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Single-stranded DNA binding of the cold-shock protein CspB from Bacillus subtilis: NMR mapping and mutational characterization

Laboratorium für Biochemie, Universität Bayreuth, D-95440 Bayreuth, Germany

Reprint requests to: Jochen Balbach, Laboratorium für Biochemie, Universität Bayreuth, D-95440 Bayreuth, Germany; e-mail: jochen.balbach{at}uni-bayreuth.de; fax: 49 921 553661.

(RECEIVED June 7, 2002; FINAL REVISION September 30, 2002; ACCEPTED October 9, 2002)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0219703.

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
Cold-shock proteins (CSPs) bind to single-stranded nucleic acids, thereby acting as a "RNA chaperone." To gain deeper insights into the rather unspecific nature of ssDNA/RNA binding, we characterized the binding interface of CspB from Bacillus subtilis to a 25-mer of ssDNA (Y-Box25) using heteronuclear 2D NMR spectroscopy. Seventeen residues, including eight out of nine aromatic amino acids, are directly involved in the Y-Box25 interaction and were identified by extreme line broadening of their cross-peaks. Eight residues belong to the earlier proposed RNP binding motifs. A second set of seven backbone amides becomes evident by major chemical shift perturbations reporting remote conformational rearrangements upon binding. These residues are located in loop ß3–ß4 and loopß4–ß5, and include Ile18. The individual contributions of the so-identified residues were examined by fluorescence titration experiments of 15 CspB variants. Phenylalanine substitutions in- and outside the RNP motifs significantly reduce the binding affinity. Unrestricted possible backbone conformations of loop ß3–ß4 also markedly contribute to binding. Stopped-flow fluorescence kinetics revealed that the different binding affinities of CspB variants are determined by the dissociation rate, whereas the association rate remains unchanged. This might be of importance for the "RNA chaperone" activity of CspB.

Keywords: Cold-shock protein; CspB; single-stranded DNA; Y-Box; protein/ssDNA complex; NMR spectroscopy

Abbreviations: ssDNA, single-stranded DNA • HSQC, 2D ¹H/¹⁵N heteronuclear single quantum coherence spectroscopy • RNP, ribonucleoprotein • OB-fold, oligonucleotide- and/or oligosaccharide-binding fold • CspA, cold-shock protein A from Escherichia coli • CspB, cold-shock protein B from Bacillus subtilis • CSPs, cold-shock proteins • CSD, cold-shock domain • D₂O, deuterium oxide • Y-Box25, 25-mer ssDNA fragment containing the Y-Box motif ATTGG

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
Rapid adaptation to changes in the environment is essential for the survival of bacteria under extreme conditions such as chemical stress and heat or cold shock. A reduction in growth temperature elicits a cold-shock response, in the course of which a number of distinct essential proteins are induced (Jones and Inouye 1994), in particular members of the family of small cold-shock proteins (CSPs). CSPs are ubiquitous proteins found in psychrotrophic, mesophilic, thermophilic, and hyperthermophilic bacteria (Jones et al. 1987; Hebraud and Potier 1999; Phadtare et al. 1999). The induction originates from an increase in transcription of their genes (Jiang et al. 1993) and from the stabilization of their mRNA (Brandi et al. 1996; Goldenberg et al. 1996). The basal concentrations and levels of induction vary strongly among the CSPs due to their different functions during cell division or during the stationary phase (Yamanaka et al. 1998; Brandi et al. 1999). CSPs stimulate the transcription of cold-shock inducible genes (Jones et al. 1992) and facilitate the initiation of translation by destabilizing nonproductive secondary structures in mRNA at low temperature (Jiang et al. 1997) without apparent sequence specificity (Lopez et al. 1999, 2001; Lopez and Makhatadze 2000) and are therefore regarded as "RNA chaperones."

