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Originally published In Press as doi:10.1074/jbc.M606057200 on August 21, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31832-31842, October 20, 2006
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Comprehensive Alanine-scanning Mutagenesis of Escherichia coli CsrA Defines Two Subdomains of Critical Functional Importance*Formula

Jeffrey Mercante{ddagger}, Kazushi Suzuki{ddagger}, Xiaodong Cheng§, Paul Babitzke, and Tony Romeo{ddagger}1

From the Departments of {ddagger}Microbiology and Immunology and §Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322 and the Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802

Received for publication, June 23, 2006 , and in revised form, August 15, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The RNA-binding protein CsrA (carbon storage regulator) of Escherichia coli is a global regulator of gene expression and is representative of the CsrA/RsmA family of bacterial proteins. These proteins act by regulating mRNA translation and stability and are antagonized by binding to small noncoding RNAs. Although the RNA target sequence and structure for CsrA binding have been well defined, little information exists concerning the protein requirements for RNA recognition. The three-dimensional structures of three CsrA/RsmA proteins were recently solved, revealing a novel protein fold consisting of two interdigitated monomers. Here, we performed comprehensive alanine-scanning mutagenesis on csrA of E. coli and tested the 58 resulting mutants for regulation of glycogen accumulation, motility, and biofilm formation. Quantitative effects of these mutations on expression of glgCA`-'lacZ, flhDC `-'lacZ, and pgaA`-'lacZ translational fusions were also examined, and eight of the mutant proteins were purified and tested for RNA binding. These studies identified two regions of the amino acid sequence that were critical for regulation and RNA binding, located within the first (beta1, residues 2-7) and containing the last (beta5, residues 40-47) beta-strands of CsrA. The beta1 and beta5 strands of opposite monomers lie adjacent and parallel to each other in the three-dimensional structure of this protein. Given the symmetry of the CsrA dimer, these findings imply that two distinct RNA binding surfaces or functional subdomains lie on opposite sides of the protein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CsrA is a 61-amino acid RNA-binding protein originally identified in Escherichia coli K-12 (1). This protein is an important regulator of gene expression for the species in which it has been studied. At least 159 putative CsrA homologues are distributed among 130 different eubacterial species (2, 3) with several species encoding more than one homologue (e.g. Coxiella burnetii, Legionella pneumophila, Pseudomonas fluorescens, and Pirellula sp.). In E. coli, CsrA represses glycogen biosynthesis, gluconeogenesis, peptide transport, and biofilm formation while activating glycolysis, acetate metabolism, and motility (4-6). CsrA is also known to be critical for the regulated expression of virulence factors in pathogens of both animals and plants. For example, L. pneumophila CsrA is essential for the repression of transmission phase genes during infection of amoebas and alveolar macrophages (7); Salmonella enterica CsrA regulates pathogenicity island (SPI1) genes required for intestinal epithelial cell invasion (8); Erwinia carotovora RsmA represses expression of extracellular enzymes needed for plant pathogenicity and development of soft rot disease as well as production of homoserine-lactone quorum-sensing signals (9).

In E. coli, CsrA controls gene expression post-transcriptionally, binding specifically and with high affinity to the 5'-untranslated leader of several mRNAs, including cstA, glgCAP, flhDC, and pgaABCD, and affecting translation and RNA stability (4, 6, 10-12). The activity of CsrA is antagonized by the untranslated RNAs CsrB and CsrC, which contain multiple binding sites (~18 and ~9, respectively) that permit sequestration of CsrA (13, 14). Regulation of gene expression by CsrA-regulated in other species (i.e. Pseudomonas, Erwinia, Salmonella, and Vibrio) is believed to occur in a similar manner as in E. coli (15-19). The affinity of CsrA for a particular RNA target depends on both the RNA sequence and secondary structure (10, 20, 21). Until recently, no protein structural or functional information existed to describe how CsrA might recognize a particular RNA target with high affinity.

A major step in understanding CsrA structure was taken when three CsrA (RsmA) proteins were independently solved, all of which exhibited the same overall topology (22-24). These studies proved CsrA to be a novel class of RNA-binding protein and confirmed previous work that suggested that CsrA forms a homodimer (4). They also highlighted the unusual way in which the dimer is formed; two interlocking CsrA monomers produce a hydrophobic core composed of 10 beta-strands and two winglike {alpha}-helices. As described previously (24), a CsrA dimer is a barrel-like structure stabilized noncovalently by an extensive network of hydrogen bonds from backbone amino and carboxyl groups. With the benefit of a CsrA three-dimensional structure onto which any particular amino acid change can be modeled, it is now possible to map protein regions essential for RNA binding and/or regulation of gene expression.

