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 §1§ Crystal structure of Bacillus subtilis YabJ, a purine regulatory protein
and member of the highly conserved YjgF family  §1§

    1. [16]Sangita Sinha [17]*,
    2. [18]Pekka Rappu [19]†,
    3. [20]S. C. Lange [21]*,
    4. [22]Pekka Mäntsälä [23]†,
    5. [24]Howard Zalkin [25]‡, and
    6. [26]Janet L. Smith [27]* , [28]§

    1.


    Departments of ^*Biological Sciences and ^‡Biochemistry, Purdue
    University, West Lafayette, IN 47907; and ^†Department of
    Biochemistry and Food Chemistry, University of Turku, Vatselankatu 2,
    FIN-20014 Turku, Finland

    1. Communicated by Michael G. Rossmann, Purdue University, West
       Lafayette, IN (received for review July 15, 1999)


   [29]Next Section

 §2§ Abstract §2§

   The yabJ gene in Bacillus subtilis is required for adenine-mediated
   repression of purine biosynthetic genes in vivo and codes for an
   acid-soluble, 14-kDa protein. The molecular mechanism of YabJ is
   unknown. YabJ is a member of a large, widely distributed family of
   proteins of unknown biochemical function. The 1.7-Å crystal structure
   of YabJ reveals a trimeric organization with extensive buried
   hydrophobic surface and an internal water-filled cavity. The most
   important finding in the structure is a deep, narrow cleft between
   subunits lined with nine side chains that are invariant among the 25
   most similar homologs. This conserved site is proposed to be a binding
   or catalytic site for a ligand or substrate that is common to YabJ and
   other members of the YER057c/YjgF/UK114 family of proteins.

   Purine biosynthesis in Bacillus subtilis is regulated by the purine
   operon repressor gene, purR. The purR operon consists of purR and yabJ,
   an ORF of unknown function. The repressor PurR binds to control regions
   upstream of the transcription start sites and regulates transcription
   of purEKBC(orf)QLFMNHD in the pur operon, of purR and yabJ in the purR
   operon, and of purA ([30]1). PurR binding to control-site DNA is
   blocked by 5-phosphoribosyl-1-pyrophosphate (PRPP), a central
   nucleotide metabolite that acts as an inducer of pur operon and purA
   transcription. 5-phosphoribosyl-1-pyrophosphate is the starting
   material for purine biosynthesis. No other metabolite, nucleotide,
   nucleoside, or nucleotide base is known to affect PurR DNA binding in
   vitro ([31]2).

   Recently, yabJ, the distal coding sequence in the purR operon, was
   shown to affect PurR function in vivo ([32]3). Regulation of
   transcription of purine biosynthetic genes is sensitive to levels of
   some nutrients and depends on the interrelated pools of
   5-phosphoribosyl-1-pyrophosphate, nucleotides, nucleosides, and
   nucleotide bases. Adenine uptake by B. subtilis indirectly increases
   PurR repression of pur operon and purA transcription through changes to
   the 5-phosphoribosyl-1-pyrophosphate pool. YabJ is also required for
   adenine-mediated repression of purA in vivo ([33]3). The mechanism by
   which YabJ stimulates adenine-mediated repression of purA by PurR is
   unknown. However, YabJ and PurR may be cotranslated and produced in
   stoichiometric quantities in vivo because the last codon of purR
   overlaps the initiation codon of yabJ.

   YabJ belongs to a widely distributed family of proteins of unknown
   function. Members of the YER057c/YjgF/UK114 family of proteins are
   identified by sequence similarity, having 20–98% pairwise identity. The
   family is named for genes or proteins in yeast (YER057c), Escherichia
   coli (yjgF), and goat (UK114). All are ≈14-kDa proteins and do not
   occur as domains of larger proteins. Many YjgF proteins are
   acid-soluble. A variety of biological processes have been reported to
   be influenced by YjgF proteins, in addition to the YabJ function in
   purine regulation. Homologs occur in bacteria, animals, and fungi but
   are absent from the genomes of most archaea and of several parasitic
   prokaryotes that also lack many biosynthetic pathways. No YjgF homolog
   has yet been detected in a plant, although its presence in a
   cyanobacterium ([34]4) suggests that it may also occur in plants. No
   three-dimensional structures of YjgF proteins have been published,
   although crystallization of a rat homolog has been reported ([35]5).
   Here we present the 1.7-Å crystal structure of B. subtilis YabJ. The
   structure reveals a conserved cleft on the protein surface that will
   aid in elucidation of a function for this family of proteins.
   [36]Previous Section[37]Next Section

