A recently determined crystal structure of Bs-CspB in complex with dT6 [24] and data from solution NMR experiments characterizing the binding of dT7to Bs-CspB [25] has shown that T-rich
Trang 1Crystal structure of hexathymidine bound to the domain-swapped form of a major cold shock protein from Bacillus caldolyticus
Klaas E A Max1, Markus Zeeb2,*, Ralf Bienert1, Jochen Balbach2,and Udo Heinemann1,3
1 Max-Delbru¨ck-Centrum fu¨r Molekulare Medizin Berlin-Buch, Germany
2 Lehrstuhl fu¨r Biochemie, Universita¨t Bayreuth, Germany
3 Institut fu¨r Chemie und Biochemie, Freie Universita¨t Berlin, Germany
When bacteria are subjected to a temperature decrease
of about 10C, they respond with an adaptive
mech-anism known as the cold shock response
Conse-quently, the expression of most cellular genes is
downregulated, and the expression of some genes
involved in cellular adaptation to cold stress is
upregu-lated [1–3] Although most genes involved in the cold
shock response vary between species, a conserved set
of genes encoding the major cold shock proteins (CSP)
has been found in more than 400 different bacteria,
including hyperthermophilic, thermophilic, mesophilic
and psychrophilic species The CSP consist of 65–70 amino acids and bind to single-stranded nucleic acids with micromolar to nanomolar dissociation constants (KD)
The precise cellular function of the CSP is under investigation In vitro, Ec-CspA, a major CSP from Escherichia coli, has been shown to prevent the forma-tion of mispaired RNA duplex structures in a sequence-unspecific manner [4] Such structures are expected to form preferentially at low temperatures and may interfere with translation or cause mRNA
Keywords
cold shock response; domain swap;
OB-fold; protein–DNA complex;
single-stranded DNA
Correspondence
U Heinemann, Max-Delbru¨ck-Centrum fu¨r
Molekulare Medizin, Robert-Ro¨ssle-Str 10,
13125 Berlin, Germany
Fax: +49 30 9406 2548
Tel: +49 30 9406 3420
E-mail: heinemann@mdc-berlin.de
Present address
*Department of Molecular Biology, The
Scripps Research Institute, La Jolla, CA, USA
Fachgruppe Biophysik, Fachbereich Physik,
Martin-Luther-Universita¨t Halle-Wittenberg,
Germany
(Received 30 October 2006, revised 22
December 2006, accepted 22 December
2006)
doi:10.1111/j.1742-4658.2007.05672.x
Bacterial cold shock proteins (CSPs) regulate cellular adaptation to cold stress Functions ascribed to CSP include roles as RNA chaperones and in transcription antitermination We present the crystal structure of the Bacil-lus caldolyticus CSP (Bc-Csp) in complex with hexathymidine (dT6) at a resolution of 1.29 A˚ Bound to dT6, crystalline Bc-Csp forms a domain-swapped dimer in which b strands 1–3 associate with strands 4 and 5 from the other subunit to form a closed b barrel and vice versa The globular units of dimeric Bc-Csp closely resemble the well-known structure of monomeric CSP Structural reorganization from the monomer to the domain-swapped dimer involves a strictly localized change in the peptide bond linking Glu36 and Gly37 of Bc-Csp Similar structural reorganiza-tions have not been found in any other CSP or oligonucleotide⁄ oligosac-charide-binding fold structures Each dT6 ligand is bound to one globular unit of Bc-Csp via an amphipathic protein surface Individual binding sub-sites interact with the DNA bases through stacking and hydrogen bonding The sugar–phosphate backbone remains solvent exposed Based on crystal-lographic and biochemical studies of deoxyoligonucleotide binding to CSP,
we suggest a common mode of binding of single-stranded heptanucleotide motifs to proteins containing cold shock domains, including the eukaryotic Y-box factors
Abbreviations
CSD, cold shock domain; CSP, cold shock protein; OB-fold, oligonucleotide ⁄ oligosaccharide binding fold.
