Crystal Structure of the Iron-dependent Regulator from Mycobacterium tuberculosis at 2.0-Å Resolution Reveals the Src Homology Domain 3-like Fold and Metal Binding Function of the Third Domain*

Michael D. FeeseDagger §, Bjarni Pàll Ingason§, Joanne Goranson-Siekierke||, Randall K. Holmes||, and Wim G. J. HolDagger §**DaggerDagger

From the Departments of Dagger  Biological Structure and  Biochemistry, § Biomolecular Structure Center, and ** Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, and the || Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Received for publication, August 18, 2000, and in revised form, October 25, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Iron-dependent regulators are primary transcriptional regulators of virulence factors and iron scavenging systems that are important for infection by several bacterial pathogens. Here we present the 2.0-Å crystal structure of the wild type iron-dependent regulator from Mycobacterium tuberculosis in its fully active holorepressor conformation. Clear, unbiased electron density for the Src homology domain 3-like third domain, which is often invisible in structures of iron-dependent regulators, was revealed by density modification and averaging. This domain is one of the rare examples of Src homology domain 3-like folds in bacterial proteins, and, in addition, displays a metal binding function by contributing two ligands, one Glu and one Gln, to the pentacoordinated cobalt atom at metal site 1. Both metal sites are fully occupied, and tightly bound water molecules at metal site 1 ("Water 1") and metal site 2 ("Water 2") are identified unambiguously. The main chain carbonyl of Leu4 makes an indirect interaction with the cobalt atom at metal site 2 via Water 2, and the adjacent residue, Val5, forms a rare gamma  turn. Residues 1-3 are well ordered and make numerous interactions. These ordered solvent molecules and the conformation and interactions of the N-terminal pentapeptide thus might be important in metal-dependent activation.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Iron, because of its ability to participate in electron transfer reactions with redox potentials ranging from +300 to -500 mV (1), is a virtually indispensable cofactor in important biological processes, including respiration, photosynthesis, nitrogen fixation, and DNA synthesis. However, despite its natural abundance, the biological availability of iron is extremely low. In neutral solution ferrous iron, Fe(II), rapidly oxidizes to ferric iron, Fe(III), and forms insoluble hydroxides (KS ~ 10-18 M). Iron is often a limiting factor for growth of living organisms, and in the case of pathogenic organisms it is a limiting factor in infection (2). Higher organisms such as humans sequester iron in tight complexes with iron storage and transport proteins such as transferrin, lactoferrin, and ferritin, making the iron unavailable to invading bacteria (3). To obtain sufficient iron from their environment, many bacteria secrete low molecular weight chelators called siderophores, such as the exochelins and mycobactins of Mycobacterium tuberculosis (4). Ferric-siderophore complexes are actively imported by membrane-spanning receptor proteins. Pathogens also secrete proteins capable of extracting iron from heme and iron transport proteins of the host (3, 5), and iron-dependent production of virulence factors has been observed in many bacteria (6).

Iron-dependent regulators (IdeRs)1 are metal-activated DNA-binding proteins found in Gram-positive and acid-fast bacteria. These proteins are transcriptional regulators of virulence factors as well as siderophore synthesis enzymes and their associated transport proteins (7, 8). If the intracellular iron concentration is sufficiently high, transcription of virulence factors and iron import pathways is repressed by the binding of Fe(II)-IdeR complexes to the promoter/operator sequences for their genes (9, 10). The observation by Pappenheimer et al. (11) that iron availability regulates production of diphtheria toxin by Corynebacterium diphtheriae led to the discovery of the first iron-dependent regulator, the diphtheria toxin repressor (DtxR) (12). DtxR, when in complex with Fe(II), represses transcription of the tox gene of corynebacteriophage beta , which encodes diphtheria toxin, by binding to a target sequence within the tox operator (8, 12). Seven other DtxR-specific iron-regulated promoter/operators (IRPs), designated IRP1-IRP6 (13-15) and hmuO (16), have also been reported.

Initial crystal structures revealed DtxR (17, 18) to be a dimer with three domains and two metal sites per monomer. Domain 1 (residues 1-74) is the DNA binding domain and contains a helix-turn-helix motif. Domain 2 (residues 75-140) is the dimerization domain and contains most of the metal binding residues. Domain 3 (residues 141-226) appeared to have an SH3-like fold and to be connected to domain 2 by a flexible linker (19). Mutational studies (20-22) have confirmed the functional importance of residues involved in metal binding, and structures have been solved that contained the transition metal ions Fe(II), Co(II), Ni(II), Zn(II), Mn(II), or Cd(II), which have been shown to act as corepressors of DtxR in vitro (23). Structures of metal-bound DtxR in complex with DNA in three distinct crystal forms (24-26) revealed that each 19-base pair pseudo-palindromic operator sequence binds two dimers of DtxR. The complex of Co(II)-DtxR-DNA by Pohl et al. (25) revealed domain 3 in a new position in which it, quite surprisingly, contributes two metal ligands to metal site 1, providing the first example of a metal binding function for an SH3-like domain and implicating this domain in the metal-dependent repressor activation mechanism.

