Recognition of the Intrinsically Flexible Addiction Antidote MazE by a Dromedary Single Domain Antibody Fragment

STRUCTURE, THERMODYNAMICS OF BINDING, STABILITY, AND INFLUENCE ON INTERACTIONS WITH DNA*

Jurij LahDagger §, Irina Marianovsky||, Gad Glaser||, Hanna Engelberg-Kulka**, Jörg KinneDagger Dagger , Lode WynsDagger , and Remy LorisDagger §§

From the Dagger  Department of Ultrastructure, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 St. Genesius Rode, Belgium, the § Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia, the Departments of || Cellular Biochemistry and ** Molecular Biology, Hebrew University-Hadassah Medical School, Ein Kerem, Jerusalem, 91120 Israel, and the Dagger Dagger  Central Veterinary Research Laboratories, P.O. Box 591, Dubai, United Arab Emirates

Received for publication, September 25, 2002, and in revised form, January 14, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Escherichia coli mazEF operon defines a chromosomal addiction module that programs cell death under various stress conditions. It encodes the toxic and long-lived MazF and the labile antidote MazE. The denaturation of MazE is a two-state reversible dimer-monomer transition. At lower concentrations the denatured state is significantly populated. This leads to a new aspect of the regulation of MazE concentration, which may decide about the life and death of the cell. Interactions of MazE with a dromedary antibody domain, cAbMaz1 (previously used as a crystallization aid), as well as with promoter DNA were studied using microcalorimetric and spectroscopic techniques. Unique features of cAbMaz1 enable a specific enthalpy-driven recognition of MazE and, thus, a significant stabilization of its dimeric native conformation. The MazE dimer and the MazE dimer-cAbMaz1 complex show very similar binding characteristics with promoter DNA, i.e. three binding sites with apparent affinities in micromolar range and highly exothermic binding accompanied by large negative entropy contributions. A working model for the MazE-DNA assembly is proposed on the basis of the structural and binding data. Both binding and stability studies lead to a picture of MazE solution structure that is significantly more unfolded than the structure observed in a crystal of the MazE-cAbMaz1 complex.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In general, programmed cell death is required for the elimination of the superfluous or potentially harmful cells (1, 2). In Escherichia coli, cell death is programmed by genetic elements called "addiction modules" (3-8). These consist of two genes encoding for a stable toxin and its labile antidote. Toxin and antidote are co-expressed. Their expression is auto-regulated at the level of transcription either by a non-covalent complex formed between toxin and antidote or by the antidote alone (3-18). In the absence of co-expression the antidote is rapidly degraded by a specific protease, enabling the toxin to attack its target. The target of the toxin is known only in the plasmidic ccdAB and kid-kis systems. In ccdAB, CcdB on plasmid F attacks the A subunit of gyrase, whereas in the kid-kis system Kid on plasmid R1 targets DnaB (19-21).

Until recently, attention was paid mainly to the extrachromosomal (plasmid) addiction modules, which are responsible for the death that occurs upon accidental plasmid loss (3-8). However, the E. coli chromosome also contains several operons homologous to those found in plasmid addiction systems (22-25). The first discovered regulable prokaryotic chromosomal addiction module is the mazEF system (or chpA), which encodes for toxic and long-lived MazF and anti-toxic and short-lived MazE (6, 9, 25). In contrast to the extra-chromosomal addiction modules that are triggered by plasmid loss, death mediated by the chromosomal mazEF is achieved under several stress conditions that prevent mazEF expression. It was initially found that the mazEF module is under the control of 3',5'-guanosinebispyrophosphate (ppGpp) (25, 26), the amino acid starvation signal produced by RelA protein (27). Overproduction of ppGpp leads to inhibition of the expression of mazEF and, thereby, to cell death (25, 26). However, inhibition of mazEF expression, and thus induction of cell death, can also be achieved by using general inhibitors of transcription and/or translation like antibiotics (rifampicin, chloramphenicol and spectinomycin) (28, 29) and the toxic protein Doc (30). In each case (ppGpp (25, 26), antibiotics (28, 29) and the Doc protein (30)), the inhibition of gene expression leads to a lack of the labile MazE and, thereby, allows the action of the more stable MazF to kill the cells.

To improve our understanding of addiction systems at the molecular level, structural and also thermodynamic characterization of the proteins involved is needed. The latter is difficult to achieve because of the problems of expression of the toxins and the labile character of the antidotes. Therefore, for some time only a crystal structure of the toxin CcdB has been known (31). The thermodynamic information on addiction proteins is also rather scarce, and often only partial answers are available (10, 32-34).

Antibodies and their derivative fragments have long been used as tools in a variety of applications in fundamental research, biotechnology, diagnosis, and human therapy (35, 36). In contrast to conventional IgG molecules, one type of the antibodies generated by camels, dromedaries, and llamas (camelids) is formed by two heavy chains but has no light chains (37). Particularly interesting are camelid single variable domain antibody fragments (VHH), which contain the smallest antigen-binding unit with a molecular size of approx 15 kDa. They are characterized by high-yield production, high solubility, and high thermodynamic stability (38, 39).

As MazE alone has a large fraction of unstructured polypeptide, a MazE-specific VHH (cAbMaz1) fragment has been used as a crystallization aid, leading to the first crystal structure of an addiction antidote.1 In the current paper we focus on the thermodynamics of the MazE-cAbMaz1 and MazE-DNA (MazE promoter) interacting systems and correlate it with the structure. The research described here is the first example of a characterization of MazE binding and denaturation energetics.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation of the Proteins and MazE Promoter DNA-- Expression and purification of MazE and the cAbMaz1 fragment are described elsewhere.1 MazE and cAbMaz1 solutions for spectroscopic and calorimetric measurements were prepared by extensive dialysis against 50 mM sodium cacodylic buffer, pH 6.9, containing 150 mM NaCl and a 10-min degassing of the sample solutions before the measurements. For DNA binding studies, the same buffer without additional salt was used. Concentrations of a 98-residue-long, His tag-fused MazE protein and a 135-residue-long cAbMaz1 antibody (Fig. 1) were determined spectrophotometrically by measuring absorbance at 280 nm. The corresponding extinction coefficients were obtained from MazE and cAbMaz1 amino acid compositions by the method introduced by Gill and von Hippel (41) (www.expasy.ch).

The 50-base pair, double-stranded oligonucleotide (5'-TGCTCGTATCTACAATGTAGATTGATATATACTGTATCTACATATGATAG-3' and 3'-ACGAGCATAGATGTTACATCTAACTATATATGACATAGATGTATACTATC-5') containing the MazE promoter sequence (underlined) was purchased from Invitrogen. To evaluate the specificity of MazE binding to the promoter DNA, the 107-bp control (not related to mazEF module) DNA was used. The same DNA was used as a control (not related) DNA in the studies of the ccdAB addiction module as well (32). Lyophilized single strands were dissolved in 50 mM sodium cacodylate, pH 6.9, and extensively dialyzed against the same buffer. The concentrations of single strands were determined by UV absorption spectroscopy at 260 nm by using extinction coefficients calculated on the basis of the nearest neighbor approximation (42). The duplexes were obtained by mixing the corresponding single strands in the 1:1 molar ratio.