Trang 2degradation Therefore, the CSP have been designated
as mRNA chaperones, which help to maintain protein
synthesis at low temperatures In a different study,
Ec-CspA, Ec-CspC and Ec-CspE, whose synthesis is
upregulated during cold stress, were shown to function
as transcription antiterminators of cold-induced genes
both in vitro and in vivo [5] Moreover, CSP have been
implicated in nucleoid condensation, the coupling of
transcription and translation, and regulation of
trans-lation [6–9]
Although it has been suggested that CSP bind to
Y-box sequences with high affinity [10], binding
experi-ments with oligonucleotides have shown that both
Ec-CspA and Bs-CspB from Bacillus subtilis
preferen-tially bind to pyrimidine-rich oligonucleotides [11,12]
Binding to the Y-box sequence, either alone or in a
poly(T) context, was negligible [13,14]; the highest
affinities of CSP were reported for thymine (T)- or
uracil (U)-rich sequences
Several CSP structures have been determined using
X-ray crystallography [15–18] and NMR spectroscopy
[19,20], including Ec-CspA, Bs-CspB and the CSP
from Bacillus caldolyticus (Bc-Csp) and Thermotoga
maritima(Tm-Csp) The peptide chains of the CSP are
arranged as five antiparallel b strands, separated by
four loops and folded into a closed b barrel [21] This
fold belongs to the oligonucleotide⁄ oligosaccharide
binding (OB) fold [22] It is conserved in eukaryotic
Y-box proteins [23], which contain structures in addition
to the cold shock domain (CSD) shared with the
bac-terial CSP
A recently determined crystal structure of Bs-CspB
in complex with dT6 [24] and data from solution
NMR experiments characterizing the binding of dT7to
Bs-CspB [25] has shown that T-rich DNA single
strands bind to an amphipathic protein surface
Sev-eral residues participating in ligand binding are located
in regions designated as RNP motifs I and II, which
can also be found in other RNA-binding proteins
[26,27]
Here we present the high-resolution crystal structure
Bc-Csp in complex with dT6 Unexpectedly, in this
crystal structure, Bc-Csp is present in a
domain-swapped dimeric structure not observed in
oligonucleo-tide⁄ oligosaccharide binding fold (OB-fold) proteins
The domain swap pairs one half, b strands 1–3, of
Bc-Csp with the other half, b strands 4 and 5, of a
second molecule and serves as proof of an
unantici-pated structural plasticity in the CSD In contrast to a
previously determined Bs-CspBÆdT6 structure [24] in
which the DNA strands bridge adjacent protein
mole-cules in the crystal lattice, each hexathymidine strand
is associated with one CSD Nevertheless, very similar
ligand-binding subsites are observed in Bs-CspB and Bc-Csp, and the mode of DNA binding is dominated
by stacking interactions between nucleobases and aro-matic protein side chains for both proteins Based on this observation and on binding assays using a set of heptapyrimidines, a model of CSPÆheptanucleotide binding is presented and a common mode of oligonu-cleotide binding to CSD is proposed
Results and Discussion
Bc-Csp, the major CSP from B caldolyticus, was crys-tallized in complex with dT6 in space group P21212, and diffraction data were collected up to 1.29 A˚ (Table 1) Initial phases were obtained by molecular
Table 1 Bc-CspÆdT6: data collection and refinement statistics Data collection
Wavelength (A ˚ ) 0.9184 Resolution (A ˚ ) 20.00–1.29 Last shell (A ˚ ) 1.40–1.29 Space group P21212 Temperature (K) 100
Unit-cell parameters
Unique reflections (last shell) 37 691 (7838)
I ⁄ r(I) (last shell) 14.8 (5.2) Data completeness (%) 97.0 (94.4)
Rmeasa (%) 6.6 (32.0) Refinement
Resolution (A ˚ ) 19–1.29
Free set (5%) 1,908
Rwork⁄ R freeb(%) 12.9 ⁄ 16.2 Number of nonhydrogen atoms 1,614 Number of protein molecules 2 Number of dT 6 molecules 2 Number of water molecules 234 Mean B factor (A˚2 ) 10.84 RMSD:
bond lengths (A ˚ ) 0.019 bond angles () 1.41 torsion angles () 4.34 planarity (A ˚ ) 0.008 Ramachandran statistics
Residues in allowed regions 95.3 Residues in add allowed regions 4.7
a
R meas , redundancy independent R factor, which correlates intensi-ties from symmetry related reflections [51].
b R work;free ¼ P jF obs jjF calc j
jF obs j , where the working and free R factors are calculated using the working and free reflection sets, respect-ively The free reflections were held aside throughout refinement.
