Evidence of a Thermal Unfolding Dimeric Intermediate for the Escherichia coli Histone-like HU Proteins: Thermodynamics and Structure

doi:10.1016/S0022-2836(03)00725-3

J. Mol. Biol. (2003) 331, 101–121

Evidence of a Thermal Unfolding Dimeric Intermediate for the Escherichia coli Histone-like HU Proteins: Thermodynamics and Structure Jean Ramstein1, Nade`ge Hervouet1, Franck Coste1, Charles Zelwer1 Jacques Oberto2 and Bertrand Castaing1* 1

Centre de Biophysique Mole´culaire, CNRS, affiliated to the University of Orle´ans rue Charles Sadron, 45071 Orle´ans cedex 02, France 2

Laboratoire de Physiologie Bacte´rienne, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie 75005 Paris, France

The Escherichia coli histone-like HU protein pool is composed of three dimeric forms: two homodimers, EcHUa2 and EcHUb2, and a heterodimer, EcHUab. The relative abundance of these dimeric forms varies during cell growth and in response to environmental changes, suggesting that each dimer plays different physiological roles. Here, differential scanning calorimetry and circular dichroism (CD) were used to study the thermal stability of the three E. coli HU dimers and show that each of them has its own thermodynamic signature. Unlike the other HU proteins studied so far, which melt through a single step (N2 $ 2D), this present thermodynamic study shows that the three E. coli dimers melt according to a two-step mechanism (N2 $ I2 $ 2D). The native dimer, N2, melts partially into a dimeric intermediate, I2, which in turn yields the unfolded monomers, D. In addition, the crystal structure of the EcHUa2 dimer has been solved. Comparative thermodynamic and structural analysis between EcHUa2 and the HU homodimer from Bacillus stearothermophilus suggests that the E. coli dimer is constituted by two subdomains of different energetic properties. The CD study indicates that the intermediate, I2, corresponds to an HU dimer having partly lost its a-helices. The partially unfolded dimer I2 is unable to complex with high-affinity, single-stranded break-containing DNA. These structural, thermodynamic and functional results suggest that the N2 $ I2 equilibrium plays a central role in the physiology of E. coli HU. The I2 molecular species seems to be the EcHUb2 preferential conformation, possibly related to its role in the E. coli coldshock adaptation. Besides, I2 might be required in E. coli for the HU chain exchange, which allows the heterodimer formation from homodimers. q 2003 Elsevier Ltd. All rights reserved

*Corresponding author

Keywords: HU; histone-like; thermal stability; DSC

Introduction High levels of precision, regulation and synchrony of DNA transactions such as replication, transcription, recombination and repair, intrinsically depend on DNA topology and dynamics which involve the formation of high-order multiprotein/DNA complexes.1 In addition to the proteins playing a direct role in gene expression and DNA topology, the DNA architectural proteins, Abbreviations used: DSC, differential scanning calorimetry; CD, circular dichroism. E-mail address of the corresponding author: [email protected]

responsible for DNA compaction, could also directly modulate DNA transactions. In bacteria, the condensed circular chromosome is associated with ten to 20 DNA-binding proteins, which altogether constitute the bacterial nucleoid.2 The relative protein composition of the nucleoid and, consequently, the resulting overall DNA structure, vary with cell growth conditions.3 The Escherichia coli HU protein is the most extensively studied DNA architectural protein. First identified as a transcriptional activator of several bacteriophage lambda genes,4 this abundant (30,000 copies per cell), small (19 kDa) and basic (pI 10.5) protein was soon considered as a histonelike protein. Indeed, it is able to induce negative

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved

102

supercoils in relaxed circular DNA in the presence of topoisomerase I.5 Similarly to eukaryotic histones, HU wraps DNA, promoting the formation of nucleosome-like structures.6 More than just a histone-like protein, several studies have shown that HU is required in various cellular processes. HU is involved for instance in the synchrony of the replication initiation of bacterial DNA at oriC,7,8 in DNA protection against gamma and UV irradiation,9 – 11 in DNA transcription and sitespecific recombination.12 – 16 At the molecular level, HU has been shown to form low or high-affinity complexes with DNA. In the low-affinity binding mode ðKA < 107 M21 Þ; HU wraps linear DNA with a stoichiometry of one dimer per nine base-pairs without sequence specificity.17,18 In the high-affinity binding mode ðKA < 109 M21 Þ; HU was shown to recognize DNA molecules containing sharp bends, kinks, branched and bulged structures19,20 or more flexible structures such as single-strand breaks9 and loops.16,21,22 The involvement of the HU protein in many specific DNA transactions probably results from its high-affinity binding mode. Cloning of the HU genes and isolation of HU defective mutants have contributed significantly to identifying the physiological roles of HU.13,15,23 In enteric bacteria such as E. coli, HU is predominantly present as a heterodimer (encoded by two genes) while, in other eubacteria, HU is a homodimer (encoded by a single gene).24,25 The E. coli HUa and HUb subunits of 90 amino acid residues share 70% sequence identity and are encoded by two distinct genes, hupA and hupB, respectively.26,27 In E. coli, disruption of the two alleles strongly affects the growth of the double mutant strain and is associated with the perturbation of the cell division, the production of anucleate bacteria (filamentation), a heat-lethal phenotype and with a hyper-sensitivity to ionising radiation.13,28,10 Under physiological conditions, bacteria disrupted for the hupA gene display a milder phenotype and those for the hupB gene exhibit a wild-type phenotype. Conversely, single and double mutants are sensitive to heat or cold-shock stresses.28,29 In vitro and in vivo, the E. coli HU protein exists in three dimeric forms, EcHUa2, EcHUb2 and EcHUab.24 A recent study showed that the relative abundance of the three HU dimers varies during cell growth: the homodimer EcHUa2 accumulates in the dividing cells whereas the heterodimer EcHUab appears during the transition phase and predominates at the end of the exponential phase and in the stationary phase.30 Only 5% of the homodimer EcHUb2 is detectable in the stationary phase.24 The HU pool composition is therefore correlated with a differential expression of the hup genes.30 In the transition phase, the HU homodimers spontaneously exchange their chains using an unknown molecular mechanism to form the EcHUab heterodimer, leading to its accumulation during the stationary phase.31

E. coli HU Thermal Stability

The asymmetrical phenotypes of the single hup mutants and the variation in the relative abundance of the HU dimers during cell growth are likely to reflect different biological functions for the three HU dimeric forms.31 Moreover, it has been shown that the cell cycle may be altered by a non-canonical ratio of HU dimers.32 In vitro, EcHUa2 and EcHUab bind with similar affinities to DNA containing single-strand breaks.33 Conversely, EcHUb2 displays a significantly lower affinity for single-strand breaks-containing DNA. Besides, the three E. coli dimers bind with comparable affinities to cruciform DNA.34 To summarize, genetic studies and DNA binding experiments strongly suggest that each E. coli HU dimer plays a different biological role. This functional disparity may reflect specific biophysical and structural features. To address this question, we undertook comparative thermodynamic studies of each E. coli dimer by differential scanning calorimetry (DSC) and circular dichroism (CD), and a structural analysis by solving the crystal structure of the EcHUa2 homodimer.

Results Purification to homogeneity of the dimeric E. coli HU proteins A high purity of the protein sample is essential to compare the thermodynamic stability parameters of very similar proteins such as the dimeric forms of the E. coli HU protein. After extraction and several purification steps, a good separation of each dimer was achieved by perfusion chromatography on the POROS-CM column (Biocad, PerSeptive Biosystem) (Figure 1A). Elution on the POROS-CM was followed by measuring absorbance at 230 nm, since the E. coli HU subunits do not contain Tyr and Trp amino acid residues. The contents of POROS-CM elution peaks were analyzed by several PAGE methods (Figure 1B, C and D). Since the SDS-PAGE analysis does not enable the different dimers to be resolved (Figure 1B), the precise HU subunit contents of each POROS-CM elution peak were estimated by denaturing polyacrylamide gel electrophoresis under acidic conditions in the presence of Triton X-100. Under these conditions, the a and b HU subunits were resolved in the gel (AUT-PAGE, Figure 1C). In addition, the presence of dimeric forms of HU was confirmed by analyzing each elution peak of POROS-CM in a non-denaturing gel (ND-PAGE, Figure 1D). The heterodimer was prepared by mixing purified EcHUa2 and EcHUb2 homodimers for 30 minutes at room temperature. Figure 2B, C and D (thick lines) show the POROS-CM chromatographic pattern of these mixtures with three EcHUa2/EcHUb2 molar ratios. For a 1:1 ratio, 100% of the heterodimer was generated at 27 8C (Figure 2C). Surprisingly, the structural rearrangement of

103

E. coli HU Thermal Stability

Figure 1. Homogeneity of the purified E. coli HU dimers. A, Elution profiles of the HU dimers on POROS-CM. After their elution, 5 mg of each dimer was analyzed by polyacrylamide gel electrophoresis (PAGE) as described in Materials and Methods: B, 16% SDSPAGE; C, 16% AUT-PAGE; and D, ND-15% PAGE analysis. Lanes 1, 2 and 3, EcHUa2, EcHUab and EcHUb2, respectively.

Figure 2. Preparation of the E. coli heterodimer EcHUab from mixtures containing the purified EcHUa2 and EcHUb2 proteins. The sample contained 100% of EcHUa2 or EcHUb2 (A and E, respectively) or a 3:1, 1:1 and 1:3 EcHUa2/EcHUb2 molar ratio (B, C and D, respectively). After 30 minutes incubation time at 4 8C or 27 8C, the mixtures were loaded onto POROS-CM and their elution by a linear NaCl gradient was followed by measuring absorbance at 230 nm. Thin and thick chromatograms were for 4 8C and 27 8C incubation temperatures, respectively.

Thermal properties of the E. coli HU dimers studied by differential scanning calorimetry (DSC) Global description at low and high ionic strengths

the HU subunits from the homodimers to the heterodimer does not need a preliminary thermal denaturation of the dimer in its unfolded monomers. Indeed, the formation of the heterodimer occurs also at 4 8C, although its formation kinetics was slowed considerably (Figure 2, thin lines). Consequently, as discussed below in greater detail, this concerted exchange between a and b HU subunits is likely to require the formation of a EcHUa2/EcHUb2 tetrameric reaction intermediate, which would decrease the global activation energy and, therefore, increase the HU subunit exchange rate. The spontaneous character of the E. coli HU chain exchange shows that one HU chain (a or b) has more affinity for the other chain than for itself and consequently, that the heterodimeric form of the E. coli HU has a greater thermodynamic stability than the homodimeric forms.