The goal of the present study was to examine the amino acid requirements for CsrA-RNA interaction and gene regulation by the systematic mutation of every nonalanine codon within the E. coli gene. Alanine replacement has an extensive history in structure-function studies (25-31) and is well suited for use in site-directed mutagenesis because it is abundant in many types of secondary structures and displays an uncharged and nonintrusive side chain that typically is compatible with native protein folding (32). In vivo examination of CsrA-repressed (glgCAP, pgaABCD) and activated (flhDC) operons in the presence of this series of mutants uncovered two regions within the primary structure of CsrA that are essential for its roles in repression and activation of gene expression. Region 1 was located at the extreme N terminus (residues 2-7); region 2 was closer to the C terminus (residues 40-47). Four CsrA mutant proteins from each of these regions (L2A, L4A, R6A, and R7A; V40A, V42A, R44A, and I47A) were found to be defective in binding to a 16-nucleotide RNA probe containing a single high affinity CsrA target site. Interestingly, critical regions 1 and 2 were aligned in parallel and adjacent to each other within the three-dimensional space of the protein. In contrast to certain other multimeric RNA-binding proteins, such as the mammalian U1A and Nova (33, 34), bacteriophage coat protein MS2 (35) and E. coli AspRS (36, 37) wherein individual monomers comprise a complete functional domain, CsrA requires that each monomer contribute N- and C-terminal portions to the creation of a functional region. This further implies that a symmetrical CsrA homodimer contains two critical surfaces or subdomains located on opposite sides of the molecule. A model for CsrA structure that integrates these data and provides a basis for understanding how CsrA interacts with its RNA targets is presented and discussed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains, Plasmids, Bacteriophage, and Growth Conditions—All strains, plasmids, and bacteriophage used in this study are listed in Table 1s in the supplemental material. For routine culture of bacteria, strains were grown in LB medium (38) at 37 °C with shaking. Cultures for biofilm assays were grown in LB medium at 26 °C for 24 h without shaking. Motility assays were carried out as previously described in semisolid tryptone medium (1% tryptone, 0.5% NaCl, pH 7.4, 0.35% agar) at 30 °C (6). Glycogen accumulation was assessed on Kornberg medium (1.1% K2HPO4, 0.85% KH2PO4, 0.6% yeast extract, 1% glucose, pH 6.8, 1.5% agar). Antibiotics were added to media as needed, at the following concentrations: ampicillin, 100 µgml-1; kanamycin, 100 µgml-1; chloramphenicol, 25 µgml-1; tetracycline, 10 µgml-1.

Glycogen Accumulation, Biofilm, and Motility Assays—Endogenous glycogen accumulation was examined by iodine vapor staining of colonies or patches grown overnight at 37 °C on Kornberg medium (1). Quantitative biofilm formation assays were performed by staining adherent cells with crystal violet (39). These assays were performed at least twice, with four replicas per experiment, and the resulting averages and S.E. values were determined. Motility was assessed as described previously (6).

beta-Galactosidase Activity and Total Protein Assays—beta-Galactosidase activity was determined using the method described previously (40) with minor modifications. Briefly, cultures were diluted 1:500 in LB medium and grown with shaking at 37 °C in 2-ml polypropylene DeepwellTM 96-well plates (Nunc, Rochester, NY). At 24 h, the plate culture was placed on ice, and chloramphenicol or tetracycline was added to a final concentration of 100 µgml-1, or 10 µgml-1, respectively. Aliquots of each culture were removed to measure cell density (A600nm) and for total protein determination. Into each well of a second deep well plate was added 1 ml of Z buffer (40 mM NaH2PO4·H2O, 60 mM Na2HPO4·7H2O, 40 mM MgSO4·7H2O, 50 mM beta-mercaptoethanol), 20 µl of 0.1% SDS, 40 µl of CH3Cl, and 50 µl of the original culture. Components were mixed by pipetting, CH3Cl was allowed to settle to the bottom of the well, and 100 µl of permeabilized cells was transferred to a 96-well microtiter plate. Either o-nitrophenyl-beta-D-galactopyranoside or 4-methylumbelliferyl beta-D-galactopyranoside were added to the permeabilized cells at 666 or 500 µM, respectively. The o-nitrophenyl-beta-D-galactopyranoside reaction was stopped after 15 min by the addition of 50 µl of 1 M Na2CO3, and the sample plate was read on a Biotech Synergy HT microplate reader (Winooski, VT) at A420 nm and A550 nm. Samples with 4-methylumbelliferyl beta-D-galactopyranoside were monitored for 1-5 h in a microplate reader, periodically reading fluorescence using an excitation wavelength of 360 ± 40 nm and an emission wavelength of 460 ± 40 nm. In vivo expression was normalized as a percentage of empty vector control levels (labeled 100%) for the glgCA'-'lacZ and pgaA'-'lacZ fusions and as a percentage of wild-type CsrA levels (labeled 100%) for the flhDC'-'lacZ fusion.

Total cellular protein was measured using the bicinchoninic acid assay (41) as developed by Pierce. The current study employed a 96-well microplate reader to monitor A562 nm and bovine serum albumin as a protein standard (Pierce).

Construction of Chromosomal pgaA'-'lacZ and flhD'-'lacZ Translational Fusions—LacZ fusion plasmids pFDCZ6 and pPGAZ4 were converted from ampicillin-resistant (ApR) to chloramphenicol-resistant (CmR) by subcloning the cat gene into a deletion created in the coding region of bla. Primers for PCR amplification of the cat gene from pKD3 are listed in Table 2s; they include sites for directional cloning using PstI and ScaI restriction enzymes. pFDCZ6 and pPGAZ4 were cut with EcoRI, made blunt-ended using T4 DNA polymerase (New England Biolabs, Ipswich, MA), and subsequently cut with PstI, which allowed ligation to the amplified cat gene. The resulting gene fusions in plasmids pFDCZ6CAT312 and pPGAZ4CAT2321, which were ApS and CmR, were integrated into the E. coli CF7789 chromosome using the {lambda} InCh1 system, as described previously (42). Chromosomal fusions were confirmed by colony PCR according to the {lambda} InCh1 protocol.

