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J. Biol. Chem., Vol. 281, Issue 16, 11366-11373, April 21, 2006
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12
From the
Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, United Kingdom and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
Received for publication, December 6, 2005 , and in revised form, January 20, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-helical bundle that buries an extensive apolar surface rich in residues invariant in CodY orthologues. The branched chain amino acid ligands reside in hydrophobic pockets of each monomer distal to the dimer-forming surface. The structure of the C-terminal DNA binding domain belongs to the winged helix-turn-helix family. The implications of the structure for DNA binding by CodY and its control by cofactor binding are discussed. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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CodY was first discovered as a repressor of the dipeptide permease operon in B. subtilis (1). Subsequently it has been recognized as having a much wider role in controlling the expression of stationary phase genes. It is now known to regulate over 100 genes distributed across some 70 or so operons (2). The CodY regulon encodes extracellular degradative enzymes, transporter proteins, catabolic enzymes, factors involved in genetic competence, antibiotic synthesis pathways, chemotaxis proteins, and sporulation proteins. During rapid growth, CodY thus represses genes whose products allow adaptation to nutrient depletion. The repressor function of CodY is activated by two different effectors, GTP and isoleucine or valine, which may be viewed as sensors of the energetic and metabolic status of the cell, respectively (3-5). These co-repressors act independently and additively to increase the affinity of CodY for its target sites on DNA. CodY binds its ligands selectively but with moderate affinities (in the millimolar range), consistent with its need to monitor the concentrations of GTP and isoleucine/valine obtaining in rapidly growing cells.
CodY is highly conserved in the low G + C Gram-positive bacteria. In Lactococcus lactis, CodY regulates the expression of genes encoding extracellular peptidases, peptide transport proteins, and intracellular enzymes involved in peptide and amino acid utilization (4). In pathogenic bacteria such as Streptococcus pyogenes, Listeria monocytogenes, Enterococcus faecalis, Bacillus anthracis, and Clostridium difficile, accumulating evidence points to a role for CodY in the regulation of virulence gene expression (6).
CodY from B. subtilis is a dimer of 29-kDa subunits. Its 259-amino acid residue sequence is unrelated to that of any proteins other than CodY orthologues. A putative helix-turn-helix (HTH)3 motif has been identified close to the C terminus of the protein and specific residues in this motif are required for high affinity DNA binding (7, 8). The only other clue to structure provided by the sequence is a set of possible GTP binding motifs again located in the C-terminal half of the molecule (3). To investigate the structural basis of GTP- and branched chain amino acid (BCAA)-dependent regulation of stationary phase gene expression, we embarked on crystallographic studies of CodY. Although we have been able to crystallize the intact protein (9), the crystals were weakly diffracting and unsuitable for structure determination. We therefore sought to identify stable proteolytic fragments of CodY as alternative targets for structure determination. Here we report the crystal structures of N- and C-terminal fragments constituting residues 1-155 and 168-259, respectively.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Partial Proteolysis of CodY—CodY-His6 protein (84 µg, purified as described above) or bovine serum albumin, Fraction V (BSA; 84 µg; Serologicals Products, Inc.), were preincubated at 25 °C for 10 min with or without BCAAs (10 mM each of isoleucine, leucine, and valine). We then added trypsin (0.26µg, Sigma) or-chymotrypsin (0.52µg, Sigma) and incubated the samples at 25 °C in 0.12 ml of a buffer containing 20 mM Tris-HCl, pH 8, 1 mM MgCl2,1mM dithiothreitol, and 0.5 mM EDTA (for trypsin) or 2 mM CaCl2 (for -chymotrypsin). After various times, 0.01-ml samples were removed, denatured, and subjected to SDS-PAGE. After electrophoresis, the proteins were electrotransferred to sheets of nitrocellulose. Bands that stained with Coomassie Blue were excised from the nitrocellulose and subjected to N-terminal sequencing by successive Edman degradation by the Tufts Protein and Nucleic Acid Core Facility.
