Báo cáo khoa học: Common mode of DNA binding to cold shock domains Crystal structure of hexathymidine bound to the domain-swapped form of a major cold shock protein from Bacillus caldolyticus pot

Common mode of DNA binding to cold shock domains Crystal structure of hexathymidine bound to the domain-swapped form of a major cold shock protein from Bacillus caldolyticus Klaas E. A. Max 1 , Markus Zeeb 2, *, Ralf Bienert 1 , Jochen Balbach 2, and Udo Heinemann 1,3 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 10 °C, 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 (K D ). 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 (dT 6 )ata resolution of 1.29 A ˚ . Bound to dT 6 , 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 dT 6 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. FEBS Journal 274 (2007) 1265–1279 ª 2007 The Authors Journal compilation ª 2007 FEBS 1265 degradation. 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 dT 6 [24] and data from solution NMR experiments characterizing the binding of dT 7 to 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 dT 6 . 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-CspBÆdT 6 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 dT 6 in space group P2 1 2 1 2, and diffraction data were collected up to 1.29 A ˚ (Table 1). Initial phases were obtained by molecular Table 1. Bc-CspÆdT 6 : 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 P2 1 2 1 2 Temperature (K) 100 Detector MAR165 CCD Unit-cell parameters a(A ˚ ) 74.34 b(A ˚ ) 64.89 c(A ˚ ) 31.20 Unique reflections (last shell) 37 691 (7838) I ⁄ r(I) (last shell) 14.8 (5.2) Data completeness (%) 97.0 (94.4) R meas a (%) 6.6 (32.0) Refinement Resolution (A ˚ ) 19–1.29 Working set 34,220 Free set (5%) 1,908 R work ⁄ R free b (%) 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 jÀjF 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. DNA single-strand binding to the cold shock domain K. E. A. Max et al. 1266 FEBS Journal 274 (2007) 1265–1279 ª 2007 The Authors Journal compilation ª 2007 FEBS replacement 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 R work ⁄ R free values 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 3 10 helix 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 functional 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 L 34 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-CspÆdT 6 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 F o ) F c difference electron density calculated from a model devoid of residues 35–38. The map (grey wire frame) was contoured at 2.5r. K. E. A. Max et al. DNA single-strand binding to the cold shock domain FEBS Journal 274 (2007) 1265–1279 ª 2007 The Authors Journal compilation ª 2007 FEBS 1267 )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). DNA single-strand binding to the cold shock domain K. E. A. Max et al. 1268 FEBS Journal 274 (2007) 1265–1279 ª 2007 The Authors Journal compilation ª 2007 FEBS Almost all of these interactions can also be found in the swapped dimers. From 15 N 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 L 34 , 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 15 N 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-CspÆdT 6 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 dT 6 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. K. E. A. Max et al. DNA single-strand binding to the cold shock domain FEBS Journal 274 (2007) 1265–1279 ª 2007 The Authors Journal compilation ª 2007 FEBS 1269 solution 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-CspBÆdT 6 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 K D val- ues of the respective Bc-CspÆoligonucleotide complexes to each other (Table 2; K D 1 ⁄ K D 2). 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 dT 7 . 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 K D value 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 K D increased 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 K D . dT 6 is 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 dT 6 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, L 34 and L 45 (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 successive 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 K D 1(nM) oligo 2 K D 2(nM) K D 2 ⁄ K D 1 a 1dT 7 (TTTTTTT) 0.9 ± 0.2 CT1 (CTTTTTT) 2.8 ± 0.9 3.1 ± 0.3 2 CT3 (C TCTTTC) 3.3 ± 0.2 CT7 (CCCTTTC) 307 ± 33 93 ± 4.4 3 CT2 (CT TTTTC) 8.3 ± 0.2 CT3 (CTCTTTC) 3.3 ± 0.2 0.4 ± 0.01 4 CT3 (CTC TTTC) 3.3 ± 0.2 CT6 (CTCCTTC) 3.5 ± 0.6 1.1 ± 0.1 5 CT3 (CTCT TTC) 3.3 ± 0.2 CT4 (CTCTCTC) 5.2 ± 0.2 1.6 ± 0.1 6 CT3 (CTCTT TC) 3.3 ± 0.2 CT5 (CTCTTCC) 36.4 ± 4.8 11 ± 0.4 7dT 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. DNA single-strand binding to the cold shock domain K. E. A. Max et al. 1270 FEBS Journal 274 (2007) 1265–1279 ª 2007 The Authors Journal compilation ª 2007 FEBS Lys39 and the O 4 and N 3 atoms of T1. Asp25, Lys7 and Trp8 contact N 3 and O 2 of T5 by hydrogen bonds in a similar way; O 4 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 O 2 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 O 4¢ 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-CspBÆdT 6 . 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 dT 6 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 L 12 , 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 C 4¢ -exo pucker, whereas the remaining sugars adopt C 2¢ -endo puckers, which are typical of double-stranded B-DNA. In ligand chain D, the terminal nucleotides adopt a C 3¢ -endo conformation, which is typical of double- stranded A-DNA and RNA, whereas all other nucleo- tides display C 2¢ -endo puckers. All sugar puckers observed in the Bc-CspÆdT 6 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-CspÆdT 6 complex, each DNA molecule binds to one globular functional unit of a swapped dimer. In contrast to the swapped dimer complex of Bc-CspÆdT 6 , closed protein monomers and ligand molecules form an interspersed arrangement in the related Bs-CspBÆdT 6 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). K. E. A. Max et al. DNA single-strand binding to the cold shock domain FEBS Journal 274 (2007) 1265–1279 ª 2007 The Authors Journal compilation ª 2007 FEBS 1271 how Bs-CspB and Bc-Csp interact with thymine-rich heptanucleotides (see Fig. 5 for Bc-Csp). In the Bc-CspÆdT 6 structure, interaction subsite 1 remains empty. In the Bs-CspBÆdT 6 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 dT 6 and 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 dotted 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 2F o ) F c difference density contoured at 1.2 r. DNA single-strand binding to the cold shock domain K. E. A. Max et al. 1272 FEBS Journal 274 (2007) 1265–1279 ª 2007 The Authors Journal compilation ª 2007 FEBS the 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-CspÆdT 6 , 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-CspÆdT 6 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, O 2 is contacted by Lys7 and Trp8 in the Bc-CspÆdT 6 complex structure instead of O 4 as observed in the Bs-CspBÆdT 6 structure. 