The thermal denaturation of the three E. coli dimers was studied by DSC at low salt concentration (0.2 M NaCl) and some preliminary experiments were also performed at high salt concentration (1 M NaCl). At low salt concentration, the apparent molar heat capacity temperature-dependence at 4.5 mg/ml protein concentration is presented in Figure 3. All the thermograms display two partly overlapping heat absorption peaks, suggesting the existence of at least two underlying melting processes with melting temperatures differing by at least 25 deg.C. In spite of the biphasic shape of these thermograms, a detailed analysis of thermogram curves shows that the thermal transition peaks for each dimer differ both by their temperature position and by their amplitudes. For both EcHUa2 and EcHUab proteins, the low and high-temperature peaks are

Figure 3. Thermogram profiles of the E. coli HU dimers. Curves of apparent excess heat capacity (top), and, experimental (red) and fitted (black) heat capacity (bottom) as a function of the temperature for protein samples at 4.5 mg/ml containing 0.2 M NaCl. The decomposition of the apparent excess heat capacity thermograms (broken curves) are calculated according to the three-state thermal denaturation (see Materials and Methods).

105

E. coli HU Thermal Stability

located at , 40 8C and 65 8C, respectively. Their low-temperature peaks are similar and have greater amplitudes than those of the high-temperature peaks. In other respects, the EcHUab has a high-temperature peak amplitude greater than that of EcHUa2. With respect to the two previous dimers, the analysis of the EcHUb2 thermogram indicates a partial decrease in the thermal stability with a low-temperature peak located between 25 8C and 30 8C, whereas the position of the hightemperature peak is nearly unchanged (located at , 65 8C). In addition, the amplitude of the low-temperature peak of EcHUb2 is now smaller than that of the high-temperature peak. As these basic proteins ðpI ¼ 10:5Þ are very positively charged at pH 7.4 (pH of DSC experiments), it was interesting to evaluate the contribution of electrostatic interactions to thermal stability.

Therefore, Figure 4A shows the thermograms of the EcHUa2 homodimer obtained at 0.2 M and 1 M NaCl. The increase in NaCl concentration is reflected by a shift in the absorption peaks toward higher temperatures and by an increase in their amplitudes. Both DSC peak positions are affected nearly equally with a shift of about 20 deg.C (Figure 4A and Table 1). In addition, the increase in ionic strength is associated with the appearance of an additional minor peak located at lower temperature, , 30 8C. At 1 M NaCl, similar results are obtained with the other two dimers (Table 1) except that the low-temperature peak of EcHUb2 is now greater than its high-temperature peak (data not shown). As already described for the HU homodimer from Bacillus stearothermophilus and Bacillus subtilis, the high ionic strength significantly stabilizes the HU proteins. This indicates that the stability of the dimer involves essentially hydrophobic rather than electrostatic interactions.34 – 37 Thermal denaturation mechanism and thermodynamic analysis To select a two-step thermal denaturation reaction compatible with the previous thermograms, it is important to determine the protein concentration effect on the two thermal transitions. The thermograms obtained for EcHUa2 at 1 mg/ml and 4.5 mg/ml are shown in Figure 4B. Despite the low signal to noise ratio at low protein concentration (1 mg/ml), we observed that upon protein concentration increase, the high-temperature DSC peak is shifted about þ 5 deg.C, whereas the position of the low-temperature peak remains unchanged. Similar results are obtained with the other two E. coli HU dimers (Table 1). Since the high-temperature peak is protein concentrationdependent, unlike the low-temperature peak, the two-step thermal denaturation of the E. coli dimers can be described by a first monomolecular thermal transition followed by a bimolecular one, according to the reaction scheme: N2 $ I2 $ 2D

Figure 4. Ionic strength and protein concentration effects on the EcHUa2 thermal stability. A, Salt-dependence. The experimental (blue and red continuous lines) and fitted (continuous and broken black lines) curves of apparent excess heat capacity for protein samples at 1 mg/ml were studied at 0.2 M and 1 M NaCl concentrations, respectively. DTpm1 and DTpm2 indicate the melting temperature shifts between 0.2 M and 1 M NaCl (see Table 1 for the temperature shift values). B, Protein concentration-dependence of the second thermal transition. The experimental (blue and red continuous lines) and fitted (continuous black lines) curves of apparent excess heat capacity were obtained with protein samples at 1 mg/ml and 4.5 mg/ml, respectively. Dtm2 indicates the second melting temperature shift between 1 mg/ml and 4.5 mg/ml (see Table 1).

N2, I2 and D stand for the three equilibrium states of the protein; the native dimer, an intermediate dimer and the denatured monomer, respectively. The structural nature of the dimeric intermediate I2 will be discussed in the last section. To analyze the previous experimental thermograms quantitatively, we used theoretical expressions according to the method of Tamura (see Materials and Methods).38 As shown in Figure 3, the best fit between the experimental (in red) and the calculated thermogram (in black) obtained by minimizing the x2 value is convincing for EcHUa2 and EcHUab, whereas it is less satisfactory in the case of EcHUb2. Using the two-step denaturation mechanism previously proposed with an additional independent monomolecular step, the calculated

106

E. coli HU Thermal Stability

Table 1. Thermodynamic parameters for the E. coli HU dimers EcHUa2, EcHUb2 and EcHUab determined by microcalorimetry EcHUa2 NaCl 0.2 M

1M

1 mg/ml tm1 Dtm1 DH1 DS1 DCp1

41

EcHUb2

4.5 mg/ml

1 mg/ml

40

27

21 52.5 167 0.9

EcHUa/b

4.5 mg/ml

1 mg/ml

26

38

44.6 148 1.3

57

56

45.7 152 1.2

40.3 124 1.4

59

53

tm2 Dtm2 DH2 DS2 DCp2

18.8 39 1.5

tm1 DH1 DS1 DCp1

59 49.7 148 0.55

50 39.3 120 0.9

60 52.4 156 0.59

tm2 DH2 DS2 DCp2

69 42.8 104 0.55

65 33.7 78 0.9

72 57.5 148 0.2

Dtm1 p Dtm2 p

þ 19 þ 20

þ 23 þ9

þ22 þ19

50 þ7

þ3 14.0 25 1.3

41 þ3

21 51.0 162 1.3

4.5 mg/ml

46.8 149 1.5 62 þ9

44.3 115 0.6

20 42 1.5

19.1 34 1.4

26.3 60 1.5

Dtma ¼ tma difference between 1 mg/ml and 4.5 mg/ml of HU dimer and Dtpma ¼ tma difference between 0.2 M and 1 M NaCl. tm ; Dtm and Dtpm are in 8C, DH (kcal mol dimer21), DS (cal K 21 mol dimer21), DCp (kcal K 21 mol dimer21); DH; DS; DCp values are for 37 8C.

thermograms obtained at 1 M NaCl also fit well with the experimental thermograms (Figure 4A). Table 1 summarizes the thermodynamic parameters obtained at 37 8C by this fitting procedure and Figure 5 shows the variation as function of the temperature of DH; DG and -TDS for both thermal transitions at 0.2 M NaCl and 4.5 mg/ml of dimers. As expected for the three proteins and for both transitions, DH and 2TDS increase and decrease, respectively, with temperature (Figure 5). It should be noted that the absolute value of these thermodynamic functions is greater for the first thermal transition than for the second one. Interestingly, the temperature-dependence of the resulting denaturation Gibbs free energy of both transitions (DG1 and DG2 ) indicates that the thermal stability of the native dimer, N2, is maximal at a temperature below 0 8C, whereas that of the intermediate I2 is located at , 37 8C (Figure 5). According to this three-state thermal unfolding model, the relative abundance of the thermal species (N2, I2, and D) of each E. coli dimer was determined as a function of the temperature (Figure 6). Thermal stability of the HU dimers studied by circular dichroism (CD) To complement the previous DSC study, the thermal denaturation of the three E. coli dimers was investigated by CD in the far-ultraviolet. The corresponding CD spectra of the EcHUa2 homo-

dimer are shown in Figure 7A. The CD spectra of the three E. coli dimers indicate that at 4 8C, their secondary structure contents are similar (Table 2). The native spectra display two negative CD bands at 208 nm and 220 nm, and one positive band at 193 nm. Such CD spectra are typical for a þ b proteins in which a and b-structures are located in

Table 2. Secondary structure contents of the native state (N2) and the intermediate state (I2) calculated from the far-ultraviolet CD spectra of each E. coli HU dimer Secondary structure content (%) Protein

a-Helix

b-Sheet

Turn

Remainder

43.2 17.0 54.8 25.1 42.7 21.0

16.7 20.7 20.5 22.6 15.8 21.5

17.1 17.6 4.7 20.3 18.2 18.8

22.6 44.2 20.1 31.6 22.9 38.2

43.8

21.1

15.7

21.4

a

CD calculated EcHUa2 Nativeb 50 8Cc Natived EcHUb2 43 8Cc EcHUab Nativeb 50 8Cc Structural modele a

From the method of Johnson.44 Between 4 8C and 10 8C. c At 4 8C. d Temperature corresponding to ðtm1 þ above 10 8CÞ: e Determined from the crystal structure of the EcHUa2 homodimer. For this calculation, we assumed that residues in the protein arms that are not visible in the density map contain essentially random coil residues. b

E. coli HU Thermal Stability

107

Figure 5. Temperature-dependence of the thermodynamic function changes. DG and DH are in cal mol21, and DS is in cal K21 mol21, protein concentration 4.5 mg/ml and 0.2 M NaCl. Dotted, broken and continuous lines are for EcHUa2, EcHUb2 and EcHUab, respectively.