Construction of CsrA-His6 Site-directed Mutants—Each nonalanine amino acid residue of E. coli CsrA was substituted with alanine, providing a library of plasmids expressing 58 site-directed mutants of CsrA-His6 (Table 1s). Mutations were constructed using the QuikChange II ® site-directed mutagenesis system (Stratagene, La Jolla, CA), employing the template plasmid pCSRH6-19 and the primers listed in Table 2s. All mutations were confirmed by DNA sequence analysis performed at SeqWright DNA Technology Service (Houston, TX).

CsrA-His6 Protein Purification—Native CsrA-His6 and eight mutant proteins were purified from E. coli strain CF7789, using nickel affinity chromatography according to a previously described method (Qiaexpressionist; Qiagen, Valencia, CA). Briefly, 1 liter of culture was grown overnight in LB + 1% glucose. The cells were concentrated by centrifugation, resuspended (1 g/ml) in wash buffer (10 mM imidazole and 1 mg ml-1 lysozyme), and lysed by sonication. The resulting lysate was cleared by centrifugation and combined with 1 ml of Ni2+-NTA2 for 1 h to allow binding of CsrA-His6 to the nickel-NTA beads. The lysate-Ni2+-NTA mix was applied to a gravity flow column, and the beads were washed with 10 ml of wash buffer containing 50 mM imidazole. CsrA-His6 was eluted with 5 ml of wash buffer containing 250 mM imidazole. Protein purity was assessed by SDS-PAGE (15%) and Coomassie Blue staining, followed by densitometry using the Bio-Rad Universal Hood and QuantityOne software package. Purity of the CsrA wild-type and alanine mutant proteins was as follows: wild type ≥ 98%; L2A ≥ 97%; L4A ≥ 97%; R6A ≥ 98%; R7A ≥ 98%; V40A ≥ 90%; V42A ≥ 90%; R44A ≥ 95%; I47A ≥ 98%.

Western Analyses of CsrA-His6—Cultures for Western blot analyses were grown at 37 °C for 24 h with shaking. Cells from 1 ml of culture were concentrated and resuspended in lysis buffer (90 mM Tris-HCl, 2% SDS, pH 6.8). Samples were boiled for 3 min, cell debris was removed by centrifugation, and the supernatant solution was saved and assayed for total protein. Five µl of SDS-PAGE loading buffer (135 mM Tris-HCl, 3% SDS, 0.03% bromphenol blue, 30% glycerol, pH 6.8) was added to 100 µg of total protein (~10 µl), and proteins were separated by SDS-PAGE (15%). Gels were equilibrated in Towbin buffer + 20% methanol (43) and electroblotted overnight to a nitrocellulose membrane (0.2 µm). Blots were washed and probed with His-probeTM horseradish peroxidase (Pierce) at 1:2000 in Tris-buffered saline with Tween 20 and developed with SuperSignal® West Pico chemiluminescent substrate (Pierce) according to the manufacturer's recommendations.

Three-dimensional Modeling—The coordinates for the three-dimensional structure of CsrA from Yersinia enterocolitica were obtained from the Protein Data Bank (accession code 2BTI). Images were rendered using the open source modeling software PyMol (44).

RNA Gel Mobility Shift Assay—Gel mobility shift assays were carried out according to previously published procedures (12, 21). All calculations were based on the mass of CsrA-His6 as a homodimer (15,357 Da). The RNA probe was designed based on a high affinity consensus CsrA binding target as determined by SELEX (systematic evolution of ligands by exponential enrichment) analysis (21). The minimal CsrA target sequence, 5'-GGCACAAGGAUGUGCC-3', was synthesized by Integrated DNA Technologies (Coralville, IA) and 5'-end-labeled using T4 polynucleotide kinase and [{gamma}-32P]ATP. Radiolabeled RNA probe was gel-purified, suspended in TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), heated to 85 °C, and slowly cooled to room temperature. Increasing concentrations of CsrA-His6 wild type or mutant protein were combined with 30 pM labeled RNA probe in 10-µl binding reactions (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM KCl, 3.25 ng of total yeast RNA, 10 mM dithiothreitol, 10% glycerol, 4 units of RNase inhibitor (Ambion, Austin, TX) for 30 min at 37 °C to allow CsrA-RNA complex formation. Binding reactions were fractionated using native PAGE (12%), and radioactive bands were visualized with an Amersham Biosciences PhosphorImager. Bound and unbound RNA species were quantified with ImageQuant software (Amersham Biosciences), and an apparent equilibrium binding constant (Kd) was calculated for CsrA-RNA complex formation according to a previously described cooperative binding equation (45), as adapted here,