The ideR gene from M. tuberculosis has been cloned (27), and the IdeR protein, which shares ~88% sequence identity to DtxR within the first 140 residues of this 230 residue protein, has been shown to be a functional homologue of DtxR. It regulates expression of beta -galactosidase under control of the tox, IRP1, or IRP2 DtxR-specific promoter/operators in response to high and low iron conditions (28). It has also been shown to regulate transcription of fxbA, a gene needed for exochelin synthesis in Mycobacterium smegmatis (29). The 2.6-Å structure of M. tuberculosis IdeR (30) revealed the first two domains and showed both metal sites fully occupied, but the intriguing third domain, which shares only ~24% sequence identity with DtxR, was invisible.

Here we present the 2.0-Å crystal structure of wild type M. tuberculosis IdeR in complex with Co(II). This structure contains, for the first time, domain 3 of IdeR and reveals its SH3-like fold and metal binding function. This is the most complete and highest resolution structure of an iron-dependent regulator to date in what we believe to be the fully active holorepressor conformation. Several features of the environments of the metal binding sites that were hinted at by previous work but remained questionable have been confirmed unambiguously. These features, which include several tightly bound water molecules and precise ordering of the N-terminal residues to form a gamma  turn centered at Val5, may be important in metal-dependent activation.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Crystallization-- Wild type M. tuberculosis IdeR was purified as described by Pohl et al. (30). For crystallization the protein was concentrated to ~8-10 mg/ml (as judged by the Bio-Rad protein assay) by evaporation in a Savant SpeedVac on low temperature and dialyzed thoroughly at 4 °C against a buffer containing only 50 mM NaCl, 10 mM Tris-HCl, pH 7.0, using the microdialysis technique described by Overall (31). This procedure was adopted because a significant amount of the protein was lost upon concentration with membrane concentrators, apparently due to adhesion to the membrane.

DNA used for crystallization was purchased from Macromolecular Resources as the reverse-phase high performance liquid chromatography-purified, single-stranded oligonucleotides. Duplex oligonucleotides were formed by mixing equimolar volumes (as judged by absorption spectroscopy at 260 and 280 nm) of complementary oligonucleotide dissolved in 5 mM MgCl2, 50 mM Tris-HCl, pH 7.5, and annealing by fully submerging in an 80 °C water bath, which was then allowed to cool gradually to room temperature. The homogeneity of the annealed duplexes was judged by capillary electrophoresis on a Bio-Rad BioFocus 3000. The oligonucleotide used for crystallization of IdeR was a blunt-ended 21-base pair duplex formed by annealing oligonucleotides with the sequences 5'-ATTAGGTTAGCCTAACCTAAA-3' and 3'-TAATCCAATCGGATTGGATTT-5'.

IdeR and annealed DNA duplex were mixed in an approximate 3:1 molar ratio (~0.3 mM IdeR monomer and ~0.1 mM DNA) with 5 mM CoCl2 and incubated on ice for 30 min prior to use. Crystallization was performed at room temperature by hanging drop vapor diffusion by mixing equal volumes of Co(II)-IdeR-DNA solution and precipitant solution on silanized coverslips over wells containing 500 µl of precipitant solution. Crystals were obtained from 2.0 M LiSO4, 0.01 M MgCl2, 0.05 M MES, pH 5.6, and grew to a maximum size of ~500 × 300 × 150 µm within 1-2 weeks. Analysis of the diffraction pattern revealed the crystals to be in space group P212121 with cell dimensions a = 46.30, b = 113.97, and c = 236.21 Å.

Data Collection-- Initial diffraction data were collected at the Advanced Light Source (ALS) beamline 5.0.2 at Lawrence Berkeley National Laboratory on a Quantum 4 CCD detector at a wavelength of 1.0 Å. The data were reduced with Mosflm (Andrew G. W. Leslie, MRC Laboratory of Molecular Biology) and Scala (Phil Evans, MRC Laboratory of Molecular Biology) and converted to structure factors with Truncate (32). A second diffraction data set was collected at the Advanced Photon Source (APS) beamline 19-ID at Argonne National Laboratory on the APS1 3 × 3 CCD detector at a wavelength of 1.0332 Å. These data were reduced at APS with HKL2000 (33) and converted to structure factors with Truncate. The crystals were flash-frozen in liquid nitrogen after immersion for ~20 s in a cryoprotectant consisting of a 20% weight/volume solution of dl-threitol in the crystal mother liquor. Diffraction from these crystals was anisotropic. Analysis of the reduced intensities by the fall-off procedure (originally from the Groningen BIOMOL package) as implemented in Truncate showed the crystals to diffract most strongly along the crystallographic b axis and most weakly along the crystallographic a axis. Data collection statistics are presented in Table I.

