, which
corresponds to a complex of 24 subunits ([13]; for
comparison with mutated variants see gel filtration profiles
in Fig 3H–J) These features were not affected if the
N-terminus was shortened by just a few amino-acid
residues HspH(D3N) and HspF(D5N) could not be
distin-guished from native HspH and HspF with regard to
chaperone activity and oligomeric state They prevented CS
aggregation (Fig 2A,E), and their apparent molecular mass
of 350–480 kDa was consistent with oligomers of 20–30
subunits (Fig 3A,E) Thus, the integrity of the extreme
N-terminus is not relevant for the assembly of functional
a-Hsp complexes
Removal of additional amino acids, however, drastically
altered the characteristics of either protein HspH(D9N),
HspH(D15N) and HspH(D20N) were devoid of chaperone
activity (Fig 2B–D) and unable to assemble into large
oligomers Instead, they formed complexes consisting of approximately eight subunits (Fig 3B–D) Oligomerization was only partially compromised, whereas chaperone activity was completely abolished Note that in many gel filtration runs [e.g HspH or HspH(D3N)], a considerable portion of the protein eluted after 12–13 min in the void volume of the column, reflecting the tendency of all B japonicum a-Hsp proteins to form large aggregates Extended truncations in the N-terminal region of HspF caused similar, but not identical effects as in HspH When 30 or more amino-acid residues were removed from the N-terminus of HspF, chaperone activity was completely lost (Fig 2F,G) and oligomer formation was impaired even more drastically than in the HspH derivatives Gel filtration analysis of HspF(D30N) and HspF(D40N) suggested that both pro-teins were only dimers (Fig 3F,G), whereas HspH deri-vatives devoid of the first half of the N-terminal region [HspH(D20N)] appeared as octameric complexes In either case, it is evident that a largely intact N-terminal region
is strictly required for the formation of active a-Hsp oligomers
In an attempt to narrowdown the critical determinants to the amino-acid level, we tested the effect of two single amino-acid exchanges (P8A and R18A) on chaperone activity and oligomeric state of HspH Although these residues are conserved throughout class A and class B proteins of B japonicum (Fig 1), neither exchange inhibited chaperone activity and oligomerization (data not shown)
Fig 2 Effect of truncations in the N-terminal
region on chaperone activity of HspH and
HspF Thermally induced aggregation of
citrate synthase at 43 C is depicted as a
function of time in the presence of various
amounts of a-Hsp proteins (A–D),
N-termi-nally truncated variants of HspH (class A);
(E–G), N-terminally truncated variants of
HspF (class B) Chaperone activity of
full-length HspH and HspF is shown for
comparison (H, I) Proteins were incubated at
43 C in a total volume of 1.0 mL of 50 m M
phosphate buffer, pH 6.9 CS aggregation was
measured by the increase of absorbance at
360 nm in the absence (r) and in the presence
of a-Hsp proteins at a final concentration of
150 n M (j), 300 n M (m), 600 n M (·) and
1.2 l M (d) The CS concentration was
600 n M Absorbance of a-Hsp proteins in the
absence of CS is also show n (S).
Trang 5Two conserved isoleucines are essential for a-Hsp
functionality
When assessing the impacts of C-terminal truncations on
oligomerization and chaperone function, we focused our
attention on the conserved I-X-I motif First, the C-terminal
extension of HspH was shortened by five and 15 amino
acids [HspH(D5C) and HspH(D15C), respectively], leaving
the I-X-I motif intact These alterations had no influence on
chaperone activity Both proteins efficiently protected CS
from thermally-induced aggregation (Fig 4A,B) and
as-sembled into large complexes (Fig 5A,B) But while
HspH(D5C) appeared similar to full-length HspH in terms
of oligomer formation, HspH(D15C) formed larger aggre-gates with an apparent molecular mass exceeding 2 MDa All C-terminally truncated a-Hsp derivatives exhibited an increased tendency to precipitate
Elimination of the entire I-X-I motif in HspH(D20C) led to a complete loss of chaperone activity (Fig 4C) and
a protein that was unable to multimerize The only species encountered in the gel filtration profile was a dimer of
29 kDa (Fig 5C) Removal of the C-terminal isoleucine from the I-X-I motif in HspF(D5C) was sufficient to completely abolish chaperone activity (Fig 4D) and
Fig 3 Oligomerization of N-terminally truncated HspH and HspF derivatives The oligomeric state of purified a-Hsp variants was determined by gel filtration over a Superdex 200 column at a flowrate of 0.6 mLÆmin)1 N-Terminally truncated derivatives of HspH (A–D) and HspF (E–G) were analyzed and compared with native HspH (H) and HspF (I) The minor peak at 12–13 min that is observed in all gel filtration profiles represents large protein aggregates eluting in the column’s void volume J, calibration curve for the gel filtration profiles in (A–I) K av ¼ (V e ) V 0 )/(V t ) V 0 ) is depicted as a function of log molecular mass (V e ¼ elution volume of the protein, V 0 ¼ column void volume, V t ¼ total bed volume) Open circles represent standard proteins listed in Experimental procedures (molecular masses given in kDa), filled circles indicate HspH and HspF variants Only major peaks aside from the void volume are indicated.