Formula(Eq.1)

where Ymax represents the maximum possible bound fraction (100%) of RNA (RNAb), and Kd is the concentration of free protein (CsrAf) at which RNAb reaches 50% bound. Isolated CsrA proteins were assumed to be 100% active for these calculations. The cooperativity of binding is described by the Hill coefficient, n.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Phenotypic Effects of CsrA Site-directed Mutations: Glycogen Accumulation, Motility, and Biofilm Formation—CsrA post-transcriptionally regulates several cellular processes in E. coli K-12 (4, 6, 10, 12, 39). To determine which CsrA residues were important for functionality in vivo, every nonalanine codon of a His-tagged csrA gene (14) was changed to alanine. The resulting library of 58 mutant csrA-expressing plasmids was introduced into the csrA-deficient strain E. coli K-12 TR1-5 MG1655 and tested for effects on the repression of glycogen accumulation and biofilm formation and activation of motility relative to the wild-type His-tagged CsrA protein and an empty vector control. Regulation of these three processes was highly dependent on the position of the mutation (Fig. 1, B, D, and F). Substitutions that caused the greatest effects on glycogen accumulation and motility were mainly clustered in two regions: the N terminus (residues 2-7) and close to the C terminus (residues 40-47). Additionally, mutation of certain amino acids between these two regions (residues 11-14 and 22) affected these phenotypes. Biofilm formation was also elevated (regulation was defective) in strains containing csrA mutations in these two main regions. However, this phenotype appeared to be more sensitive to changes throughout the CsrA sequence, and mutations in two other regions (residues 18-23 and 31-38) also increased biofilm formation.

Effects of CsrA Alanine Substitutions on Expression of Chromosomally Encoded glgCA'-, flhDC'-, and pgaA'-lacZ Translational Fusions—We next determined the effects of CsrA alanine substitutions on the regulation of the glgCAP, flhDC, and pgaABCD operons using glgCA'-'lacZ, flhDC'-'lacZ, and pgaA'-'lacZ translational fusions (Fig. 1, A, C, and E). The gene expression effects of alanine substitutions were well correlated with the phenotypic effects on glycogen accumulation, motility, and biofilm formation. Once again, the extreme N terminus (residues 2-7), and a region closer to the C terminus (residues 40-47) were important. Alanine substitution of Thr11, Leu12, Ile14, and Val22 also led to defects in regulation. Based on a composite graph developed from these findings (see Fig. 6, In vivo regulatory defect), mutations in two regions affected expression of all three fusions: region 1 (Leu2, Ile3, Leu4, Arg6, and Arg7) and region 2 (Val40, Val42, Arg44, Glu46, and Ile47). Only modest defects were exhibited by mutation of Thr5, His43, or Glu45 in these regions. In addition, although pgaA'-'lacZ expression exhibited greater effects of most of the alanine mutations when compared with glgCA'-'lacZ or flhDC'-'lacZ, regulation of all fusions followed the same trends (Fig. 1). The pgaABCD leader contains six confirmed CsrA binding sites, more than any other known CsrA-regulated operon (12). Perhaps efficient binding at all six sites is needed for full repression, and therefore a small reduction in CsrA binding affinity may have greater potential to derepress pgaABCD expression.


Figure 1
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  		FIGURE 1.
Phenotypic and regulatory effects of CsrA alanine replacement mutations. Glycogen accumulation, biofilm formation and motility phenotypes, and expression of chromosomally encoded glgCA'-'lacZ, pgaA'-'lacZ, and flhDC'-'lacZ translational fusions, respectively, were examined in the presence of each alanine-scanning csrA mutant. The amino acid residue that was changed to alanine is indicated in each panel. The phenotypic effects were tested in strain TRMG1655 (csrA::kan). A, C, and E, expression from a glgCA'-'lacZ fusion in strain TRKSGA18 (A), pgaA'-'lacZ fusion in strain TRCFPGACAT2321 (C), and flhDC'-'lacZ fusion in strain TRCFFDCAT312 (E) are shown. All values are shown relative to the wild type (WT) and vector controls, located to the right. B, intracellular glycogen levels were assessed by staining with iodine vapors after growth on Kornberg media with glucose. D, Biofilm formation was assessed by staining adherent cells with crystal violet, and motility was determined in semisolid tryptone medium (see "Experimental Procedures"). Values for expression analyses are reported as average ± S.E. All experiments were performed at least twice and each time in triplicate or quadruplicate.

 
Surprisingly, at least one alanine substitution, T19A, caused CsrA to become a stronger regulator of gene expression from all operons that were tested (Fig. 1). N35A also appeared to make CsrA a slightly better regulator of gene expression from glgCA'- and flhDC'-'lacZ.

Steady-state Protein Levels of CsrA Site-directed Mutant Proteins—Altered CsrA function in vivo might reflect a change in either the intrinsic activity or intracellular concentration of a mutant protein. Therefore, the steady-state levels of mutant proteins were determined by Western blot analysis (Fig. 2). This analysis revealed that the defective proteins I3A, T11A, L12A, I14A, V22A, and E46A failed to accumulate properly. Levels of most of the mutant proteins (59%) were within 2-fold of the wild type protein. A substantial percentage of mutants exhibited either very low (≤25%) or very high (≥175%) levels. Interestingly, of the three mutants that accumulated to ≥175% of wild type protein levels, two were functionally impaired (R6A and R44A). Conversely, among the nine proteins that exhibited ≤25% of wild type protein levels, five displayed ≥50% of wild type activity (Q52A, V18A, K55A, V20A, and V34A).

