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Protein Science (2001), 10:592-598.
Copyright © 2001 The Protein Society

Solution structure of the DNA-binding domain of the TyrR protein of Haemophilus influenzae

Yunjun Wang1, Shimin Zhao2, Ronald L. Somerville2 and Oleg Jardetzky1

1 Department of Molecular Pharmacology, Stanford University, Stanford, California 94305–5174, USA
2 Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907–1153, USA

Reprint requests to: Dr. Oleg Jardetzky, Department of Molecular Pharmacology, Stanford University, Stanford, California 94305–5174, USA; e-mail: jardetzky{at}stanford.edu; fax: (650) 723-2253.

(RECEIVED October 24, 2000; FINAL REVISION December 13, 2000; ACCEPTED December 13, 2000)

Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.45301.

Supplemental material: See www.proteinscience.org.


   Abstract
TOP
 Abstract
Introduction
 Results and discussion
 Materials and methods
 References
 
The TyrR protein of Haemophilus influenzae is a 36-kD transcription factor whose major function is to control the expression of genes important in the biosynthesis and transport of aromatic amino acids. Using 1H and 15N NMR spectroscopy, we have determined the 3D solution structure of the TyrR C-terminal DNA-binding domain (DBD) containing residues from 258 to 318 (TyrR[258–318]). The NMR results show that this segment of TyrR consists of a potential hinge helix at its N terminus (residues 263–270) as well as three well-defined {alpha}-helices extending from residues 277–289 (HR-2), 293–300 (HR-1), and 304–314 (HR). Helix HR-1 and HR fold in a typical helix–turn–helix (HTH) motif. The three helices and the hinge helix are tightly bound together by hydrophobic interaction and hydrogen bonds. Several hydrophilic residues whose side chains may directly interact with DNA are identified. A hydrophobic patch that may be part of the interaction surface between the domains of TyrR protein is also observed. Comparisons with the structures of other HTH DNA-binding proteins reveal that in terms of the spatial orientation of the three helices, this protein most closely resembles the cap family.

Keywords: TyrR; protein structure; NMR; DNA-binding domain; helix-turn-helix motif

Abbreviations: 2D and 3D, two- and three-dimensional • DBD, DNA-binding domain • DQF-COSY, double quantum-filtered correlation spectroscopy • DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid • E. coli, Escherichia coli • RMSD, root mean square deviation • gHSQC, gradient heteronuclear single-quantum coherence • HTH, helix-turn-helix structural motif • TyrR, tyrosine repressor • TyrR(258-318), residues 258-318 of TyrR • NOESY, nuclear Overhauser enhancement spectroscopy • NMR, nuclear magnetic resonance • TOCSY, total correlation spectroscopy


   Introduction
TOP
 Abstract
 Introduction
Results and discussion
 Materials and methods
 References
 
The tyrosine repressor (TyrR) proteins are transcription factors that belong to the prokaryotic NtrC superfamily. Their primary biological role is to control the expression of several genes important in the biosynthesis or transport of L-tyrosine (Pittard and Davidson 1991; Kristl et al. 2000). In addition to its ability to engage operator targets in DNA, TyrR protein has ATPase activity (Cui et al. 1993) and phosphomonoesterase activity (Zhao et al. 2000). Limited proteolysis shows that TyrR proteins contain two major functionally distinct domains: a larger N-terminal ligand-binding domain and a smaller C-terminal DNA-binding domain (DBD). An additional N-terminal domain, shown to be critical for positive transcriptional regulation in E. coli, is also present in the TyrR protein of E. coli (Cui and Somerville 1993). The DBD of TyrR proteins is typically composed of ~60 amino acids, having substantially high sequence similarity and the characteristics of putative HTH DNA-binding motif (Yang et al. 1993; Zhao and Somerville 1999).