Trang 3replacement using a crystal structure of Bc-Csp
with-out a ligand (1C9O) The crystal’s asymmetric unit
contains a swapped dimer of Bc-Csp (chains A and B)
in contact with two DNA molecules (chains C and D)
(Fig 1) Two methylpentanediol molecules from the
crystallization setup, which are associated with the
protein–DNA complex, were included in the structural
model The structure was refined using refmac
v 5.1.24 to final Rwork⁄ Rfreevalues of 13.1 and 16.3%
The electron density is well defined, all heavy atoms
from protein and ligand molecules could be placed
Bc-Csp overall structure and domain swap
In the Bc-Csp structure the two protein chains form a
swapped dimer with two globular functional units
which are composed of residues 1–35 from one protein
chain and residues 38–65 from another protein chain
(Figs 1A, 2) The architecture of the functional units
closely resembles that of all other structural models of
CSP, featuring five highly curved antiparallel b strands
connected by four loops and a short 310helix at the
C-terminus of b3 In the nonswapped (closed monomeric)
structures the respective b strands 1–3 and 4+5 are
arranged as two b sheets which form a closed b barrel
In the domain-swapped structure, the first b sheet of
one chain assembles with a second b sheet from a dif-ferent chain and vice versa The swapped chains can
be interrelated by a noncrystallographic twofold rota-tion axis The funcrota-tional units align well with the structures of the two models of the closed protein (1C9O) giving RMSD values of < 0.5 A˚ for all a-car-bon atoms Both functional units superimpose with an rmsd of 0.1 A˚, and the phosphorus atoms of the sugar–phosphate backbone from both DNA chains superimpose with an rmsd of 0.23 A˚ Formation of a swapped dimer reduces the solvent-accessible surface per subunit by 5.4% (493 A˚2)
The Bc-Csp domain swap provides insight into the folding and misfolding of the CSP
The Bc-Csp domain swap occurs in the middle of loop
L34 and is promoted by a unique combination of torsion angles Glu36w and Gly37/, compared with closed monomers Interestingly, crystal structures of monomeric Bc-Csp show a two-state conformational variability at these torsion angles: of 10 protein mod-els from six different Bc-Csp crystal structures [17,18], six models display Glu36w⁄ Gly37/ mean torsion angles of 162 ± 14 ⁄ 99 ± 5, designated as state 1 The other models show mean torsion angles of
A
B
Fig 1 Crystal structure of The Bc-CspÆdT6
complex (A) The DNA strands (red ¼
back-bone, beige ¼ bases) bind to globular units
of a swapped Bc-Csp dimer (green ¼ chain
A, blue ¼ chain B) Each globular unit is
composed of residues 1–35 and 38–66 of
two different protein chains The base of
the terminal nucleotide T6 occupies two
dif-ferent positions in chain D (dotted arrow).
(B) The region of the domain swap in chain
B revealed by Fo) F c difference electron
density calculated from a model devoid of
residues 35–38 The map (grey wire frame)
was contoured at 2.5r.
Trang 4)15 ± 15 ⁄ )65 ± 8, designated as state 2
(Fig 2A,B) In all crystal structures featuring two
pro-tein molecules, one molecule maintains Glu36w⁄
Gly37/ torsion angles according to state 1, whereas
the other molecule adopts a conformation according to
state 2 All other torsion angles in Bc-Csp represent
single states with standard deviations of < 20, except
for the terminal residues The torsion angle differences
between states 1 and 2, 183 for Glu36w and)164 for
Gly37/, roughly compensate for each other and do
not result in noticeable tertiary structural deviations
between monomers in state 1 and state 2 In contrast
to closed Bc-Csp structures, the two polypeptide chains
of the swapped dimer show Glu36w⁄ Gly37/ torsion
angle combinations of 141 ± 4 (similar to state 1)
and )85 ± 1 (similar to state 2) This results in
effective 180 rotations of their main chains at Glu36w, causing the Bc-Csp structures to open up and allowing them to re-associate as domain-swapped di-mers Apart from differences in the course of the pro-tein backbone at the point of transition, in the swapped dimer the overall structure of the functional units is not altered significantly as compared to the closed monomers in state 1 and 2
In the open state, the monomer of Bc-Csp is parti-tioned into two subdomains of similar length, which are separated by a long loop Subdomain 1 is a sheet including b strands 1–3, subdomain 2 is a b ladder comprising strands 4 and 5 In closed monomers, these two subdomains are stabilized by 26 backbone hydro-gen bonds; the interface between the subdomains con-tains eight backbone hydrogen bonds (Fig 3)
A
B
C
Fig 2 Comparison of open (domain-swapped) and closed states of Bc-Csp (A) Torsion angle distribution of Glu36w (left) and Gly37/ (right) from 14 closed models of Bc-Csp and Bs-CspB and two domain-swapped Bc-Csp molecules (yellow trian-gles) The closed structures feature a two-state conformational variability involving Glu36w ⁄ Gly37/ mean torsion angles of either 162 ± 14 and 99 ± 5 (state 1, green squares) or )15 ± 15 and )65 ± 8 (state