B. subtilis contains three homologous CSPs (CspB, CspC, CspD), which can complement each other in vivo (Graumann et al. 1997). The structure of the small acidic protein CspB (67 residues, Fig. 1) has been solved both in crystal and in solution, and revealed a very similar protein backbone conformation to CspA from Escherichia coli (Schindelin et al. 1993, 1994; Schnuchel et al. 1993; Newkirk et al. 1994; Feng et al. 1998). CspB comprises a five-stranded antiparallel ß-barrel with perpendicularly arranged strands of subdomain I (ß-strands 1, 2, and 3) and subdomain II (ß-strands 4 and 5). It belongs to the OB-fold superfamily (Murzin 1993), which also includes ribosomal proteins S1 and S17, and translational initiation factor IF1 of E. coli (Sette et al. 1997; Draper and Reynaldo 1999). CSPs recognize DNA and RNA with the two conserved binding motifs RNP1 and RNP2, which comprise amino acids K13-V20 and V26-H29 in CspB, respectively. Investigations by mutagenesis and gel retardation experiments revealed that several aromatic and basic residues within the RNP motifs constitute the putative nucleic acid binding surface (Schröder et al. 1995). Until now, no structure of a cold-shock protein in complex with a single-stranded nucleic acid has been reported. CspB shows low sequence specificity with a preference for thymidine and uridine rich stretches (Schröder et al. 1995; Lopez et al. 1999, 2001; Phadtare and Inouye 1999). U-rich regions can be found upstream of the promotor in the unusually long 5'-untranslated region (5'-UTR) of the CspB mRNA, and T-rich stretches can be located at factor-independent transcription termination signals (Richardson 1993; Henkin 1996; Phadtare and Inouye 1999).

View larger version (49K): [in this window] [in a new window]   Figure 1. Ribbon drawing of the cold-shock protein B from B. subtilis. The three-dimensional structure consists of two perpendicularly arranged subdomains (I: ß-strands ß1–ß3, II: ß-strands ß4 and ß5), which form a five-stranded ß-barrel. The MOLMOL (Koradi et al. 1996) representation is based on the coordinates from the protein data bank file 1csp.pdb (Schindelin et al. 1993).

In the present article we characterized the structural basis of CspB binding to a 25-mer ssDNA fragment termed Y-Box25, containing the Y-box core motif ATTGG, which is a cis-element in the promoter region of mammalian MHC II genes (Didier et al. 1988). Oligodeoxynucleotides containing Y-Box25 are the standard substrates of CspB used in previous studies (Schnuchel et al. 1993; Graumann and Marahiel 1994; Schröder et al. 1995; Schindler et al. 1999). Seventeen CspB residues involved in ssDNA binding were identified by line broadening of their backbone amides in a titration experiment followed by heteronuclear 2D NMR. They include eight residues of the proposed RNP motifs. Interestingly, seven amino acid residues in two loop regions that are not part of the classical RNP motifs show substantial changes in the chemical shifts of their backbone resonances upon binding. They report on remote conformational rearrangements in the nucleoprotein complex. The contributions of residues, identified by NMR, to the Gibbs free energy of binding were quantitatively characterized by equilibrium and kinetic fluorescence quenching experiments of 15 protein variants containing single amino acid substitutions. All examined aromatic and positively charged residues are necessary for a tight binding of Y-Box25. In addition, conformational restriction in loop ß3–ß4 decreases the binding affinity significantly. Reduced binding correlates with increased dissociation, rather than association, rate constants compared to the wild-type protein. This might be of importance for the "RNA chaperone" function of CspB.

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

 
Identification of residues involved in the ssDNA binding of CspB by NMR spectroscopy
Cold-shock proteins bind to both ssDNA and RNA. To gain insights into contributions of individual residues, we used NMR spectroscopy. NMR signals of the backbone amides provide two probes to monitor complex formation: extreme resonance line broadening and chemical shift perturbation. Therefore, we performed a titration experiment by successively adding aliquots of a highly concentrated solution of Y-Box25 (Graumann and Marahiel 1994) to a solution of uniformly ¹⁵N-enriched CspB. After each titration step a 1D ¹H and a 2D ¹H/¹⁵N-HSQC spectrum was recorded. Figure 2 shows a stacked plot of 1D spectra acquired during the titration. In the absence of Y-Box25 both a good dispersion and sharp lines of the NMR signals are prevalent (Fig. 2, top spectrum). As expected, in the presence of high concentrations of Y-Box25 all resonances are broadened (Fig. 2, lower spectra) because of the increased molecular mass of the complex leading to decreased Brownian motion. Some resonances, for example, Trp8 H¹, are extremely broadened and further completely lost at high Y-Box25 concentrations. The signal of Ile18 H¹ shifts to higher field upon ssDNA binding.

View larger version (39K): [in this window] [in a new window]   Figure 2. Sections of 1D 1H NMR spectra recorded during the titration of CspB with Y-Box25. Spectra are shown for ssDNA concentrations between 0 and 330 µM in 20 mM sodium cacodylate/HCl pH 7.0 at 15°C and a fixed CspB concentration of 0.6 mM. The 1H resonance of H1 of the Trp8 side chain (W8sc) undergoing extreme line broadening and the shifting signal of Ile18 H1 (I18sc) to higher field upon Y-Box25 binding have been specified.