Structure Determination-- The structure was solved by molecular replacement in AMoRe (34) with the ALS data set from 20.0 Å to 3.5 Å, using as a search model the Zn(II)-IdeR structure (30). This dimeric structure contains only the first two domains (residues 4-140) of IdeR. AMoRe located two clear solutions. The first solution had an initial correlation coefficient (CC) of 15.8 and R-factor of 54.8%, which was improved to a CC of 38.4 and R-factor of 50.9% after rigid body refinement. Addition of the second solution gave a CC of 35.6 and R-factor of 48.5%, which improved to a CC of 43.6 and R-factor of 46.8% after rigid body refinement. These solutions represented the 21 helical packing arrangement of IdeR along the crystallographic a axis, with an ~40-Å wide solvent channel running through the center. This two-domain solution provided no crystal contacts along the b and c axes, suggesting that a significant portion of the model, presumably domain 3, was yet to be included.

The initial two-domain solution was refined briefly by the conjugate direction method with TNT (35). Each domain (residues 4-74 and 75-140) was restrained by the 4-fold improper noncrystallographic symmetry. Clear and unbiased density for domain 3 was then revealed by density modification and averaging; a combination of solvent flattening, histogram matching, and NCS averaging was performed with DM (36) beginning with sigma A-weighted (37) observed structure factors phased on the partially refined model. NCS averaged electron density maps were then calculated with FFT (Lyn F. Ten Eyck) and Maprot (38). These maps allowed residues 151-207 and 216-228 (~30% of the total protein mass of IdeR) to be built de novo. This model for domain 3 packed perfectly into the unit cell to provide the missing contacts along the b and c axes of the unit cell.

Examination of sigma A-weighted Fobs - Fcalc maps with Peakmax (Phil Evans) allowed placement of 13 apparent Co(II) atoms in the asymmetric unit. At this point refinement was continued with the APS data set, and NCS restraints were removed. Maximum Likelihood refinement with Refmac (39) was interspersed with rounds of model building with XtalView (40). Several applications of the wARP protocol (41) were used to identify potential solvent molecules. A number of significant electron density features were noted that had a tetrahedral shape and an appropriate size to accommodate a sulfate or orthophosphate ion. Because the crystal mother liquor contained 2.0 M LiSO4, these peaks were modeled as sulfate ions. One of these sites, formed by Arg27 and Arg60, approximates the binding of a phosphoryl group of the DNA backbone as observed in the Co(II)-DtxR-DNA complex (25). A total of 17 sulfate ions were included in the final model.

Rfree decreased steadily as the model building and refinement progressed, and plots of log(sigma A) versus resolution (37) for the final model were linear from medium to the highest resolution. Val5, as discussed below, is a severe outlier in the Ramachandran plot for all four IdeR monomers. Arg60 is also a consistent but less severe outlier in the Ramachandran plot. This might be functionally significant because Arg60 is known to bind to a phosphoryl group of the DNA backbone in the DtxR-DNA complexes. Residues Ala148 and Asp149 of monomer A are also outliers in the Ramachandran plot. This, however, seems to be due to limitations of the data because these residues are in the linker region between domain 2 and domain 3, which has very poor electron density. Residues Asp147 and Asp148 of monomer A and residue Arg194 of monomers C and D were truncated to their Cbeta atoms because of poor electron density. Final refinement statistics for the APS data set are presented in Table I.

Solvent-accessible surface area calculations, superimpositions, and other coordinate analyses were performed with EDPBD (42). Figures were made with MOLSCRIPT (43), BOBSCRIPT (44), RASTER3D (45), and GRASP (46).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Overall Structure-- The crystal structure of wild type M. tuberculosis IdeR in complex with Co(II) has been solved at 2.0-Å resolution and refined to an R-factor of 21.4% with an Rfree of 27.2% and good geometry (Table I). The electron density is of excellent quality and reveals nearly the complete polypeptide chain for four 25-kDa IdeR monomers (designated as A, B, C, and D) in the asymmetric unit that form two functional dimers. As determined by PROCHECK (47), 99.2% of peptide phi -psi angles fall within allowed regions of the Ramachandran plot with 92.6% within the most favored regions (Fig. 1). Val5 falls into a severely disallowed region of the Ramachandran plot (phi  = ~75°, psi  = ~-60°) in all four IdeR monomers. The electron density revealed that this residue participates in a gamma  turn (48) from residues Leu4 to Asp6 (Fig. 2). The implications of this unusual feature are discussed further below.


                              
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Table I
Data collection and refinement statistics



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Fig. 1.   Ramachandran plot for the final refined model of the M. tuberculosis Co(II)-IdeR complex as produced by PROCHECK. Valines 5 from all four monomers in the asymmetric unit, labeled in red, are tightly clustered in a disallowed region of phi -psi space at about (+75°, -60°).



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Fig. 2.   Stereo diagram of the electron density of the gamma  turn at Val5. The sigma A-weighted Fobs - Fcalc omit map was calculated after 20 cycles of omit refinement with Refmac in Maximum Likelihood mode and is contoured at 1.0 sigma .