2, red circles) The domain-swapped struc-tures show torsion angles of 141 ± 4 for Glu36w (similar to state 1) and )85 ± 1 for Gly37/ (similar to state 2) (B) Superposi-tions of L 34 residues from Gln34 to Lys39 involving all backbone atoms (Left) Super-position of two models featuring Glu36w ⁄ Gly37/ torsion angle combinations
of state 1 (green) and state 2 (red) (Centre) Superposition of a model featuring Glu36w ⁄ Gly37/ torsion angles of state 1 (green) and a domain-swapped structure (yellow) Residues 34–37 were used for su-perposition (Right) Superposition of a model featuring Glu36w ⁄ Gly37/ torsion angles of state 2 (red) and a domain-swapped struc-ture (yellow) Residues 36–39 were used for superposition (C) Comparison of an open monomer CSP structure (yellow) (2HAX, monomer A) with two closed monomeric CSP structures (1HZA monomers A & B) featuring Glu36w ⁄ Gly37/ torsion angles according to state 1 (green) and state 2 (red).
Trang 5Almost all of these interactions can also be found in
the swapped dimers From 15N relaxation and H⁄
D-exchange NMR experiments of Bs-CspB, which shares
86% sequence identity and a closely similar tertiary
fold with Bc-Csp, we expect that loop L34, which
divides the subdomains, remains flexible even in the
folded state of the protein [25] A state similar to that
of the open monomer may reflect a substate in the
folding pathway of Bc-Csp In such an arrangement,
the subdomains may form independently from an
unfolded chain (Fig 2C) Formation of the
subdo-mains would contribute two thirds of all backbone
hydrogen bonds and stimulate the organization of a
bipartite hydrophobic core, which would be solvent
exposed at this stage The association of subdomains
results in the formation of the closed b barrel burying
the hydrophobic core At present there is no experi-mental evidence for the occurrence of the domain-swapped form of Bc-Csp or any other CSP in solution
or inside bacterial cells However, the Bc-Csp crystal structure reveals an unanticipated structural poly-morphism The domain-swapped form of the protein must be close in energy to the globular monomeric state, because otherwise these crystals could not have formed We cannot discount the possibility that its for-mation has been overlooked in previous biochemical studies of CSPs Further studies are required to evalu-ate its physiological relevance
It has been shown that Ec-CspA, which shares 57% sequence identity with Bc-Csp, aggregates forming amyloid fibrils under acidic conditions [28] Analysis of this amyloid formation using NMR techniques has revealed time-dependent changes in 15N T2 relaxation accompanying the exponential phase of polymeriza-tion, which suggest that the first three b strands may form association interfaces that promote aggregate growth In the late stage of amyloid formation, signals from the N-terminal half of the molecules (equivalent
to residues 5–36 in Bc-Csp) appear to be more severely broadened than those from the C-terminal half This may be relevant for folding, because Ec-CspA in this experiment shows a bipartite organization resembling that of the open state of Bs-CspB
Domain swapping of a further b-sheet protein, ribo-nuclease A, has recently been implicated in amyloido-genesis [29] For this enzyme, swapped dimers may be formed in solution, which can be isolated from closed monomers using chromatographic techniques [30,31]
A swapped dimer was formed from two different defective ribonuclease A variants by complementation involving the swapping of functional subdomains [29,32] Using a similar approach, amyloid fibres of ribonuclease A were generated, for which enzymatic activity could be demonstrated [29] It has thus been suggested that amyloid cross-b spines consisting of extended b sheets may also be formed from domain-swapped protein assemblies with retained native struc-ture This model of amyloid protofilament formation may also be relevant for the CSP and may explain why a bipartite organization can be observed in the late stage of Ec-CspA amyloidogenesis [28] To form extended linear fibrils by swapping subdomains, an arrangement different to that seen in the Bc-CspÆdT6 crystal structure, in which two chains form a closed dimeric arrangement, would be required
Because the domain-swapped dimer of Bc-Csp has only been observed in the presence of a bound dT6 strand, the question remains whether DNA binding to the CSP is responsible for domain swapping To date
Fig 3 Topology plot of Bc-Csp b-Strands are depicted as blue
(subdomain 1) and green (subdomain 2) arrows Intra- and
intersub-domain hydrogen bonds from the protein backbone are indicated as
black and grey arrows (donor acceptor) All hydrogen bonds can be
observed in the closed as well as domain-swapped Bc-CspB
struc-tural models In the latter case, intersubdomain hydrogen bonds
are formed between different protein chains Residues 36 and 37
are depicted in pink Their backbone torsion angles display a
two-state conformational variability in closed CSP structures and enable
the domain swap observed in the Bc-CspBÆdT 6 structure.