Complex formation was investigated in more detail by 2D ¹H/¹⁵N-HSQC NMR spectroscopy. The cross-peaks of all 65 backbone amides in the 2D spectrum of 0.6 mM uncomplexed CspB (fine contours in Fig. 3) were assigned according to earlier studies (Schnuchel et al. 1993). Therefore, changes in line widths and chemical shifts upon ssDNA binding could be followed in a residue-specific fashion. Cross-peaks of backbone amides located in the hydrophobic core (e.g., V6, F49, I51) show nearly unmodified resonance frequencies and sharp lines. Therefore, we can conclude that the hydrophobic packing in the interior of the protein is not affected by the complex formation. This holds also for the backbone amides of all residues indicated in blue in Figure 4 and the side-chain amides of Q23, Q45, N55, and N62.

View larger version (24K): [in this window] [in a new window]   Figure 3. 2D 1H/15N-HSQC spectrum of 0.6 mM CspB in the absence and in the presence of 70 µM Y-Box25 at pH 7.0 and 15°C. The assigned cross-peaks of the amide backbone are labeled using the one-letter amino acid code and the sequence position. The indole 1H/15N cross-peak of the W8 side chain and the side-chain amides of N10 and Q59 are also labeled. Fine lines represent the spectrum in the absence of Y-Box25, and solid lines, the spectrum at 70 µM Y-Box25. The boxes indicate resonance signals, which vanished completely at a Y-Box25 concentration of 70 µM due to extreme line broadening. Nonlabeled cross-peaks with low signal intensities from impurities of probably unfolded peptide chains become visible at the lower contour level of the spectrum at 70 µM Y-Box25. They show no obvious dependence on the ssDNA concentration.

View larger version (174K): [in this window] [in a new window]   Figure 4. Identification of residues involved in ssDNA binding of CspB. Amino acid residues with cross-peaks undergoing extreme line broadening upon ssDNA binding at 70 µM Y-Box25 are represented in yellow (K7, W8, F9, N10, S11, K13, G14, F15, G16, F17, F27, V28, H29, F30, S31, F38, Q59). Residues with cross-peaks of backbone amide protons, which exhibit a substantial chemical shift perturbation during the NMR titration experiment, are indicated in green (I18, I33, G35, G37, E53, G54, G57). Residues of the RNP motifs are depicted with an increased bond radius. This representation was drawn in the same orientation as Figure 1 using the program MOLMOL (Koradi et al. 1996).

This concentration range was found around a 10-fold excess of CspB over the Y-Box25 oligodeoxynucleotide. The 2D ¹H/¹⁵N-HSQC NMR spectrum of CspB at an ssDNA concentration of 70 µM is plotted with solid contours in Figure 3. The boxes in Figure 3 indicate cross-peaks of residues with signal intensities broadened beyond the detection level in the presence of 70 µM Y-Box25. They include backbone amide resonances of K7, F9, N10, S11, K13, G14, F15, G16, F17, F27, V28, H29, F30, S31, F38, and the side-chain resonances of W8, N10, and Q59. These 17 residues are indicated in yellow in the three-dimensional structure representation of CspB in Figure 4. All of them are located at one side of the CspB molecule, with eight of them belonging to the classical RNA binding motifs RNP1 (K13–V20) and RNP2 (V26–H29). The backbone amides of seven out of the nine aromatic residues of CspB are extremely broadened (F9, F15, F17, F27, H29, F30, F38) as well as residues with positively charged side chains (K7, K13).

Chemical shift perturbations report about alterations in the chemical environment of protein nuclei upon binding to the nucleic acid. Changes in chemical shifts could be followed only for residues lacking the above-mentioned extreme line broadening. We found a gradual shift for some CspB residues with an increasing Y-Box25 concentration without extreme line broadening. The most likely reason for this observation is a small difference in the resonance frequencies between exchanging protein conformations.