It is noteworthy that although total protein (SDS-extractable) for R44A was high, the quantity of soluble protein isolated by nickel affinity chromatography was similar to that of the wild type (data not shown). This suggests that R44A may have formed aggregates that were relatively resistant to proteolysis in the cell, which were removed by the centrifugation during purification. Such behavior was not reported for the Pseudomonas R44A mutant protein but may have been missed because the yield of this protein was determined only after purification under native conditions (24).


Figure 2
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  		FIGURE 2.
Quantitative immunoblotting of TRMG1655 (csrA::kan) expressing wild type and 58 alanine-scanning mutant CsrA proteins. E. coli whole cell lysates were examined by SDS-PAGE and Western analysis, using a nickel-conjugated horseradish peroxidase to detect CsrA-His6 (see "Experimental Procedures"). CsrA mutant protein levels were measured by densitometry and are reported as a percentage of wild type (WT). Experiments were repeated at least twice, and values are reported as average ± S.E.

 
RNA-binding Studies of Eight CsrA Mutant Proteins—To determine whether the in vivo regulatory defects of selected mutant proteins were due to defective RNA binding, eight of the mutant proteins were purified and examined by gel mobility shift assays (Fig. 3). Proteins I3A, T11A, L12A, I14A, V22A, and E46A, whose defects were likely to have been caused by altered accumulation (Fig. 2), were avoided. Mutant proteins L2A, L4A, R6A, R7A, V40A, V42A, R44A, and I47A were purified by nickel affinity chromatography and tested for binding to a 5'-end-labeled 16-nucleotide RNA probe, which contained a high affinity binding site (21). We chose this minimal RNA sequence for the binding studies, rather than using a native transcript containing multiple CsrA target sites, in order to simplify the analyses and focus on direct protein-RNA binding while attempting to avoid cooperativity effects. Binding of wild type CsrA and the eight mutant proteins to the small RNA oligomer led to the formation of shifted complexes (Fig. 3, A-I, left panels). Binding curves, Hill coefficients (n), and apparent equilibrium binding constants (Kd) for the wild type and mutant proteins (Fig. 3, A-I, right panels) were calculated as described under "Experimental Procedures." The affinities of these mutant proteins for RNA were substantially decreased. Their apparent Kd values were increased from 10- to 150-fold with respect to the wild type CsrA protein, and their affinities for RNA were as follows, in order from highest to lowest: wild type > R7A > R6A > L4A > V40A > I47A > L2A > V42A > R44A. In addition, the observed binding affinities of these proteins were roughly correlated with their abilities to regulate gene expression in vivo (Fig. 4). The cooperativity of binding (n) was also increased for all mutant proteins tested as compared with wild type. Currently, we cannot account for this difference in cooperativity.

Mapping Critical Amino Acid Residues of CsrA onto a Three-dimensional Model—Critical regions 1 and 2 were mapped to the recently published, high resolution three-dimensional structure of CsrA (RsmA) from Yersinia enterocolitica (Fig. 5) (24). CsrA from Escherichia and Yersinia are 95% identical, differing only at amino acids 58-60, which have not been structurally defined in any model (22-24) and which did not affect regulation (Fig. 1). The important residues of region 1 were all located within the first beta-strand (beta1) of the protein. Region 2 residues were distributed from T3 through beta5 to the beginning of the {alpha} helix (Figs. 5, A and C, and 6, Secondary Structure). As revealed by the crystal structure, strand beta1 of one CsrA monomer is located adjacent and parallel to strand beta5 from the other monomer (Fig. 5, A and C). This suggests that region 1 of one monomer and region 2 of the other monomer together define a functional subdomain, and alanine substitutions created in either strand alter the same critical area of the dimer. The side chains from Leu2, Leu4, Val40, and Val42 all appear in hydrophobic surroundings (Fig. 5, F and G). Mutation of these residues probably compromises the core by creating space that results in improper folding or affects solvent access. Specifically, the side chain of Val40 is directed into the hydrophobic core (Fig. 5G) while Leu2, Leu4, and Val42 form a separate but adjacent hydrophobic pocket (Fig. 5F) that may stabilize the interactions between beta1', beta2', and beta5 (and beta1, beta2, and beta5'). The solvent-exposed side chains of Arg7, Ile47, and Arg44 are all oriented in the same direction on each side of the dimer and do not appear to be involved in intra- or intermolecular contacts (Fig. 5, D and E). Arg6 and Glu46 are also exposed to solvent and appear to be connected via a salt bridge (data not shown), as suggested previously (24).