In comparison with other transcription factors, the TyrR protein has several unique features including, first, the fact that the normally dimeric TyrR protein undergoes self-association to form a hexamer in the presence of ATP and tyrosine. The hexamer but not the dimer is able to bind to its operator DNA (Bailey et al. 1996; Katayama et al. 1999). Second, distinctions between strong high-affinity and weak low-affinity TyrR targets reside in the central six bases (AT rich in strong boxes) and in the overall agreement with the consensus sequence (Hwang et al. 1999). A recent study also shows that the N-terminal ligand-binding domain and the C-terminal DNA-binding domain of the TyrR protein of Haemophilus influenzae can associate to yield a nicked species, whose operator DNA-binding properties closely resemble those of the intact protein (Zhao and Somerville 1999).

Despite extensive studies aimed at elucidating the mechanism of TyrR-mediated repression and activation at tyrP as well as other promoters regulated by TyrR (Hwang et al. 1997, 1999; Bailey et al. 1998; Zhao and Somerville, 1999), limited structural information is available either for TyrR or other proteins in the NtrC superfamily. The 3D structural analysis of TyrR DBD alone or complexed to its operator would help to clarify the basic rules of DNA recognition applicable to this family of transcription factors and should extend our knowledge of specific and reversible protein–DNA binding mechanisms. We report here the first step toward this goal with the solution structure of the DNA-binding domain of the TyrR protein from H. influenzae deduced from 1H and 15N NMR experiments, as well as detailed structural comparisons with other DNA-binding proteins. We hope that this work will provide deeper insight into the mechanistic basis of the regulatory function of TyrR proteins as well as the NtrC superfamily.


   Results and discussion
TOP
 Abstract
 Introduction
 Results and discussion
Materials and methods
 References
 
Characteristics of the solution structure
Nearly complete 1H and 15N assignments of TyrR(258–318) were obtained by analyzing homo- and heteronuclear spectra. An assigned (1H-15N) HSQC spectrum of TyrR(258–318) is shown in Figure 1Go. The secondary structure was first determined from an analysis of {alpha}-proton chemical shifts (Wishart et al. 1992), amide proton exchange rates, and a qualitative analysis of the sequential, medium-range NOEs. As shown in Figure 2Go, up-field {alpha}-proton chemical shifts indicate the existence of the {alpha}-helical structure.



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  		Fig. 1. 1H-15N HSQC spectrum of TyrR (258–318). Assignments for the backbone amides are labeled. Cross peaks connected by solid lines correspond to glutamine and asparagine side-chain NH2 groups.

 


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  		Fig. 2. Net structural {alpha}-proton chemical shift (measured {alpha}-proton chemical shift minus the random coiled value, Wishart et al. 1995) versus the residue number for TyrR (258–318). Numbering starts at residue 258.

 
Of the 120 NMR structures calculated, 42 showing no interproton distance restraint violation >0.5 Å, no dihedral angle restraint violation >5°, no 3JHNH{alpha} coupling constant restraint violation great than 1.5 Hz, no root mean square (rms) deviation from ideal bond length >0.02 Å, and no rms deviation from ideal bond angle >2° were first selected. Of the selected 42 structures, a subset of the 32 lowest-energy structures was accepted into the final structural ensemble of TyrR(258–318). Backbone superposition of the 36 NMR structures is shown in Figure 3Go. As seen in this figure, the whole structure of TyrR(258–318) is very well-defined. The overall average RMSD relative to the mean coordinates was calculated to be 0.61 Å for the backbone atoms and 1.06 Å for all the heavy atoms. These RMSD values fall to just 0.17 and 0.77 Å for the region from residues 277 to 314. These results show good convergence of the calculated structures and the presence of only one family of structures. The stereochemical properties of the final structural ensemble were assessed with the program PROCHECK-NMR (Laskowski et al. 1996). The average G-factor was -0.5. The average hydrogen bond energy was calculated to be 0.8 kcal/mol, and the number of bad contacts per 100 residues was determined to be <1.5. With regard to the {phi}/{psi} distribution of backbone dihedral angles, nearly 90% were found within the most favorable or additional allowed regions. These parameters indicate that the quality of the NMR structures is comparable to a 2.5-Å X-ray structure. A complete summary of the structural statistics for the final set of structures is provided in Table 1Go.