A section of a HSQC spectrum in both the absence and the presence of an equimolar concentration of Y-Box25 is shown in Figure 5. All possible responses of the protein resonances to complex formation are evident here: minor (G4), medium (V47), and large (G35, G37) chemical shift perturbations as well as extreme line broadening (G14). Most resonances detectable during the entire titration indeed exhibit very small chemical shift perturbations ([¹H] < 0.05 ppm, [¹⁵N] < 0.25 ppm), suggesting minor changes in the structural environment of the respective nuclei. In contrast, cross-peaks of Ile 18 at the C-terminal end of ß-strands ß2 and amino acids forming the loop between the two subdomains (I33, G35, G37) and the loop connecting the ß-strands ß4 and ß5 (E53, G54, G57) display larger chemical shift changes ([¹H] > 0.05 ppm, [¹⁵N] > 0.5 ppm) in their resonance frequency. These residues are highlighted in green in Figure 4.

View larger version (20K): [in this window] [in a new window]   Figure 5. Chemical shift perturbation upon ssDNA binding. Expansions of HSQC spectra in the absence (fine contours) and in the presence of an equimolar Y-Box25 concentration (strong contours) are overlaid. Alterations of the resonance frequency are small (G4), medium (V47), or large (G35, G37). The respective cross-peak of G14 in the complexed protein is broadened beyond detection.

To determine the number of CspB molecules bound to one Y-Box25 molecule, we performed a titration experiment at 10 µM CspB (Fig. 6A). It yields an almost linear change in fluorescence up to 2 µM Y-Box25, indicating stoichiometric binding conditions. The intersection of the two lines in the titration profile at 3 µM Y-Box25 allows estimation of the ratio between CspB and Y-Box25 as 3:1, implying that one Y-Box25 molecule is able to bind three CspB molecules. A 3:1 stoichiometry was confirmed in the titration experiment monitored by NMR by analyzing changes in the chemical shifts of 11 residues at various Y-Box25 concentrations up to 0.6 mM (Fig. 6B). The coincidence indicates that the stoichiometry and mechanism of complex formation remains the same over a wide range of experimental conditions (0.25 to 10 µM CspB for fluorescence quenching or 0.6 mM for NMR spectroscopy).

View larger version (15K): [in this window] [in a new window]   Figure 6. Stoichiometric titration of CspB with Y-Box25 probed by (A) fluorescence quenching and (B) NMR chemical shifts. An intersection at (A) 3 µM Y-Box25 (10 µM CspB) and (B) 0.2 mM Y-Box25 (0.6 mM CspB) implies that one Y-Box25 molecule binds three CspB molecules. (B) The mean weighted difference in the chemical shifts for 11 residues of ß-strands ß4 and ß5 (E43, G44, A46, V47, S48, I51, G54, N55, G57, N62, K65) were calculated according to MW(1H,15N) = [((1H)2 + 1/25•(15N)2)/2]0.5 (Grzesiek et al. 1996).

View larger version (21K): [in this window] [in a new window]   Figure 7. Quenching effect on the Trp8 fluorescence of CspB upon binding to Y-Box25. The hyperbolic binding isotherms of 0.25 µM wild-type CspB (open circles), G35A (filled circles), F38A (open triangles), and G54P (filled triangles) depict different Y-Box25 binding affinities in 50 mM sodium cacodylate/HCl, 100 mM KCl, pH 7.0 at 15°C. The resulting KD values from fits using Equation 1 (solid lines) are given in Table 1.

Aromatic residues located outside the RNP1 and RNP2 motif also contribute to the affinity for Y-Box25. The K_D of F9A (in the C-terminal part of ß-strand ß1), F30A (in the C-terminal part of ß-strand ß3) and F38A (in the subdomain connecting loop between ß3 and ß4) are four- to tenfold increased compared to the wild-type protein. The importance of F9 and F38 has not been identified and analyzed in previous binding studies.

Cold-shock proteins from thermophilic bacteria such as Bacillus caldolyticus or Thermotoga maritima bind more tightly to Y-Box25 at 15°C with respective K_D values of 0.7 and 0.01 µM (M. Zeeb and J. Balbach, unpubl.). Sequence comparisons revealed that both thermophilic proteins differ only at one or two positions (BcCsp or TmCsp) in the RNP1 and RNP2 motif from CspB. They contain Tyr15 and a Trp30 instead of Phe15 and Phe30. Therefore, we produced CspB variants F15Y and F30W. Both proteins revealed a twofold enhanced binding affinity to Y-Box25 compared to wild-type CspB, showing that the composition of aromatic residues involved in binding can still be improved.