The SH3-like Third Domain-- Clear and unbiased electron density obtained by density modification and averaging, as described under "Experimental Procedures," revealed domain 3 of IdeR for the first time (Fig. 3). This domain, like domain 3 of DtxR determined by Qiu et al. (19) and Pohl et al. (25), has an SH3-like fold with six beta -strands and three alpha -helices. Superimposing 77 equivalent Calpha atoms from domain 3 of IdeR monomer A and domain 3 of DtxR monomer 2 from the Co(II)-DtxR-DNA complex (25) resulted in an r.m.s. deviation of 1.38 Å. The linker region (residues 141-150) between domain 2 and domain 3 is visible in one IdeR monomer, although the electron density is generally poor, but it is not discernible in the other three IdeR monomers.



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Fig. 3.   Stereo diagram of the unbiased electron density for domain 3 of IdeR revealed by density modification and 4-fold improper NCS averaging. The Calpha trace for the final refined model is shown. The modified map was produced as described in detail under "Experimental Procedures" and is contoured at 1.0 sigma .

Domain 3 is positioned in the groove between domains 1 and 2 (Fig. 4) and contributes two ligands, Glu172 and Gln175, to metal site 1. The interactions of domains 3 with domains 1 bury 307-357 Å2 of solvent-accessible surface for domain 1 and 295-347 Å2 for domain 3. The interactions of domains 3 with domains 2 bury 537-656 Å2 for domain 2 and 532-566 Å2 for domain 3. This interaction of domain 3 of IdeR with the other two domains is essentially identical to that of the "wedge" position of domain 3 of DtxR as observed in the Co(II)-DtxR-DNA complex (25).



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Fig. 4.   Interface of domain 3 with domains 1 and 2. One monomer of IdeR is shown with its molecular surface calculated by GRASP. The DNA binding helix, residues 38-52, is shown as a ribbon. Co(II) atoms are shown as yellow spheres and water molecules, labeled Sol, are shown as blue spheres. Domain 3 is rotated out and away from domains 1 and 2 for clear viewing, but the Co(II) atom and tightly bound solvent molecule at metal site 1 are included in both orientations for reference. Residues, excluding direct ligands to the Co(II) atom at metal site 1, are shown whose centers make contacts within 4.0 Å at the interface.

As suggested by the range of buried surface areas reported above, the position of domain 3 is slightly different in each IdeR monomer. The Calpha atoms for domains 1 and 2 (residues 1-140) were overlaid for each possible pair of monomers, and the orientation of the domains 3 were then compared. The difference in orientations is due to overall rotations of 3.0-7.3° with the Co(II) atom at metal site 1 acting as the approximate pivot point. Because all domains 3 are involved in crystal contacts, it is difficult to assess the significance of this observation. A similar analysis of domain 3 in the Co(II)-DtxR-DNA complex (25), in which only one domain 3 is involved in a crystal contact, showed differences in relative orientation ranging from 1.1° to 8.0° (note that in this case three of the domains 3 are incomplete so only residues 168-187 and 215-226 could be used in the comparison). Taken together, these analyses indicate a certain degree of flexibility in the interactions of domain 3 with domains 1 and 2.

Metal Site 1-- The Co(II) atoms at metal sites 1 were identified in sigma A-weighted (37) Fobs - Fcalc electron density maps as a 13.3, 24.1, 17.7, and 9.9 sigma  peaks for the A, B, C, and D monomers after the protein portion of the model was completely built and partially refined. The site appears to be fully occupied with refined B-factors for the Co(II) atoms of 21.3, 27.5, 32.1, and 30.5 Å2. The Co(II) atom is liganded by five protein side chains; His79, Glu83, and His98 are contributed by domain 2, and Glu172 and Gln175 are contributed by domain 3 (Fig. 5). The liganding atoms from each side chain form direct coordinate bonds to the Co(II) atom (Table II). Furthermore, there is no sixth ligand; the Co(II) atom at metal site 1 is pentavalently coordinated with distorted trigonal bipyramidal geometry. This geometry is a common, fairly low energy state for Co(II), Fe(II), and other first row transition metals (49) known to act as corepressors of DtxR (23).



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Fig. 5.   Metal site 1. Stereo diagram of the sigma A-weighted Fobs - Fcalc omit map calculated after 20 cycles of omit refinement with Refmac in Maximum Likelihood mode and contoured at 1.0 sigma . Residues of domain 2 are shown in orange, and residues of domain 3 are shown in green. The Co(II) atom, labeled 1, is shown as a yellow sphere, and "water 1" bound in the anion site, labeled Sol, is shown as a blue sphere.


                              
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Table II
Co(II)-ligand atom distances for metal binding sites

Additional peaks of 6.6, 6.7, 5.4, and 4.2 sigma  were identified near metal sites 1 of the four IdeR monomers after refinement of the Co(II) atoms. The peaks were identified as a water molecule by the wARP protocol (41) and refined with B-factors of 19.9, 26.3, 26.8, and 33.3 Å2. This solvent-inaccessible Water 1 occupies an polar pocket formed by the side chains of Arg80, Glu83, Ser126, and Asn130 from domain 2 and Glu172, and Gln175 from domain 3. Because of its pivotal position in bridging the two metal binding ligands of domain 3 with domain 2, Water 1 may have an important role in metal-dependent activation of IdeR.