separated protein domains. The 208 nm band has a greater intensity than the 220 nm band at low ionic strength (here, 0.2 M sodium fluoride) but the reverse situation has been observed for the HU homodimer of B. subtilis at high ionic strength.36 Figure 7B shows the melting curve obtained for HUa2 (Figure 7A) by plotting the ellipticities at 200 nm and 222 nm as a function of the temperature. The melting curve at 200 nm is biphasic, indicating, in agreement with the DSC study, a two-step denaturation process characterized by two melting temperatures of , 37 8C and , 54 8C. Using the predictive method of Johnson, the secondary structures content of each dimer in their native and intermediate states (N2 and I2) were estimated from CD spectra at 4 8C and 10 8C above the first melting temperature, respectively (Table 2).39 In the native state, (N2), the percentage of secondary structures for each dimer is in good agreement with that obtained from the crystal structure of the EcHUa2 protein (see below). In addition, the thermal transition N2 versus I2 for each dimer is associated with the loss of 50% of

the a-helices, initially present in the native state (Table 2). Crystal structure of the E. coli HUa2 homodimer The E. coli homodimer a2 of HU (EcHUa2) crystallized as a single monomer in the asymmetric unit.40 The overall structure is highly similar to the X-ray structure of HU from B. stearothermophilus (BstHU, monomer 3) with an r.m.s. deviation of ˚ between all the Ca atoms of the molecules.41 0.6 A The structure of the HU monomer can be divided into two separate halves (Figure 8A and B). The N-terminal half of the molecule is composed of two long a-helices (a1 and a2), which are folded as a helix-turn-helix (HTH) motif, whereas the C-terminal half contains a threestranded antiparallel b-sheet (b1, b2 and b3) including the DNA-binding arm (residues from 58 to 71 are missing) and a short C-terminal a-helix (a3). The tertiary fold of the monomer is stabilized by two intra-helical salt-bridges (K22 ) E26 and K83 ) D87), an inter-helical salt-bridge (K18 ) D8) and three hydrogen bonds involving

108

E. coli HU Thermal Stability

Figure 7. Thermal denaturation of EcHUa2 followed by circular dichroism. A, CD spectra of EcHUa2 incubated at different temperatures. B, Melting curve of the EcHUa2 protein followed by CD at 200 nm and 222 nm.

Figure 6. Temperature-dependence of the thermal specie populations of each E. coli HU dimer at 0.2 M and 1 M NaCl. Curves in broken line, continuous line and dotted line indicate the relative abundance of the native dimer (N2), the intermediate dimer (I2), and the denatured monomer (D), respectively. Thin and thick lines are for 0.2 M and 1 M NaCl, respectively.

S17, S35 and S81, which link turn 1 to helix a2, turn 2 to helix a2 and turn 5 to helix a3, respectively (Figure 8B). As in the B. stearothermophilus HU Xray structure, several water molecules are closely associated with parts of the monomer. The water molecules W103 and W133 link turn 2 with the strand b2. In the same way, W107 and W109 link turn 3 to the helix a3.41 The biologically relevant homodimer is generated by a crystallographic 2-fold rotation axis parallel with the c crystal parameter. A cartoon of this dimeric structure is shown in Figure 8C. The EcHUa2 homodimer is very compact and is stabil-

ized by a pronounced hydrophobic core located underneath the b-strands involving six phenylalanine residues (F47, F50 and F79 from each monomer) and by several aliphatic residues filling in the gap between the HTH motifs (Figure 9A). The dimer structure is also stabilized by a hydrogen bond between the amine N-terminal atom of the M1 residue and the carbonyl O atom of the A14 residue (Figure 9B) of the other monomer. Site-specific features related to the thermal stability of the dimers will be described in Discussion and will focus mainly on the differences between the EcHUa2 and BstHU structural models. Temperature-dependence of the Gap-DNA binding (GDB) activity of each E. coli dimer In a previous work, we have shown that singlestrand breaks-containing DNA molecules are high-affinity ligands for the E. coli HU protein.9 We termed this property the HU GDB activity. Here, we used this assay to examine the temperature-dependence of the apparent dissociation constant ðKD appÞ of each dimer for a two nucleotide gap-containing DNA. Protein/DNA mixtures varying by their protein concentrations were

E. coli HU Thermal Stability

109

Figure 8. Primary, secondary, tertiary and quaternary structures of EcHUa2. A, Sequences of the EcHUa and EcHUb subunits and sequence alignment of the unique sequence subunit of the B. stearothermophilus (BstHU) and B. subtilis homodimers (BsuHU). Residues that are identical in all the sequences are shown in white on a red background. Homologies are highlighted in red. Arrows and rectangles indicate the a-helices and the b-stands of EcHUa2, respectively. Alignments were performed by CLUSTALW and are represented with ESPrit.67,68 B, Description of the secondary structure elements of the monomer. This stereo view shows the principal intra-chain H-bonds stabilizing the monomer secondary structure in the homodimer. C, Overall structure of the EcHUa2 dimer. This stereo view shows the monomer interlocking which forms the dimer. The dimer can be defined as a compact body, which constitutes a scaffold to anchor two flexible arms proposed to interact with DNA (partially missing from the electron density map, broken lines). The protein body can be subdivided into two domains: the a-subdomain (blue), rich in a-helices (a1 an a2 of the HTH motif and their symmetric partners) and the b-subdomain (orange), rich in b-strands (b1, b2, b3 and a3 and their dimer partners).

110

E. coli HU Thermal Stability

equilibrated at 5 8C, 15 8C and 20 8C, respectively, and analyzed by EMSA in a non-denaturing polyacrylamide gel equilibrated at the sample temperature. From the resulting titration curves at each incubation temperature, we extracted the KD app values assuming that the protein concentration needed to bind 50% of the DNA probe corresponds to KD app (Table 3).9 Even in its native state (5 8C), EcHUb2 binds DNA less efficiently than EcHUa2 and EcHUab, as previously described.33 Interestingly in the 5 8C to 20 8C temperature range, the GDB activity of EcHUa2 and EcHUab was not significantly modified while that of EcHUb2 was highly affected. This rapid loss of EcHUb2 GDB activity seems to correlate with the I2 concentration increase, suggesting that only the native state, N2, binds preferentially to nickedDNA (Table 3). The physiological relevance of these results will be discussed below.

Discussion E. coli HU dimers thermal stability

than in its native state. Another consequence of the change in hydration interactions upon denaturation is the increase of the specific heat, Cp ; at constant pressure, which explains the marked temperature-dependence of the denaturation enthalpy and entropy. It appears experimentally that the heat capacity of the unfolded state is always greater than that of the native state, as illustrated by the thermograms presented in Figure 3 (bottom). On the other hand, the denaturation entropy is dominated by the so-called configurational entropy change, which is a measure of the conformational freedom increase of both the polypeptide backbone and residue side-chains upon denaturation. Therefore, to fold a protein, this entropic barrier must be slightly overcome by optimizing a favorable internal van der Waals packing and hydrogen bonding enthalpy. In addition to this conformational entropy, there is an entropic term reflecting the ordering of the water molecules around the hydrophobic protein residues. This hydration entropy decreases upon denaturation and consequently, contributes to stabilizing the native state of the protein.

Globular protein thermal stability: a survey Before discussing the DSC results, it is worth summarizing the most important aspects of protein thermal stability, which have been reviewed by Privalov.42 Generally, the equilibrium of a protein between the native and denatured state is governed by two thermodynamic functions termed denaturation enthalpy and denaturation entropy. The denaturation enthalpy of a protein in solution at a given temperature results from the change in the interplay of several interactions, which can be classified into two groups: (i) interactions between the atomic groups shielded from water and buried inside the hydrophobic core of the protein; and (ii) interactions between the protein residues exposed at the surface of the protein and the surrounding solvent molecules. These latter interactions, termed the hydration interactions, are dependent on the solvent accessibility area of the protein atomic groups and, therefore, are energetic components more important in the unfolded state of the protein

Thermodynamic analysis of the E. coli HU dimers thermal unfolding The DSC experiments showed a clear biphasic thermal denaturation for the three E. coli HU dimers, indicating a three-state thermal unfolding process (Figure 3). In contrast to the E. coli proteins, the other HU proteins studied so far are proposed to unfold according to a two-state thermal mechanism in which the native dimer (N2) melts cooperatively into two unfolded monomers (D) with a melting temperature range between 60 8C and 90 8C.35,36 This monophasic thermal process is not a thermodynamic feature specific to thermostable HU proteins such as HU from B. stearothermophilus, since the mesophilic HU protein from B. subtilis also unfolds in a unique step.36 Whereas previous studies of the HU thermal stability were carried out only on homodimeric HU forms, our study exemplifies that whatever the homo- or

Table 3. Apparent dissociation constants ðKD appÞ of each E. coli HU dimer for a two-nucleotide gap-containing-DNA at different temperatures Dimer

5 8C Thermal species (%)

EcHUa2 EcHUab EcHUb2

N2 I2 N2 I2 N2 I2

100 0 100 0 90 10

15 8C KDapp(nM) 10 15 40

Thermal species (%) N2 I2 N2 I2 N2 I2

100 0 100 0 77 20

20 8C KDapp (nM) 15 15 70

Thermal species (%) N2 I2 N2 I2 N2 I2

100 0 100 0 65 30

KDapp (nM) 15 25 130

KD app was determined by EMSA.9 The relative abundance of the thermal dimeric species of each dimer was estimated from the titration curves of Figure 6.