Structure Determination—Native data taken from crystals of CodY-(1-155) to 1.7-Å resolution were collected on beamline 10.1 (
= 0.9800 Å) at the SRS (Daresbury, UK), using a MAR Research CCD165 detector. Three-wavelength CodY-(1-155) SeMet data were collected to 2.3-Å resolution at beamline BM14 at the ESRF (Grenoble, France). Data were processed and scaled using the program HKL2000 (11). The crystals were assigned to space group P21212 with one molecule per asymmetric unit. The structure was solved by MAD phasing using the program HKL2MAP (12). The model was built using the program ARP/WARP (13) with manual refitting in the program COOT (14) and refined with REFMAC (15). The electron density maps were of excellent quality and all 155 CodY residues of the single molecule in the asymmetric unit of the crystal were defined together with an isoleucine ligand and 175 solvent molecules. The final free R factor is 21.2%, Rwork is 15.3%, and the root mean square deviations of bond lengths and bond angles from ideal geometry are small (Table 1).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2 and 5 pack against a central 5-stranded anti-parallel 
-pleated sheet, with a strand order 2-1-5-4-3 (Fig. 1, A and B). The top layer, which does not lend itself to such simple description, is formed by two extended loops that connect strands 2 and 3, and 3 and 4. The 2-3 segment contains two -helices 3 and 4, whereas the 2-3 loop lacks any recognizable secondary structure. These loops form the walls of a cavity whose base is formed by the -sheet itself.
Amino Acid Binding—The electron density maps clearly define the presence of an isoleucine ligand consistent with the inclusion of this amino acid in the crystallization drops (Fig. 1D). No evidence for the binding of the GTP analogue, Gpp(NH)p was seen, however. The isoleucine is bound above the -sheet in such a way that the 2-3 and 3-4 loops effectively clasp the ligand. The isobutyl group of the ligand projects downwards toward the sheet so that it is enclosed in a hydrophobic cavity circumscribed by the side chains of Met62, Met65, Phe71, Pro72, Tyr75, and Pro99 (Fig. 1E) and main chain atoms from residues 97-99. Valine binds to CodY in a very similar manner to isoleucine, the only significant difference in binding is caused by the absence of a methylene group in valine relative to isoleucine, which may be associated with a small rotation of the phenyl ring of Phe71 (Fig. 1F). The -amino and -carboxylate groups of the amino acid ligand form polar interactions with the protein and solvent. The former forms charge-dipole interactions with the main chain carbonyl groups of Thr96 and Phe98 and a further hydrogen bond to the surface water molecule WAT24. The carboxylate group forms a two-pronged ion-pairing interaction with the guanidinium group of Arg61 and further polar interactions with the amide group of Val100 and the water molecule WAT143.
The residues making up the isoleucine-binding pocket are strongly conserved in the Bacillus, Listeria, Enterococcus, Staphylococcus, Streptococcus, and Lactococcus spp., although surprisingly they are less well conserved in the Clostridium spp. (Fig. 2). Arg61, which binds the ligand carboxylate, is replaced by apolar side chains in CodY from C. difficile and Clostridium perfringens, whereas the apolar Met65, which contacts the apolar side chain of the ligand, is replaced by Glu or Lys in these species. This suggests differences in the mode of ligand binding.
Quaternary Structure—Analysis of the molecular packing suggests an obvious dimer consistent with observations made from gel filtration of CodY-(1-155) during purification, and with the knowledge that intact CodY is a dimer (9).4 In this dimer, the subunits are related by a 2-fold crystallographic symmetry axis. Intermolecular contacts are mediated principally through the first and the last-helices of the respective domains (Fig. 3). These helices come together to form a four-helix bundle that is extended to a six-helix bundle by the adjacent helices 2 from the respective subunits (Fig. 3). Each subunit contributes 
1050 Å2 of its surface area to the contact interface, corresponding to 13% of the total accessible surface area and typical of a dimer interface (16).