Metal Site 2 and the N-terminal Residues-- The Co(II) atoms at metal sites 2 of the four IdeR monomers were identified as 23.8, 23.3, 11.5, and 9.3 sigma  peaks in sigma A-weighted Fobs - Fcalc electron density maps. This site appears to be fully occupied with refined B-factors for the Co(II) atoms of 23.2, 26.9, 31.1, and 31.4 Å2. The Co(II) atom is liganded by four protein side chains: Met10 from domain 1 and Cys102, Glu105, and His106 from domain 2 (Fig. 6). Cys102, which was identified first by Tao and Murphy (22) as a crucial residue, forms a bidentate interaction with the Co(II) atom; its Sgamma and carbonyl oxygen atoms form adjacent ligands in the octahedral coordination sphere with an average angle of 88.0° between their coordinate bonds. This is only slightly more acute than the ideal 90° angle for octahedral coordination. The Sgamma to metal ion distances range from 2.43 to 2.50 Å (Table II) and are within the range for metal-liganding sulfur atoms (50).



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Fig. 6.   Metal site 2. Stereo diagram of the sigma A-weighted Fobs - Fcalc omit map calculated after 20 cycles of omit refinement with Refmac in Maximum Likelihood mode and contoured at 1.0 sigma . Residues of domain 1 are shown in blue, and residues of domain 2 are shown in orange. The Co(II) atom, labeled 2, is shown as a yellow sphere, and water 2, which bridges the Co(II) atom to the main chain carbonyl of Leu4, labeled Sol, is shown as a blue sphere.

In a number of previous structural determinations of wild type DtxR, metal site 2 has been disrupted by an apparent oxidative modification of Cys102 Sgamma (17, 19). In this current structure of IdeR, there is no sign of modification. In fact, this is the only refined crystal structure of an iron-dependent regulator, aside from the Co(II)-DtxR-DNA complex at 3.2 Å (25), in which the Sgamma is unambiguously making a direct coordinate bond to the metal atom.

The sixth ligand to the Co(II) atom in metal site 2, which completes the octahedral coordination, is a solvent molecule that is a water or possibly a hydroxyl ion. This solvent appeared as 6.9, 6.6, 6.2, and 6.2 sigma  peaks after refinement of the Co(II) atoms, was identified as a water molecule by the wARP protocol, and refined to an average B-factor of 29.4 Å2. Like the newly identified Water 1 molecule at metal site 1, this Water 2 also appears to play a role in the mechanism of metal-dependent repressor activation. Water 2 bridges the cobalt atom at metal site 2 to the N-terminal pentapeptide via the main chain carbonyl oxygen of Leu4. The Co(II)-water distance is ~2.2 Å, and the Water 2-Leu4 carbonyl distance is ~2.8 Å.

In our new structure of IdeR, in contrast to most previous structures of iron-dependent regulators, the complete N terminus is well ordered in all four monomers (Fig. 7) with excellent electron density (Figs. 2, 6). Met1 folds into the functional dimer interface, making hydrophobic contacts with Val5 and the aliphatic portion of Asp6 from its dimer mate. Residues 2-5 form a type I hydrogen-bonded beta  turn (51) that reverses the main chain and brings Val5 back to the dimer interface, where it packs above Val107. Residues 4-6 form a hydrogen-bonded gamma  turn (48) that immediately reverses the polypeptide chain again. Although the gamma  turn forces the polypeptide main chain into the disallowed region of phi -psi space according to early studies (52), later studies show that this is in fact an allowed conformation (53).



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Fig. 7.   Stereo diagram of the interaction of the N-terminal residues with metal site 2. The Co(II) atom, labeled 2, is shown as a yellow sphere, and the water molecule that bridges the Co(II) atom to the main chain carbonyl of Leu4, labeled Sol, is shown as a blue sphere. Side chains of residues discussed in the text are colored blue and labeled. Electrostatic interactions discussed in the text are shown as thick red dotted lines. The hydrogen bonds of helix 1 are shown for reference as thin gray dotted lines.

It should be mentioned here that the unusual phi -psi conformation of Val5 was noted in relation to the Co(II)-water-Leu4 carbonyl interaction by White et al. (24) for the Ni(II)-Cys102 right-arrow AspDtxR-DNA and Ni(II)-Cys102 right-arrow AspDtxR structures and was also noted by Pohl et al. (25) for the Co(II)-DtxR-DNA complex.