 §2§ Materials and Methods §2§

 §3§ Cloning and Overexpression of yabJ.  §3§

   The yabJ gene was amplified by PCR from chromosomal DNA of a wild-type
   B. subtilis strain. The 446-bp PCR product was confirmed by sequencing
   and was cloned into a T7 expression vector to produce plasmid pDR1. E.
   coli strain BL21(DE3)/pLysS was transformed with pDR1. Strain
   BL21(DE3)/pLysS/pDR1 was grown at 37°C in LB medium supplemented with
   100 μg/ml ampicillin to an OD[600] of 0.6, was induced with 0.4 mM
   isopropyl β-d-thiogalactoside, and was grown for 10 hr at 30°C. Cells
   were harvested by centrifugation and were resuspended in 50 mM Tris⋅HCl
   (pH 8.0), 2 mM EDTA, 10 mM MgCl[2], and 10 μg/ml DNase I (5 ml of
   buffer per gram of wet cells). The cell pellet was frozen at −20°C.

 §3§ Purification of YabJ. §3§

   Frozen BL21(DE3)/pLysS/pDR1 cells were thawed and incubated at room
   temperature for 15 min. The lysate was clarified by centrifugation at
   20,000 × g for 30 min. All purification steps were performed at room
   temperature. The lysate soluble fraction was stirred during addition of
   70% perchloric acid to a final concentration of 5%. Precipitated
   proteins were removed by centrifugation at 12,000 × g for 15 min. The
   perchloric acid extract was neutralized with 1.5 volumes of 1 M
   Tris⋅HCl (pH 8.0), and the extract was dialyzed against Buffer A (40 mM
   sodium phosphate buffer, pH 7.4). The ≈95% pure YabJ sample was
   chromatographed by using the resin Poros HQ/M (PerSeptive Biosystems,
   Framingham, MA). The column was washed with 5 volumes of Buffer A. YabJ
   was eluted with 0.2 M NaCl, was dialyzed against Buffer A, and was
   concentrated by ultrafiltration. N-terminal sequence analysis showed
   that Met1 was not present in the purified protein. The protein was
   stored at 4°C or was frozen in small aliquots for long-term storage at
   −70°C. The overall yield was 120 mg of purified YabJ per liter of E.
   coli culture.

 §3§ Crystallization and Data Collection. §3§

   YabJ was crystallized by hanging-drop vapor diffusion from a 1:1
   mixture of protein (6.3 mg/ml YabJ in Buffer A) and reservoir (20%
   polyethylene glycol 4,000/0.45 M ammonium acetate/0.1 M sodium acetate,
   pH 4.6) solutions. Crystals grew to a maximum size of ≈0.1 × 0.1 × 0.6
   mm in 4–5 days and were stabilized in reservoir solution. A Hg
   derivative was prepared by soaking for 3 hr in 0.5 mM ethyl Hg
   phosphate in reservoir solution, followed by back soaking for 0.5 hr.

   Crystals were cryoprotected by a 3-min soak in 15% polyethylene glycol
   400 in reservoir solution and were flash-frozen in a N[2] cold stream
   at 100 K. Unit cell parameters in space group P6[5] are a = b = 53.3 Å
   and c = 204.9 Å, which is consistent with either two (V [m] = 3.1
   Å^3/Da, ≈60% solvent) or three (V [m] = 2.0 Å^3/Da, ≈40% solvent)
   copies of the YabJ polypeptide per asymmetric unit. All data used for
   structure determination and refinement were collected from a single,
   Hg-derivatized, frozen crystal. Multiwavelength anomalous diffraction
   (MAD) data were collected at BioCARS beam line BM14D at the Advanced
   Photon Source (Argonne National Laboratory). MAD data (λ[1], λ[2],
   λ[3]; Table [38]1) were recorded on an Q1 CCD detector (Area Detector
   Systems, Poway, Ca) as two 80° sweeps of data at each wavelength.
   Resolution was limited by detector size and the 205-Å c axis, resulting
   in incomplete coverage of reciprocal space at the detector edge.
   Several weeks later, complete, high resolution, 1.7-Å data (λ[4], Table
   [39]1) were collected from the same crystal by using BioCARS beam line
   BM14C at the Advanced Photon Source. The data were recorded on a Mar
   345 imaging plate detector (X-ray Research GmbH, Hamburg, Germany) as a
   single, 80° sweep of data. Data integration and reduction were done by
   using the hkl package ([40]6). The MAD data set used for phasing was
   produced by scaling the λ[1], λ[2], and λ[4] data sets to the merged
   data for λ[3], using scaleit from the CCP4 suite ([41]7). For phase and
   model refinement, a complete high-resolution data set (λ[3] + λ[4],
   Table [42]1) was generated by merging data at λ[3] (30–2.5 Å) and λ[4]
   (3–1.7 Å).
   View this table:
     * [43]In this window
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   Table 1