Isothermal Titration Calorimetry (ITC)2-- The heat accompanying MazE + cAbMaz1, DNA + MazE, and DNA + MazE-cAbMaz1 associations was measured by an Omega isothermal titration calorimeter (MicroCal, Northampton, MA). In MazE + cAbMaz1 experiments at 25, 35, 45, and 55 °C the MazE dimer solution (1.33 ml) was titrated by cAbMaz1 solution in the same buffer using a motor-driven 250-µl syringe. cAbMaz1 concentration was about 100 µM, whereas the MazE dimer concentration in the titration cell was 4.8 µM. DNA + MazE and DNA + MazE-cAbMaz1 experiments were performed at 25 °C. MazE dimer and MazE-cAbMaz1 concentrations were around 50 µM, whereas the DNA concentration in the titration cell was about 50 times lower. Each injection generated a heat burst, with the area under the curve being proportional to the heat of interaction (Fig. 5a). The titration curves (Figs. 5b and 8) were constructed by subtraction of the heat effects that accompany the ligand dilution.

Fluorescence Spectroscopy (FL)-- FL spectra were recorded using an AMINCO-Bowman Series2 luminescence spectrometer (Spectronic Instruments Rochester, NY) equipped with a thermally controlled cell holder and a cuvette of 1 cm path length. MazE + cAbMaz1 titrations were performed at 25 °C and 45 °C by incrementally injecting 4-20-µl aliquots of cAbMaz1 solution into 2 ml of 0.5-1 µM MazE dimer solution in the same buffer. After each injection, FL emission spectrum was recorded between 290 and 480 nm (lambda ext = 280 nm). In the case of DNA titration with MazE at 25 °C the emission spectrum was recorded between 310 and 380 nm (lambda ext = 300 nm). The 1.5 µM DNA oligomer containing the promoter sequence was titrated by a 21 µM solution of MazE dimer.

Circular Dichroism (CD) Spectropolarimetry-- CD spectra were recorded on J-715 spectropolarimeter (JASCO, Tokyo, Japan). MazE + cAbMaz1 titrations were performed at 25 and 45 °C by incrementally injecting 4-20 µl aliquots of cAbMaz1 solution into 2 ml of ~1 µM MazE dimer solution (1-cm cuvette) in the same buffer. After each injection, the ellipticity, theta , was monitored between 210 and 260 nm (Fig. 3). The denaturation of MazE, cAbMaz1, and their tetrameric complex was followed by recording the far UV (205-260 nm) CD spectra at different temperatures with the temperature step of 2 or 1 °C at the transition region (Fig. 7). The temperature of the sample was controlled by a sensor built into the cuvette holder and connected to a Haake N3 (Gebrueder Haake, Karlsruhe, Germany) circulating bath which adjusted the temperature of the sample with an accuracy of 0.1 °C. The concentrations of MazE, cAbMaz1, and their tetrameric complex in a 0.1- or 0.2-cm cuvette were about 10 µM.

Differential Scanning Calorimetry (DSC)-- The thermally induced transitions of MazE, cAbMaz1, and their tetrameric complex were measured using a Nano-II DSC differential scanning calorimeter (Calorimetry Sciences, Provo, UT). The heating rate was 1 °C/min, and the concentration of proteins in the measuring cell (0.33 ml) was about 10 µM. To obtain the presented thermograms (Delta CP versus T curves, Fig. 6a), the heat capacity of the protein in the native state was subtracted from the raw signal (corrected for buffer contribution). The transition enthalpies Delta Hcal were obtained by integration of Delta CP versus T curves. Two consecutive temperature scans were carried out to observe the extent of reversibility, which was higher than 0.8 for all the proteins.

FL, CD, and Calorimetric Titrations-- Changes in the spectral properties (Figs. 3 and 4) suggest that the cAbMaz1-MazE complex formation is accompanied by structural alterations of MazE or/and cAbMaz1. Moreover, binding of the first cAbMaz1 molecule to one MazE dimer binding site influences the binding of the second one to the other available site. Thus, the spectral changes (CD and FL) and the heat effects seen during titration experiments may contain contributions from direct binding and structural changes. To describe the cAbMaz1-MazE association, a mass action model that includes the mentioned contributions is proposed, as shown below in Reaction 1,


<UP>   M<SUB>2</SUB></UP>+<UP>A</UP> <LIM><OP><ARROW>↔</ARROW></OP><UL>K<SUB>1</SUB></UL></LIM> <UP>M′<SUB>2</SUB>A′</UP>+<UP>A</UP> <LIM><OP><ARROW>↔</ARROW></OP><UL>K<SUB><UP>2</UP></SUB></UL></LIM><UP> M″<SUB>2</SUB>A″<SUB>2</SUB></UP>

<UP>R<SC>eaction</SC> 1</UP>
with K1 and K2 defined in Equations 1 and 2, respectively,
K<SUB><UP>1</UP></SUB>=<FR><NU>[<UP>M′<SUB>2</SUB>A′</UP>]</NU><DE>[<UP>M<SUB>2</SUB></UP>][<UP>A</UP>]</DE></FR> (Eq. 1)

K<SUB><UP>2</UP></SUB>=<FR><NU>[<UP>M″<SUB>2</SUB>A″<SUB>2</SUB></UP>]</NU><DE>[<UP>M′<SUB>2</SUB>A′</UP>][<UP>A</UP>]</DE></FR> (Eq. 2)
and where M2, A, M'2A', and M"2A"2 represent the MazE dimer, the cAbMaz1 monomer, and their trimeric and tetrameric complexes in the native state, respectively. Quantities in the square brackets are the equilibrium molar concentrations; K1 and K2 are the corresponding apparent association constants. Overall change in the thermodynamic quantity (standard Gibbs free energy, Delta G°; standard enthalpy, Delta H°; standard entropy, Delta S°) for the presented process can be expressed as a sum of contributions of the M"2 + 2A" association as rigid bodies in the final conformation and other contributions which involve the conformational changes,
&Dgr;G°=&Dgr;G<SUB><UP>rb</UP></SUB>°+&Dgr;G<SUB><UP>other</UP></SUB>° (Eq. 3)

&Dgr;G<SUB><UP>rb</UP></SUB>°=<UP>−</UP>RT<UP>ln</UP>([M″<SUB>2</SUB>A″<SUB>2</SUB>]/[M″<SUB>2</SUB>][A″]<SUP>2</SUP>) (Eq. 4)

&Dgr;G<SUB><UP>other</UP></SUB><UP>°</UP>=<UP>−</UP>RT<UP>ln</UP>([<UP>M″<SUB>2</SUB></UP>]<UP>/</UP>[<UP>M<SUB>2</SUB></UP>])+<UP>2ln</UP>([<UP>A″</UP>]<UP>/</UP>[<UP>A</UP>])] (Eq. 5)
as shown above in Equations 3, 4, and 5. It follows that the contributions of conformational effects (other) can be estimated as a difference between experimentally obtained Delta G°, Delta H°, and TDelta S° and the corresponding Delta Grb°, Delta Hrb°, and TDelta Srb°, which were estimated by M''2A''2-structure-based calculations.