Additional Metal Binding Sites-- Examination of the sigma A-weighted Fobs - Fcalc electron density maps revealed an average 15.0-sigma peak adjacent to the Nepsilon 2 atoms of residue His219 and His223 in domain 3 (which we will designate as metal site 3) in all four IdeR monomers in the asymmetric unit. Because of the peak intensity and its proximity to residues known for their excellent metal liganding properties, these peaks were also modeled as full occupancy Co(II) atoms that refined to an average B-factor of 39.4 Å2. The separation of His219 and His223 by a single turn of alpha -helix, much like Cys102 and His106 at metal site 2, positions them to provide two adjacent ligands to the octahedrally coordinated Co(II) atoms with an average 101.6° angle between their coordinate bonds. The remaining four ligands are water molecules that were identified by the wARP protocol, with the exception of metal site 3 in the B monomer for which only three water ligands were identified.

In monomers A, B, and C, two of the coordinating water molecules from the metal site 3 Co(II) atom bridge it to a tightly bound sulfate ion ~5 Å away. These sulfate ions were identified as roughly tetrahedral peaks of average 8.4 sigma  in the sigma A-weighted Fobs - Fcalc maps. The interaction of each sulfate ion with IdeR is somewhat different.

The importance of this newly identified metal site is unknown. Metal binding at the corresponding site has not been reported for any DtxR structure. This, together with the inconsistent binding interactions of the associated sulfate ions, suggests that the occupation of metal site 3 is a result of crystallization in the presence of Co(II) and a high concentration of lithium sulfate. Further investigation might prove otherwise.

Crystallographically related domains 3 from monomers B, C, and D form a fourth Co(II) binding site (metal site 4) that is probably important for the growth of this crystal form. The Nepsilon 2 atoms of His212 from each of these three domains 3 converge upon an oblong 11.6-sigma peak in sigma A-weighted Fobs - Fcalc electron density maps. This peak was modeled as a Co(II) atom, which refined to a B-factor of 49.5 Å2, with two flanking water molecules. A sixth ligand appears to be a partially occupied or partially ordered water lying between the Co(II) atom and the main chain carbonyl of Asp186 from monomer D. This interaction highlights one notable difference between the monomers within the asymmetric unit; the loop (residues ~207-217) containing His212 in domain 3 appears in two distinct conformations. In monomers A and D, this loop packs closely against domain 3 with Ile207, Ile209, and Val215 making hydrophobic contacts with Leu180, Leu184, Ala187, and Val189. However, in monomers B and C, this loop is rotated outward by ~25°. This Co(II) binding site might be necessary for crystallization, but does not seem likely to be important for repressor function.

Domain Orientation: Comparison to Apo-DtxR and Co(II)-DtxR-DNA-- Qiu et al. (17) proposed that activation of DtxR occurs by a reorientation of the DNA binding domains by a metal binding induced hinge motion at about residue 74. This hypothesis was upheld by the comparison of the structures of apo-DtxR and Zn(II)-DtxR determined by Pohl et al. (54), which showed a clear hinge bending motion in the predicted region. To further investigate the hinge bending properties of iron-dependent regulators, the relative domain orientations for this current Co(II)-IdeR structure were examined by superimposing the Calpha atoms of one domain 2 (residues 75-120) for each functional dimer of the apo-DtxR (54) and the Co(II)-DtxR-DNA complex (25) onto one domain 2 of the current Co(II)-IdeR structure (Fig. 8). The r.m.s. deviation in Calpha positions between the current Co(II)-IdeR structure and the superimposed domains 2 is 0.40 Å for apo-DtxR and 0.39 Å for Co(II)-DtxR-DNA. The r.m.s. deviation in Calpha positions for the domains 2 of the nonsuperimposed monomer is 0.70 Å for apo-DtxR and 0.56 Å for Co(II)-DtxR-DNA, indicating that the relative positions of domains 2 do not change significantly between these three structures. On the other hand, the r.m.s. deviation in Calpha positions for the domains 1 of the superimposed monomer is 1.63 Å for apo-DtxR and 1.34 Å for Co(II)-DtxR-DNA, and for the nonsuperimposed monomer it is 2.06 Å for apo-DtxR and 1.90 Å for Co(II)-DtxR-DNA. The DNA-binding domains among these structures are thus moving considerably with respect to the core of the dimer formed by the dimerization domains.



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Fig. 8.   Superimposition of the apo-DtxR and Co(II)-DtxR-DNA structures onto the Co(II)-IdeR structure. Superimposition was performed as described in the text. The Calpha trace of Co(II)-IdeR is shown in blue, apo-DtxR in green, and Co(II)-DtxR-DNA in orange. For Co(II)-IdeR and Co(II)-DtxR-DNA the Co(II) atoms are also shown in the corresponding colors. The hinge bending region near residue 74 is indicated by black arrows, and the DNA-binding helices and N termini are labeled for reference.