E. coli HU Thermal Stability

hetero-dimeric form of the protein studied, at least two steps for the thermal unfolding pathway are needed to read the DSC experimental thermograms of the E. coli HU dimers. The biphasic behavior as well as the protein concentration-dependence of the thermograms suggest a three-state denaturation mechanism characterized by a first monomolecular step in which the native dimer, N2, yields an intermediate dimer, I2, followed by a second bimolecular step in which I2 leads to the unfolded monomers, D (Figures 3 and 4(B), Table 1). Using this minimal three-state model, the experimental thermograms can be fitted (Figure 3). A perfect fit is obtained for EcHUa2 and EcHUab, whereas the fit for EcHUb2 is less satisfactory for the first peak (bottom of Figure 3). For EcHUa2 and EcHUab, the first thermal transition is associated with a cooperative monomolecular reaction. In the case of EcHUb2, it is likely that the first peak does not correspond to a simple two-state cooperative transition and probably additional intermediate states have to be taken into account to fit properly the first denaturation peak. For the proposed two-step thermal denaturation model, the temperature-dependence of the enthalpy change ðDHÞ and of the related entropy change ð2TDSÞ for both thermal transitions are presented in Figure 5. DH for both transitions are positive and increase with temperature. Thus, the native and intermediate states N2 and I2 are mainly stabilized by internal interactions and this stabilization is increasing upon heating. This is a situation which holds for all small globular proteins studied so far.42 As expected, the 2TDS values are negative and decrease as a function of temperature, indicating that the N2 and I2 protein states are globally more and more entropically destabilized as the temperature increases. Despite the overall similarities of the temperature-dependence of the two thermodynamic functions DH and 2TDS in both transitions, it should be pointed out that the enthalpy and entropy changes of the first transition (DH1 and 2TDS1 ) at a given temperature are significantly larger than those of the second one (DH2 and 2TDS2 ) (Table 1). This may indicate that more protein residues are involved in the first transition than in the second one. If we assume that DH1 and DH2 are approximately proportional to the number of residues involved in the corresponding thermal transitions, it is then possible from DSC data to roughly estimate the number of residues involved in each thermal transition. Neglecting the residue contribution of the loosely structured protein arms to the denaturation enthalpy, the average denaturation enthalpy change per mole of residue of the dimers is of the order of 0.5 kcal mol21 at 37 8C. This value compares well with the average value of the denaturation enthalpies obtained for small globular proteins, which ranges between 0.25 kcal mol21 and Dividing the denaturation 0.7 kcal mol21.42 enthalpies of the first and second transitions by

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this mean value should give an estimate of the corresponding number of protein residues involved in each thermal transition. For example, in the case of the EcHUa2 dimer, the values of 108 and 30 residues were calculated for the first and the second transition, respectively. Interestingly, these values are in good agreement with the value of 96 and 48 residues obtained for the residues involved, respectively, in a-helices (a1 þ a2 þ a3) and bstrands (b1 þ b2 þ b3) of the dimer body (the dimer without its arms), calculated from the crystal structure of the EcHUa2 (Figure 8). Assuming that the other two dimers have the same overall fold as EcHUa2, similar results were obtained for the EcHUab and EcHUb2 dimers. In the same way, we can analyze the denaturation entropies. Neglecting also the contribution of the poorly structured protein arms, we obtain the average denaturation entropy of the protein by dividing the total measured entropy corrected for the dimerization association entropy change by the number of residues involved in the protein body.43 For example, a value of 1.3 cal K21 mol21 was obtained at 37 8C for the EcHUa2 dimer, which compares well with the corresponding values expected for small globular proteins range between 0.25 cal K21 mol21 and 1.9 cal K21 mol21.42 With respect to the denaturation entropy per residue, the entity formed by the protein body of E. coli dimers is comparable to a globular protein. On the other hand, the denaturation entropy per residue of the low and high-temperature transitions, calculated by dividing the corresponding measured entropy change per the number of residues involved in the transition, is markedly different. For EcHUa2 at 37 8C, the denaturation entropy change per residue is lower for the hightemperature transition than for the low-temperature one (0.43 cal K21 mol21 and 1.7 cal K21 mol21, respectively). As seen in Table 1, similar results are obtained for the EcHUab and EcHUb2 dimers. The denaturation entropy values per residue are approximately four times greater in the low-temperature transition than in the high-temperature one. Two consequences result from this observation: the first concerns the structure of the dimer and the other, the relative stability of ahelices and b-sheets of these proteins (structural domains defined with respect to their crystal structures, see below). The low value of DS2/residue indicates that, on average, the residues implicated in the second transition must have a greater conformational freedom than those involved in the first transition. Consequently, the secondary, tertiary and quaternary structure elements which melt in the first transition (concerning maybe the a-helices) are more compact and less flexible than those that melt in the second transition (concerning maybe the b-sheets). In addition considering the free Gibbs energy, the E. coli dimers are constituted by two energetic entities, the last one which melts is more thermally stable and consequently, is entropically stabilized.

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Thermal stability and structure of the E. coli HU dimer The first thermal transition corresponds to the loss of a-helices As discussed above, the DSC experiments support a denaturation model with two temperature transitions corresponding to the successive melting of the a-helices and b-sheets. In the intermediate state, I2, most of the a-helices would therefore be melted. To support this structural hypothesis, we studied the thermal unfolding of the E. coli HU dimers by circular dichroism (Figure 7). Depending on the wavelength, the experimental melting curves obtained from CD spectra can display either a mono- or a biphasic shape (Figure 7B). At 222 nm, the melting curve of EcHUa2 seems monophasic rather than biphasic with a melting temperature of , 40 – 45 8C. This thermal transition corresponds well to the first transition identified by DSC, but this single transition was slightly cooperative and extended over 40 8C, suggesting a more complex thermal process than the monophasic one. At 200 nm, the melting curve of EcHUa2 is clearly biphasic, which is compatible with a three-state denaturation process with melting temperatures of , 37 8C and 53 8C. These tm estimations compare well with the tm1 and tm2 values obtained by DSC (Table 1). Considering the limit CD spectra of a peptide folded in a-helix, b-sheet or in random coil structures, secondary structures contribute in the same range to the CD signal at 200 nm whereas the signal of the a-helices predominates at 222 nm.44 In this case, the melting curve at 200 nm is therefore more informative concerning the melting process of the E. coli HU dimers. The EcHUa2 secondary structure content, estimated from CD data (method of Johnson) was in good agreement with that expected by the crystal structure of the dimer (Table 2). Similar secondary structure contents have been estimated for the native forms of the other two dimers. Once this procedure has been validated for the native state of EcHUa2, the CD spectra obtained 10 deg.C above the first melting temperature were used to estimate the secondary structure content of the intermediate dimer, I2 (Table 2). Indeed at tm1 plus 10 deg.C, the dimers predominate in their I2 conformation (Figure 6). If we consider that the protein arms of the dimer are expected to contribute to the CD signal as unfolded peptides, each E. coli I2 dimer is then a dimer having lost 50% of its ahelices. As suggested by DSC experiments, the first thermal transition likely corresponds to the melting of a-structures of the E. coli dimers.

Structural determinants for the E. coli HU partition into two energetic subdomains DSC and CD experiments suggest that the E. coli HU proteins are constituted by two energetic

E. coli HU Thermal Stability

entities, which unfold separately according to a three-state mechanism characterized by a dimeric intermediate. This type of sequential subdomain melting has already been described for several low and high-molecular mass monomeric globular proteins. Thus, the E. coli HU dimer unfolding exemplifies that such a mechanism can be extended to the case of dimeric proteins. Interestingly, this thermal unfolding model is specific for the E. coli HU proteins, since the B. stearothermophilus and B. subtilis HU homodimers (BstHU and BsuHU, respectively) were shown to reversibly unfold according to a two-state model.35,36 Unlike E. coli dimers, these latter proteins have therefore a “dimer body” (the globular part of the dimer without its arms) constituted by a single energetic entity. Consequently, BstHU and BsuHU must have additional interactions linking physically the a-subdomain to the b-subdomain, which are absent from the E. coli dimers. Thus, we now address the question of how the energetic description of HU proteins studied so far can correlate with their three-dimensional structure. To this end, we solved the crystal structure of the EcHUa2 homodimer. Its overall structure consists of a very compact structure formed by the precise interlocking of the two EcHUa subunits. The globular part of the dimer (called the “protein body”) constitutes a scaffold to anchor two very flexible and extended DNA-binding arms (missing in the electron density map). The EcHUa2 overall fold is very similar to that of the B. stearothermophilus HU homodimer (BstHU).41 Like BstHU, the EcHUa2 protein body can be structurally subdivided in an a-helix rich-(a-) and a bstrand rich-(b-) subdomains (blue and orange, respectively, Figure 8). By comparing the crystal structure of EcHUa2 with that of BstHU, we have investigated physical interactions that are responsible for the strong association between the a-subdomain and the bsubdomain and which can explain, to some extent, the two-state denaturation behavior of BstHU. We observed in BstHU such structural interactions, which are absent from EcHUa2. One of these interactions is located in a hydrophobic core of BstHU, involving a remarkable cluster of eight phenylalanine residues (Phe29, Phe47, Phe50, Phe79 and their symmetrical partners), which is extended by a buried hydrophobic network of aliphatic residues filling in the space between the a-helices a1 and a2 (the highly conserved HU residues Leu6, Ileu32, Leu36, and Leu44: Figures 8A and 9A). This aromatic cluster determines a strong monomer –monomer interaction linking the top of the a-subdomain to the hydrophobic inner surface of the b-subdomain (Figures 8C and 9A). In EcHUa2, this aromatic cluster contains only six phenylalanine residues (Phe47, Phe50, Phe79), instead of eight, belonging exclusively to the b-subdomain. Phe29 located in the helix a2 of BstHU is replaced by Leu in EcHUa2 (Figure 8A).41 In BstHU, Phe29 and its dimeric partner Phe290 appear to be

E. coli HU Thermal Stability

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Figure 9. Some structural features of EcHUa2 homodimer for the protein body partition in two energetic subdomains. A, Structural comparison of the hydrophobic cores of the B. stearothermophilus (BstHU) and the EcHUa2. This stereo view shows a superimposition of the aromatic cluster of EcHUa2 (red) and BstHU (green). The residue 29 of EcHUa2 and BstHU (Ileu and Phe, respectively) in each dimer are underlined by their side-chains and their van der Waals spheres. B, The N-terminal extremity of BstHU is more anchored at the protein body than that of EcHUa2. This stereo view shows the absence in EcHUa2 (green backbone) of the salt-bridge between Met10 and Asp40 (blue arrow), previously observed in BstHU (pink backbone). In EcHUa2, this inter-monomer salt-bridge is replaced by an intra-monomer hydrogen bond between the carbonyl group of the Asp40 main-chain and the hydroxyl group of Ser35 side-chain (gray arrow). Hydrogen bonds and salt-bridge are indicated by broken lines (yellow).