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  		Fig. 3. Backbone superposition of 32 selected NMR structures of TyrR(258–318).

 

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  		Table 1. Structural and stereochemical statistics of the selected 32 NMR structures of TyrR(258–318)
 
As shown in Figure 4Go, TyrR(258–318) consists of three well-defined {alpha}-helices extending from residues 277–289 (HR-2), 293–300 (HR-1), and 304–314 (HR). It has been shown that the existence of a helix cap can stabilize a {alpha}-helix by forming additional hydrogen bonds and hydrophobic interactions (Harper and Rose 1993; Aurora et al. 1994). There is no obvious helix cap motif in TyrR(258–318). However, typical helix-capping hydrogen bonds and hydrophobic interaction patterns are identified respectively for helices HR-1 and HR. The Ccap-like hydrogen bond (C" to C3) between the HN of Val 302 and CO at Ala 297 in HR-1 and the hydrophobic interaction Ile 316 and Leu 311 (C3 to C") in HR are observed. As expected from sequence homology with other DNA-binding proteins, HR-1 and HR, which are linked by an {alpha}GBB{alpha} turn composed of residues Gly 301–Val 302–Ser303, fold into a typical HTH motif. A detailed examination of the structures shows that the three helices as well as the N-terminal residues are tightly packed together. Residues Ile 261, Leu 263, Phe 266, Leu 271, Ile 275, Tyr 278, Val 282, Leu 283, Phe 286, Tyr 287, Tyr 290, Leu 300, Ile 307, and Leu 311 form a strong hydrophobic cluster, which could play an important role in stabilizing the folding of this protein. Among these hydrophobic residues, Phe 266, Tyr 278, Phe 286, and Tyr 290 further form an aromatic stack. Inspection of the final structures reveals that the whole molecule is also highly structured and stabilized by a network of hydrogen bonds. Except for the characteristic helical pattern of i,i+4 hydrogen bonds observed, the following hydrogen bonds connecting different segments of the molecule are found in most of the structures: Tyr 287 HH and Thr 293 O{gamma}1, Thr 293 OH and Thr 270 H{gamma}1, Thr 270 O and Glu 273 HN, Thr 270 H{gamma}1 and Glu 272 O{varepsilon}, and Leu6 263 and Tyr 290 HH. Hydrogen bonds between Tyr 314 OH and Gly 276 HN, Tyr 314 HH and Gly 276 O, and Tyr 314 HH and Gly 279 O{varepsilon} are also detected in some structures. In addition, the close proximity between the side-chain amino group of Lys 310 and the side-chain carboxyl group of Asp 272 indicates the presence of a salt bridge.



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  		Fig. 4. Ribbon representation of the restrained minimized mean structure of TyrR(258–318). The side chains of residues forming the hydrophobic (cyan) and hydrophilic (green) are displayed, and some of them are labeled.

 
The existence of a hinge helix has been demonstrated for several repressor proteins (Penin et al. 1997; Spronk et al. 1999). This type of helix, which is known to play a crucial role in the induction of several operons, is formed only on repressor oligomerization and the formation of a complex with operator DNA. Helix–turn structures observed between N-terminal residues 261–270 indicate the presence of a hinge helix. As shown in Figure 4Go, in this hinge-helix region Asp 264, Glu 265, Glu 267, and Asn 268 form a hydrophilic cluster facing toward the DNA-binding side. On binding to DNA, this region (residues 261–270) could possibly become converted to a well-defined {alpha}-helix from its helix–turn state and undergo some interaction with DNA.