Highly conserved and/or invariant residues among CodY orthologues cluster at the dimer interface. There are two intermolecular salt bridges formed between the pairs of Arg8 and Glu144 residues, which engage in two-pronged interactions with each other. These interactions would appear to be crucial for CodY structure and/or function as both residues are invariant in the CodY orthologues (Fig. 2). There is an additional pair of hydrogen bonding interactions between Gln15 and Thr148, the former residue being strongly conserved whereas the latter is invariant.
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atoms are separated by 57 Å (Fig. 3). The C-terminal Arg155 residues that in the intact protein are connected to the DNA binding domains are close in space.
CodY Has a GAF Domain—Despite the absence of any substantial sequence homology, a number of structures in the Protein Data Bank contain domains that can be superimposed onto CodY-(1-155) to yield positional root mean squared deviations of 2.5-4.0 Å for 80-120 matching C atoms (17, 18). The shared characteristic of these proteins is a GAF domain, named after its discovery, on the basis of sequence analysis, in cGMP-stimulated phosphodiesterases, adenylate cyclases and a bacterial transcription regulator FhlA (19). It is now recognized that GAF domains are present in numerous signaling and sensory proteins.
The structure of the GAF domain was first defined in a study of the yeast protein YKG9 (Protein Data Bank entry code 1f5m (18)) and subsequently other GAF domain structures in complex with ligands have been determined (Protein Data Bank entry codes 1mc0 and 1ykd (20, 21)). The superposition of CodY and YKG9 shows clearly that CodY belongs to the GAF domain family (Fig. 4, A and B). The -sheet and two of the -helices that pack against it are superimposable in all GAF domain proteins, with family members distinguished by differences in the loops connecting -strands on the face of the sheet distal to these helices. In the ligand-responsive members of the family (20, 21), these loops shape the binding pocket.
Structure of the C-terminal Domain—CodY-(168-259) has a compact globular structure with an anti-parallel -sheet of topology 6-8-7 and five -helices arranged with respect to the strands in the order 6-7-6-8-9-7-8-10 (Fig. 1C). Helices 8 and 9 correspond to the predicted helix-turn-helix spanning residues 203-226. The sequence in this region is exceptionally highly conserved in the CodY orthologous set (Fig. 2). The role of this HTH in DNA binding has been established by site-directed mutagenesis studies, which have shown that non-conservative substitutions of Ala207, Arg214, Ser215, and Val218 dramatically lower the affinity of CodY for ilvB and dpp promoter DNAs (8). The structure presented here shows that the side chains of Arg214, Ser215, and Val218 are situated at the beginning of the putative recognition helix with their side chains pointing outwards from the molecule, consistent with a role in DNA recognition. Ala207 resides on the preceding helix at a position where a small side chain would be required if the HTH helices are to be arranged in the current manner.
Structural comparisons reveal that the DNA binding domain of CodY belongs to the winged HTH family of nucleic acid-binding proteins (17, 22). These domains consist of three -helices (7, 8, and 9 in CodY) packing against a three-stranded anti-parallel -sheet. The so-called "wings," W1 and W2, are constituted by the loop connecting the second and third strands (7-8) and a looping segment following the third strand (8), respectively. In three dimensions, these segments flank the second helix of the HTH apparently like the wings of a butterfly.
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-chymotrypsin yielded at least seven fragments (Fig. 5E). Of these, only cleavages after Tyr51, after Phe80, and near Tyr181 occurred in the presence of BCAAs (Fig. 5F). Cleavage at other sites, such as Tyr95, Tyr145, and Met173 was completely inhibited by BCAAs. Again, cleavage of BSA was unaffected by BCAAs (Fig. 5, G and H). It is interesting to note that the presumed conformational change in CodY that accompanies binding of BCAAs can affect residues far removed in space from the co-repressor binding site.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The exact mechanism by which binding of co-repressors increases the affinity of CodY for its target sites remains mysterious (see below), but it seems clear that interaction with BCAAs alters the conformation of CodY enough to change the susceptibility of certain residues to proteolytic enzymes (Fig. 5). These residues include some that are outside the N-terminal domain, suggesting that binding of BCAAs induces a conformational change that is translated to the C-terminal, DNA-binding domain.