Trang 6solution data have only indicated the formation of
complexes consisting of one protein and one ligand
molecule for ligands in the range of hexa- or
heptanu-cleotides (data not shown) The Bs-CspBÆdT6 structure
[24] clearly demonstrates that a domain swap is not
required for ligand binding to the CSP Most protein–
ligand contacts of the two structures are in good
agree-ment (see below), suggesting that domain swapping
does not change the ligand interaction sites of the
CSP
Preferential binding of different pyrimidine-based
oligonucleotides to Bc-Csp
It has been shown that homologous Bs-CspB has a
general binding preference for polypyrimidines over
polypurines, and its binding site has been suggested to
interact with 6–7 nucleotides [12–14] In order to
further analyse the preferential binding of
heptanucleo-tides, we performed binding studies using
deoxyhepta-pyrimidines (Table 2) The selected oligonucleotides
differ from each other only by single pyrimidine bases
and thus allow us to determine the effect of
thymine-to-cytosine base changes at different sequence positions
in a heptanucleotide sequence by relating the KD
val-ues of the respective Bc-CspÆoligonucleotide complexes
to each other (Table 2; KD1⁄ KD2) To prevent slippage
of oligonucleotides within the binding site, caused by
single nucleotide changes within homogenous T-rich
surroundings, we used a less degenerate
oligonucleo-tide (CTCTTTC) as a scaffold, which is bound with
similar affinity as dT7
The binding experiments show that there is only a
small preference for T over C at positions 1, 4, 5 and
7, associated with an increase in the KDvalue of up to
threefold At positions 2 (Table 2, CT3, CT7) and 6
(Table 2, CT3, CT5) the decrease in affinity was
signi-ficantly stronger: When C was introduced at these
positions the KDincreased 93- or 11-fold By contrast,
at position 3 (Table 2, CT2, CT3) C was preferred
slightly over T, with an associated 2.5-fold decrease
in KD
dT6is bound to a hydrophobic platform on the protein surface
Globular functional units of Bc-Csp have a strongly dipolar surface (Fig 4B) One side has a prominent negative surface potential which is derived from acidic side chains On the opposite side, several solvent-exposed aromatic side chains form a hydrophobic plat-form surrounded by basic and by polar groups This amphipathic interface associates with dT6 via various hydrophobic- and hydrogen-bonding interactions In the following description, the interactions between pro-tein and ligand are observed in the ligand-binding interfaces of both functional units unless stated other-wise Protein groups forming the ligand-binding sur-face originate from the first three b strands and loop 3; many are located within the RNP motifs RNP1 (Lys13–Val20) and RNP2 (Val26–Phe30), which are conserved in various RNA-binding proteins [27,33] Further groups participating in ligand binding are located in b5, L34and L45(Fig 5)
Oligonucleotide binding to Bc-Csp is dominated by stacking interactions that involve single stacks between the side chains of Trp8, Phe17 and Phe27, and the nucleobases of T6, T5 and T4, respectively An impres-sive five-member stack is formed by succesimpres-sive side chain and nucleobase groups from T3, His29, Phe30, T1 and Phe38 T2 is contacted through an edge-on stack by Phe30 Shielding of Val26, Val28 and Tyr15 also may contribute to ligand binding, because the sol-vent-exposed location of these side chains in the absence of a ligand is expected to be thermodynamic-ally unfavourable
The polar contribution to ligand binding involves eight hydrogen bonds and five water-mediated interac-tions between protein and DNA groups Two hydro-gen bonds are formed between backbone groups of
Table 2 Relative increases in affinity associated with nucleobase exchanges at individual positions within a heptapyrimidine sequence Position oligo 1 KD1 (n M ) oligo 2 KD2 (n M ) KD2 ⁄ K D 1 a
1 dT7(TTTTTTT) 0.9 ± 0.2 CT1 (CTTTTTT) 2.8 ± 0.9 3.1 ± 0.3