Alanine scanning and missing contact tests on the TyrR protein of E. coli identified several residues (Arg 484, Arg 489, His 494, Lys 500, and Leu 501) critical for binding to DNA (Hwang et al. 1997, 1999). The TyrR proteins of H. influenzae and E. coli have a high degree of sequence similarity in their C-terminal DNA-binding domains. These critical residues are well conserved in the TyrR protein of H. influenzae, the corresponding residues being Arg 291, Arg 296, His 301, Lys 307, and Leu 308. The side chains of Arg 484 and His 494 in the TyrR protein of E. coli could play an important role by forming hydrogen bonds directly with DNA (Hwang et al. 1997). A close examination of the structure of TyrR(258–318) reveals that R284, H294, T295, N309, and K312 form a hydrophilic cluster that is completely accessible to solvent and that could possibly be involved in the interaction with DNA. On the opposite side of the molecule, residues Leu 261, Val 282, Leu 285, and Tyr 290 form a hydrophobic cluster. The existence of a hydrophobic patch, which may be important for communicating with the TyrR ligand response domain, has been demonstrated using anilinonaphthalene-8-sulfonate (ANS) probes (Zhao and Somerville, 1999). The hydrophobic patches are also shown in Figure 4Go.

Structural comparison with other HTH DNA-binding proteins
There are dozens of HTH DNA-binding proteins in the PDB (protein data bank), however their sequence identity with TyrR is quite low (<25%). This motivated us to compare the structures of TyrR(258–318) with other DNA-binding proteins. On the basis of the spatial arrangement of the three helices, HTH DNA-binding proteins were classified into seven structural families (Wintjens and Rooman 1996). Careful superimposition of the three helix elements with those of each of the families show that TyrR(258–318) shares highest structural similarity with the cap family in terms of the spatial orientation of the three helices. Superimposition of the backbone atoms of the helices HR-2, HR-1, and HR of TyrR(258–318) with that of several members in the cap family gave RMSD values ranging from 1.7 to 2.0 Å. The second closest family is the 434 Cro family, for which the backbone RMSD values were found to be 2.0–2.4 Å. It was of interest to find that after backbone atom superimposing the well-defined helices HR-2, HR-1, and HR of Tyr(258–318) onto that of the 434 Cro family, the hinge helix of TyrR(258–318) fit well into the HR+1 position of the 434 Cro family.


   Materials and methods
TOP
 Abstract
 Introduction
 Results and discussion
 Materials and methods
References
 
Materials
The TyrR(258–318) protein was prepared by a modification of a previously described method (Zhu et al. 1997; Zhao and Somerville 1999). By site-directed mutagenesis, colon 257 was charged from Q to M. The resulting protein, fully active in operator binding, was cleaved with CNBr to yield the desired 61-mer. Uniformly 15N-labeled full-length TyrR protein was produced by growing plasmid bearing E. coli BL21 (DE3) pLysS in the minimal medium containing 15NH4Cl as the sole nitrogen sources. Samples of unlabeled and 15N labeled TyrR(258–318) were prepared for NMR experiments at protein concentrations of ~2.0 mM in 90% H2O,10% D2O, 50 mM phosphate buffer (pH 6.5) containing 1 mM EDTA, 0.02% NaN3, and 1 mM DSS as chemical shift reference (Wishart et al. 1995).

NMR spectroscopy
All spectra were recorded on a Varian 800 MHz spectrometer at the Stanford Magnetic Resonance Laboratory (SMRL). Homonuclear 2D experiments, DQF-COSY (Piantini et al. 1982), TOCSY (Braunschweiler and Ernst 1983), NOESY (Jeener et al. 1979), and PE-COSY (Mueller 1987), were performed on unlabeled TyrR(258–318) sample. For NOESY experiments, mixing times in the 70–250-msec range were used. Spectra were recorded at different temperatures (20°–35°C) to resolve chemical shift degeneracy. To identify slowly exchanging amide protons, the same amount of lyophilized sample was dissolved in 500 µL D2O, and then a complete NOESY spectrum was acquired within 5 h. Heteronuclear experiments, gHSQC (Kay et al. 1992), HMQC-J (Kay and Bax 1990), 3D sensitivity enhanced 15N-NOESY-HSQC, and 15N-TOCSY-HSQC (Zhang et al. 1994) were carried out on the 15N-labeled sample. VNMR (Varian), NMRPIPE (Delaglio et al. 1995), and NMRView (Johnson and Blevins 1994) software packages were used for data processing and analysis. MOLMOL (Koradi et al. 1996) was used to visualize the structures and to calculate RMSD values.