By assuming linear dependence of a measured physical property (F) on the concentration of individual components in ideal solution, it is possible, by subtracting the contributions of M2 and A, to obtain the difference in physical property (Delta F), which can be expressed as shown below (43) in Equation 6,
&Dgr;F=<LIM><OP>∑</OP><LL>i=1</LL><UL>2</UL></LIM>&Dgr;f<SUB><UP>i</UP></SUB>[<UP>M′<SUB>2</SUB>A′<SUB>i</SUB></UP>]<UP> where &Dgr;</UP>f<SUB><UP>i</UP></SUB>=f<SUB><UP>M′<SUB>2</SUB>A′<SUB>i</SUB></UP></SUB>−f<SUB><UP>M<SUB>2</SUB></UP></SUB>−<UP>i</UP>f<SUB><UP>A</UP></SUB> (Eq. 6)

<UP>and if i</UP>=1 ⇒ ′=′<UP> and if i</UP>=2 ⇒ ′=″
where fM2, fA, and fM'2A'i are concentration-independent physical properties of M2, A and M'2A'i, respectively. In each of the techniques used in this work, the described properties have specific physical meaning as defined in the following: (i) FL (Fig. 3b), where Delta F is difference fluorescence and Delta fi depends on the optical path length, quantum yields, intensity of incident light, and molar extinction coefficients of M2, A, and M'2A'i; (ii) CD, where Delta F = Delta theta  = difference ellipticity and Delta fi is the product between difference molar ellipticity of M'2A'i and optical path length; (iii) ITC (Fig. 8), where Delta F = Q = cumulative heat effect given per mole of added ligand at single injection and Delta fi is the product between Delta Hi° (standard enthalpy of M'2A'i complex formation) and the volume of solution in the measuring cell divided by the amount of added ligand per injection. In the ITC, the differential form of Equation 6 is usually used where the signal is given as Delta H (enthalpy change given per mole of added ligand at single injection) and Delta fi as Delta Hi° (Fig. 5b).

In the case of DNA + M2 titration, the model that assumes binding of M2 on identical sites on DNA was used. The model was derived from Reaction 1 and Equations 1 and 6 for i = 1.

It follows from Reaction 1 and Equation 6 that spectroscopic and calorimetric titration curves (Figs. 3b, 5b, and 8) can be at given total concentrations of M2 and A described only in terms of parameters Delta fi and Ki. Their values were obtained by fitting the model functions (Equation 6) to the experimental titration curves using the non-linear procedure based on Levenberg-Marquardt minimization of the non-weighted chi 2 function (44).

Temperature-dependent CD and DSC-- Our results reveal that all monitored denaturation processes can be adequately described as reversible two state transitions, as presented in Reaction 2 and Equation 7,


<UP>N</UP><SUB>n</SUB> <LIM><OP><ARROW>↔</ARROW></OP><UL>K</UL></LIM> n<UP>D</UP>

<UP>R<SC>eaction</SC> 2</UP>

&Dgr;G°=−RT<UP>ln</UP>K=−RT<UP>ln</UP><FENCE><FR><NU>(n&agr;)<SUP>n</SUP> [P]<SUP>n−1</SUP></NU><DE>1−&agr;</DE></FR></FENCE> (Eq. 7)
where Nn represents M2 (n = 2), A (n = 1) and M"2A"2 (n = 4) in the native state, while D corresponds to the MazE and cAbMaz1 in the denatured state. K is the equilibrium constant of denaturation, Delta G° is the corresponding standard Gibbs free energy change, n is the number of subunits to which each protein dissociates upon denaturation, alpha  is the degree of denaturation, and [P] is the total protein concentration given per mole of protein in its native state. Delta G° can also be expressed by the integrated Gibbs-Helmholtz equation shown below in Equation 8,
&Dgr;G°=T<FENCE><FR><NU>&Dgr;G°(T<SUB>1/2</SUB>)</NU><DE>T<SUB>1/2</SUB></DE></FR>+&Dgr;H°(T<SUB>1/2</SUB>)<FENCE><FR><NU>1</NU><DE>T</DE></FR>−<FR><NU>1</NU><DE>T<SUB>1/2</SUB></DE></FR></FENCE></FENCE> (Eq. 8)

+&Dgr;C<SUB>P</SUB>°<FENCE>1−<FR><NU>T<SUB>1/2</SUB></NU><DE>T</DE></FR>−<UP>ln</UP><FENCE><FR><NU>T</NU><DE>T<SUB>1/2</SUB></DE></FR></FENCE></FENCE>}
in which Delta H°(T1/2) is the standard enthalpy of denaturation at the reference temperature T1/2 (transition temperature at alpha  = 0.5), and Delta CP° is the corresponding standard heat capacity change assumed to be independent of temperature. According to the model (Reaction 2 and Equation 7), alpha  can be expressed as a function of ellipticity, theta  (measured at single wavelength), as shown in Equation 9,
<UP>&agr; = </UP><FR><NU><UP>&thgr; − &thgr;<SUB>N</SUB></UP></NU><DE><UP>&thgr;<SUB>D</SUB> − &thgr;<SUB>N</SUB></UP></DE></FR> (Eq. 9)
where theta N and theta D are the ellipticities of the native and denatured state, respectively, which are assumed to be linear functions of temperature. In the case of DSC, the measured Delta CP (native state is a reference state) can be expressed as (45-47) as shown in Equation 10,
  <UP>&Dgr;</UP>C<SUB><UP>P</UP></SUB>=&agr;&Dgr;C<SUB><UP>P</UP></SUB>°+ <FR><NU>&agr;(1 − &agr;)</NU><DE>n<UP> − &agr;</UP>(n<UP>−1</UP>)</DE></FR> <FR><NU>(<UP>&Dgr;</UP>H°(T<SUB>1/2</SUB>) + &Dgr;C<SUB>P</SUB>°(T−T<SUB><UP>1/2</UP></SUB>))<SUP><UP>2</UP></SUP></NU><DE>RT<SUP><UP>2</UP></SUP></DE></FR> (Eq. 10)
where all quantities are defined by Reaction 2 and Equations 7 and 8. Taking into account Reaction 2 and Equations 7 and 8, the temperature profile can be described in terms of parameters Delta H °(T1/2), Delta CP°, and T1/2. Their values were obtained by fitting of the model function (CD = Equation 9; DSC = Equation 10) to the experimental temperature profiles using the previously mentioned non-linear chi 2 regression procedure (44).

Structure-based Thermodynamic Calculations-- The non-polar (ASAN) and polar accessible surface areas (ASAP) of proteins were calculated with the program NACCESS, version 2.1 (48). The ASA of native proteins was obtained from the crystal structure of the MazE-cAbMaz1 complex (probe size = 1.4 Å). ASA of the denatured proteins was estimated as the sum of the accessibilities of the protein residues in an extended Ala-X-Ala tripeptide. Delta CP,rb° values were calculated from changes in non-polar and polar accessible areas from the equation introduced by Murphy and Freire (49),


<UP>&Dgr;</UP>C<SUB><UP>P,rb</UP></SUB><UP>° = 0.45</UP>[<UP>cal mol<SUP>−1</SUP>K<SUP>−1</SUP><A><AC>A</AC><AC>˚</AC></A><SUP>−2</SUP></UP>]<UP>·&Dgr;</UP>ASA<SUB><UP>N</UP></SUB> (Eq. 11)

<UP> − 0.26</UP>[<UP>cal mol<SUP>−1</SUP>K<SUP>−1</SUP>Å<SUP>−2</SUP></UP>]<UP>·&Dgr;</UP>ASA<SUB><SUB>P</SUB></SUB>
which is shown above in Equation 11. For binding and denaturation of the proteins studied here, the combination of Equation 11 and similar relations introduced by Spolar and Record (50), Myers et al. (51), and Makhatadze and Privalov (52) results in the same average Delta CP,rb° values as those obtained by Equation 11 alone. The enthalpy change for M"2 + A" (rigid body) association was calculated as (53, 54) as shown in Equation 12,
<UP>&Dgr;</UP>H<SUB><UP>rb</UP></SUB><UP>° = −8.44</UP>[<UP>cal mol<SUP>−1</SUP><A><AC>A</AC><AC>˚</AC></A><SUP>−2</SUP></UP>]<UP>·&Dgr;ASA<SUB>N</SUB></UP> (Eq. 12)