2 CT3 (CTCTTTC) 3.3 ± 0.2 CT7 (CCCTTTC) 307 ± 33 93 ± 4.4
3 CT2 (CTTTTTC) 8.3 ± 0.2 CT3 (CTCTTTC) 3.3 ± 0.2 0.4 ± 0.01
4 CT3 (CTCTTTC) 3.3 ± 0.2 CT6 (CTCCTTC) 3.5 ± 0.6 1.1 ± 0.1
5 CT3 (CTCTTTC) 3.3 ± 0.2 CT4 (CTCTCTC) 5.2 ± 0.2 1.6 ± 0.1
6 CT3 (CTCTTTC) 3.3 ± 0.2 CT5 (CTCTTCC) 36.4 ± 4.8 11 ± 0.4
7 dT 7 (TTTTTTT) 0.9 ± 0.2 CT9 (TTTTTTC) 1.5 ± 0.2 1.7 ± 0.1
a
The binding propensities for thymine and cytosine at individual positions are compared by relating K D values of two different Bc-CspÆoligopyrim-idine complexes, which differ only at the position of interest (underlined) Their errors were estimated by Gaussion propagation of mean errors.
Trang 7Lys39 and the O4 and N3 atoms of T1 Asp25, Lys7
and Trp8 contact N3 and O2 of T5 by hydrogen bonds
in a similar way; O4 of T5 is connected to the Asp25
backbone carbonyl group via a water-mediated
inter-action The side chain of Gln59 contacts both
nucleo-bases of T3 and T4 Asn10 forms a hydrogen bond
with O2 of T6 In chain D this interaction is observed
for both conformers of the terminal nucleobase The
DNA backbone is contacted by a small number of
interactions One direct hydrogen bond is formed
between the side chain of His29 and the sugar O4¢ of
T2 In one functional unit, Arg56 interacts with the
phosphate group connecting T3 and T4 This side
chain shows great conformational flexibility in the set
of ligand-free CSP structures and interacts with the nu-cleobase group in the structure of Bs-CspBÆdT6 Upon ligand binding the solvent-accessible surface from a functional unit is reduced by 16.2% (696 A˚2), of which
65 and 35% can be assigned to hydrophobic and hydrophilic areas, respectively
Structural organization of the ligand The dT6 ligand adopts an extended, irregular confor-mation Looking from the protein surface towards the ligand, the sugar–phosphate backbone appears curved like a ‘C’ (Fig 6), with the nucleobases pointing towards the protein surface The 5¢-to-3¢ polarity of the ligand follows that of most other nucleic acid com-plexes of OB-fold proteins, starting in the vicinity of
L12, proceeding along the N–C polarity of b2 and pointing towards the kink in b1 (Fig 4A) There is no stacking between nucleobases, and all nucleosides are
in the anti conformation The solvent-exposed sugar– phosphate backbone shields the hydrophobic nucleo-bases and the hydrophobic-binding platform of the protein below them from the polar solvent (Fig 2B) The sugar of T1 from chain C maintains a C4¢-exo pucker, whereas the remaining sugars adopt C2¢-endo puckers, which are typical of double-stranded B-DNA
In ligand chain D, the terminal nucleotides adopt a
C3¢-endo conformation, which is typical of double-stranded A-DNA and RNA, whereas all other nucleo-tides display C2¢-endo puckers All sugar puckers observed in the Bc-CspÆdT6 structure are within ener-getically favourable regions of the pentose pseudorota-tion cycle [34] and the exocyclic angles of the sugar– phosphate backbone are within limits observed in tRNA structures [35]
Assignment of seven common interaction subsites to Bc-Csp and Bs-CspB
In the Bc-CspÆdT6 complex, each DNA molecule binds
to one globular functional unit of a swapped dimer In contrast to the swapped dimer complex of Bc-CspÆdT6, closed protein monomers and ligand molecules form an interspersed arrangement in the related Bs-CspBÆdT6 complex [24] (Figures 7 and 8 give a schematical and structural comparison of both hexa-thymidine complex structures.) Many interactions between protein and DNA ligand molecules are com-mon to both structures, yet certain interactions can only be observed with either Bs-CspB or Bc-Csp Based on the two structures, we can now define a com-mon interaction interface that allows us to understand
A
B
Fig 4 Binding of hexathymidine to amphipathic platforms of a
Bc-Csp swapped dimer (A) Topological representation of a functional
unit of the swapped dimer associated with a single dT 6 molecule.