Implications for GTP Binding—The GAF domain complexes described here define the BCAA binding site in CodY leaving open the question of how GTP is bound. In the absence of a structure of a GTP complex, we have examined other GAF domain proteins and CodY sequence alignments for other clues to nucleotide binding. Two of the known GAF domain protein structures contain nucleotide ligands, cyclic guanosine monophosphate (cGMP) in mouse phosphodiesterase 2A (PDE-2A) and a pair of cyclic adenosine monophosphate (cAMP) ligands in the tandem GAF domains of a cyanobacterial adenylyl cyclase (20, 21). The topologies of the ligand-binding sites are very similar and the ligands and the surrounding protein backbones are closely superimposable.
Compared with isoleucine in CodY, the cGMP in PDE-A2 is more deeply embedded in the GAF domain (Fig. 4C). Following superposition, the guanine base lies close to a pair of buried water molecules that occupy the largest cavity in the CodY structure (Fig. 4D). These are flanked by the side chains of Phe40 and Phe98 waters, which are therefore well placed to make ring-stacking interactions with the guanine base of a putative GTP ligand. Phe98 is invariant, whereas Phe40 is conserved in all of the CodYs except those from L. lactis and the Streptococcus spp. Interestingly the CodYs from L. lactis and Streptococcus pneumoniae (and by inference respond the other Streptococcus spp.) do not to GTP (23, 24)5. The side chains of residues Gln38, Gln55, and Ser129 provide hydrogen-bonding opportunities for the buried waters and potentially for the polar groups of a guanine base. Ser129 is conserved as a small residue (Ser, Gly, or Ala) in the CodY orthologues in Fig. 2, with the exception of those from L. lactis and the Streptococcus spp., which may again relate to their failure to respond to GTP. It is tempting to suggest that the Trp replacements in these CodY proteins provide an indole group to fill the volume that would otherwise be available to the guanine base of GTP.
The cavity containing these waters is contiguous with the isoleucinebinding pocket although it tapers considerably before opening out into the latter. However, given that ligand binding is probably accompanied by closure of the 2-3 and 3-4 loops by a claw-like action, it is possible that these elements could adapt their conformation so as to embrace the GTP ligand. Thus, the nucleotide base may bind in this cavity with the ribose and triphosphate moieties extending toward the surface where Arg61 may mediate important interactions with the 
-phosphate, which is known to be a determinant of the action of GTP because GDP is not an effector of CodY (3). This speculative model for nucleotide binding predicts overlapping binding sites for GTP and isoleucine. The implied mutually exclusive binding of corepressors, however, is not easily reconcilable with the observation that GTP and isoleucine have independent and additive effects on DNA binding by CodY. Although we have thus far been unable to grow crystals of CodY-(1-155) in the absence of isoleucine/valine, the full-length protein has been crystallized both in the absence of effectors and in the presence of GTP as sole effector (9) leaving open the possibility that at least one of the GTP binding determinants, such as the putative G motifs (3) resides in the C-terminal domain.
Implications for Cofactor Control in CodY—In other transcriptional regulatory systems, ligand binding promotes relative domain motions and/or quaternary structural changes. In CodY, the isoleucine-binding site is not linked to any obvious hinge that could mediate long-range conformational change. As the isoleucines are bound in pockets on the faces of the -sheets distal to the dimer interface, it seems unlikely that the effects of ligand binding would alter subunit interactions. Indeed there is no evidence for ligand-dependent quaternary structural changes in CodY.
The organization of the CodY-(1-155) subunits in the dimer places the C termini in close apposition, allowing for the possibility that the emerging DNA binding domains reside side by side. However, 12 linker residues between 155 and 168, a high proportion of which have ionizable side chains (Fig. 2), are not present in either of the structures and an appreciation of the juxtaposition of the domains in intact CodY will have to await the solution of the structure of the intact protein.