Gly35 and Gly37 experience a very strong chemical shift perturbation of amide crosspeaks upon complex formation (Fig. 5). They are located in the loop between ß-strand ß3 and ß4. To modulate the possible backbone conformations of this loop Gly35 was mutated to Ala or Pro. The G35A substitution restricts the expected backbone conformations to negative angles at this position but left the affinity for Y-Box25 virtually unchanged (Table 1). A further restriction by introducing a proline reduces the K_D by a factor of 6.5 comparable to the extent of complex destabilization by mutations inside RNP1 (F15A, sevenfold) or RNP2 (H29Q, sevenfold). The same substitutions were performed for Gly54 in the loop between ß-strands ß4 and ß5. The G54A mutation left the K_D for Y-Box25 almost constant, and inserting a proline at position 54 leads only to a threefold increase of the dissociation constant. The affinity was also unchanged when the possible backbone conformations of this loop were enhanced by a P58A substitution. Together, these results indicate that conformational restrictions of the CspB backbone in the loop between ß4 and ß5 are of minor importance for the Y-Box25 binding affinity whereas conformational freedom in the loop between ß3 and ß4 is necessary for a tight binding.

Heat-induced unfolding transitions
To investigate whether variations in the ssDNA binding affinities of the examined CspB variants are caused by different thermodynamic stabilities, thermal transitions were measured for all variants. The transition midpoints and the extrapolated Gibbs free energies of unfolding (G_U) at 15°C are given in Table 1. All protein variants depict a positive G_U, and all variants have reached the native baseline in the thermal transitions at 15°C. Therefore, we can conclude that they all were fully native under the experimental conditions of the Y-Box25 binding studies. Additionally, we found no correlation between the thermodynamic stability and the binding affinity.

Association and dissociation kinetics of protein/ssDNA complexes
To elucidate whether the variations in binding affinity of the CspB variants originate from changes in the rate of association or of dissociation of the nucleoprotein complex, we measured the kinetics of complex formation by stopped-flow fluorescence. The association (k_on) and dissociation (k_off) rate constants were determined from the concentration dependence of the observed rate constant (k_obs) under pseudo first-order conditions (Bernasconi 1976) as displayed in Figure 8. All kinetic curves are well described by a simple one-step mechanism P + D PD, where P represents the protein, D the nucleic acid, and PD the nucleoprotein complex. Considering the 3:1 stoichiometry of the CspB/Y-Box25 complex it is assumed that the binding sites are equivalent and noninteracting. Analyzing the data of Figure 8 according to the equation k_obs = k_on•[P] + k_off provides the association rate constant as the slope and the dissociation rate constant as the intercept with the ordinate (Fig. 8, inset). Because k_off is measured in the k_on•[P] >> k_off regime, the extrapolation to the intersection with the ordinate is not very accurate, compared to the slope to determine k_on (Bernasconi 1976). Even though the k_off values from the intercept (e.g., 28.9 ± 3.3 sec^-1 for wild-type CspB) were only marginally affected by extrapolation errors, we calculated the k_off value as the product of k_on and the equilibrium dissociation constant K_D determined by the fluorescence quenching titration experiment (e.g., 26.9 ± 1.9 sec^-1 for wild-type CspB). The association and dissociation rate constants of five protein variants are shown in Table 2.

View larger version (27K): [in this window] [in a new window]   Figure 8. Association kinetics of CspB with Y-Box25. Final CspB concentrations in 50 mM sodium cacodylate/HCl, 100 mM KCl pH 7.0 at 15°C were 9 µM (open circles) and 15 µM (filled circles) in the presence of 1.5 µM Y-Box25. Continuous lines represent the best fits of a single exponential equation to the data. The inset shows the concentration dependence of the observed pseudo first-order rate constant of the wild-type protein (open circles) and the F15Y mutant (filled circles) including the linear fits (results are listed in Table 2).

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods References

 
We identified by an NMR titration experiment those residues of the cold-shock protein CspB, which are involved in binding of the single-stranded DNA fragment Y-Box25. The individual contributions of these residues to the binding affinity have been elucidated by various single residue substitutions.

The 3:1 stoichiometry of the CspB/Y-Box25 complex was found by two independent probes: quenching of the fluorescence of Trp 8, and NMR resonances of backbone amides. Both datasets are well explained by assuming three independent binding sites for CspB at Y-Box25. The apparently single exponential binding kinetics supports this simple model and virtually identical K_D values are derived from the ratio of the measured k_on and k_off rates and from the equilibrium titration. There is no evidence for more complex behavior, but of course, protein–protein interactions found for example in the CopG operator complex (Gomis-Rüth et al. 1998) and mechanisms such as "sliding" (Berg et al. 1981) found essential for the DNA replisome (Hingorani and O’Donnell 2000) or for RecB (Phillips et al. 1997) cannot be excluded rigorously.