<UP>+ 31.4</UP>[<UP>cal mol<SUP>−1</SUP><A><AC>A</AC><AC>˚</AC></A><SUP>−2</SUP></UP>]<UP>·&Dgr;</UP>ASA<SUB><UP>P</UP></SUB><UP> + &Dgr;</UP>C<SUB><UP>P,rb</UP></SUB><UP>°</UP>(T<UP> − 333.15</UP>)
and the entropy change upon rigid body association was calculated as a sum of three contributions (53-55),
&Dgr;S<SUB><UP>rb</UP></SUB>°=&Dgr;S<SUB><UP>sol</UP></SUB>°+&Dgr;S<SUB><UP>sc</UP></SUB>°+&Dgr;<UP>S<SUB>mix</SUB>°</UP> (Eq. 13)
which are shown above in Equation 13. The solvation term, Delta Ssol°, was obtained as: Delta CP,rb°ln(T/385.15) (55, 56). The term that reflects the change in side chain conformational entropy, Delta Ssc°, was calculated as the sum over each amino acid in the protein-protein interface (excluding alanine, proline, and disulfide-bonded cysteine), scaling its side chain conformational entropy by its change in ASA normalized to its ASA in Ala-X-Ala tripeptide (54), i.e. Sigma (Delta ASAsc ssc°/ASAAla-X-Ala). ssc° was taken from Lee et al. (57). Delta Smix° was calculated as a "cratic" term (58) that reflects the mixing of solvent and solute molecules and effectively accounts for the entropy changes due to changes in translational/rotational degrees of freedom upon binding (54, 55). For the M"2 + A" left-right-arrow M"2A" reaction using a 1 M standard state, this equals Rln(1/55.5).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Structure of cAbMaz1 and Its Interaction with MazE

Initially, we raised an immune response against MazE in a dromedary in order to identify VHH domains that could be used as aids for the crystallization of MazE. Cloning the VHH repertoire in a phage display vector and isolating the MazE binders resulted in the identification of a single gene fragment that encodes a VHH (cAbMaz1) that interacts with MazE with high affinity.

The amino acid sequence of cAbMaz1 is shown in Fig. 1 and contains all the VHH-specific features that distinguish VHH domains from classic VH domains. We have recently reported the successful crystallization of MazE in complex with cAbMaz1 and discussed the structure with emphasis on MazE.1 Here we analyze the complex in terms of its inter-protein contacts. All three hypervariable regions (H1, H2, and H3) of cAbMaz1 are involved in the interaction with MazE. However, H1 plays only a marginal role (60 Å2 buried), whereas H2 (190 Å2 buried) and H3 (270 Å2 buried) have the largest contribution. In contrast to many other VHH domains, the interactions involve mainly side chain rather than main chain atoms. It was suggested previously (59) that the preferential use of main chain atoms by VHH domains could compensate for the lack of contribution to binding from H1 as well as the missing VL domain.


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Fig. 1.   Amino acid sequence of cabmaz1 aligned with types of other camelid VHH domains of known three-dimensional structure. The limits of the framework regions were determined based on the superposition of the structures of all VHH fragments. The C-terminal His tails are not shown. beta -strands are indicated by arrows. Residues that make up the specific signature for a VHH domain are indicated.

Like most other camelid VHH domains, the H1 conformation is unique, resembling neither the canonical structures observed in classical VH domains nor any of the H1 conformations of other camelid VHHs with the known three-dimensional structure. The H2 conformation, on the other hand, is of canonical type 2A and closely resembles that of cabhcg (60) and cabamb9 (61). This conformation is the one most commonly observed for camelid VHH's.

The MazE dimer contains two structurally identical binding sites (Fig. 2a) for the antibody fragment. cAbMaz1 recognizes a cluster of hydrophobic, mainly aromatic residues that extend from the hydrophobic core of MazE (Fig. 2b). The epitope recognized by cAbMaz1 consists of two polypeptide stretches. Most of the interactions involve the short alpha -helix (18-24), of which about 340 Å2 gets buried upon complexation. The remaining 180 Å2 of buried surface comes from residues (35-40). It should be noted that these two stretches come from two different monomers forming the MazE dimer. Thus, cAbMaz1 specifically recognizes the dimer and is, as such, expected to stabilize the dimeric folded conformation of MazE.


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Fig. 2.   Panel a, overall view of the crystal structure of the MazE-cAbMaz1 complex. Two cAbMaz1 molecules (green) are bound to identical sites on opposite sides of the MazE dimer (red and blue). Panel b, stereo view of the epitope on MazE recognized by cAbMaz1. Residues interacting with the antibody are shown as ball-and-stick figures. The epitope consists of two stretches (18-24 and 35-40) coming from two different MazE monomers. Panel c, left, Trp-9 (in green) of MazE, which is involved in creating the MazE dimer via planar stacking with itself. The MazE dimer consists of two monomers presented in light and dark tones, respectively. The cAbMaz1 is not shown because it does not come close to Trp-9. Panel c, right, Trp-102 (in green) of cAbMaz1 (light tone) interacting with MazE dimer (dark tone). It interacts with Pro-18, Leu-21 Ala-24, and Thr-20 of one MazE monomer and with Leu-37 of the other.

Thermodynamics of cAbMaz1 Binding to MazE

It can be seen from a crystal structure that MazE exists as a dimer in a solid state. It was proven by gel filtration in 50 mM sodium cacodylic buffer (pH 6.9) that MazE exists as a dimer also in solution and that its dimeric state does not change under the experimental conditions applied in the binding studies.

Fluorescence Spectroscopy-- Fig. 3a shows raw FL emission spectra that correspond to the titration of the MazE dimer (M2) by cAbMaz1 (A) at 45 °C. It can be seen that the intensity changes more rapidly when the A/M2 molar ratio (r) is <= 1, whereas at r > 1 the changes are less pronounced. The MazE + cAbMaz1 association is accompanied by a blue shift of FL maximum. The shift is about 8 nm in comparison with the corresponding sum of FL spectra of "total MazE" and "total cAbMaz1" at r = 1 (Fig 3a). At r = 2 the blue shift is reduced to ~5 nm. The FL spectral changes (induced intensity, blue shift) are a consequence of different environments of Trp residues in the bound and free state of MazE and cAbMaz1. According to the crystal structure, the ordered part of MazE contains a single tryptophane (Trp-9) that does not interact with the antibody but is involved in creating the MazE dimer via planar stacking with itself (Fig. 2c). By titration of MazE to the buffer solution, we observed linear dependence of FL intensity on MazE concentration with no spectral shift. This is an additional proof that the oligomeric state of MazE stays the same in the concentration range used in FL measurements. cAbMaz1 contains three tryptophanes of which one, Trp-102, is important for binding MazE. In the complex, it interacts hydrophobically with Pro-18, Leu-21, Ala-24, and Thr-20 of one MazE monomer and with Leu-37 of the other (Fig 2c). Therefore, the blue shift and the intensity changes can be interpreted as arising from the shielding of Trp-102 from its contact with water. Because of the fact that FL spectral properties accompanying binding are different at r < 1 and r > 1 and that both cAbMaz1 binding sites on MazE are structurally identical, the differences are most probably caused by structural changes in MazE and/or cAbMaz1.