Fig 4 Effect of truncations and single amino acid exchanges in the C-terminal extension on chaperone activity of HspH and HspF Chap-erone activity of purified a-Hsp proteins was determined by the CS aggregation assay as outlined in the legend of Fig 2 (A–C) C-Terminally truncated variants of HspH (D) C-Terminally truncated variant of HspF; E-G, point mutated variants of HspH Depicted is
CS aggregation in the absence (r) and in the presence of a-Hsp proteins at final concen-trations of 150 n M (j), 300 n M (m), 600 n M
(·) and 1.2 l M (d); as well as the absorbance
of a-Hsp proteins in the absence of CS (S).
Trang 6severely impaired oligomer formation (Fig 5D) The
apparent molecular mass of 44–48 kDa probably
repre-sents a dimer
As both truncations affecting the I-X-I motif impaired
chaperone activity and oligomer formation, we replaced the
two isoleucine residues in HspH by alanines and analyzed
whether these mutations had a similar effect In fact,
HspH(I133A), HspH(I135A) and HspH(II133,135AA)
were devoid of chaperone activity (Fig 4E–G) and
appeared as small, dimeric structures of 43–50 kDa
(Fig 5E–G) Summarizing the gel filtration data, the
calibration curve in Fig 5H illustrates that all modifications
affecting the I-X-I motif resulted in the formation of small,
presumably dimeric complexes Taken together, these
results demonstrate that the isoleucine motif plays a crucial
structural and functional role in the assembly of functional
HspH or HspF oligomers
The a-crystallin domain alone is not sufficient
for dimer formation
All HspF and HspH variants investigated so far acquired
dimeric structures, indicating that the dimerization motif
remained untouched In order to determine the a-Hsp
region responsible for dimerization, we initially attempted
to purify the isolated a-crystallin domain As the protein
repeatedly showed a strong tendency to precipitate and
could only be recovered in concentrations too lowfor size
exclusion chromatography, we used an alternative approach
instead The interaction of full-length HspH with a series of
truncated His6-tagged HspH variants, i.e HspH(a)–His6,
HspH(Na)–His6, HspH(aC)–His6 and HspH(HD20N)–
His6 was tested Crude extracts containing tagged and
untagged protein were mixed, denatured and renatured
before being applied to Ni-nitrilotriacetic acid affinity
columns The eluted proteins were subsequently analyzed
by SDS/PAGE Co-elution of the untagged protein together
with the His6-tagged species was indicative of protein–
protein interactions, whereas proteins that did not interact
with the His6-tagged species were not retained on the column [13]
Untagged HspH did not coelute with the His6-tagged a-crystallin domain (Fig 6A), suggesting that the isolated a-crystallin domain was unable to dimerize The a-crys-tallin domain carrying the C-terminal extension appeared
to enable weak interactions with native HspH, because after copurification of HspH(aC)–His6with HspH, a faint band corresponding to HspH was visible on SDS gels In contrast, HspH(Na)–His6, an HspH derivative consisting
of the a-crystallin domain plus the N-terminal region, strongly interacted with native HspH After co-affinity purification, both proteins were detected on SDS gels in a
1 : 1 ratio A similarly efficient copurification of HspH was observed with HspH(D20N)–His6 This suggests that the a–Hsp interaction is in part mediated by a portion of the N-terminal region in vicinity of the a-crystallin domain
To ensure that efficient dimerization requires solely a-crystallin domain and N-terminal region and not the C-terminal extension, HspH(Na)–His6was also subjected to
a copurification assay with untagged HspH(Na) The latter protein alone did not bind to the Ni-nitrilotriacetic acid resin (Fig 6B, lane 5) However, it co-eluted with HspH(Na)– His6 (Fig 6B, lane 4) Size exclusion chromatography confirmed that HspH(Na) was present as small, presumably dimeric species, which were, as expected, devoid of chaper-one activity (data not shown) Thus, dimer formation does not depend on the integrity of the C-terminal extension but requires part of the N-terminal region
D I S C U S S I O N
Up to now, systematic a-Hsp structure–function studies have almost exclusively focused on a-crystallins from higher organisms Introduction of mutations often had little impact
on oligomerization and chaperone activity, as mammalian a-Hsp proteins are very resistant to mutational changes owing to their intrinsic plasticity [20,26–30,39] In
compar-Fig 5 Oligomerization of HspH and HspF derivatives with alterations in the C-terminal extension Gel filtration profiles of a-Hsp derivatives with truncations or point mutations in the C-terminal extension are shown (A–C) C-Terminally truncated variants of HspH; (D) C-terminally truncated variant of HspF; E-G, point mutated variants of HspH See Fig 3H,I for the gel filtration profiles of HspH and HspF The minor peak at 12–13 min observable in most gel filtration profiles represents large protein aggregates eluting in the column’s void volume (H) calibration curve for the gel filtration profiles depicted in (A–G) Standard proteins (molecular masses given in kDa) are represented by open circles, HspH and HspF variants by filled circles HspH(D15C) eluting in the void volume of the column was not included.