Extremely broadened NMR resonances were observed for 17 contiguous residues at the surface of CspB (yellow residues in Fig. 4). They include backbone amides of seven out of the nine aromatic residues (F9, F15, F17, F27, H29, F30, F38), two residues with positively charged side chains (K7, K13), and also the side chains of W8, N10, and Q59. Only eight of these residues belong to the earlier proposed RNP motifs derived from an alignment of different CSPs and CSDs. The RNP motifs are drawn by an increased bond radius in Figure 4, indicating the importance of the NMR titration data to map the entire binding interface. Schröder et al. classified the aromatic residues W8, F15, F17, F27, H29, F30, and basic residues K7 and K13 (all belong mainly to the RNP1 and RNP2 motifs) as essential for tight ssDNA binding (Schröder et al. 1995). They could not detect complex formation for CspB variants with F A substitutions (F15, F17, F27) and for H29Q in gel retardation experiments. Here we could show that these mutations lead to a 4–10-fold increase in the respective dissociation constant. Newkirk et al. probed the DNA binding epitope of CspA from E. coli in complex with a 24-mer corresponding to the 5' leader region of cspA mRNA (Newkirk et al. 1994). They used one single high ssDNA concentration and found strong perturbations of chemical shifts and/or line broadening for 26 residues, including six residues from the RNP motifs. Corresponding residues 7, 11, 13, 14, 16, 27, 29, 33, 35, 38, and 57 (CspB numbering) show perturbed NMR resonances in both CspA and CspB upon ssDNA binding. This comparison suggests that different residues close to the conserved RNP motifs facilitate the substrate specificity of the cold-shock proteins for different nucleic acid fragments.

Our NMR titration experiment is restricted to analyses of the backbone and side-chain amides, and well-resolved side-chain resonances of W8 and I18. Therefore, we complemented the NMR study by a mutational analysis of residues that are supposedly involved in Y-Box25 binding. Substitutions in the nucleic acid binding motifs RNP1 or RNP2 reduced the binding affinity. Phe Ala mutations of the highly conserved phenylalanines lead to a loss of key interactions for complex stabilization. Outside the RNP motifs, F9 and F38 are important for binding.

Undetectably broad resonance signals might originate from an intermediate exchange between free and bound forms of CspB on the NMR chemical shift time scale. Broadened interface resonances have indeed been detected for entropically advantageous fluctuations in the network of protein–DNA hydrogen bonds (Billeter et al. 1993; Foster et al. 1997). Changes in the dynamics of aromatic rings also caused line broadening of proximal nuclei (Iwahara et al. 2001), which might apply for the CspB/Y-Box25 interaction dominated by aromatic residues.

The cross-peaks of several amino acid residues shift gradually with increasing Y-Box25 concentration. This suggests that different CspB conformations are in fast exchange on the NMR time scale with rates above about 300 sec^-1. An extrapolation of the observed binding rates to NMR concentrations (Fig. 8, inset) supports this assumption.

The residues with perturbed backbone chemical shifts in the Y-Box25 complex (highlighted in green in Fig. 4) are located approximately in a plain behind residues with extremely broadened lines (yellow in Fig. 4). One reason for perturbed chemical shifts without extreme line broadening might be that the chemical shifts between exchanging conformations are very small. The perturbed nuclei include the backbone amide of I18 and its side chain H¹ proton (Fig. 2), which is located in the hydrophobic core of CspB. Therefore, it is unlikely that I18 interacts directly with Y-Box25. We suggest that I18 and most of the residues with perturbed chemical shifts report conformational changes in CspB remote from the ssDNA binding interface.

Several glycine residues of CspB in the loops ß3–ß4 and ß4–ß5 show strong perturbations of their ¹H and ¹⁵N chemical shifts upon binding. The G54A, G54P, and P58A variations in loop ß4–ß5 lead only to marginal changes in binding, but the G35P mutation in loop ß3–ß4 reduces the Y-Box25 affinity of CspB by a factor of 7. Therefore, complex formation may induce conformational tension, which can be compensated by readjusting the protein backbone at less constraining glycine positions.