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Fig. 3.   FL spectra accompanying MazE (M2) + cAbMaz1 (A) titration at 45 °C at A/M2 molar ratio r varying between 0 and 3.5. Dotted lines represent the spectra up to r = 1 and full lines at r > 1. The bold line represents a sum of the spectra of "total" M2 and "total" A at r = 1 (panel a). The corresponding titration curves at 350 nm measured at 25 °C () and 45 °C (open circle ) are presented in panel b. Lines are graphs of the best fitted model function (Equation 6). Speciation diagrams at 25 °C (dotted line) and 45 °C (full line) calculated from the best fitted values of apparent binding constants (Reaction 1 and Equations 1 and 2) are presented in panel c. alpha  is the fraction of each MazE species.

By subtraction of the contributions of MazE dimer (M2) and cAbMaz1 (A) at each titration point, the difference spectra and the corresponding titration curve at 350 nm (Fig. 3b) were constructed. Observations of spectra and titration curves suggest that A binds to M2 in two distinctive binding modes. This hypothesis was tested and proved by fitting of the model function (Equation 6) to the experimental titration curve. It can be seen in Fig. 3b that model function with K1 and K2 as adjustable parameters correlates well with the experimental curves measured at 25 and 45 °C. The obtained K1 values are about one order of magnitude higher than the values of K2 (Table I), which strongly suggests that the binding of two A molecules to M2 is anti-cooperative. This allosteric effect can be seen more clearly in speciation diagram (Fig. 3c) calculated from the fitted K1 and K2 values at 25 and 45 °C. Up to r approx  0.5 lowering of free M2 concentration is a consequence of M'2A' formation, whereas at r > 0.5 the M"2A"2 complex starts to form and finally becomes the predominating species in solution at r > 1.5 (Fig. 3c). In our FL titration experiments the initial concentrations of M2 were in the 0.5-1 µM range, which enabled us to obtain reliable K1 and K2 in cases when their values were lower than approx 108 M-1. A significant dependence of the model function (Equation 6) on K1 (K2) is lost at K1 (K2) >108 M-1 and, therefore, the value of K1 determined at 25 °C (Table I) should be considered only as a best lower estimate of the apparent binding constant.


                              
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Table I
Comparison of the binding parameters K1 and K2
Apparent equilibrium constants K1 and K2 accompanying binding of the first and the second cAbMaz1 molecule to MazE dimer obtained by model analysis of calorimetric (ITC), fluorimetric (FL), and spectropolarimetric (CD) titration curves.

CD Spectropolarimetry-- The far UV CD spectra that accompany the titration of M2 by A at 25 °C are presented in Fig. 4. The intensity of the CD signal around 220 nm decreases with r up to r approx  1 and increases at r > 1. This fully supports the conclusions made in the case of FL spectroscopy that binding of A to M2 occurs in two different binding modes. The changes in the far UV CD spectra induced by binding correspond to the changes in the secondary structure of M2 and/or A upon formation of M'2A'  and M"2A"2. Theoretically, the changes of each type of the secondary structure upon binding can be estimated from the far UV CD spectra. Fig. 4 shows that CD spectrum of A has an "unusual" shape, which has often been observed in the antibody family but never explained in terms of secondary structure (38). Proteins with such unusual CD spectra are not involved in the databases of proteins (with known conformations and CD spectra) used for estimation of the secondary structure. Therefore, a comparison of the measured CD spectra with those calculated from the data base would be meaningless. Raw CD spectra (Fig. 4) were analyzed in the same way as the corresponding FL spectra. From the shapes of the corresponding titration curves we were able to detect two different binding modes of A both at 25 and 45 °C. Because of high affinities of A, the starting concentration of M2 was chosen as low as possible to get reliable K1 and K2 values from the model analysis (Equation 6). The changes in CD signal upon titration were still well measurable at an M2 concentration of ~1 µM (Fig. 4). However, the quality of the titration curves obtained by subtraction was too low for trustworthy model analysis. Therefore, we were only able to estimate K1 and K2 from CD titration at 45 °C (Table I).


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Fig. 4.   CD spectra accompanying MazE (M2) + cAbMaz1 (A) titration at 25 °C at A/M2 molar ratio r varying between 0 and 3. Dotted lines represent the spectra up to r = 1 and solid lines at r > 1. The bold line is the spectra of free cAbMaz1 (A) in its 1 µM solution.

Isothermal Titration Calorimetry-- ITC experiments presented in Fig. 5 were performed at 25, 35, 45, and 55 °C and analyzed in terms of the same model as the corresponding FL and CD titrations (Reaction 1 and Equations 1, 2, and 6). As an advantage over spectroscopic methods, the model function for description of the ITC signal besides K1 and K2 (Table I) also contains two other thermodynamic parameters, Delta H1° and Delta H2° (apparent standard enthalpies of binding). This enabled us to describe the binding of the first and the second A molecule to M2 with a thermodynamic profile (Fig. 5c) that includes apparent standard Gibbs free energies of binding Delta G1° and Delta G2° (Equation 8) and the corresponding apparent standard entropies of binding Delta S1° and Delta S2° calculated from Gibbs equation (Delta Gi° = Delta Hi° - TDelta Si°; i = 1, 2). From the temperature dependence of Delta H1° and Delta H2° (Fig. 5c) the standard heat capacity changes Delta CP,1° and Delta CP,2° were calculated as the slopes of the linear regression Delta Hi° versus T lines (Delta CP,1° = (partial Delta Hi°/partial T)P; i = 1, 2). Because of the very high affinity binding of the first A molecule to M2 at 25, 35, and 45 °C and the applied concentration conditions, almost all added A is bound at r < 1 and, therefore, the evaluation of K1 by a straightforward fitting procedure was not possible (see also FL spectroscopy). At all applied temperatures the fitting was successful in obtaining all other model parameters, i.e. K2, Delta H1°, and Delta H2°. At 55 °C the value of K1 (Table I) was low enough to be accurately determined by the model analysis. The obtained K1 and Delta H1° at 55 °C in combination with Delta CP,1° enabled us to calculate K1 values at 25, 35, and 45 °C from Equation 8. Table I shows that K1 values are higher than K2. They are in good agreement with the values determined by the fitting of FL and CD titration curves with the same model. This supports the conclusion made in the case of FL and CD that the binding of A to M2 is anti-cooperative. Furthermore, the anti-cooperative binding effect is lowered when the temperature rises (the difference between K1 and K2 is reduced; Table I). By extrapolation of K1 and K2 via Equation 8 it can be shown that this allosteric effect is lost above 80 °C. The binding of both A molecules is highly exothermic with Delta H1° being 1-2 kcal/mol lower than Delta H2°. The Delta H1° and Delta H2° values become more exothermic at higher temperatures, resulting in negative Delta CP,1° and Delta CP,2° values of -0.25 ± 0.04 and -0.18 ± 0.01 kcal/mol, respectively.


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Fig. 5.   Typical calorimetric MazE (M2) + cAbMaz1 (A) titration (panel a) and the corresponding binding isotherm at 55 °C () (panel b), where the solid line represents the best fitted model function (Equation 6). Delta H values are expressed in kcal/mol of added A. The corresponding thermodynamic profile of M2 + A association is presented in panel c, where the symbols open circle  (Delta G1°),  (Delta H1°), and Delta  (TDelta S1°) correspond to the binding of the first A molecule, and  (Delta G2°), black-square (Delta H2°) and black-triangle (TDelta S2°) correspond to the binding of the second one. In the case of Delta Hi° solid lines are linear regression lines from which Delta CP,i° values were calculated, whereas in the cases of Delta Gi° and TDelta Si° the lines serve just as a guide to the eye. The thermodynamic parameters of M2 + A association as rigid-bodies (Delta Grb°, Delta Hrb°, and TDelta Srb°) obtained from the structure-based calculations (Equations 11-13) are presented as dotted lines.