Role of DNA Oligonucleotides in Crystallization-- The P212121 crystal form of IdeR was obtained while attempting to crystallize a complex of IdeR with DNA. The 21-base pair duplex DNA oligonucleotide present in the crystallization trials was never visible in electron density maps, but it seems to have been essential as an additive for the crystal growth or nucleation. After the P212121 crystal form was discovered, controlled crystallization experiments showed that these crystals could not be obtained unless the DNA was included. Because all components of the crystallizations were carefully buffered, the pH was not significantly altered by the presence of the DNA. Therefore, we speculate that some interaction of DNA with IdeR is required for crystallization. The only region in the crystal capable of accommodating the DNA is an ~40-Å-wide solvent channel formed along the a unit cell direction. The DNA-binding helices from domains 1 of all monomers of IdeR in the unit cell project into this solvent channel, suggesting that the DNA is involved in forming or stabilizing the helical array of IdeR that forms this channel. However, due to the close packing interactions between domains 1 of every monomer, it would be impossible for the DNA to be bound in this crystal form in the same manner as is observed for DNA bound to DtxR (24-26). If DNA does exist in this solvent channel, it must be highly disordered, for there were no features in the electron density maps that could be reasonably interpreted as DNA in either the ALS or APS data sets.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The 2.0-Å structure of IdeR from M. tuberculosis represents another significant step toward an understanding of the mechanism of activation of iron-dependent regulators. The high resolution data revealed the first detailed picture of the metal binding sites and N-terminal residues in the fully activated conformation of an iron-dependent regulator, with the benefit of four crystallographically independent observations. In addition, the SH3-like fold and metal binding function of domain 3 were confirmed by unbiased electron density. Because the ionic radii of Co(II) and Fe(II) are very similar even in different coordination states (55), and because Co(II) has been shown to be an excellent corepressor of DtxR (23), we propose that the Co(II)-IdeR and Fe(II)-IdeR structures are very similar and that domain 3 also displays its metal binding function in vivo in the Fe(II)-IdeR complex.

At metal site 1 a buried water molecule, Water 1, was shown to participate in a network of electrostatic interactions involving the Glu83 and Gln175 ligands to the metal site 1 Co(II) atom and residues Arg80, Ser126, and Asn130. Because of its pivotal position in bridging the metal binding ligands of domain 3 with domain 2, Water 1 might have an important role in metal-dependent activation of IdeR. Such a role would be consistent with recent mutagenic experiments by Goranson-Siekierke et al. (21), in which it was shown that alanine substitution of either Arg80, Ser126, or Asn130 disrupted repressor activity in C. diphtheriae DtxR even more severely than alanine substitution of the actual metal liganding residues at metal site 1. Arg80 is particularly crucial as it bridges all three domains by making direct electrostatic interactions with the metal ligand Glu172 from domain 3 and with Glu20 from domain 1. Ser126 and Asn130, however, interact with the metal site 1 ligands Glu83 and Gln175 only indirectly via Water 1.

Residues Arg80, Ser126, and Asn130 were previously postulated to form an anion binding site that allowed orthophosphate to act as a co-corepressor in concert with the bound metal ion (19). The Co(II)-DtxR-DNA (25) complex revealed that this site is able to accommodate the metal binding ligands from domain 3, and this current high resolution Co(II)-IdeR complex has revealed the participation of the tightly bound Water 1 and its potential importance in the regulator's function.

At metal site 2, the Sgamma of Cys102 has been confirmed to be the fifth ligand to the Co(II) atom. The sixth ligand at metal site 2 was confirmed to be a tightly bound water molecule, Water 2, that bridges the Co(II) atom to the main chain carbonyl of Leu4. This Co(II)-water-Leu4 carbonyl interaction has been previously observed by White et al. (24) in their 3.0-Å resolution structure of the Cys102 right-arrow Asp variant of DtxR in complex with Ni(II) and DNA. White et al. proposed that this bridging interaction was responsible, as a crucial aspect of metal-dependent activation, for a helix-coil transition that removed a severe steric clash between the N-terminal helix and DNA, allowing DNA to dock properly to the DNA binding helices of domain 1. However, neither apo-DtxR solved in two crystal forms to ~2.3 Å by Pohl et al. (54) nor any of the previously determined structures of DtxR with partially occupied or unoccupied metal sites 2 (17, 19) show a helical conformation of the N-terminal residues. The poor electron density and typically high B-factors for the N-terminal residues of these structures suggests that they are rather flexible and disordered when metal site 2 is not occupied, and might therefore be unable to maintain a rigid enough conformation to provide a severe steric hindrance to DNA binding.

The Co(II)-water-Leu4 carbonyl interaction is part of a network of electrostatic and hydrophobic interactions that stabilize the distinct conformation of the N-terminal residues, including the gamma  turn conformation of Val5 (Fig. 7). There is a helix capping interaction between Odelta 2 of Asp6 and the main chain nitrogen atoms of residues Thr8 and Glu9 at the N terminus of helix 1. There are hydrogen bonds between Ogamma 1 of Thr7 and Ndelta 2 of Asn2, and between the main chain carbonyl of Asn2 and the main chain nitrogen of Val5. The directional nature of these electrostatic interactions might rigidify the conformation of the N-terminal residues. Leu4 packs into a hydrophobic surface created by the side chains of Met10, Tyr11, and Leu34. Met1 is anchored at the dimer interface, making hydrophobic contact with Val5 from both monomers of the functional dimer. These hydrophobic interactions might enforce the overall conformation and position of the N-terminal residues. Interestingly, Leu4, Thr7, Tyr11, and Leu34 are all highly conserved residues among the known IdeR homologues (56). We propose that this represents the correct conformation of the N-terminal residues for the active holorepressor form of IdeR, and possibly for the active holorepressor form of DtxR as well.