involved in edge-to-face aromatic interactions with Phe470 and Phe47, respectively (Figure 8A). Phe29 and Phe47 are located in the a-helix a2 of the HTH motif and in turn 3 connecting the b-strand b1 to the b-strand b2, respectively. Therefore, the Phe29/Phe47 aromatic interactions create physical links between the a-subdomain and the b-subdomain. Such inter-monomer aromatic interactions are absent from the hydrophobic core of EcHUa2 (Figure 9A), as well as in that of EcHUb2, where an Ileu residue replaces Phe29 (Figure 8A) and subsequently, in the hydrophobic core of EcHUab. On the contrary, Phe29 is conserved in the mesophilic BsuHU protein and, like BstHU, BsuHU was also proposed to thermally unfold through a two-state mechanism.36 Furthermore, the Phe ! Trp conservative exchange at position 29 in BsuHU was shown to associate with the larger loss of the dimer stability rather than the same exchange of the Phe residues belonging to the bsubdomain (Phe47, 50, and 79).36 Thus, the amino acid residue at position 29 may be one of the key structural elements that modulate the energetic coupling between the a-subdomain and b-subdomain in the BstHU and BsuHU proteins. These additional aromatic interactions present in the

hydrophobic core of BstHU and BsuHU may contribute to maintaining their protein body as a single energetic entity, which cooperatively unfolds via a two-state mechanism. There is another quaternary interaction, only present in BstHU, which clamps the N terminus of one monomer to the b-strand b1 of the other monomer, and can be regarded as a structural feature participating in the cooperativity of the thermal unfolding. This interaction consists of an inter-monomer salt-bridge (and its symmetric counterpart) between the carboxyl group of Asp40 of one monomer and the N-terminal amino group of Met1 of the other one.41 Surprisingly, this interaction is not observed in the crystal structure of EcHUa2, in spite of the local sequence identities between EcHUa2 and BstHU (Figures 8 and 9B). Actually, the backbone conformation of turn 2 in EcHUa2 is slightly different from that of BstHU. In EcHUa2, this backbone remodeling probably results from the formation of an intra-monomer hydrogen bond between the carbonyl group of the Asp40 main chain and the hydroxyl group of the Ser35 side-chain, which stabilizes turn 2 of each monomer in a different conformation (Figures 8A and 9B). Such a hydrogen bond cannot exist in

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BstHU, since Ser35, which is present in EcHUa2 is replaced by an Ala residue (Figure 8A). In EcHUa2, the Asp40 side-chain has rotated and therefore adopts a conformation that prevents the formation of a salt-bridge with Met10 (gray arrow, Figure 9B). Interestingly, Ser35 is conserved in the EcHUb subunit, whereas BsuHU has an Ala at position 35, suggesting that BsuHU displays also these inter-monomer salt-bridges. This structural comparative analysis has revealed additional aromatic interactions and inter-monomer salt-bridges contributing to reinforcing the physical association between the a-subdomain and the b-subdomains in BstHU. These additional structural features present in BstHU provide a reasonable explanation for its highly cooperative two-state denaturation process, in contrast to the E. coli HU proteins where these peculiar interactions are absent and for which we observe a three-state thermal unfolding. This does not exclude the possibility that other unidentified structural elements might also contribute to stabilizing BstHU and BsuHU in a single energetic entity. Structural model for the E. coli HU dimer thermal unfolding Taking energetic and structural data together, we can propose a possible structural model for the thermal unfolding of the E. coli HU proteins (Figure 10). During the first monomolecular thermal transition, a large part of a-helix residues of the native protein (N2) cooperatively unfolds, prior to the rest of the protein, leading to a less structured dimer intermediate (I2) having lost at least 50% of the native a-helix. The choice of the a-helix residues that unfold shown in Figure 10 (essentially, those of helix a2) is arbitrary and it cannot be excluded that these residues may belong also to a-helices a1 and a3 of the protein. Sequence alignments of Figure 8A indicate that the a-subdomain (structurally defined by the HTH motifs, in blue in Figure 8C) is the less conserved region of these proteins, especially at the end of the helix a2

E. coli HU Thermal Stability

(Figure 8A). This suggests that the intrinsic stabilities of the three forms of the E. coli HU protein can be related to the different stabilities of this ahelix. On the contrary, the sequence of the helix a3 is highly conserved and consequently, can energetically contribute in the same way for all the HU proteins (Figure 8A). If we ignore the HU protein arms, the intermediate I2 has therefore a protein body having conserved at least 60% of its native secondary structures. During the bimolecular transition, I2 melts into two unfolded monomers, D (Figure 10). According to this structural unfolding model, EcHUb2 (tm1 ¼ 27 8C at 0.2 M NaCl) has a less stable a-subdomain than EcHUa2 and EcHUab (tm1 around 40 8C at 0.2 M NaCl). In the absence of an EcHUb2 structural model, it is difficult to correlate energetic observations to particular structural features of each homodimer. However, the intra-monomer salt-bridge between K22 and E26 identified in the crystal structure of EcHUa2 contributes, probably, to the stability of a-helix a2 and, therefore, to the stability of the EcHUa2 a-subdomain (Figure 8B). Such an interaction in EcHUb2 is not expected, since, in its primary structure, Lys22 and Glu26 are replaced by Gly and Asp residues, respectively (Figure 8A). On the other hand, we have no structural evidence to explain the enhanced thermal stability of EcHUab as compared to the homodimers in the absence of its structural model. We are now in a position to discuss, in the frame of our minimal thermal denaturation equilibrium mechanism, a possible folding kinetic scenario that holds as far as the intermediate equilibrium state I2 is unaffected by the experimental renaturation conditions. As previously described for other proteins, it is expected that in the first phase, the unfolded monomers form an initial dimer, which results in a very fast hydrophobic collapse of hydrophobic HU residues (see above) followed by a slower second phase leading to the intermediate state, I2.45 For many proteins, these transient kinetic intermediates are very similar to equilibrium intermediates and result from the hierarchical nature of the protein folding.46 According to such

Figure 10. Structural model for the E. coli HU dimer thermal unfolding.

E. coli HU Thermal Stability

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a hierarchical folding model, the protein partition function is defined in terms of multiple levels of interacting cooperative units. This formalism applied to the melting of myoglobin has revealed the importance of hydrophobic interactions for the cooperativity in protein folding.47 In the resulting intermediate state, I2, the conformational entropy of the partly denatured polypeptide chains is now sufficiently reduced to allow the formation of tertiary interactions, which open the way to the folding of the a-subdomain, yielding the fully protein native state, N2. Thus, the inter-monomer hydrophobic interactions along the 2-fold symmetrical axes of the protein described above play a major role in the thermal unfolding mechanism of the E. coli HU proteins. This exemplifies further, how the spatial distribution of hydrophobic interactions between two subunits can modulate the extent of thermal cooperativity in a protein. Ionic strength effect on the denaturation thermodynamic parameters: a major conformational change for I2 In contrast to DNA, which is stabilized by high ionic strength, the salt effect upon protein stability is difficult to predict.48,49 This is mainly a consequence of the fact that ion concentration changes are likely to affect in different ways both enthalpy and entropy. The effect of the NaCl concentration increase on the stability of the E. coli HU dimers is illustrated by the DH and 2TDS temperaturedependence at 0.2 M and 1 M NaCl (Figure 11). Whereas both subdomains are stabilized by an increase of about 20 deg.C of the melting temperatures (Figure 4A, Table 1), the origins of these stabilizations are quite different (Figure 11). For example, at 37 8C, the salt-stabilization of the native EcHUa2 dimer during the first transition results from the entropic component change ð-TDDS1 ¼ þ6 kcal mol21 Þ; which is more important than the small destabilization effect of the enthalpic component change ðDDH1 ¼ 23 kcal  mol21 Þ (Figure 11). In contrast, the stabilization of the intermediate I2 of EcHUa2 (second thermal transition) is due to an increase in the enthalpic component change ðDDH2 ¼ þ23 kcal mol21 Þ; which slightly dominates the destabilization entropic component change ð2TDDS2 ¼ 220 kcal mol21 Þ (Figure 11). Consequently, the a-subdomain and b-subdomain of EcHUa2 are, respectively, entropically and enthalpically stabilized by the ionic strength increase. In the case of EcHUab, both subdomains are slightly enthalpically stabilized. On the contrary, the EcHUb2 subdomains are both slightly entropically stabilized rather than enthalpically upon NaCl concentration increase (Figure 11). Due to this entropic effect, EcHUb2 predominates in its N2 conformation at 37 8C and 1 M NaCl (a situation already true for the other dimers whatever the salt concentration), whereas at 0.2 M NaCl, I2 is its more favorable thermodynamic conformation (Figure 6). In all cases, the salt effects

Figure 11. Temperature-dependence of the denaturation enthalpy and entropy changes for the three E. coli dimers at low and high NaCl concentrations. Energetic component changes (DDH and 2TDDS) at 37 8C of the thermodynamic functions between 0.2 M and 1 M NaCl concentrations (thin and thick curves, respectively) are indicated by arrows. Red and green curves were for, respectively, DH1 and 2TDS1 of the first thermal transition and, blue and magenta curves were for, respectively, DH2 and 2TDS2 of the second one.

are more important on the intermediate I2, i.e. on the stability of the b-subdomain. Besides the different origin of the salt stabilization effects (entropic or enthalpic), DH for both subdomains of EcHUa2 and EcHUab at 1 M NaCl and 37 8C are now comparable, differing only by about 6 kcal mol21, whereas at 0.2 M NaCl, they differ by as much as 20 kcal mol21 (Figure 11). The same remark holds