   Crystallographic summary

 §3§ Structure Determination and Refinement. §3§

   Positions of three Hg atoms were determined by inspection of a Bijvoet
   difference Patterson map using the λ[2] data set. Refinement of the Hg
   partial structure and MAD phasing were performed by the
   pseudo-isomorphous replacement approach using mlphare ([45]8).
   Noncrystallographic 3-fold symmetry was clearly visible in the
   unmodified MAD map at 2.7-Å resolution. A C3 noncrystallographic
   symmetry operator was located and refined by using ncs6d ([46]9).
   Phases were refined and extended to 2.0 Å by 3-fold averaging,
   histogram matching, and solvent flattening with a 32% solvent mask
   using dm ([47]10) and the λ[3] + λ[4] data set. The overall figure of
   merit for all reflections to 2.0 Å was 0.63.

   All model building was done in the program o ([48]11). The model was
   refined against the λ[3] + λ[4] data set using maximum likelihood
   amplitude and phase probability targets using cns ([49]12). The initial
   model had R [work] = 0.355 and R [free] = 0.351 for all data between
   30.0 and 2.0 Å. During the initial cycles of refinement, the model was
   restrained by 3-fold noncrystallographic symmetry. The last iteration
   of refinement and model building was followed by one round of atomic
   occupancy refinement for residues in dual positions and for the ethyl
   Hg ions. Final R [work] = 0.166 and R [free] = 0.197 for all data
   between 30.0 and 1.7 Å. The model, including 124 residues in each of
   three YabJ monomers, one ethyl Hg ion covalently linked to Cys104 in
   each of two monomers, two partially occupied Hg sites in the third
   monomer, six acetate ions, and 474 water molecules, has been deposited
   in the Protein Data Bank and is available with ID code [50]1qd9. A
   crystallographic summary is presented in Table [51]1.
   [52]Previous Section[53]Next Section

 §2§ Results and Discussion §2§

 §3§ Protein Production and Structure Determination. §3§

   B. subtilis YabJ was readily produced in E. coli from an inducible
   plasmid. Purification was simplified by the acid extraction step. We
   tried this approach because several YabJ homologs were reported to be
   acid stable ([54]13, [55]14), and acid solubility was exploited in the
   purification of the rat homolog ([56]15), which is 45% identical to
   YabJ. The crystal structure was determined by Hg MAD, based on
   modification of the single cysteine residue in YabJ. A complete,
   high-quality model of YabJ resulted from refinement against complete
   1.7-Å data. Electron density is clear for all parts of the protein. The
   model agrees well with the diffraction data and with stereochemical
   criteria (Table [57]1). No residues are in forbidden regions of the
   Ramachandran plot ([58]16). The estimated coordinate error of the final
   model is 0.19 Å ([59]17).

 §3§ Protein Structure. §3§

   The YabJ monomer is a 124-residue polypeptide folded into a single
   domain. The YabJ fold is a six-stranded, mostly antiparallel β-sheet
   with one parallel connection (Fig. [60]1). Two α-helices pack against
   one face of the β-sheet. The core of each monomer includes no ionizable
   groups or polar side-chain interactions. This structural feature may
   contribute to the acid stability of YabJ. Preliminary refinement of the
   YabJ model against data from Hg-free crystals at pH 6.5 indicates no
   significant structural change from the Hg complex at pH 4.6 apart from
   rotation of the Cys104 side chain from χ[1] = 180° to χ[1] = −60°.
   [61]Figure 1
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   Figure 1

   Stereo view of the YabJ monomer. The ribbon diagram is rendered in
   rainbow colors from blue at the N terminus to red at the C-terminus.
   The figure was produced with molscript ([65]31) and raster3d ([66]32).