Resonance assignment and structure calculation
1H chemical shift assignments were completed according to well-established protocols (Wuthrich 1986). Spin system and sequential assignments were achieved using 1H TOCSY, DQF-COSY, and NOESY spectra collected at various mixing times and temperatures. The heteronuclear 1H-15N experiments, HSQC, TOCSY-HSQC, and NOESY-HSQC, allowed the confirmation of the sequential assignments and the resolution of ambiguous assignments. The 1H and 15N chemical shifts were deposited at the BioMagResBank under accession code 4784.

For the structure determination, interproton distance restraints were derived from 2D 1H NOESY and 3D NOESY–(1H, 15N)–HSQC spectra acquired with a mixing time of 80 msec. The assigned NOE intensities were classified as strong, medium, weak, and very weak, corresponding to interproton restraints of 1.8–2.8, 1.8–4.0, 1.8–5.0, and 1.8–6.0 Å, respectively. Upper distance limits for those involving methyl and non-stereo-specifically assigned methylene protons were corrected by adding 0.5 Å. Backbone hydrogen bonds were identified from a combined analysis of the previously obtained secondary structure, the crude initial NMR structures, and the data from amide exchange measurements. Hydrogen bonds were implemented by using an upper limit distance restraint of 1.8–2.3 Å between the corresponding carbonyl oxygen and amide proton and a restraint of 1.8–3.3 Å between the carboxyl oxygen and amide nitrogen. A total of 23 {phi} backbone torsion angle restraints were derived from 34 3JHNN{alpha} coupling constants measured from (1H-15N) HMQC-J using the line-width measurement technique (Wishart and Wang 1998). Specifically, for those residues with 3JHNN{alpha} >= 8.5 Hz, {phi} angle restraints were set to -120 ± 30°; for those residues in well-defined helical regions and with 3JHNN{alpha} <= 5.5 Hz, {phi} angle restraints were set to -65 ± 30°. The remaining 11 coupling constants, for which the {phi} angle could not be explicitly determined, were used directly as coupling constant restraints with an uncertainty of 1.5 Hz. Backbone {psi} dihedral angle restraints were obtained from an analysis of dN{alpha}/d{alpha}N ratio. The {psi} dihedral angle restraints were set to 120 ± 100° and -30 ± 110° for dN{alpha}/d{alpha}N ratio <1 and >1, respectively (Gagne et al. 1994). Throughout the calculations, all {omega} angles were set to 180 ± 10°. {chi}1torsion angle restraints from DQF-COSY, E-COSY, and NOESY spectrum were set to 180°, -60°, or 60°, with an uncertainty of ±30°.

Simulated annealing protocols as implemented in the X-PLOR package were used for structural generation. In the calculation, 996 distance restraints, 119 backbone dihedral angle ({phi}, {psi}, and {omega}) restraints, 11 3JHNN{alpha} coupling constant restraints, 16 {chi}1 angle restraints, and 142 proton chemical shift restraints were used. Of the 120 NMR structures calculated, a subset of 36 structures satisfied the criteria of lowest energy and was accepted into the final ensemble. The coordinates were deposited at the Protein Data Bank (PDB) under accession code 1G2H.


   Acknowledgments
 
We thank Corey Liu (SMRL, Stanford University) for maintaining the NMR facility. This work is supported by NIH grant GM22131 (R.S.).

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


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TOP
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 Introduction
 Results and discussion
 Materials and methods
 References
 
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