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 O 2 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 O 2 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 dT 7 with a similar affinity of K D values 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 K D 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-CspÆdT 6 crystal 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. K. E. A. Max et al. DNA single-strand binding to the cold shock domain FEBS Journal 274 (2007) 1265–1279 ª 2007 The Authors Journal compilation ª 2007 FEBS 1273 T-to-C changes at positions 4, 5 and 7 did not signi- ficantly influence the affinity of dT 7 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 15 °C to allow comparison. Although for B. subtilis this temperature is close to its growth conditions, the temperature optimum for B. caldolyticus is more than 40 °C 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 CSPÆdT 6 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 dT 6 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-CspBÆdT 6 (grey, black font) and Bc- CspÆdT 6 (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. DNA single-strand binding to the cold shock domain K. E. A. Max et al. 1274 FEBS Journal 274 (2007) 1265–1279 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... transcribed, the affinity of the dA–U base pairs is insufficient to stabilize the DNA RNA duplex, and the transcript, together with the RNA polymerase, disassembles from the template It is expected that the obstruction of stem–loop formation prevents termination and leads to transcription of cistrons on the 3¢ side of termination sites The ability of the CSP to prevent doublestrand formation in RNA may... the absence of a ligand (Fig 8) The nucleotide -binding site of the CSP thus DNA single-strand binding to the cold shock domain appears to be a conserved preformed platform, which does not undergo major reorientations upon ligand binding A similar degree of conservation can also be found in the sequences of CSP homologues from other bacteria (Fig 9) Individual amino acids aligned to residues from the binding. .. Umbach P (2003) Facilities and methods for the high-throughput crystal structural analysis of human proteins Acc Chem Res 36, 157–163 DNA single-strand binding to the cold shock domain 45 Kabsch W (1993) Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants J Appl Crystallogr 26, 795– 800 46 Navaza J (2001) Implementation of molecular replacement... Y-box proteins and the CSP not only have a common ancestor but also share a closely similar DNA- binding mode Experimental procedures Data collection and processing Bc-Csp was purified, and the Bc-CspÆdT6 complex was formed and crystallized as previously described [43] A crystal was frozen in liquid nitrogen, and X-ray diffrac˚ tion data were collected at a wavelength of 0.9184 A at the Protein Structure. .. interface are conserved at a level of at least 75% similarity, whereas amino acids aligned with residues from other surface areas do not show a similar degree of conservation This suggests that binding modes and preferences of homologous CSP in bacteria are very similar and may be attributed to the same function, a finding that has already been demonstrated for the CSP paralogues of B subtilis and E... single-stranded DNA Importance of the T base content and position within the template J Biol Chem 276, 15511–15518 Zeeb M & Balbach J (2003) Single-stranded DNA binding of the cold- shock protein CspB from Bacillus subtilis: NMR mapping and mutational characterization Protein Sci 12, 112–123 Schindelin H, Marahiel MA & Heinemann U (1993) Universal nucleic acid -binding domain revealed by crystal structure of the. .. protein, YBAP1, mediates the release of YB-1 from mRNA and relieves the translational repression activity of YB-1 Mol Cell Biol 25, 1779–1792 Bienert R , Zeeb M, Dostal L, Feske A, Max KEA, Welfle H, Balbach J & Heinemann U (2004) Singlestranded DNA bound to bacterial cold- shock proteins: preliminary crystallographic and Raman analysis Acta Crystallogr D Biol Crystallogr 60, 755–757 Heinemann U, Bussow K,... [3,41] Apart from eubacteria, proteins with CSD can also be found in eukaryotic proteins A high degree of sequence identity (> 45%) was reported between the CSP and the nucleic acid -binding domains of the eukaryotic Y-box factors [10] Y-Box proteins have been implicated in transcriptional activation and repression, regulation of alternative splicing, regulation of mRNA stability, translational activation... Structure Factory beamline BL 14.1 [44] of the Free University of Berlin at BESSY (Berlin, Germany) using a MAR165 CCD camera A complete data ˚ set was collected to a maximal resolution of 1.29 A The xds package [45] was used to integrate reflection intensities The quality of the data set is summarized in Table 1 The Bc-CspÆdT6 complex crystallized in space group ˚ P21212 with unit cell parameters of a ¼ 74.34... structure of the B subtilis major cold- shock protein Nature 364, 164–168 Schindelin H, Jiang W, Inouye M & Heinemann U (1994) Crystal structure of CspA, the major cold shock protein of Escherichia coli Proc Natl Acad Sci USA 91, 5119–5123 Mueller U, Perl D, Schmid FX & Heinemann U (2000) Thermal stability and atomic-resolution crystal structure of the Bacillus caldolyticus cold shock protein J Mol Biol 297, . Common mode of DNA binding to cold shock domains Crystal structure of hexathymidine bound to the domain-swapped form of a major cold shock protein from. 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