Trang 7ison with mammalian a-crystallins, the oligomerization
principles of bacterial a-Hsp proteins have received little
attention Available data indicate that a-Hsp proteins from
prokaryotes and plants may differ significantly from their
mammalian counterparts in structure and function [40,41]
Some prokaryotic a-Hsp proteins possess remarkably rigid
structures [11,14] that render them more susceptible to point
mutations and truncations We therefore chose two
dis-tantly-related bacterial a-Hsp proteins from B japonicum in
order to study, in detail, the contribution of their subregions
to chaperone activity and oligomerization
Tight coupling of chaperone activity
and complex assembly
B japonicum HspH and HspF oligomers contain 24
subunits, as shown previously with crude extracts [13] and
confirmed with purified proteins in the present study
Transmission electron microscopical analysis of both
pro-teins revealed roughly spherical structures (data not shown) similar to those observed for other members of the a-Hsp family [10,12,20,21]
We have demonstrated that assembly of this native structure is severely impaired by truncations in the N-terminal region and the C-terminal extension Interest-ingly, all of the resulting lowmolecular mass complexes were devoid of chaperone activity Although we cannot rule out that the truncated proteins had lost substrate binding regions in addition to oligomerization sites, this finding illustrates that chaperone activity and oligomerization are tightly coupled The ability to bind unfolded proteins and prevent them from aggregation clearly requires fully assembled multimeric a-Hsp complexes Even complexes that exceed the size of the wild-type protein, e.g HspHD15C, retained chaperone activity Much like our a-Hsp variants, naturally occurring multimerization-incom-petent a-Hsp proteins have poor or lacking chaperone activity [24,25,42] The chaperone activity of mammalian a-crystallins, on the other hand, appears to be much more resistant against structural modifications Human aB-crys-tallin does not require a multimeric a-Hsp complex for chaperone activity [43] The functional, substrate-binding entity might actually be an a-Hsp dimer rather than the fully oligomerized particle, which is regarded as a transient storage form of the chaperone [34,44]
Prerequisites for dimer formation The initial step in the oligomerization process is apparently the formation of dimers Even the most drastic modifications within the N- and C-terminal regions of HspF and HspH did not impair dimerization This observation argues for the presence of additional interaction sites in or near the a-crystallin domain Crystallographic data from M janna-schiiHsp16.5 [14] and wheat Hsp16.9 [34], as well as spin labeling studies with aA-crystallin and Hsp27 [45] indicate that the dimeric building block of many a-Hsp proteins is formed by interacting b strands in the a-crystallin domain Although this may also apply to the B japonicum a-Hsp proteins, the isolated a-crystallin domain of HspH was not sufficient for dimer formation Only the presence of a C-terminal portion of the N-terminal region enabled the a-crystallin domain to dimerize It is conceivable that the a-crystallin domain, as it is defined on the basis of sequence similarities, does not reflect the actual functional and structural entity This assumption is supported by the fact that a b strand overlaps the junction of N-terminal region and a-crystallin domain of M jannaschii Hsp16.5 [14] However, even the addition of four N-terminal amino acids
to the deduced a-crystallin domain of HspH (Fig 1) in order to avoid disruption of a predicted b strand was not sufficient for dimer formation This argues that more residues towards the N-terminus are involved in dimeriza-tion
Importance of the N-terminal region Additional portions of the N- and C-terminal regions are required for assembly into functional multimeric complexes Interestingly, class A (HspH) and class B (HspF) proteins exhibited similar, yet not identical assembly properties Truncations in the N-terminal region of HspF interfered
Fig 6 Interaction of truncated HspH variants with full-length and
truncated HspH demonstrated by coaffinity purification Crude extracts
with overexpressed native HspH were mixed with extracts containing
truncated His 6 -tagged HspH variants according to the schematic
representation on the right half of the figure After denaturation and
renaturation, the extracts were applied to Ni-nitrilotriacetic acid
affinity columns, and eluates were analyzed by tricine SDS/PAGE [49].