Other alanine substitutions outside of regions 1 and 2, which compromised (T11A, L12A, I14A, and V22A) or improved (T19A and N35A) regulation were also mapped onto the structural model (Fig. 5, D and G). Residues Ile14 and Val22 clearly point into and stabilize the hydrophobic core Fig. 5G). Leucine 12 (Fig. 5G) is positioned at the boundary between the core proper and the extended hydrophobic pocket created by Leu2, Leu4, and Val40 (Fig. 5F). According to the three-dimensional model, Thr11 is solvent-exposed and potentially makes a polar contact with Thr21 (Fig. 5D). Additionally, the polar side chain of Thr19 is solvent-exposed and is located directly adjacent to Asn35 (Fig. 5D). These two residues appear to interact with each other both directly and through a water molecule (data not shown). At present, it is difficult to explain why alanine substitutions of the latter residues (T19A and N35A) should cause a gain of function.

Sequence Alignment of Critical Residues across 30 Representative Species—Orthologs of CsrA are well represented in the Proteobacteria but are also known from the Actinobacteria, Thermotogae, Planctomycetes, Spirochaetes, and Firmicutes (2). A sequence alignment of 30 CsrA representatives, including at least one member from each of the six phyla listed above, is shown in Fig. 6. Among the representative sequences listed here and in the collection of 126 CsrA orthologs at the Sanger Pfam alignment data base (available on the World Wide Web at pfam.wustl.edu/cgi-bin/getdesc?name=CsrA), 14 CsrA amino acid residues (23%) are ≥80% identical across all species. Conservation analysis was performed on the CsrA alignment using the algorithm Analysis of Multiply Aligned Sequences (AMAS) (46), as it is implemented in the JalView Sequence alignment viewer (47) (Fig. 6, Conservation). AMAS grades positional physiochemical amino acid conservation on a scale from 0 to 10 where 10 represents complete conservation. Based on this analysis, 21 (34%) of the CsrA residues aligned here and 23 (38%) residues in the Pfam CsrA alignment data base are positionally identical (scored 10). Highly conserved residues (scoring 7-9) were distributed throughout the CsrA primary structure but were especially concentrated at the extreme N terminus, residues 2-7. A majority of the identical or highly conserved residues found between regions 1 and 2 (i.e. beta2-beta4) are located within constrained environments, where they may contribute to the stability of the three-dimensional structure. These positions show a large number of neutral substitutions (i.e. Leu -> Ile or Val -> Leu) (48). These residues include Leu12, Ile14, Val18, Val20, Val22, Val25, Val30, Ile32, Ile34, and Ala36, which point into the hydrophobic core, and Gly15 and Pro37, which are found within loops 2 and 4, respectively.


Figure 3
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  		FIGURE 3.
Gel mobility shift assays for the binding of wild type and mutant CsrA proteins to a 16-nucleotide high affinity RNA target. 5'-End-labeled RNA (30 pM) was incubated with increasing concentrations of CsrA, shown below each lane. The positions of bound (B) and free (F) RNA are indicated. A-I, graphs to the right of each gel shift display the best fit curve, apparent equilibrium binding constant (Kd), and Hill coefficient (n) for each protein (see "Experimental Procedures"). J, graphical comparison of CsrA mutant best fit binding curves. Graphpad Prism version 4 for Mac (San Diego, CA) software was used for calculations and graphical displays.

 


Figure 4
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  		FIGURE 4.
Correlation between the "in vivo regulatory defect" (Fig. 6) and the equilibrium binding constant (Kd) (Fig. 3) of eight CsrA mutant proteins. The correlation coefficient (R2) of a linear best fit comparison of these values was 0.63.

 
The two regions that were critical for regulation and RNA binding are highlighted in Fig. 6 (black boxes), and a composite graph (In vivo regulatory defect) allows a direct comparison of sequence conservation and these data. There was a strong correlation between amino acids that were identical or highly conserved in regions 1 and 2 and those that were important for CsrA function. The residues labeled region 1, found entirely within the first beta-strand, were given an AMAS score of 7 or above. This value increased to 9+ when comparing the entire Pfam group of 126 (data not shown). Specifically, positions 3 and 4 contained neutral substitutions to aliphatic amino acids (Ile, Val, or Leu), whereas positions 5 and 7 encoded polar (Ser or Thr) and basic residues (Lys or Arg), respectively. Both Leu2 and Arg6 were 100% identical, and Leu4 was ≥96% identical across all species in the data base. Unlike region 1, every residue within region 2 was not highly conserved; however, amino acids that proved to be critical for CsrA function (Val40, Val42, Arg44, Glu46, and Ile47) scored 8 or above by AMAS. Both Arg44 and Glu46 were ≥98% identical across all species in the Pfam data base (data not shown). Positions Val40, Val42, and Ile47 were between 63 and 73% identical and contained mostly neutral substitutions to aliphatic amino acids.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study has taken advantage of recent advances in our understanding of CsrA structure in order to define its structure-function relationship. CsrA represents a novel class of RNA-binding protein, and our experiments define a new type of subdomain for RNA interaction and regulation of gene expression. The CsrA homodimer contains two of these functional subdomains, located on opposite sides of the protein. Each subdomain is composed of primary sequences (regions 1 and 2) contributed by each polypeptide chain of the dimer (Fig. 5, A and C). The beta-strands of these two regions are positioned adjacent and parallel to each other within the dimer. As discussed below, this structure appears to account for the stoichiometry of CsrA within the CsrA-CsrB ribonucleoprotein complex, which heretofore could not be explained.