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for their 2TDS; with the differences of at the most 13.8 and as much as 30 kcal mol21 at 1 M and 0.2 M NaCl, respectively. These results indicate clearly that at 1 M NaCl, the b-subdomain of EcHUa2 and EcHUab has a much more compact overall structure than at 0.2 M NaCl, which may partly result from an important structuring of the protein arms. For EcHUb2, DH and 2TDS differences for both transitions between 1 M and 0.2 M NaCl are less pronounced (as much as 5.4 kcal mol21 and 13.4 kcal mol21 for DH and 2TDS change, respectively, Figure 11) and suggest that the salt concentration increase has less effect on the global compaction of the b-subdomain. Altogether, these data indicate that the thermodynamic differences between E. coli dimers partly result from significant differences in the intermediate I2 thermal stabilities. As previously for the thermodynamic parameters obtained at 0.2 M NaCl, this latter observation can be discussed in terms of average DH and 2TDS per residue at 1 M NaCl, considering here again that these quantities are roughly proportional to the number of residues participating in the denaturation process. Thus, dividing DH1 of EcHUa2 (at 1 M NaCl and 37 8C) by the number of residues of the a-helices (92 residues), the average denaturation enthalpy per residue ðDH1 =mol residue) is 0.54 kcal mol21. Assuming that the average value per residue of the DH2 is close to the average DH1 =mol residue; the number of residues involved in the b-subdomain melting is 80 at 1 M NaCl and 37 8C. This is exactly the total number of residues involved in b-sheets and in the protein arms, which indicates again that at 1 M NaCl, I2 adopts a very compact structure, which is associated with the formation of additional interactions involving, probably, residues of the protein arms. Similar results are obtained with the two other HU dimers. Therefore, it is safe to conclude that the intermediate, I2, must be more sensitive to the ionic strength than the native state, N2, whereas such a salt effect is very unlikely for the unfolded state of the protein, D. The compaction of the bsubdomain of EcHUa2 and EcHUab is further evidenced by the increase of DS2 at 1 M NaCl (Table 1). On the contrary, for EcHUb2, DS2 decreases between 0.2 M and 1 M NaCl (Table 1). Such DS2 salt-dependent variations simply reflect the high conformational entropy change of the I2 conformation upon denaturation. It is tempting to speculate that such a salt concentration effect on the structure of I2 can mimic the effect of the negative charges of the phosphate-DNA backbone through HU binding. We shall comment on this remark in the following paragraph when we examine the temperature-dependence of the E. coli dimer DNA binding. Through DNA binding, HU dimers must be stabilized by DNA, probably in their N2 conformation. The nature of the additional interactions induced by the ionic strength in the intermediate I2 is unknown and this point needs further investigation.

E. coli HU Thermal Stability

Possible consequences of the existence of a dimeric intermediate for the E. coli HU physiology I2 might be the major physiological conformation of the homodimer EcHUb2, in vivo The most remarkable result of this study is the possible equilibrium of at least two dimeric conformations for each dimer: the so-called “native dimer”, N2, and the intermediate dimer, I2, having undergone some unfolding event (Figure 6). Our thermodynamic study has demonstrated that their relative abundance is not dependent on the total dimer concentration (Figure 4B and Table 1). On the contrary, the N2 $ I2 equilibrium may be strongly shifted in response to environmental changes such as ionic strength and temperature changes (Figures 4, 6 and 11). Using the threestate unfolding model, we can estimate the relative abundance of this dimeric conformation for each E. coli dimer at an ionic strength close to physiological conditions (Figure 6). At 37 8C (the physiological temperature for E. coli), EcHUb2 is essentially in its I2 conformation (, 85% of thermal species) while EcHUa2 and EcHUab predominate in their N2 conformations (, 60%). Thus like N2, I2 seems to be one of the physiological conformations of E. coli dimers and especially, represents the EcHUb2 preferential conformation. This result may be correlated with an in vivo observation: although the HUa and HUb subunits are very similar (70% sequence identity) and the corresponding dimers probably adopt a similar global fold, EcHUb2, as compared with other dimers, is very sensitive to the bacterial protease Lon.50 Under physiological conditions, EcHUb2 does not accumulate in bacteria and is degraded and/or in the presence of EcHUa2, is quickly recombined into the more stable heterodimer.30,31 Under physiological conditions, we suggest that the Lon hypersensitivity of EcHUb2 is directly related to its less compact preferential conformation, I2. The fact that EcHUb2 predominates in its I2 conformation is very intriguing. While EcHUa2 and EcHUab were shown to maintain negative supercoils in the bacterial chromosome, the physiological role of EcHUb2 remains unclear. Under normal growth conditions, bacteria deleted for the hupB gene have no detectable phenotype (see Introduction) and no observable deficiency other than the lack of EcHUab heterodimers. In a recent work, Giangrossi and collaborators have proposed that EcHUb2 is actually a cold-shock protein of E. coli.29 During cold shock, the expression of the hupB gene is stimulated while the hupA gene is repressed, suggesting that EcHUb2 might play an important role during cold adaptation. The more flexible conformation of EcHUb2 could be related to its implication in the resistance to cold shock. For instance, it has been shown that bonds across the subunit – subunit interface in the cold-active

E. coli HU Thermal Stability

alkaline phosphatase dimer (AP) of Atlantic cod are considerably weaker than in mammalian calf AP dimer.51 A higher flexibility at the dimer interface and, possibly, at around the active site in cold-adapted AP may be an important feature in enhancing the reaction rate which permits catalysis at low temperature. We surmise that like cod AP, the high flexibility of EcHUb2 could be more appropriate to cold shock response than those of EcHUa2 and EcHUab, by an unknown molecular mechanism. In its I2 conformation, HU seems unable to bind to nicked DNA preferentially From a functional point of view, we examined the preferential affinities of the different E. coli HU proteins for a two nucleotide gap-containing DNA in the 5 8C to 20 8C temperature range (Table 3).9,33 Under these conditions, the DNA binding of EcHUa2 and EcHUab seems slightly affected by temperature changes. On the contrary, even in its N2 conformation (4 8C), EcHUb2 binds three- to fourfold less efficiently to single-strand break-containing DNA than the other dimers. According to our model, this suggests that, in addition to the protein arms, stable a-helices are required for the HU gap-DNA binding. At 20 8C, EcHUb2 has fiveto tenfold less affinity for gap-DNA than the other dimers (Table 3). An increase in the apparent dissociation constant ðKD appÞ is unambiguously associated with a significant increase in I2 concentration (Table 3). Thus, the I2 conformation of HU does not seem appropriate to bind preferentially to DNA. Since we can expect that DNA has a similar salt effect, which, through DNA binding, stabilizes the HU dimers in their N2 conformations (Figures 4A, 6 and 11, Table 1), the alternative I2 conformation of the HU dimers may contribute to maintain continuously a pool of free HU proteins in bacteria. Such a molecular mechanism by which a protein undergoes a physiological unfolding event has been described.52 – 55 For example, SecA, the principal component of the protein export of E. coli, was shown to partially unfold and to change its dimeric conformation (in a thermal dimeric intermediate) in order to insert itself into the membrane or to interact with SecB, another partner of the protein export system.55 Similarly, the E. coli HU dimers might be associated in their I2 conformations with still unknown biological functions. I2, a flexible conformation possibly required in vivo for the formation of the E. coli heterodimer, EcHUab The data we present allow us to speculate on the physiological dimeric intermediate I2. While most bacteria have selected a unique and a more stable HU homodimer, only enteric bacteria, on the other hand, have evolved a heterodimeric protein whose subunits unfold through this intermediate.34,36 We

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propose that an additional role of I2 would be to facilitate the de novo formation of the heterodimer EcHUab from homodimers. In vitro, the formation of EcHUab is slower at 4 8C than at room temperature since after 30 minutes only 50% of heterodimers are generated from homodimers (Figure 2). At 4 8C, the native form N2 predominates and, according to our hypothesis, the HU subunit exchange is reduced considerably. Thus, the very stable and compact N2 structure of each homodimer is probably inappropriate to a quick exchange reaction between the a and b HU subunits. On the contrary, the difference in the denaturation enthropies of the two melting processes is likely to reflect a high overall flexibility of the I2 conformation, which might be more adapted to perform the HU chain exchange between the two homodimers (Table 1). Since this chain exchange mechanism is irrelevant in the case of the unique homodimeric form of HU from B. stearothermophilus and B. subtilis, the I2 conformation has not been selected by these bacteria. The precise N2 $ I2 equilibrium of the E. coli HU proteins would be regarded as an example of a post-translational regulation system that finely regulates under physiological conditions the relative abundance of each E. coli HU dimer at a constant HU concentration and quickly adapts it in response to environmental condition changes. This system thus allows a fast reorganization of the E. coli nucleoid structure and/or the change in gene expression. Furthermore, the HU chain exchange mechanism between homodimers must be highly specific, fast and easy at the physiological temperature (37 8C). Indeed, the integration host factor (IHF), required for lambda site-specific recombination in E. coli, is a structural homologue of HU, which can also exist as homo- and heterodimeric forms and may interfere with the HU subunit exchange.56 This suggests that IHF has its own chain exchange molecular mechanism, using perhaps partially unfolded homodimers. Indeed, it has been shown by mass spectroscopy that it was possible to obtain small amounts of interspecies heterodimer between BstHU and BsuHU mixing both their corresponding thermal unfolding homodimers followed by a slow cooling.57 Under physiological conditions, the possibility of such an inter-species chain exchange is energetically highly improbable. In vivo, to avoid non-cognate chain exchange between HU and IHF, the monomeric forms of these proteins are probably not appropriate. Thus, we suggest that the free HU homodimers in their I2 forms (their N2 forms being probably trapped in nucleic acid complexes) specifically interact through a highly specific complex. In this putative heterotetrameric complex, EcHUa2 and EcHUb2 in their I2 conformations expose structural determinants, initially buried in their N2 conformations, which allow the precise recognition of the a-helices between each E. coli HU subunit. The requirement of the I2 conformations for the E. coli HU chain exchange still remains a fascinating hypothesis, which must be supported by further

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genetic, biochemical, structural and energetic studies.