Stability of MazE, cAbMaz1, and Their Complexes

The thermodynamic stability of MazE, cAbMaz1, and their tetrameric complex was studied by DSC and temperature-dependent CD spectropolarimetry. Usually it is described in terms of the standard Gibbs free energy change, Delta G°, which corresponds to the reversible denaturation of a given protein. Delta G° as a function of temperature, T, was calculated from Equation 8.

Differential Scanning Calorimetry (DSC)-- Thermograms for M2, A, and M"2A"2 (Fig. 6a) were described in terms of the equilibrium two-state model (Reaction 2 and Equation 7). For M2 and A, the model function (Equation 10) correlates well with the experimental data. Moreover, model-independent transition enthalpy (Delta Hcal) values for M2 and A show good agreement with Delta H°(T1/2), indicating that the two-state approximation is applicable for description of their denaturation processes (Table II). Therefore, the parameters T1/2, Delta H °(T1/2), and Delta CP° (Table II) derived from model analysis of DSC data were used in interpretation of the thermodynamic stability of M2 and A (Fig. 6b). In the case of M"2A"2, Delta H°(T1/2) is somewhat lower than Delta Hcal, indicating that the model that takes into account only native M"2A"2 and denatured M and A is too simple for description of the DSC thermogram. Namely, because of high affinities of A to M2, at applied concentrations and lower T M"2A"2 is practically the only protein species in solution. However, from extrapolation of binding constants by Equation 8, it follows that at T, where the denaturation occurs (Fig. 6a), the presence of M'2A' as well as free M2 and A may not be neglected any more. Because the thermogram is the sum of the contributions of all the species in solution and only the denaturation of M"2A"2 involves the interaction enthalpy of two A molecules, the Delta H°(T1/2) of M"2A"2 is higher than predicted by the described analysis (Equation 7). As the denaturation transitions of all species occur in the same T interval, the deconvolution of the thermogram based on multiple contributions is not possible. However, because the enthalpy of A binding to M2 was determined by ITC, we were able to estimate that the integrated Delta Hcal is <5% (experimental error) different from the denaturation enthalpy of M"2A"2. Apparently, the multiple species contributions affect the shape of the thermogram (Delta H°(T1/2)) more than the corresponding integrated area (Delta Hcal). Therefore, Delta Hcal instead of Delta H°(T1/2) was used in the calculation of the thermodynamic stability of M"2A"2 (Fig. 6b).


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Fig. 6.   Panel a, DSC thermograms of MazE (M2), cAbMaz1 (A) and their tetrameric complex (M"2A"2). Delta CP is expressed in kcal/Kmol of monomeric unit. Full lines represent graphs of the best fitted model function (Equation 10). Panel b, comparison of the thermodynamic stability of M2, A and M"2A"2. The standard Gibbs free energy change upon denaturation versus temperature diagrams is determined on the basis of parameters obtained by the analysis of the DSC transition curves (Equations 7, 8 and 10). The dotted line is a sum of stability curves, M2 + 2A. Panel c, the transition temperature, T1/2, as a function of decadic logarithm of protein concentration, log[P], calculated for reversible two-state denaturation of M2 and M"2A"2 (Equation 7).


                              
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Table II
Comparison of thermodynamic parameters of denaturation
Thermodynamic parameters of MazE (M2), cAbMaz1 (A), and their tetrameric complex (M"2A"2) denaturation obtained by model analysis of DSC thermograms and spectropolarimetric (CD) temperature profiles.a

Temperature-dependent CD Spectropolarimetry-- The denaturation of M2, A, and M"2A"2 (Fig. 7a) was monitored by measuring far UV CD spectra at different temperatures. At the end of the M"2A"2 transition, the corresponding CD spectrum (Fig. 7a) is equal to the sum of individual CD spectra of denatured M and A, suggesting that two M and two A domains dissociate from M"2A"2 upon melting. The denaturation temperature profiles (Fig. 7b) constructed from the corresponding melting curves at a single wavelength were described in terms of an appropriate two state model (Equations 7-9) to obtain Delta H°(T1/2) and T1/2 values. They are very close to those derived from DSC (Table II). Despite the fact that the fitted model functions (Equation 9) display good agreement with experimental melting curves (Fig. 7b) we were able to estimate Delta CP° only for denaturation of M2 (Delta CP° = (1.9 ± 0.5) kcal/mol K). It can be shown by simulation of the model melting curves that their shapes do not depend significantly on parameter Delta CP° in the case of sharp transitions (high Delta H°(T1/2)), so Delta CP° cannot be obtained by the model analysis of A and M"2A"2 melting curves.


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Fig. 7.   Panel a, far UV CD spectra of tetrameric MazE-cAbMaz1 complex (M<UP><SUB>2</SUB><SUP>A<UP><SUB>2</SUB><SUP>) measured from 40 to 95 °C. The  symbols represent the sum of the CD spectra of denatured MazE and cAbMaz1 (2 M+ 2A). Inset, the corresponding melting curve constructed at single wavelength (332 nm). Panel b, degree of denaturation alpha  obtained from CD melting curves as a function of T for M2 (black-square), A (), and M"2A"2 (). Full lines are graphs of the best fitted model function (Equation 9).

Binding of MazE to Its Promoter DNA and the Influence of cAbMaz1

To characterize the binding of M2 to its promoter DNA at 25 °C, FL spectroscopic titrations and ITC were employed. In addition, a significant difference between the far UV CD spectra of free M2 and M2 bound to DNA in the 230-220 nm wavelength range was observed. However, due to high absorption of DNA, the spectra below 220 nm were not good enough even for qualitative analysis. The M2 promoter consists two alternating palindrome sequences (Fig. 9a), which were previously found to be responsible for binding of a MazE-MazF complex as well as MazE (M2) alone (9).

Fluorescence Spectroscopy-- M2 binding to promoter DNA was characterized by a significant quenching of Trp fluorescence accompanied by a small blue shift of FL maximum (2 nm at M2/DNA ratio of 3). The emission FL spectra were analyzed in the same way as in M2 + A titrations (Fig. 3). The resulting titration curve is presented in Fig. 8. The model function based on M2 dimer binding to the independent sites on DNA (Equation 6) gave the best agreement for the value of apparent binding constant, K = (2.8 ± 0.8)·106 M-1, and the number of binding sites on DNA, n = 3.1 ± 0.1. We have tried to employ more complicated models (including different types of binding sites and cooperativity) for description of FL titration curves. However, the reciprocal correlation between adjustable parameters was too high, meaning that the data could be equally well fitted by different sets of model parameters. Because the physical meaning of parameters obtained by these models is completely lost, we stick to a model of independent identical sites. The titration of the "control" (not mazEF related) DNA by M2 resulted in the induced FL intensities that are negligible in comparison to those resulted from the binding of M2 to the promoter DNA (Fig. 8).