   YabJ is a symmetric trimer in crystals (Fig. [67]2). The three β-sheets
   of the trimer form a triangular barrel, with the β-strands
   approximately parallel to the barrel 3-fold axis. The six α-helices of
   the trimer pack on the outside of the barrel. Residues in strands β3–β6
   of each monomer participate in the trimer interface, which is
   constructed of alternating hydrophobic and polar regions. At the top of
   the barrel, the three backbones form a ring of nine intersubunit
   hydrogen bonds involving residues 106, 108, and 109. Below this polar
   region is an extensive hydrophobic interface including six residues
   from each monomer (Phe77, Met81, Val105, Val107, Leu110, Pro111). In
   the center of the trimer is a polar cavity filled with 21 ordered water
   molecules and ringed with six intersubunit polar contacts, consisting
   of a salt bridge from Lys73 (β4) and a hydrogen bond from Ser103 (β5)
   in each monomer to Glu119 (β6) in an adjacent monomer. Each water
   molecule in the cavity forms at least three hydrogen bonds to other
   waters or to protein atoms (Tyr28, Ser29, Lys73, Thr75, Glu119,
   Val120), and the constellation of 21 waters has ≈3-fold symmetry.
   Another 12 intersubunit hydrogen bonds are formed by nearby residues
   (Ser18, Ser30, and Gly31 with Lys99, Pro100, and Arg102). The floor of
   the polar cavity is formed by three Tyr28 (β3) side chains, which are
   hydrogen-bonded to a cavity water molecule. The cavity waters would be
   accessible to external bulk solvent by slight conformational breathing
   of the Tyr28 side chains. The Tyr28 residues are the beginning of
   another hydrophobic interface, which includes eight residues from each
   monomer (Ile21, Val23, Met26, Tyr28, Val72, Ala101, Ile121, Leu123). At
   the bottom of the barrel is a ring of six hydrogen bonds between the
   amides of Asn24 and Asn25 in adjacent subunits.
   [68]Figure 2
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   Figure 2

   YabJ Trimer. Subunits are rendered in different colors in this ribbon
   diagram viewed along the molecular threefold axis. The position of the
   Hg ion used to solve the structure and bound to Cys104 is indicated
   with a sphere.

   The YabJ quaternary structure has not been established in solution.
   Several features of the crystalline trimer, including the extent and
   character of buried monomer surface and the dehydration of subunit
   interfaces, are consistent with a YabJ trimer in solution. The total
   monomer-accessible surface buried in the trimer is very large, a total
   of 1,850 Å^2, ≈28% of the total ([72]18). Two extensive hydrophobic
   contact regions are part of the trimeric aggregate. Apart from the
   central cavity described above, the subunit interfaces do not bury any
   water molecules. Thus, the subunit interfaces of the crystalline trimer
   are much more like biological aggregates than like crystal contacts,
   which are extensively hydrated, rather small and very polar. Some YabJ
   homologs have been reported to be dimeric ([73]13, [74]19, [75]20,
   [76]29). However, residues forming the polar and hydrophobic zones of
   the subunit interface are very well conserved among the YabJ homologs,
   suggesting that all are trimers.

   Neighboring subunits of the trimer create three clefts on the outer
   surface of YabJ. The clefts are narrow (8–9 Å) and deep (12–13 Å).
   Glu117 forms the innermost surface of each cleft, and several
   hydrophobic side chains ring the outermost surface.

 §3§ YabJ in the purR operon of B. subtilis.  §3§

   Transcription of purA is repressed ≈10-fold in vivo by addition of
   adenine to cells ([77]3). This repression is mediated by PurR. However,
   disruption of YabJ results in the loss of adenine-mediated PurR
   repression by an unknown mechanism. The predominance of acidic residues
   on the surface of YabJ (calculated pI = 5.3) suggests that it does not
   interact directly with DNA. Preliminary soaking experiments with YabJ
   crystals and various purine-containing molecules and calcium ions,
   which are reported to bind a YabJ homolog ([78]20), have not resulted
   in binding in the cleft or anywhere else on YabJ. Further experiments
   are underway to identify a ligand or substrate.