E. coli HU Thermal Stability

Theoretical expressions and analysis of the experimental Cp(T ) thermograms The fundamental starting expression is the following:

Materials and Methods Proteins and DNA The overproduction and the purification of EcHUa2 have been described.40 The same experimental procedure was used to prepare to homogeneity the homodimer EcHUb2. The concentration of the homogeneous homodimers was measured by the Biuret method.58 The EcHUab heterodimer was prepared at 27 8C by mixing the two purified HU homodimers (1:1 molar ratio) in 50 mM Tris – HCl (pH 7.5), 1 mM Na2EDTA, 0.1 mM PMSF, 50 mM NaCl. The homogeneity of the purified HU dimers was confirmed by mass spectroscopy and N-terminal micro-sequencing.9 The preparation and sequence of the two nucleotide gap-containing DNA probe used for binding experiments has been described.9 Apparent dissociation constants ðKD appÞ were determined by electrophoretic mobility shift assay (EMSA).9 Polyacrylamide gel electrophoresis (PAGE) The contamination of the active fractions of one HU dimer by another HU dimer was estimated by a denaturing (SDS-PAGE and acid/urea/Triton (AUT)-PAGE) and non-denaturing polyacrylamide gel electrophoresis (NDPAGE). SDS-PAGE and AUT-PAGE were performed as described.41 For ND-PAGE, protein samples were diluted in 0.06 M KOH, 0.06 M acetic acid, 0.01% (w/v) pyronine Y, 10% (v/v) glycerol and loaded onto polyacrylamide gels (a 3.75% stacking gel in 0.096 M KOH, 0.1 M acetic acid (pH 6.8) over a 15% separating gel in 0.06 M KOH, 0.36 M acetic acid (pH 4.3)). Electrophoresis was carried out in 0.35 M 6-amino caproı¨c acid, 0.14 M acetic acid (pH 4.5). Differential scanning calorimetric measurement (DSC) Calorimetric measurements were performed by differential microcalorimetry with temperature scanning using a Microcal MC-2 instrument. Protein concentrations of 1 mg/ml and 4.5 mg/ml determined by Biuret titration were used. The calorimetric scans were performed in high or low ionic strength buffer (10 mM sodium phosphate (pH 7.4), 0.1 mM dithiotreitol and 1 M or 0.2 M NaCl, respectively). All samples were scanned three times. The nearly perfect reproducibility of the scans showed that the denaturation of all the proteins was fully reversible under the chosen experimental conditions. To correct the experimental data for the small error introduced by the slight characteristic differences of the two cells, the instrumental base-line determined with buffer in both cells was subtracted from the experimental apparent molar heat capacity at constant pressure ðCp Þ curve obtained with buffer and protein solution, respectively in the reference and sample cells. We did not take into account the fact that the heat capacity of the proteins in solution is smaller than that of the same volume of the solvent leading to a measured negative heat capacity, which is termed therefore apparent heat capacity.59

½D

½N2

½I2

HN2 þ HI þ 2 2 HD khDim l ¼ CDim CDim 2 CDim

ð1Þ

It relates at a given temperature t the average enthalpy per mole of protein expressed in dimer concentration to the molar enthalpies of the protein, in the native state (N2), in the intermediate state (I2) and in the denatured state (D), respectively. Each dimer molar enthalpy is weighted by a factor representing the corresponding percentage of dimer of each thermal species at a given temperature t. Making use of the classical relation between the molar heat capacity at constant pressure and the corresponding molar enthalpy: dH dt

Cp ¼

ð2Þ

And from (1), we can write: dkhDim l ð3Þ dt where kCpDim l is the average heat capacity per mole of protein expressed in dimer. This is an experimental quantity that we measure as a function of temperature. To relate expression (3) to the temperature, the computation steps given by Sturtevant38,60 were used. Express the equilibrium concentrations N2, I2 and D as a function of the two dissociation coefficients defined by: kCpDim l ¼

a1 ¼

½I2 þ

½D

2

CDim

ð4Þ

½D

2 ð5Þ a2 ¼ ½D

½I2 þ 2 We have defined the melting temperature, t1 and t2 as the temperature where K1 ðt1 Þ ¼ 1 and K2 ðt2 Þ ¼ 2 CDim : Finally, we have assumed a linear temperature dependence for CpN2 ; CpD ;38,61 and for CpI2 we assumed a weighted average between CpN2 and CpD according to the relation: CpI ¼ wCpN2 þ ð1 2 wÞ2CpD

ð6Þ

where w and ð1 2 wÞ are, respectively, the percentage of the native and denatured protein states. Circular dichroism measurements (CD) Measurements were performed in 0.01 cm thermostated quartz cuvettes in a Jobin-Yvon CD6 circular dichroism spectrometer and a range of wavelengths from 185 nm to 260 nm. Thermal denaturations were carried out in 10 mM sodium phosphate buffer (pH 7.4), 0.1 mM dithiotreitol, 0.2 M sodium fluoride and 1 mg/ ml of the HU dimer. All the spectra were averaged over six scans and then processed for base-line subtraction using standard software. The spectra are presented in molar mean residue ellipticity [u] expressed in (deg. cm21 mol21), where the concentration refers to amino acid residues concentration of HU dimers (using a mean residue molecular mass of 107.5 Da). Secondary

119

E. coli HU Thermal Stability

Table 4. Refinement and model statistics ˚) Resolution range (A No. of reflections s Cut-off Completeness (%) Test set (% of data) R factor (%) Rfree (%)64 No. of atoms Non-H protein atoms Solvent atoms Final G factor66 r.m.s.d. from ideal values ˚) Bond lengths (A Bond angles (deg.) Dihedral angles (deg.) Improper torsion angles (deg.) ˚ 2) Mean B factors (A Main-chain atoms Side-chain atoms Solvent atoms

20.0–2.3 4607 2.0 96 10.5 23.4 26.7 539 50 0.4 0.006 1.20 21.10 0.68 26.99 27.73 33.95

structure contents were calculated from the far ultraviolet CD spectra with the method of Johnson adapted in the CDpro programm packaging by Screerama using the program CDSSTR and the bank 6 of CD spectra.44,62 Crystal structure determination of the EcHUa2 homodimer The crystallization and the X-ray analysis of the EcHUa2 protein have been described.40 The structure was solved by the molecular replacement method using the HU crystal structure of B. stearothermophilus (PDB code 1HUU) as the search model.41,63 After rigid-body refinement, the structure was subjected to several cycles of molecular dynamics simulated annealing from 3000 K using the program CNS and manual rebuilding with the graphics program TURBO-FRODO.64,65 Incorporation of water molecules was done according to 2mFo 2 DFc and mFo 2 DFc electron density maps, geometry of the hydrogen bonds and temperature-factor values. Towards the end of the refinement, series of annealed omit maps were calculated with ten residues deleted each time to inspect the position of amino acids without model bias. The final model has an R factor of 23.4% and an Rfree ˚ resolution shell. Residues of 26.7% within the 20 – 2.3 A from 58 to 71 are missing due to the flexibility of the molecule arms. The lateral chains of the residues E12, K13, K18, T19, K51 and R55 are incomplete. The biologically relevant homodimer is generated by a crystallographic 2-fold rotation axis parallel with the c parameter. The quality and geometry of the final model were analyzed using the PROCHECK program.66 Refinement and model statistics are included in Table 4. Accession numbers The atomic coordinates of the EcHUa2 homodimer have been deposited in the RCSB Protein Data Bank under the accession code ID 1mul

Acknowledgements We are greatly indebted to Dr A. Sanson (CEA,

Saclay, France) for help in CD data collection; and to Dr J. -L. Maurizot and Dr F. Culard (CBM, CNRS, Orle´ans) for helpful discussion. This work was supported by Electricite´ de France (EDF).

References 1. Echols, H. (1990). Nucleoprotein structures initiating DNA replication, transcription, and site-specific recombination. J. Biol. Chem. 265, 14697– 14700. 2. Pettijohn, D. E. (1996). Escherichia coli and Salmonella (Neidhardt, F. C., ed.), pp. 158– 166, ASM, Washington, DC. 3. Almiron, M., Link, A. J., Furlong, D. & Kolter, R. (1992). A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli. Genes Dev. 6, 2646– 2654. 4. Rouvie`re-Yaniv, J. & Gros, F. (1975). Characterization of a novel, low-molecular-weight DNA-binding protein from Escherichia coli. Proc. Natl Acad. Sci. USA, 72, 3428– 3432. 5. Rouvie`re-Yaniv, J. (1978). Localization of the HU protein on the Escherichia coli nucleoid. Cold Spring Harbor Symp. Quant. Biol. 42, 439– 447. 6. Rouvie`re-Yaniv, J., Yaniv, M. & Germond, J. E. (1979). E. coli DNA binding protein HU forms nucleosomelike structure with circular double-stranded DNA. Cell, 17, 265– 274. 7. Dixon, N. E. & Kornberg, A. (1984). Protein HU in the enzymatic replication of the chromosomal origin of Escherichia coli. Proc. Natl Acad. Sci. USA, 81, 424 –428. 8. Hwang, D. & Kornberg, A. (1992). Opposed actions of regulatory proteins. DnaA and IciA, in opening the replication origin of Escherichia coli. J. Biol. Chem. 267, 23087– 23091. 9. Castaing, B., Zelwer, C., Laval, J. & Boiteux, S. (1995). HU protein of Escherichia coli binds specifically to DNA that contains single-strand breaks or gaps. J. Biol. Chem. 270, 10291– 10296. 10. Boubrik, F. & Rouve`re-Yaniv, J. (1995). Increased sensitivity to gamma irradiation in bacteria lacking protein HU. Proc. Natl Acad. Sci. USA, 92, 3958– 3962. 11. Li, S. & Waters, R. (1998). Escherichia coli strains lacking protein HU are UV sensitive due to a role for HU in homologous recombination. J. Bacteriol. 180, 3750– 3756. 12. Flashner, Y. & Gralla, J. D. (1988). DNA dynamic flexibility and protein recognition: differential stimulation by bacterial histone-like protein HU. Cell, 54, 713 –721. 13. Huisman, O., Faelen, M., Girard, D., Jaffe´, A., Toussaint, A. & Rouvie`re-Yaniv, J. (1989). Multiple defects in Escherichia coli mutants lacking HU protein. J. Bacteriol. 171, 3704 – 3712. 14. Wada, M., Kotsukabe, K., Komano, T., Imamoto, F. & Kano, Y. (1989). Participation of the hup gene product in site-specific DNA inversion in Escherichia coli. Gene, 76, 345– 352. 15. Dri, A.-M., Moreau, P. L. & Rouvie`re-Yaniv, J. (1992). Role of the histone-like proteins OsmZ and HU in homologous recombination. Gene, 120, 11 – 16. 16. Aki, T. & Adhya, S. (1997). Repressor induced sitespecific binding of HU for transcriptional regulation. EMBO J. 16, 3666–3674. 17. Broyles, S. & Pettijohn, D. E. (1986). Interaction of the Escherichia coli HU protein with DNA: Evidence for

120

18.

19. 20.

21.

22.

23. 24. 25.

26.

27. 28.

29.

30. 31.

32.

33.

34.