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Fig. 8.   FL (open circle ) and calorimetric () titration curves accompanying DNA + MazE (M2) association at 25 °C in comparison with corresponding control (unrelated) DNA + M2 FL (triangle ) and ITC (black-triangle) curves. ITC curve for DNA + MazE-cAbMaz1 (M"2A"2) binding at the same temperature is presented by black squares (black-square). Dotted lines are graphs of the best fitted model function (Equation 6). The difference FL at 360 nm (Delta F360) as a function of M2/DNA molar ratio, r, was obtained from the corresponding emission spectra. Q is a cumulative heat effect at each titration point expressed in kcal/mol of added M2 (M"2A"2) per injection.

Isothermal Titration Calorimetry-- Calorimetric titration curves describing the binding of M2 and the complex M"2A"2 to DNA are presented in Fig. 8. For the same reason as FL data, ITC measurements were analyzed only in terms of M2 (M"2A"2) binding to the independent sites on DNA (Equation 6). The model analysis describes M2 binding as highly exothermic with the enthalpy of binding Delta H° = -71 ± 4 kcal/mol, apparent binding constant K = (2.5 ± 0.6)·106 M-1, and the number of binding sites for an oligomer, n = 3.0 ± 0.1. Binding of the M"2A"2 complex (Fig. 8) is characterized by Delta H° -80 ± 9 kcal/mol, K = (1.9 ± 0.6)·106 M-1 and n = 3.2 ± 0.3, values which are very similar to those obtained in the case of free M2. The heat effects accompanying the M2 + "control DNA" titration are negligible in comparison to the effects that correspond to the M2 + promoter DNA titration (Fig. 8).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MazE-cAbMaz1 Interactions-- The observed changes in FL and CD spectra upon MazE + cAbMaz1 titrations strongly suggest that their association is accompanied also by conformational effects. Thus, the thermodynamic parameters obtained from model analysis of FL, CD, and ITC signals contain contributions from both direct binding (rigid body association) and conformational changes of MazE and/or cAbMaz1. In this study an attempt was made to separate the two contributions. It was shown that the structure-based thermodynamic calculations give reliable estimates of thermodynamic parameters of binding in the systems where the conformational effects are negligible (54, 62). Therefore, they were used to describe the "rigid body" association of cAbMaz1 with MazE. In the calculations, the conformational states of cAbMaz1 and MazE were defined by the crystal structure of their complex. Because both binding sites on MazE dimer are structurally identical, the calculated parameters are the same for binding of the first and the second cAbMaz1 molecule. Fig. 5c shows that the MazE dimer + cAbMaz1 rigid body association is an entropy-driven process characterized by a small negative Delta Hrb° and a negative Delta CP,rb°. Such energetics may be considered as hydrophobic in nature. However, interpretation of rigid body association solely in terms of hydrophobic effect may be misleading, because Delta Hrb°, Delta Srb°, and Delta CP,rb° are produced from large compensating effects (Equations 11-13). In MazE, the hydrophobic core is small but extends to the surface on two sides of the dimer (Fig. 2b). It is exactly this hydrophobic patch that is recognized by two cAbMaz1 molecules. In addition, we have also found several hydrogen-bonding contacts between MazE and cAbMaz1. The small exothermic Delta Hrb° (Equation 12) value shows that its part that is associated with the hydrogen-bonding polar groups is only slightly dominant over the unfavorable (endothermic) non-polar contribution. The largest contribution that makes TDelta Srb° (Equation 13) the major driving force in stabilizing the rigid body complex comes from hydrophobic effect (Delta Ssol°). On the other hand, the "freezing" of side chains (Delta Ssc°) and the lowering of translational/rotational degrees of freedom (Delta Smix°) upon binding cause the entropic loss that reduces the Delta Srb° to nearly half of the Delta Ssol° value. In some studies, much higher values for the loss of translational/rotational entropy were taken into account (63, 50); however, it has been suggested that they are overestimates (54, 64, 65). It was shown that when Delta Ssol° and Delta Ssc° are taken into account the cratic correction (Delta Smix°) accounts well for the loss of translational/rotational entropy upon rigid body association (54, 55).

By contrast, the overall thermodynamic profiles obtained from model analysis of FL, CD, and ITC data (Fig. 5c) indicate that the binding of both cAbMaz1 molecules to MazE dimer is an enthalpy-driven process accompanied by unfavorable entropic contributions. Fig. 5 shows that the overall Delta Gi° is in the same range with Delta Grb°. However, Delta Hi° is much more exothermic than Delta Hrb°, indicating that additional energetically favorable contacts (due to structural alternations) are formed upon cAbMaz1 binding to MazE. The favorable enthalpy difference is compensated by a large conformational entropy loss (TDelta Si° < TDelta Srb°). Thermodynamic studies of biomolecular associations and protein unfolding have demonstrated that Delta CP,i° may be considered as the most reliable distinctive feature of site-specific binding (49-52, 66-68). For cAbMaz1 binding to MazE Delta CP,1° + Delta CP,2°, the value of -0.43 kcal/mol K obtained by ITC is significantly lower than the corresponding 2Delta CP,rb° value of -0.28 kcal/mol K, which is in accordance with the removal of large amounts of nonpolar surface from water and with the structural alternations MazE or/and cAbMaz1. The observed binding anti-cooperativity, due to the structural alterations of the involved proteins, is also visible from the thermodynamic parameters. ITC results show that Delta H2° > Delta H1° (more energetically favorable contacts or/and less unfavorable contacts are formed upon binding of the first cAbMaz1 molecule) and Delta CP,2° Delta CP,1° (larger part of a non-polar surface is buried or/and more polar surface is exposed upon binding of the first cAbMaz1 molecule).

Thermodynamic Stability of MazE, cAbMaz1, and Their Complexes-- The stability curves (Delta G° versus T) of MazE, cAbMaz1, and their tetrameric complex are presented in Fig. 6b. The comparison of the stability curve of MazE to that of another addiction antidote, CcdA (32), shows that both proteins have very similar thermodynamic stability. Because MazE and the toxin MazF are co-expressed, and their expression is auto-regulated on the level of transcription (6, 9), the total concentration of MazE in the cell will never be higher than a certain level set by equilibrium constants of MazE-MazF promoter and MazE promoter complex formation and the concentration of promoter DNA. Consequently, the concentration of free MazE (not bound in the MazE-MazF, MazE-MazF promoter, and MazE promoter complexes) may be rather low. As lowering the concentration shifts the T1/2 (Fig. 6c) to lower values, it follows that, under physiological temperatures, the free MazE might be partially dissociated and unfolded. The native dimer-denatured monomer equilibrium may therefore play an important role in regulation of the mazEF system as a whole, because it is known that fluctuations in the concentrations of the system products may result in cell death (28). The thermodynamic stability of the cAbMaz1 was also compared with the corresponding Delta G° values of some other members of the camelid VHH antibody family determined at 25 °C. The Delta G° value of our cAbMaz1 at 25 °C is 8.7 kcal/mol, which places it somewhere in the middle of the list of VHH stabilities (38, 39). However, its transition temperature T1/2 (Table II) is among the highest observed for any VHH (38, 39). The high T1/2 and high degree of reversibility (0.85) are the properties of VHH that may be highly appreciated in processes where transient heating may take place. Fig. 6b displays the extent to which MazE dimer is stabilized by the binding of two cAbMaz1 molecules. The difference between the curve obtained as a sum of Delta G° versus T curves for MazE and cAbMaz1, and the curve of their tetrameric complex is a measure of apparent binding affinity of the two cAbMaz1 molecules (Delta G1° + Delta G2°). Delta G1° + Delta G2° values obtained from DSC are in good agreement with those obtained by model analysis of the ITC, FL, and CD titration curves (Table I, and Fig. 5c). As cAbMaz1 is also relatively small and well soluble, it proved to be an ideal crystallization and phasing aid for MazE.1 It follows that VHHs may be applicable also for stabilization of other proteins with short shelf life and relatively low thermodynamic stability.