 §3§ Stuctural Homolog. §3§

   YabJ has no detectable sequence similarity to any other protein in the
   structure database ([79]21). Unexpectedly, it has a fold ([80]22) and
   aggregation state in common with chorismate mutase from B. subtilis
   ([81]23). The core fold, β3–β6 and α1, is present in both structures.
   The second α-helix of YabJ is replaced by a shorter 3[10]-helix in
   chorismate mutase. Strands β1 and β2 of YabJ and a fifth, C-terminal
   strand of chorismate mutase lie on the same end of the core β-sheet but
   are not superimposable. Based on the structure alignment, the sequences
   of YabJ and chorismate mutase are only 8% identical. The rmsd for the
   83 superimposable C[α] atoms per monomer is 2.6 Å (Fig. [82]3 A). YabJ
   and chorismate mutase also form very similar trimers with an rmsd of
   3.3 Å for 249 C[α] atoms. However, the trimer interface of chorismate
   mutase is entirely hydrophobic and lacks the polar central cavity of
   YabJ.
   [83]Figure 3
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   Figure 3

   Comparison of YabJ and B. subtilis chorismate mutase. (A) Superposition
   of subunits. C[α] traces of YabJ (thick lines) and chorismate mutase
   (thin lines) are overlaid. Orientation is similar to Fig. [87]1. (B)
   Ribbon diagram of the chorismate mutase trimer. The reaction product
   prephenate is shown in the active site cleft. Orientation is as in Fig.
   [88]2.

   Chorismate mutase catalyses the unimolecular, pericyclic rearrangement
   of chorismate to prephenate, a reaction at the branch point of the
   biosynthetic pathway for aromatic amino acids. The three active sites
   of the chorismate mutase trimer are located in clefts between subunits,
   analogous to the clefts at the subunit interfaces of the YabJ trimer
   (Fig. [89]3 B). Eleven residues in the active site of chorismate mutase
   have been implicated in substrate binding and transition state
   stabilization ([90]23). Two of these side chains, Phe 57 and Cys 75,
   have chemical and topological equivalents, Phe 84 and Cys 104, in YabJ.
   However, elsewhere in the cleft, YabJ and chorismate mutase do not have
   common functional groups. We conclude that YabJ and chorismate mutase
   probably have a common ancestor but have evolved to perform different
   functions.

 §3§ Sequence Homologs. §3§

   YabJ belongs to the YER057c/YjgF/UK114 family of small proteins
   (ProSite accession nos. PS01094 and UPF0076). We identified a total of
   37 homologs having 20–53% sequence identity with YabJ in the sequence
   database ([91]21). This level of identity indicates a common ancestor,
   a common fold, and possibly a common function for the proteins.

   No invariant residues result from multiple alignment ([92]24) of the 38
   sequences of YabJ and its homologs. However, if the homologs have
   evolved to more than one function, invariance would not be expected. To
   probe the sequence data for clues to biochemical function, we looked
   for a subset of more closely related sequences. Based on the multiple
   sequence alignment, the homologs were divided into two groups—a “high
   identity” group of 25 more closely related sequences, each having at
   least 43% identity with another sequence in the group, and a “low
   identity” group of 13 more divergent sequences, each having generally
   <30% identity to any member of either group. The high-identity group,
   including YabJ, has nine invariant residues (Fig. [93]4). Although they
   are spread throughout the primary sequence, all of these residues map
   to the narrow, deep cleft between subunits of the YabJ trimer (Fig.
   [94]5). This site is structurally analogous to the active site of
   chorismate mutase. The clustering of invariant residues in the cleft
   strongly suggests a common molecular function of catalysis or binding
   for members of the high-identity group.
   [95]Figure 4
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   Figure 4

   Comparison of sequences of YabJ and 24 homologs in the high-identity
   group. Residues in red boxes are invariant; in yellow boxes are sites
   of conservative substitution. Secondary structures are indicated above
   the alignment. Residues in the subunit interface are indicated by
   triangles below the alignment, and those with main-chain interactions
   by circles. Multiple sequence alignment was done with clustalw
   ([99]24). The figure was produced with alscript ([100]33).
   [101]Figure 5
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   Figure 5

   Stereo view of the intersubunit cleft of YabJ. The translucent
   molecular surface is in gray, with surfaces of invariant residues from
   the two subunits in rust and cyan. C[α] wire diagrams for two subunits
   are yellow and white, as are the side chains of invariant residues
   (Phe84, Asn88, and Arg102 in the yellow subunit, and Pro11, Tyr17,
   Gly31, Asn56, Pro111, and Glu117 in the white subunit). The figure was
   produced with dino (ref. [105]34;
   [106]http://www.bioz.unibas.ch/∼x-ray/dino).