Recently, the solution structure and DNA-binding properties of the cold shock domain of human Y-Box protein YB-1 have been determined by NMR spectroscopy (Kloks et al. 2002). This CSD has 43% sequence identity to CspB and a very similar structure. An NMR titration with the 5-mer ATTGG (Y-box core motif) revealed a K_D of about 150 µM and fast chemical exchange between free and complexed protein. The authors identified 21 residues involved in ssDNA binding and the following 15 residues correspond to our findings: 8, 9, 13, 16, 17, 18, 35, 36, 37, 38, 39, 50, 52, 55, and 57 (CspB numbering). This suggests that the binding interface of the prokaryotic cold-shock proteins is conserved in eukaryotic cold-shock domains. It is remarkable that residues at position 19 and 20 (indicated by blue bonds with increased radius in Fig. 4) of the proposed RNP1 motif (Schröder et al. 1995) show no interaction with ssDNA in the NMR titration experiments of CspA, CspB, and YB-1.

Cerdan et al. performed a similar NMR titration experiment of the high-mobility group domain of Drosophila melanogaster with a bulge DNA, and found as well strong line broadening for protein and DNA nuclei directly located at the binding interface (Cerdan et al. 2001). These protein/DNA complexes are also in fast exchange on the NMR chemical shift time scale and, therefore, show gradual shifts of the resonances during the NMR titration. In contrast, proteins such as the transcriptional coactivator PC4 revealed complexes in the slow exchanging regime, where two separate sets of resonances appear, one for the free protein and one for the protein/DNA complex (Werten et al. 1999).

It has been proposed that proteins have evolved to balance their function and stability (Meiering et al. 1992; Schreiber et al. 1994; Shoichet et al. 1995). For the mutants of this study there is no coupling between these properties. F30 and F38 of CspB are essential for tight ssDNA binding but not for protein stability (Schindler et al. 1998). G54A on the other hand, has a decreased Gibbs free energy of unfolding but the effect on Y-Box25 binding is small. Finally, the substitution of F15 or F17 with alanine reduces both affinity and stability.

Many complexes of proteins with nucleic acids have been investigated to date, and changes upon point mutations have been observed in k_off (Hoopes et al. 1998; Katsamba et al. 2001), k_on (Dong et al. 1999), or both rates (Neylon et al. 2000). The binding affinities of the examined CspB variants vary due to altered dissociation rate constants whereas the association rates remain nearly constant. This might be related to the "RNA chaperone" function of CspB by a rather fast, but unspecific interaction with stretches of mRNA. Productive interactions with U-rich stretches would result in a tight binding because of a reduced dissociation rate.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

 
Materials
The ssDNA fragment Y-Box25 was purchased from Interactiva AG (Germany) and was used throughout the entire study. The Y-box core motif is included and underlined in the sequence 5'-ATCCTACTGATTGGCCAAGGTGCTG-3' (Graumann and Marahiel 1994). Y-Box25 concentrations were calculated from the absorbance at 260 nm. The extinction coefficient at 260 nm (268330 M^-1cm^-1) was calculated by adding the extinction coefficients of the individual nucleotides (Wallace and Miyada 1987).

Protein purification
CspB from B. subtilis and CspB variants were overexpressed in E. coli K38 using the bacteriophage T7 RNA polymerase promotor system (Schindelin et al. 1992) and purified as described previously (Schindler et al. 1995) with minor modifications. The respective molar extinction coefficients were determined as 5800 M^-1cm^-1, 7300 M^-1cm ^-1, and 11,700 M^-1cm ^-1 for wild-type, F15Y, and F30W CspB according to (Gill and von Hippel 1989).

NMR spectroscopy
NMR experiments were performed at 15°C on a Bruker DRX600 spectrometer. The protein concentration was 0.6 mM uniformly ¹⁵N enriched CspB dissolved in 20 mM sodium cacodylate/HCl, 3 mM MgCl₂, pH 7.0 in 9:1 H₂O:D₂O. The protein–ssDNA titration experiment was carried out by successive addition of small aliquots of a 14-mM unlabeled Y-Box25 stock solution dissolved in the same buffer as the protein. Complex formation was monitored by recording a ¹⁵N-decoupled 1D ¹H and a 2D ¹H/¹⁵N-HSQC spectrum with WATERGATE solvent suppression (Piotto et al. 1992) after each Y-Box25 addition step. For the 2D spectra a combination of sensitivity enhancement and pulsed-field gradient coherence selection was applied with 1024 x 256 complex points (Palmer et al. 1991; Kay et al. 1992; Schleucher et al. 1994). All spectra were processed on a Silicon Graphics O₂ workstation using Felix 97 (MSI).