A careful examination of the three-dimensional structure of CsrA permitted predictions of the manner in which specific alanine substitutions affected protein function. The residues most likely to interact directly with RNA are Arg7, Arg44, and Ile47. The side chains of these critical amino acids are solvent-exposed and are not associated with other residues in the CsrA dimer. They are clustered on each side of the protein within areas of positive surface potential (Fig. 5B), forming two possible RNA binding surfaces (Fig. 5, D and E). R44A substitution caused the greatest defect in RNA binding and eliminated regulation by CsrA. These results were consistent with a previous study (24), demonstrating that this amino acid was important for RsmA-mediated regulation of several Pseudomonas aeruginosa phenotypes as well as RNA binding. I47A also exhibited low affinity for RNA and retained only ~5% of its ability to regulate gene expression. Of these proteins, R7A showed the mildest RNA binding defect and regulated gene expression at ~30% of wild-type levels. Originally, the side chains of Thr5 (24), Lys26, Arg31, Asn28 (22, 24), Val30, Glu10, or Gln29 (22) were proposed to be components of an RNA binding surface based on their conservation, solvent accessibility, charge, or proximity to Arg44. However, we found no evidence for critical involvement of these residues in regulation of gene expression. These cases highlight the pitfalls of using structural data alone to deduce the important features of a novel protein fold.

The basic residue Arg6 appears to span the gap between beta1 and beta5' to make an important structural contact with the side chain of Glu46. This bond probably stabilizes the relative positions of beta1 and beta5 ', in turn correctly orienting Arg7, Arg44, and Ile47. We cannot eliminate the possibility that Arg6 might also contact RNA directly. The importance of the Arg6-Glu46 interaction is also supported by the finding that these amino acids are ≥98% identical across the 126 species listed in Pfam. Mutation of Arg6 to alanine probably abolishes this critical interstrand connection, perhaps increasing the flexibility of beta5' and beta1 and altering the hydrophobic pocket located around Leu2, Leu4, and Val42. It is interesting to note that Arg6 is the main outlier when comparing RNA binding affinity with in vivo regulation (Fig. 4). Thus, relative to other substitutions, R6A was able to bind RNA with a greater affinity than its in vivo function would predict. This protein also accumulated ~2-fold greater than wild type (Fig. 2). A possible explanation for both observations is that this protein might form protease-resistant aggregates in the cell, which accumulate but do not participate in regulation in vivo. The tendency of CsrA to aggregate has been documented since the protein was first purified (14, 22).

The extended hydrophobic pocket formed by Leu2, Leu4, and Val42 that was disrupted by alanine substitution may serve as an RNA binding surface. There is precedence for this type of mechanism; human splicing factor protein U1A binds nucleic acids via variable loops but also creates an enlarged hydrophobic core when it packs RNA bases against nonpolar residues found on a conserved beta-sheet (49). It is not uncommon for nonpolar amino acids to participate in nucleic acid binding, and occasionally hydrophobic interactions are the central mechanism of association. For instance, Nova-2 KH3 binds RNA via an extended {alpha}/beta platform that is composed entirely of hydrophobic amino acids. Interestingly, both human U1A, which recognizes RNA structure and charge distribution, and Nova-2 KH3 preferentially bind RNA stem-loops, similar to CsrA (49, 50).


Figure 5
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  		FIGURE 5.
Critical residues of CsrA mapped onto the three-dimensional model of Y. enterocolitica CsrA. A, ribbon diagram of CsrA highlighting functional regions 1 and 2 (red) identified in the present study. The individual CsrA monomers are colored white and green. The second functional region found on the opposite side of the homodimer is not highlighted. B, identical view angle of CsrA, as shown in A, displaying electrostatic surface potential. Electropositive areas are represented in blue, whereas the electronegative areas are in red. C, CsrA displaying critical residues found in regions 1 (Leu2, Leu4, Arg6, and Arg7) and 2 (Val40, Val42, Arg44, and Ile47). The corresponding critical residues found on the opposite side of the protein are not illustrated. D, front view of a semitransparent CsrA displaying residues that are probably involved directly in RNA binding (Arg44, Ile47, and Arg7) and several residues found outside regions 1 and 2 (Thr11, Thr19, Thr21, and Asn35) that affect regulation. E, bottom-up view of the likely RNA binding residues listed in D. F and G, top-down view illustrating amino acids that compose a separate but adjacent hydrophobic pocket (Leu2, Leu4, and Val42) (F) and those that point inward and stabilize the CsrA hydrophobic core (Leu12, Ile14, Val22, and Val40) (G).

 
Not every amino acid residue within the two critical regions of CsrA was vital for regulation of gene expression (e.g. Thr5 and Ser41 of regions 1 and 2, respectively), and some residues were more important than others (R7A exhibited the mildest regulatory defect, R44A the most severe). Interestingly, the majority of the important residues among the region 2 mutations exhibited an alternating pattern, consistent with their location within a beta-strand. Residues directed to one side of the strand were critical (Val40, Val42, Arg44, and Glu46), whereas others (Glu39, Ser41, His43, and Glu45) were not.