   Two structural databases were queried for similar residue
   constellations in YabJ and any protein of known structure and function.
   procat ([107]25) is a database of functional groups in enzyme active
   sites. None of these active site templates matches any constellation of
   residues in the YabJ structure. Conversely, the nine YabJ side chains
   that are invariant in the high-identity group do not have an analog
   among protein structures deposited in the Protein Data Bank, as probed
   with the program spasm ([108]26). Thus, it appears that the arrangement
   of residues in the conserved cleft of YabJ does not exist in any
   reported protein structure.

   Biological functions attributed to YabJ homologs do not point to a
   common biochemical activity. Most of the homologs are hypothetical
   proteins of unknown function. The genetic context of yabJ (purR operon)
   correlates with the observed phenotype of yabJ ^− (loss of purA
   repression by adenine). Similarly, Lactococcus lactis aldR has been
   implicated in regulation of isoleucine biosynthesis ([109]27).
   Salmonella typhimurium yjgF may have a role in regulation of the
   isoleucine and thiamine biosynthetic pathways ([110]28). In cases in
   which protein products have been studied, no certain biochemical
   activity has been identified, and a wide variety of biological
   functions has been proposed. The rat and human homologs are putative
   translation inhibitors ([111]14, [112]29), goat UK114 is implicated in
   tumor antigenicity ([113]30), mouse Hrp12 is thought to be a heat
   response protein ([114]19), and the bovine and rat homologs are
   proposed to affect activation of the calcium-dependent protease calpain
   ([115]20). Sequences for all of these homologs are in the high-identity
   group. The strong conservation of side chains in the YabJ cleft implies
   that the homologs of the high-identity group achieve their variety of
   biological functions by a common biochemical mechanism.

   Any common biochemical mechanism for the broadly distributed YabJ
   homologs would necessarily involve ligand or substrate binding to the
   12-Å deep, 8-Å wide conserved cleft at the subunit interface (Fig.
   [116]5). We screened broadly for a small-molecule ligand by soaking
   crystals at neutral pH in a stabilizing solution augmented with B.
   subtilis cell extract. No electron density for a ligand was identified
   in difference electron density maps computed by using diffraction data
   from the soaked crystals. This negative result does not distract us
   from investigation of the conserved cleft. Studies are underway to
   identify a ligand or substrate in solution and in vivo.

   We propose that YabJ and the high-identity homologs use the conserved,
   deep, narrow cleft for catalysis or binding of a common chemical
   entity. Molecular mechanisms by which these proteins could use a common
   biochemistry to influence a variety of biological processes include,
   among others, binding a regulatory macromolecule, altering the
   concentration of a key small-molecule metabolite through catalysis, and
   binding another macromolecule after a ligand-induced conformational
   change. Use of the conserved, deep, narrow cleft for binding implies
   recognition of an appropriately shaped small-molecule ligand or a
   single amino acid residue in a protein ligand. The structure of YabJ
   should be an important guide for identification of this molecule. This
   work also illustrates the challenges of deducing protein function from
   sequence and three-dimensional structure.
   [117]Previous Section[118]Next Section

 §2§ Acknowledgments §2§

   The authors thank the staff of BioCARS at the Advanced Photon Source,
   Argonne National Laboratory, for their advice during the data
   collection and Carol Greski for expert preparation of the manuscript.
   This work was supported by National Institutes of Health Grants
   DK-42303 to J.L.S. and GM-24658 to H.Z. and by the Finnish Ministry of
   Education (P.M.).
   [119]Previous Section[120]Next Section

 §2§ Note §2§

   After completion of this manuscript, a structure of E. coli YjgF, a
   YabJ homolog, was presented at the annual meeting of the American
   Crystallographic Association ([121]35). The function of E. coli YjgF is
   unknown. In E. coli, yjgF is not linked to purR, and E. coli purR is
   unrelated to B. subtilis purR.
   [122]Previous Section[123]Next Section

 §2§ Footnotes §2§

     * [124]↵ § To whom reprint requests should be addressed. E-mail:
       smithj{at}purdue.edu.
     * Data deposition: The atomic coordinate has been deposited in the
       Protein Data Bank, [125]www.rcsb.org (PDB ID code [126]1qd9).
     * Abbreviation:

        rmsd,
                rms deviation

     * Copyright © 1999, The National Academy of Sciences

   [127]Previous Section

 §2§ References §2§

    1. [128]↵
         1. Weng I ,
         2. Nagy P ,
         3. Zalkin H
       (1995) Proc Natl Acad Sci USA 92:7455–7459, pmid:7638212.
       [129]Abstract/FREE Full Text
    2. [130]↵
         1. Shin B S ,
         2. Stein A ,
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