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The homoserine lactone ligand is believed to stabilize TraR against intracellular proteolysis (28). Consistent with this idea, it is even more deeply recessed in the hydrophobic cavity of the GAF domain (25, 26) than the isoleucine in CodY (Fig. 4C). This suggests that CodY too may be susceptible to proteolysis as BCAA/GTP levels drop when the cells enter the stationary phase. Although BCAA binding alters the susceptibility to in vitro proteolysis (Fig. 5), in vivo measurements indicate that CodY protein is present at the same concentrations throughout growth and stationary phase (3).
Implications for DNA Binding—A winged HTH domain with similarity to that observed in CodY occurs in the fatty acid-responsive transcription factor FadR. The structure of FadR in its complex with FadR operator DNA is shown in Fig. 6B (29, 30). The winged HTH DNA binding domain is connected to a C-terminal seven-helix bundle domain containing a hydrophobic pocket for acyl-CoA binding. FadR binds to DNA as a dimer using its recognition helix to make contacts with the major groove of the DNA and one of its wings, W1, to penetrate the minor groove.
The winged HTH domains of FadR and CodY can be superposed on one another to give a positional root mean square deviation of 2.7 Å for the 55 matching C atoms. This comparison shows that the invariant residues among CodY orthologues lie on the face of the molecule corresponding to the DNA binding surface in FadR. These are located in the segment L179SYSE183 in the turn between the helices 6 and 7, residues 211-228, which span the recognition helix and the preceding turn, and residues 232-240, which constitute the wing, W1 (Fig. 2). In the latter instance residues Arg233 and Lys238 appear to be well placed for forming interactions with the backbone phosphates of the DNA.
It is interesting to note that there is no tertiary structural similarity between CodY and the leucine-responsive regulatory protein, which performs in Gram-negative bacteria many of the functions attributed to CodY in B. subtilis (31, 32). It would appear that amino acid and guanine nucleotide regulation of stationary phase gene expression has evolved independently in these two systems. Finally in the process of manuscript review, a referee drew our attention to a bioinformatics analysis where it is shown that the GAF plus winged HTH domain composition of CodY is predictable by bioinformatics analysis.
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FOOTNOTES |
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* This work was supported by the Biotechnology and Biological Science Research Council, United Kingdom, Grant BBS/B/1213X, European Union SPINE Contract QLG2-CT-2002-00988, and United States Public Health Service Grant GM042219. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
1 Present address: Laboratoire de Microbiologie et Pathologie Cellulaire Infectieuse, INSERM U431, Universite de Montpellier II, Place E. Bataillon, 34095 Montpellier, France.
2 To whom correspondence should be addressed. Tel.: 44-1904-328261; Fax: 44-1904-328366; E-mail: ajw{at}ysbl.york.ac.uk.
3 The abbreviations used are: HTH, helix-turn-helix; BCAA, branched chain amino acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; SeMet, selenium methionine; BSA, bovine serum albumin; PDE-2A, phosphodiesterase 2A; Gpp(NH)p, guanosine 5'-(,-imido)triphosphate).
4 K. Matsuno and A. L. Sonenshein, unpublished data.
5 R. P. Shivers and A. L. Sonenshein, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and displayed on the bound isoleucine (magenta) and surrounding residues. Polar interactions are indicated by dashed lines. E, ball-and-stick representation with the isoleucine ligand in green and surrounding residues colored according to atom type. Hydrogen bonding/electrostatic interactions are indicated by the dotted lines. For Phe98, atoms other than the carbonyl group that forms an interaction with the amino group of the ligand have been omitted for clarity. F, comparison of valine and isoleucine binding to CodY-(1-155). The ligands are colored by atom type and protein atoms are blue in the CodY-Val complex and red in the CodY-Ile complex. Figures were drawn with CCPMG and MOLSCRIPT (