Fluorescence spectroscopy
Fluorescence intensities were measured with a Hitachi F-4010 spectrofluorometer equipped with a thermostated cell holder, which was attached to a circulating water bath. All experiments were carried out at 15°C in 50 mM sodium cacodylate/HCl, 100 mM KCl, pH 7.0. For the determination of the dissociation constant (0.25 µM protein) the fluorescence of Trp8 was measured at 343 nm and excited at 280 nm. An excitation wavelength of 300 nm was used for a titration under stoichiometric conditions (10 µM CspB). The initial volume of the titration experiments was 1.5 mL so that the dilution effect of the successive addition of highly concentrated Y-Box25 solutions was below 4%. Samples were gently stirred during the titration. The tryptophan fluorescence intensity was corrected for inner filter effects, buffer fluorescence and dilution. The changes of the fluorescence intensity with increasing ssDNA concentration reveal hyperbolic binding isotherms, which were analyzed according to the binding equation 1 (Lohman and Bujalowski 1991; Eftink 1997):

1

with A = K_D + [P]₀ + n[Y]₀, where Q is the quenching of the intrinsic fluorescence intensity of Trp8 after each addition of Y-Box25; Q_max represents the maximum quenching upon complete saturation of the protein with ssDNA; [P]₀ and [Y]₀ are the overall CspB and Y-Box25 concentration, respectively; n is the stoichiometry of the protein–ssDNA complex (number of protein molecules bound to one molecule Y-Box25); and K_D is the equilibrium dissociation constant of the complex. The binding model (Equation 1) does not take the degeneracy of multiple binding sites within one ssDNA molecule into account, and therefore, the obtained results depict apparent equilibrium constants.

To exclude the influence of random collisions to the fluorescence quenching effect a titration experiment with N-acetyl-tryptophan-amide was performed under the same experimental conditions (data not shown). There was no substantial quenching of the N-acetyl-tryptophan-amide fluorescence indicating that the decrease of the fluorescence intensity of Trp8 results from specific interactions between CspB and Y-Box25.

Heat-induced equilibrium unfolding transitions
Thermal denaturation curves were recorded with a Jasco J600A spectropolarimeter equipped with the Jasco Peltier element PTC-348WI. The temperature was measured with a sensor, which was directly inserted into the cell and the accuracy was verified by using a calibrated precision thermometer (Brand). The heating rate was 1°C per minute and the protein concentration typically 4 µM in 50 mM sodium cacodylate/HCl, 100 mM KCl, pH 7.0. Unfolding was monitored by the decrease of the ellipticity at 222.6 nm. Reversibility was examined by subsequent cooling of the sample to the starting temperature. The temperature transition curves were analyzed according to a two-state mechanism between the folded and unfolded conformation as described previously (Mayr et al. 1993). To derive the Gibbs free energy of unfolding at 15°C a linear extrapolation method was used.

Stopped-flow kinetic experiments
A DX.17MV sequential mixing stopped-flow spectrometer from Applied Photophysics was used for all kinetic experiments. The observation chamber has a path length of 2 mm. To absorb scattered light from the excitation beam a 5-mm cell with acetone was inserted between the observation chamber and the emission photomultiplier. The association kinetics was observed by the change in fluorescence intensity above 320 nm after excitation at 295 nm to account for the inner filter effect of Y-Box25. The zero time point and the dead time of mixing of the stopped-flow instrument were determined by using the procedure suggested by Tonomura et al. (1978).

All experiments were performed in 50 mM sodium cacodylate/HCl, 100 mM KCl, pH 7.0 at 15°C. Association reactions were initiated by a rapid 1:10 mixing of a 16.5 µM Y-Box25 solution (end concentration 1.5 µM) with a solution containing 6.6 µM to 23.1 µM protein (end concentration 6 to 21 µM) to obtain pseudo first-order conditions. Kinetic traces were collected at least eight times at identical conditions and averaged. Each set of stopped-flow data was analyzed according to a single exponential equation.

	Acknowledgments

 
We thank P. Rösch for NMR time at the DRX 600 in Bayreuth, R. Jaenicke for the clone of Csp from Thermotoga maritima, D. Perl and T. Schindler for several CspB variants, and R. Maier, A. Martin, M. Rape, and F.X. Schmid for helpful discussions. This research was supported by grants from the Deutsche Forschungsgemeinschaft (Ba 1821/1–1: Ba 1821/2–1).

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

	References

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