The general architecture of globular proteins is such that a hydrophobic core is surrounded by hydrophilic shell. This compartmentalization is the main driving force of the protein folding. To qualitatively describe to what extent buried hydrophilic groups as well as exposed hydrophobics destabilize the folded conformation of the studied proteins, we compared the experimental Delta CP° values with those calculated from the changes in the non-polar and polar solvent accessible surface areas upon denaturation (Equation 11). The calculated Delta CP° values for the denaturation of the tetrameric MazE-cAbMaz1 complex and cAbMaz1 agree well with the experimental ones, whereas in the case of MazE, the calculated Delta CP° is significantly higher (Table II). As the opposite effect was observed upon MazE-cAbMaz1 binding, this discrepancy may be ascribed to the more non-polar (or/and less polar) residues exposed to solvent in the MazE native state in the solution than is predicted by MazE-cAbMaz1 crystal structure.

Binding of MazE to the Promoter DNA-- It was reported recently, that two alternating palindrome sequences in the promoter (Fig. 9) are crucial for DNA binding of the MazE-MazF complex and thus for the regulation of MazE and MazF expression (9). Footprint analysis of the promoter revealed protection of DNA against DNase I by the MazE-MazF complex. On the other hand, it was suggested that the binding of MazE to DNA is weaker than the binding of the MazE-MazF complex (9). In the present work, we were able to estimate thermodynamic parameters of MazE association on the basis of a simple model (Reaction 1 and Equations 1 and 6), which shows that up to three MazE dimers are bound at the promoter sequence and that the binding is highly exothermic (Delta H° = -71 kcal/mol) with the apparent binding constant in the micro-molar range (Delta G° = -RTlnK = -8.7 kcal/mol). MazE-promoter DNA interactions may be characterized as specific, because the induced ITC and FL signals upon MazE + control DNA titration were negligible in comparison to those resulted from the MazE binding to the promoter DNA. Rather low K can be ascribed to the highly unfavorable entropic contribution (TDelta S° = Delta H°- Delta G° = -62 kcal/mol). As DNA is relatively rigid and the displacement of water from interacting surfaces is entropically favorable, this highly negative value most probably results not just from lowering of degrees of freedom due to association reaction (MazE + DNA = MazE-DNA) but also from folding of an otherwise unstructured part of the MazE dimer.1 This makes MazE one of many recently identified proteins that are intrinsically flexible but become more ordered upon binding a specific partner (40). We have observed that the binding of the MazE-cAbMaz1 complex to DNA, just like in the case of MazE, is a highly enthalpy-driven (Delta H° = -80 kcal/mol) process accompanied by negative entropy contributions (TDelta S° = -69 kcal/mol). The apparent affinity of MazE-cAbMaz1 (Delta G° = -8.6 kcal/mol) is practically the same as that of MazE alone. This indicates that the binding of two cAbMaz1 molecules to the MazE dimer does not significantly influence its interactions with the promoter DNA. Because the residues of MazE dimer to which the two cAbMaz1 molecules are bound cannot interact with DNA, only a limited number of orientations of the MazE dimer in the complex with DNA are possible.


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Fig. 9.   Panel a, 45 bp of ideal B-DNA showing the alternating palindrome structure (9) found in the MazE promoter. Panel b, model for MazE binding to its promoter DNA. Because of the length of the DNA, large spaces remain between the individually bound MazE dimers, in agreement with independent binding. Sufficient room is available for positioning MazF and/or the disordered part of MazE. Panel c, equivalent model of the binding of the MazE-cAbMaz1 complex to the mazEF promoter DNA. In this model, the antibody fragments touch neither each other or the DNA.

Based on this observation as well as on results from site-directed mutagenesis and the observation of residue conservation within the MazE family of antidotes, we have recently suggested a structural model for MazE-DNA binding.1 When we consider the 3-fold repeat in the promoter DNA sequence and dock the MazE dimer into the center of each repeating unit, we see that all MazE molecules are too far apart from each other to directly interact. This is in full agreement with our model of three non-interacting, quasi-equivalent binding sites (Fig. 9b). Sufficient space is available between the bound MazE dimers to position the otherwise unstructured MazE domain (which we assume to become more structured upon DNA binding). The spacing between the bound MazE dimers is also of the right magnitude to allow them to be bridged by MazF dimers. In the presence of bound cAbMaz1, no additional protein-DNA contacts or steric hindrance of any sort is observed in our structural model (Fig. 9c), which is also in agreement with the experimental results.

The general property of autoregulation of transcription in all described addiction systems is that the antidote is the main DNA-binding protein of which affinity to promoter DNA is significantly enhanced in the presence of the toxin. On the other hand, the details such as the number of palindrome DNA sequences involved in binding, the protection of DNA against DNase I by the complex and also by the antidote alone, and the length of the protected region differ from system to system. In the case of phd-doc, under physiological conditions unfolded Phd monomers are stabilized by binding to promoter DNA, a process that is accompanied by dimer formation (10-11). The additional stabilization of Phd by dimer formation is most probably the reason why Phd binding to each of the two distinct palindromes is more energetically favorable than the described binding of MazE, where the dimers already exist in solution. CcdA and ParD (the antidote from parDE system) are, like MazE, dimeric proteins in the micromolar concentration range at physiological temperatures. Under these conditions they bind to DNA as dimers. However, the binding affinities and other thermodynamic parameters have not been reported (32-34).

    ACKNOWLEDGEMENTS

We acknowledge the use of synchrotron beam time at the European Molecular Biological Laboratory (EMBL) beamlines at the DORIS storage ring (Hamburg, Germany) and the European Synchrotron Radiation Facility (ESRF; Grenoble, France). We thank Joris Messens and Karolien Van Belle for purifying the MazE-cAbMaz1 complex and performing quantitative gel filtration experiments with MazE. Professor Gorazd Vesnaver from University of Ljubljana (where the DSC measurements were performed) and Patrick Van Gelder are gratefully acknowledged for critical reading of the manuscript and useful suggestions.

    FOOTNOTES

* This work was supported by the Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO), and Israel Science Foundation Grants 467/99-19 (to G. G.) and 215/99-2 (to H. E.-K.), which were administered by the Israel Academy of Science and Humanities.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a short-term fellowship as visiting scientist from the Fonds voor Wetenschappelijk Onderzoek Vlaanderen and to whom correspondence should be addressed: University of Ljubljana, Faculty of Chemistry and Chemical Technology, Askerceva 5, 1000 Ljubljana, Slovenia. Tel.: 386-1-2419-414; Fax: 386-1-2419-437; E-mail: jurij.lah@uni-lj.si

§§ Postdoctoral fellow of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen.

Published, JBC Papers in Press, January 17, 2003, DOI 10.1074/jbc.M209855200

1 R. Loris, I. Marianovsky, J. Lah, T. Laermans, H. Engelberg-Kulka, G. Glaser, S. Muyldermans, and L. Wyns, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: ITC, isothermal titration calorimetry; FL, fluorescence spectroscopy; CD, circular dichroism; DSC, diffferential scanning calorimetry; ASA, accessible surface area.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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