(B) Electrostatic surface potential of a Bc-Csp functional unit All
fig-ures were drawn using PYMOL [52], the electrostatic surface
poten-tial was calculated with APBS [53] for pH 7 with a range from )10
(red) to +10 kT (blue).
Trang 8how Bs-CspB and Bc-Csp interact with thymine-rich
heptanucleotides (see Fig 5 for Bc-Csp)
In the Bc-CspÆdT6 structure, interaction subsite 1
remains empty In the Bs-CspBÆdT6 complex, an
edge-on stack between Phe38 and a nucleobase is observed
at this subsite In contact subsite 2, Phe30 and Phe38 form a three-membered stack with the T1 base This base is specifically bound via two hydrogen bonds to
A
B
Fig 5 Hydrophobic and polar interactions between dT6and Bc-Csp (A) Stereoscopic representation The contact surface of Bc-Csp is shown as a semitransparent grey object, protein groups involved in stacking interactions and hydrogen bonding are col-oured according to CPK with the exception
of carbon which is green (monomer A) and light blue (monomer B) Hydrogen bonds between protein and DNA groups are depic-ted as dotdepic-ted lines (B) Schematic overview
of intermolecular interactions: DNA (black) and protein groups (grey) interact through stacking interactions (solid lines) and hydro-gen bonds (dashed lines) Some contacts are mediated by water molecules (circles) Interactions observed in only one functional unit of the structure are in light grey, whereas a common set of interactions also observed in the Bs-CspB crystal structure is highlighted in red Nucleobase binding sub-sites (numbers below the schemes) are defined as discussed in the text Subsites not occupied by bases are parenthesized The numbers of the contact subsites for individual nucleobases are given at the bottom.
Fig 6 DNA single strands adopt an irregular conformation upon binding to Bc-Csp The sugar–phosphate backbone appears curved like a ‘C’ character All nucleobases are unstacked with respect to each other The nucleotides are in anti conformation The stereo view is from the protein surface towards DNA strand D The DNA is surrounded by its 2Fo) F c difference density contoured at 1.2 r.
Trang 9the backbone of Lys39 in a geometry reminiscent of a
Watson–Crick TA base pair The existence of a third
contact site has been hypothesized in the Bs-CspB
structure based on missing electron density for the 5¢
nucleotide, which was expected to be located at this
position In Bc-CspÆdT6, a third subsite was found as
anticipated It involves the side chain of His29, which
reveals an edge-on contact with the nucleobase and a
hydrogen bond with the deoxyribose ring oxygen of
the nucleoside in subsite 3 In contact subsites 4 and 5,
the side chains of His29 and Phe27 stack with bases
from adjacent nucleotides Gln59 contacts their
nucleo-base head groups via hydrogen bonds In the
Bc-CspÆdT6 structure an additional hydrogen bond
provided by the backbone carbonyl group of Pro58
contacts the nucleobase In subsite 6, a nucleobase
stacks against Phe17 while its head groups interact
with the side chains of Asp25, Lys7 and Trp8 via
hydrogen bonds Interestingly, the orientations of the
nucleobases in this subsite differ between the
oligo-thymidine complexes of Bc-Csp and Bs-CspB They
may be related by a 180 rotation Consequently, O2is
contacted by Lys7 and Trp8 in the Bc-CspÆdT6
complex structure instead of O4 as observed in the
Bs-CspBÆdT6structure
The most prominent difference between the two CSPÆoligothymidine complexes involves interaction subsite 7 In the Bc-Csp structure, the 3¢ nucleotide stacks against Trp8, its O2 is contacted by Asn10 In the Bs-CspB structure, Trp8 is inaccessible due to a crystal contact An alternative seventh binding site was attributed to a hydrogen bond between Arg56 and the
O2 of the nucleobase However, after evaluating both crystal structures we conclude that the alternative ori-entation of the base and sugar–phosphate backbone of the nucleotide in subsite 6 and the formation of the alternative subsite 7 are a consequence of the inaccessi-bility of Trp8 in this crystal form
In addition to their structures, Bc-Csp and Bs-CspB also share functional similarities Both proteins bind
dT7 with a similar affinity of KDvalues of 0.9 ± 0.2 1.8 ± 0.2 nm (Table 2) [24] In solution, their highest preference for T was observed for positions 2 and 6 in
a heptanucleotide Replacement of T by C at these positions results in significantly decreased affinities as observed by 93- and 11-fold increased KD values for Bc-Csp These distinct preferences are in good agree-ment with the deduced binding mode, in which the most specific contacts for thymine head groups are formed at nucleobase subsites 2 and 6
Fig 7 Schematic overview of CSPÆoligonucleotide interactions Protein molecules (grey) interact with bases from oligonucleotides at distinct binding subsites (A) In the Bc-CspÆdT6crystal structure, two protein chains (light and dark grey) form two functional units each of which binds a DNA molecule (B) In the Bs-CspBÆdT 6 crystal [24], a continuous arrangement of protein and DNA molecules is formed A gap between the 3’ nucleotide (bound to subsite 2) and the first structured 5’ nucleotide (bound to subsite 4) exists, which is expected to bind the unstructured T1 nucleotide (grey) (C) In solution, all seven subsites are occupied by a single oligonucleotide molecule.