35.

formation of nucleosome-like structures with altered DNA helical pitch. J. Mol. Biol. 187, 47 – 60. Bonnefoy, E. & Rouvie`re-Yaniv, J. (1991). HU and IHF, two homologous histona-like proteins of Escherichia coli, form different protein – DNA complexes with schort DNA fragments. EMBO J. 10, 687– 696. Pontiggia, A., Negri, A., Beltrame, M. & Bianchi, M. E. (1993). Protein HU binds specifically to kinked DNA. Mol. Microbiol. 7, 343– 350. Bonnefoy, E., Takahashi, M. & Rouvie`re-Yaniv, J. (1994). DNA binding parameters of the HU protein of Escherichia coli to cruciform DNA. J. Mol. Biol. 242, 116 –129. Lavoie, B. D., Shaw, G. S., Millner, A. & Chaconas, G. (1996). Anatomy of a flexer –DNA complex inside a high-order transposition intermediate. Cell, 85, 761– 771. Lyubchenko, Y. L., Shlyakhtenko, L. S., Aki, T. & Adhya, S. (1997). Atomic force microscopic demonstration of DNA looping by GalR and HU. Nucl. Acids Res. 25, 873– 876. Oberto, J., Drlica, K. & Rouvie`re-Yaniv, J. (1994). Histones, HMG, HU, IHF: Meˆme combat. Biochimie, 76, 901– 908. Rouvie`re-Yaniv, J. & Kjeldgaard, N. O. (1979). Native Escherichia coli HU protein is a heterotypic dimer. FEBS Letters, 106, 297– 300. Oberto, J. & Rouvie`re-Yaniv, J. (1996). Serratia marcescens contains a heterodimeric HU protein like Escherichia coli and Salmonella typhimurium. J. Bacteriol. 178, 293– 297. Kano, Y., Yoshimno, S., Wada, M., Yokoyama, K., Nobuhara, M. & Imamoto, F. (1985). Molecular cloning and nucleotide sequence of the HU-1 gene of Escherichia coli. Mol. Gen. Genet. 201, 360– 362. Kano, Y., Osato, K., Wada, M. & Imamoto, F. (1987). Cloning and sequencing of the HU-2 gene of Escherichia coli. Mol. Gen. Genet. 209, 408–410. Wada, M., Kano, Y., Ogawa, T. & Okazaki, T. (1988). Construction and characterization of the deletion mutant of hupA and hupB genes in Escherichia coli. J. Mol. Biol. 204, 581– 591. Giangrossi, M., Giuliodori, A. M., Gualerzi, C. O. & Pon, C. L. (2002). Selective expression of the b-subunit of nucleoid-associated protein HU during cold shock in Escherichia coli. Mol. Microbiol. 44, 205– 216. Claret, L. & Rouvie`re-Yaniv, J. (1996). Regulation of HU alpha and HU beta by CRP and FIS in Escherichia coli. J. Mol. Biol. 263, 126– 139. Claret, L. & Rouvie`re-Yaniv, J. (1997). Variation in HU composition during growth of Escherichia coli: the heterodimer is required for long term survival. J. Mol. Biol. 273, 93 – 104. Bahloul, A., Boubrik, F. & Rouvie`re-Yaniv, J. (2001). Roles of Escherichia coli histone-like protein HU in DNA replication: HU-beta suppresses the thermosensitivity of dnaA46ts. Biochimie, 83, 219– 229. Pinson, V., Takahashi, M. & Rouvie`re-Yaniv, J. (1999). Differential binding of the Escherichia coli HU, homodimeric forms and heterodimeric form to linear, gapped and cruciform DNA. J. Mol. Biol. 287, 485– 497. Kawamura, S., Abe, Y., Ueda, T., Masumoto, K., Imoto, T., Yamasaki, N. & Kimura, M. (1998). Investigation of the structural basis for thermostability of DNA-binding protein HU from Bacillus stearothermophilus. J. Biol. Chem. 273, 19982– 19987. Wilson, K. S., Vorgias, C. E., Tanaka, I., White, S. W. & Kimura, M. (1990). The thermostability of DNA-

E. coli HU Thermal Stability

36.

37.

38.

39. 40.

41.

42. 43. 44. 45. 46.

47. 48.

49.

50.

51.

52.

53.

binding protein HU from bacilli. Protein Eng. 4, 11– 22. Welfle, H., Misselwitz, R., Welfle, K., Schindelin, H., Scholtz, A. S. & Heinemann, U. (1993). Conformations and conformational changes of four Phe ! Trp variants of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis studied by circular dichroism and fluorescence spectroscopy. Eur. J. Biochem. 217, 849– 856. Christodoulou, E. & Vorgias, C. E. (2002). The thermostability of DNA-binding protein HU from mesophilic, thermophilic, and extreme thermophilic bacteria. Extremophiles, 6, 21 – 31. Tamura, A. & Sturtevant, J. M. (1995). A thermodynamic study of mutant forms of Streptomyces subtilisin inhibitor. I. Hydrophobic replacements at the position of Met103. J. Mol. Biol. 249, 625– 635. Johnson, W. C. (1999). Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins: Struct. Funct. Genet. 35, 307– 312. Coste, F., Hervouet, H., Oberto, J., Zelwer, C. & Castaing, B. (1999). Crystallisation and preliminary X-ray diffraction analysis of the homodimeric form a2 of the HU protein from Escherichia coli. Acta Crystallog. sect. D, 55, 1952– 1954. Tanaka, I., Appelt, K., Dijk, J., White, S. W. & Wilson, K. S. (1984). 3-A resolution structure of a protein with histone-like properties in prokaryotes. Nature, 310, 376– 381. Makhatadze, G. I. & Privalov, P. (1995). Energetics of protein structure. Advan. Protein Chem. 47, 307– 425. Tamura, A. & Privalov, P. L. (1997). The entropy cost of protein association. J. Mol. Biol. 273, 1048– 1060. Johnson, W. C. (1990). Protein secondary structure and circular dichroism: a practical guide. Proteins: Struct. Funct. Genet. 7, 205– 214. Baldwin, R. L. (2002). Making a network of hydrophobic clusters. Science, 295, 1627– 1658. Chamberlain, A. K. & Marqusee, S. (2000). Comparison of equilibrium and kinetic approches for determining protein folding mechanisms. Advan. Protein Chem. 53, 283– 323. Freire, E. & Murphy, K. P. (1991). Molecular basis of cooperativity in protein folding. J. Mol. Biol. 222, 687– 698. Dominy, B. N., Perl, D., Schmid, F. X. & Brooks, C. L., 3rd (2002). The effects of ionic strength on protein stability: the cold shock protein family. J. Mol. Biol. 319, 541– 554. Pace, C. N., Hebert, E. J., Shaw, K. L., Schell, D., Both, V., Krajcikova, D. et al. (1998). Conformational stability and thermodynamics of folding of ribonucleases Sa, Sa2 and Sa3. J. Mol. Biol. 279, 271– 286. Bonnefoy, E., Almeida, A. & Rouvie`re-Yaniv, J. (1989). Lon-dependent regulation of the DNA binding protein HU in Escherichia coli. Proc. Natl Acad. Sci. USA, 86, 7691– 7695. Asgeirsson, B., Hauksson, J. B. & Gunnarsson, G. H. (2000). Dissociation and unfolding of cold-active alkaline phosphatase from atlantic cod in the presence of guanidinium chloride. Eur. J. Biochem. 267, 6403– 6412. Staniforth, R. A., Giannini, S., Higgins, L. D., Conroy, M. J., Hounslow, A. M., Jerala, R. et al. (2001). Threedimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily. EMBO J. 20, 4774– 4781. Canals, A., Pous, J., Guasch, A., Benito, A., Ribo, M., Vilanova, M. & Coll, M. (2001). The structure of

121

E. coli HU Thermal Stability

54.

55. 56.

57. 58. 59. 60.

61.

engineered domain-swapped ribonuclease dimer and its implications for the evolution of proteins toward oligomerization. Structure, 9, 967– 976. Reme´nyi, A., Tomilin, A., Pohl, E., Lins, K., Philippsen, A., Reinbold, R. et al. (2001). Differential dimer activities of the transcription factor Oct-1 by DNA-induced interface swapping. Mol. Cell, 8, 569–580. Doyle, S. M., Braswell, E. H. & Teschke, C. M. (2000). SecA folds via a dimeric intermediate. Biochemistry, 39, 11667 –11676. Zulianello, L., de la Gorgue de Rosny, E., van Ulsen, P., van de Putte, P. & Goosen, N. (1994). The HimA and HimD subunits of integration host factor can specifically bind to DNA as homodimers. EMBO J. 13, 1534– 1540. Vis, H., Dobson, C. M. & Robinson, C. V. (1999). Selective association of proteins molecules followed by mass spectroscopy. Protein Sci. 8, 1368– 1370. Gornall, A. C., Bardawill, C. J. & David, M. (1949). Determination of serum proteins by the biuret reaction. J. Biol. Chem. 177, 751– 766. Privalov, P. L. & Potekhin, S. A. (1986). Scanning microcalorimetry in studying temperature-induced changes in proteins. Methods Enzymol. 131, 4 – 51. Tamura, A., Kojima, S., Miura, K. & Sturtevant, J. M. (1995). A thermodynamic study of mutant forms of Streptomyces subtilisin inhibitor. II. Replacements at the interface of dimer formation, Val13. J. Mol. Biol. 249, 636– 645. Sturtevant, J. M. (1987). Biochemical applications of

62.

63. 64. 65. 66.

67.

68.

differential scanning calorimetry. Annu. Rev. Phys. Chem. 38, 463–489. Sreerama, N. & Woody, R. W. (2000). Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal. Biochem. 287, 252– 260. Rossman, M. G. & Blow, L. H. (1962). The detection of sub-unit within the crystallographic asymetric unit. Acta Crystallog. 15, 24 – 31. Bru¨nger, A. (1992). Free R value: a novel statistical quantity for assessing the accuracy of crystal structure refinement. Nature, 355, 472– 475. Roussel, A. & Cambillau, C. (1991). Silicon Graphics Geometry Partners Directory Silicon Graphics, pp. 77 – 88, Mountain View, CA. Laskowski, R. A., MacArthur, W., Moss, D. S. & Thornton, J. M. (1993). Procheck: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673 –4680. Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. (1999). ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics, 4, 305– 308.

Edited by J. O. Thomas (Received 7 March 2003; received in revised form 28 May 2003; accepted 2 June 2003)