Coomassie-stained eluates on 13% polyacrylamide gels are shown on
the left half of (A) and (B) The expected positions of relevant proteins
are indicated by arrows (A) Copurification of untagged HspH with
His 6 -tagged HspH derivatives Lane 1, purification of the His 6 -tagged
HspH variant; lane 2, His 6 -tagged HspH variant and untagged HspH
after copurification (B) Interaction of tagged and untagged
HspH(Na) Lane 3, HspH(Na)-His after purification; lane 4,
copuri-fication of HspH(Na) and HspH(Na)-His; lane 5, untagged
HspH(Na) application to the Ni-nitrilotriacetic acid affinity column.
Trang 8more drastically with oligomerization than comparable
alterations in HspH (compare Fig 3D,F) In line with
clearly deviating N-terminal sequences (Fig 1),
oligomer-ization of HspH and HspF might be mediated by different
interacting regions Such distinctions most likely explain
why a-Hsp proteins from different classes are unable to
interact [13,36,46]
In both classes, the region near, but not directly at the
N-terminus is evidently critical Likewise, in Hsp16–2 from
Caenorhabditis elegans, removal of the N-terminal 15
residues was sufficient to inhibit chaperone activity and
oligomerization [44] In a-crystallin, on the other hand,
numerous point mutations and even truncation of the first
half of the N-terminal region did not affect complex
formation and functionality [26–30] Only complete removal
of the N-terminal region resulted in the formation of an
inactive lowmolecular mass species [26,32] These data
suggest that subunit interactions of mammalian a-Hsp
proteins may differ from their counterparts in prokaryotes
and plants The crystal structure of wheat Hsp16.9
demon-strates that the N-terminal region of this protein is involved
in numerous subunit contacts [34] It is puzzling that only
every other N-terminal region in the Hsp16.9 complex
but all N-termini in M jannaschii Hsp16.5 are highly
disordered [14,34] Possibly, the N-terminal regions of
B japonicum a-Hsp proteins are also disordered, which
might explain why subunit contact sites in the N-terminal
region could not yet be narrowed down to individual
conserved residues
A crucial motif in the C-terminal extension
Wheat Hsp16.9 and M jannaschii Hsp16.5 are distantly
related proteins with distinct quaternary structures
Never-theless, the role of their C-terminal extensions in oligomeric
assembly is strikingly similar The C-termini reach out to
neighbouring subunits, where the I-X-I motif undergoes
intramolecular hydrophobic interactions with a b strand in
the a-crystallin domain [14,34] Our mutational studies
strongly suggest that the C-terminal extension and the
conserved I/V-X-I/V motif in particular plays a similar
structural role in both classes of bacterial a-Hsp proteins
Partial or entire removal of the two conserved isoleucines
led to a dramatic reduction in complex size and solubility
and a complete loss of chaperone activity All modifications
touching the motif resulted in dimers On the other hand,
truncations that left the motif intact did not inhibit
multimerization and chaperone activity Similarly,
alter-ations outside of the I/V-X-I/V motif did not impair
oligomer formation of vertebrate a-Hsp proteins [31,33],
though some of them lowered chaperone activity [31,35] So
far, very fewmutational studies have included the isoleucine
motif C-Terminally truncated C elegans Hsp16–2 lacking
the I-X-I motif still formed high molecular mass complexes
and retained full chaperone activity [47] In aA-crystallin,
C-terminal truncations touching the I/V-X-I/V motif did
not inhibit multimerization, but reduced chaperone activity
[27] Only in one particular plant a-Hsp, Hsp17(II) from
pea, were C-terminal truncations, including the I/V-X-I/V
motif, associated with a significant reduction in oligomeric
mass [36] However, whether the motif itself or other
residues in the C-terminal extension were responsible for
this effect was not determined
The data reported in the present study further substan-tiate that oligomerization of prokaryotic a-Hsp proteins is a complicated multistep process that differs from the assem-bly of eukaryotic a-Hsp proteins and merits further investigation
A C K N O W L E D G E M E N T S
We thank Hauke Hennecke for support and encouragement This study was supported by a grant from the Swiss National Foundation.
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