The N-terminal residues might serve to properly orient and stabilize the DNA-binding helices of domain 1 to bind to DNA. In the current IdeR structure, the conformation of the N-terminal residues leads to a substantial interaction between domains 1 that buries an additional ~320 Å2 of solvent-accessible surface area at the dimer interface. In most previous structures, the interaction between domains 1 was much less substantial and buried only ~87 Å2 of accessible surface area. In the Ni-Cys102 right-arrow AspDtxR-DNA complex (24) and in the Co(II)-DtxR-DNA complex (25), the interaction between domains 1 buried merely ~32 Å2 and ~12 Å2 of solvent-accessible surface area, respectively. However, this comparison is impaired because in all of these previously determined DtxR structures the N termini are incomplete, relatively poorly defined in the electron density, and have in general very high B-factors. Thus, it is unclear what role the ordering of the N-terminal residues might play in metal-dependent activation.

The current Co(II)-IdeR structure provides an opportunity to further evaluate the hinge bending properties of iron-dependent regulators. The relative domain orientations among the known structures are consistent with the previous proposals by Qiu et al. (17) and Pohl et al. (54) of a hinge-binding motion at about residue 74 being involved in metal-dependent activation. The Co(II)-IdeR structure presented here is the first structure of a non-DNA-bound regulator with the SH3-like domain in the "wedge" position, the N-terminal residues well ordered, and both metal sites fully occupied. It is surprising, therefore, that it does not show the same orientation of the DNA-binding domains as do the Co(II)-DtxR-DNA (25) and Ni(II)-Cys102 right-arrow AspDtxR-DNA (24, 26) complexes. In fact, the DNA binding helices of the functional dimers of Co(II)-IdeR are shifted by 3.7-4.6 Å and rotated overall by 4.3-6.6° relative to the of DNA-binding helices of these two DtxR-DNA complexes. The DNA-binding helices of Co(II)-IdeR are, stated most simply, twisted somewhat into a more open conformation than those in the DtxR-DNA complexes. The differences to the non-DNA-bound apo- and metal-containing structures of DtxR solved previously are even larger, ranging from a relative shift of 3.3 Å and relative rotation of 7.1° for Zn(II)-DtxR (54) to a relative shift of 5.5 Å and relative rotation of 13.4° for Ni(II)-Cys102 right-arrow AspDtxR (24). The orientation of the DNA-binding helices in the Co(II)-IdeR is most similar to the described previously structure of Zn(II)-IdeR by Pohl et al. (30), with a relative shift of 2.3 Å and relative rotation of only 2.7°.

The variation in relative domain orientations among these structures presents the possibility that the fully metal-activated holorepressor conformation is not an exact fit to the DNA, but that a further conformational change occurs upon binding to target DNA. In the known complexes of DtxR with DNA (24-26), the DNA itself is notably distorted, raising the intriguing possibility that the inherent flexibility of the DtxR target sequences is a crucial aspect of recognition. This hypothesis is inspired by recent work by Lee et al. (57), which has shown that substitutions of nucleotides in DtxR-specific operators that are not contacted by DtxR in the crystal structures can have dramatic effects on DtxR binding and promoter strength. Additionally, studies of the effect of noncontacted bases on DNA recognition by the 434 repressor (58) and P22 repressor (59) support the importance of "indirect readout" in recognition of DNA by proteins (60). Further biochemical experimentation and structural studies will be required to fully reveal the importance of conformational flexibility of both iron-dependent promoter/operator sequences and iron-dependent regulators.


    ACKNOWLEDGEMENTS

We thank Stewart Turley for maintaining the in-house x-ray data collection equipment, Francis Athappilly for maintaining the Hol laboratory computing environment, and Joshua Gable for assistance with protein purification. We thank Keith Henderson at ALS and Rongguang Zhang at APS for assistance with synchrotron data collection.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants R01CA65656 (to W. G. J. H.) and R01AI14107 (to R. K. H.) and by a major equipment grant from the Murdock Charitable Trust (to the Biomolecular Structure Center at the University of Washington).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.

The atomic coordinates and the structure factors (code 1fx7) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Dagger Dagger To whom correspondence should be addressed. E-mail: hol@gouda.bmsc.washington.edu.

Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M007531200


    ABBREVIATIONS

The abbreviations used are: IdeR, iron-dependent regulator; DtxR, diphtheria toxin repressor; SH, Src homology domain; NCS, noncrystallographic symmetry; IRP, iron-regulated promoter/operator; APS, Advanced Photon Source; ALS, Advanced Light Source; r.m.s., root mean square; CC, correlation coefficient; MES, 4-morpholineethanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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