Trang 10T-to-C changes at positions 4, 5 and 7 did not
signi-ficantly influence the affinity of dT7 for Bc-Csp,
whereas in binding studies with Bs-CspB weak
prefer-ences for T were observed At sequence position 3, a
cytosine base is preferred to thymine by both CSPs
Although the preference of Bc-Csp for individual
nu-cleotides at most positions was not as pronounced as
with Bs-CspB, they clearly follow the same trend The
smaller base discrimination by Bc-Csp may be related
to the fact that all fluorescence titration measurements
were performed at 15C to allow comparison
Although for B subtilis this temperature is close to its
growth conditions, the temperature optimum for
B caldolyticusis more than 40C higher
Functional implications
The common features of most nucleobase interaction
subsites suggest that both Bc-Csp and Bs-CspB share
identical ligand-binding interfaces (Fig 8), whereas
dif-ferences in binding involving subsites 6 and 7, as well
as the arrangement of protein and DNA molecules,
appear to arise from different crystal-packing
environ-ments Despite the fact that heptanucleotides rather
than hexanucleotides fully occupy the CSP binding site
[12,25], we have not yet found suitable crystallization
conditions for CSP in complex with
heptadeoxynucleo-tides Likewise, attempts to grow crystals of Bs-CspB
in the presence of oligoribonucleotides have remained
unsuccessful In contrast to the individual Bs-CspB
and Bc-CspÆhexathymidine complex structures, the combined structural models allow us to understand how both CSPs bind thymine-rich heptanucleotide motifs and explain binding preferences seen in bio-chemical binding studies in solution
Although the CSPÆdT6 crystal structures contain an single-strand DNA ligand, they support the assump-tion that single-strand RNA ligands bind the same way, because the exposed sugar 2¢OH groups and the missing methyl groups of uridines would not enhance
or impair ligand binding, and the backbone torsion angles of the DNA ligands are compatible with data obtained from tRNA crystal structures The extended irregular conformation of dT6 oligonucleotides in the binding site, the unstacking of bases with respect to each other upon binding, and the shielding of the nu-cleobase head groups by the protein suggest that the CSP may counteract double-strand formation in nucleic acids CSP surface properties favour the bind-ing of thymine-rich sequences, with the exception of nucleobase binding subsite 2, which favours cytosine The biological functions of CSP are still under inves-tigation Certain CSP have initially been reported to function as transcriptional activators of cold-induced genes such as hns [36] and gyrA, encoding a subunit of DNA gyrase [37] The ability of CSP to bind to single-stranded nucleic acids and prevent their association to double strands in vitro led to the assumption that these proteins may function as RNA chaperones [4], which may prevent the formation of mRNA double strands
Fig 8 Stereoview of the structurally con-served CSP ligand-binding surface Struc-tures of the nucleobase ligands from the Bs-CspBÆdT6(grey, black font) and Bc-CspÆdT6(green) complexes have been shif-ted (upper) to allow a better view on the CSP binding site (lower) Lines schemati-cally relate individual nucleobases to their position in the complex structures Protein groups involved in nucleobase interactions
in the complex structures are shown as sticks in corresponding colours Their equiv-alents from CSP structures without an oligo-nucleotide ligand are displayed as blue lines.