Figure 6
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  		FIGURE 6.
Comparison of CsrA orthologs from six phyla, highlighting regions that were crucial for RNA binding and in vivo regulation with predicted amino acid functions. Secondary structure and residue conservation are displayed above and below the aligned sequences, respectively. The "In vivo regulatory defect" for each mutant was calculated from an equal weighted mean of the gene expression data from the three reporter fusions (Fig. 1). Mutant CsrA proteins that were experimentally tested for RNA binding, CsrA primary structure that was important for function, and functional predictions for individual amino acids (P) are as shown at the bottom. Conservation analysis was performed as described under "Results" using the algorithm AMAS (a score of 10 indicates complete conservation) and displayed using JalView (47). CsrA protein sequences were collected from the Sanger Pfam data base (2) and aligned with ClustalW (65). *, positions that show 100% identity; +, an AMAS score of 10.

 
Although the previous x-ray and NMR analyses suggested similar structures for the three CsrA orthologs, all of these proteins contained amino-terminal modifications, and none was documented to be biologically active (22-24). Studies with N-terminally tagged CsrA derivatives3 and results from the current study demonstrate that the N terminus of CsrA is critical for its function. The N-terminal four amino acids in almost every species are hydrophobic (Fig. 6), and the N-terminal fusions, which introduced charged or polar residues, almost certainly affect function (32). In fact, modeling of the Y. enterocolitica and P. aeruginosa CsrA (Protein Data Bank accession code 1VPZ) structures revealed two and three possible nonnative hydrogen bonds, respectively, between the backbones of beta1 and beta'4 in proteins containing these tags (data not shown). Thus, the structure of the native protein around the important beta1, beta'4-T-beta'5 region may be slightly different from the existing models.

The finding that a symmetric CsrA dimer contains two functional subdomains on opposite sides of the protein may explain the longstanding observation that CsrB RNA, which contains ~18 CsrA target sequences, binds to ~18 CsrA subunits (nine functional dimers) to form a globular complex (14). Due to the flexibility of RNA, CsrA could theoretically bridge two target sites (e.g. within two hairpin loops). Additional studies will be required to clearly define the precise stoichiometry of this and other CsrA-RNA complexes. Furthermore, CsrA itself may react to RNA in an adaptable, plastic manner, as suggested by solution NMR studies, which found that almost every amide signal of CsrA exhibits a shift upon the addition of RNA (22). The dimeric structure of CsrA also dictates that for each mutation that was constructed, two alanine substitutions resulted in the homodimer. Although pairs of substitutions may serve to further destabilize the core when they involve an interior hydrophobic residue, those that affect CsrA-RNA contact may actually have removed some ambiguity from the results by decreasing RNA binding affinity simultaneously at both subdomains.

RNA-binding proteins utilize several distinct secondary structures to construct a wide array of tertiary folds that mediate protein-RNA contact (51-54). Detailed structural information exists for protein-RNA complexes formed by several motif classes, including the RNA recognition motif (51, 55), KH (56), Sm-like (57), and OB fold (52). The 5beta-1{alpha} configuration of CsrA, however, does not fit within any of these protein families. The RNA recognition motif is a 4beta-2{alpha} structure, where the central two strands in a four-stranded antiparallel sheet are involved in RNA binding (55); the OB fold is characterized by a flattened beta-barrel composed of two beta-sheets, where beta-strands 2 and 3 interact with nucleic acid (52). In both the RNA recognition motif and OB, a beta-sheet comprises the primary RNA binding surface displaying aromatic side chains involved in intermolecular stacking. However, the OB-fold also stabilizes the RNA-protein complex via packing interactions with nonpolar residues and the aliphatic regions of polar side chains (52, 56, 58). The CsrA functional region defined in this study contains hydrophobic, acidic, and basic but not aromatic residues, which appear to be critical for binding and biological function.

The number and arrangement of RNA recognition motifs in a protein is critical for proper function. Such motifs can function individually (e.g. human HNRNPC and U1-70K) or in multiples (e.g. Drosophila ELAV and human U2B'') (53, 59). Tandem motifs can bind single-stranded RNA across the face of both motifs, as exemplified by poly(A)-binding protein (60), or within clefts formed between the motifs, as in Drosophila Sxl (61). The former type of protein-RNA relationship is also seen for the Thermus thermophilus SerRS dimer, where a single tRNA binds across the surface of both protein subunits (62), and for the Bacillus subtilis TRAP undecamer, a toroid-shaped protein that contacts each of a series of RNA triplet repeats in the trp leader transcript using sequential subunit interfaces around its circumference (63, 64). CsrA may employ its two distinct subdomains simultaneously in binding to RNA. Ultimately, a CsrA-RNA co-crystal or solution NMR structure will be necessary to define the critical contacts between CsrA and its RNA substrates and would provide new insights into protein-RNA molecular interactions.


   FOOTNOTES
 
* 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1s and 2s. Back

1 To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Emory University School of Medicine, 3105 Rollins Research Center, 1510 Clifton Rd. N.E., Atlanta, GA 30322. Tel.: 404-727-3734; Fax: 404-727-3659; E-mail: romeo{at}microbio.emory.edu.

2 The abbreviations used are: NTA, nitrilotriacetic acid; AMAS, Analysis of Multiply Aligned Sequences. Back

3 T. Romeo, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Alex Yakhnin for technical assistance and help with binding equations.



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