Báo cáo khoa học: Structure of FocB – a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli pdf

Structure of FocB – a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli Ulrika W. Hultdin 1 , Stina Lindberg 2 , Christin Grundstro ¨ m 1 , Shenghua Huang 1 , Bernt Eric Uhlin 2 and A. Elisabeth Sauer-Eriksson 1 1 Department of Chemistry, Umea ˚ University, Sweden 2 Department of Molecular Biology, The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea ˚ University, Sweden Introduction Fimbriae, which are long, adhesive, polymeric protein structures, are expressed on the surface of many patho- genic bacteria. Uropathogenic Escherichia coli (UPEC), the primary cause of urinary tract infections, express various types of fimbriae for adhering to (and some- times invading) host cells [1,2]. Because the interaction between the fimbrial adhesins and their receptors is specific, the expression of different types of fimbriae makes infection progress in different niches of the urinary tract. Keywords fimbriae; FocB; repressor protein; uropathogenic Escherichia coli; X-ray crystallography Correspondence A. E. Sauer-Eriksson or B. E. Uhlin, Department of Chemistry, Umea ˚ University, SE-90187 Umea ˚ , Sweden; Department of Molecular Biology, Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea ˚ University, SE-901 87 Umea ˚ , Sweden Fax: +4690 7865944; +4690 772630 Tel: +4690 7865923; +4690 7856731 E-mail: elisabeth.sauer-eriksson@ chem.umu.se; bernt.eric.uhlin@molbiol.umu.se Database The atomic coordinates and structure factors for the Escherichia coli FocB protein are available in the Protein Data Bank database under the accession number 3M8J (Received 28 January 2010, revised 5 May 2010, accepted 17 June 2010) doi:10.1111/j.1742-4658.2010.07742.x In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involved in the regulation of fimbriae gene clusters. Recent findings suggest that members from this family of proteins may form homomeric or heteromeric complexes and exert both positive and negative effects on the transcription of fimbriae genes. To elucidate the detailed function of FocB, we have determined its crystal structure at 1.4 A ˚ resolution. FocB is an all a-helical protein with a helix- turn-helix motif. Interestingly, conserved residues important for DNA-binding are located not in the postulated recognition helix of the motif, but in the preceding helix. Results from protein–DNA-binding stud- ies suggest that FocB interacts with the minor groove of its cognate DNA target, which is indicative of a DNA interaction that is unusual for this motif. FocB crystallizes in the form of dimers. Packing interactions in the crystals give two plausible dimerization interfaces. Conserved residues, known to be important for protein oligomerization, are present at both interfaces, suggesting that both sites could play a role in a functional FocB protein. Structured digital abstract l MINT-7901626: focB (uniprotkb:Q93K76) and focB (uniprotkb:Q93K76) bind (MI:0407)by x-ray crystallography ( MI:0114) Abbreviations CRP, cAMP receptor protein; HNF, hepatocyte nuclear factor; HTH, helix-turn-helix; PDB, Protein Data Bank; UPEC, uropathogenic Escherichia coli. 3368 FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS To balance metabolic efficiency, the expression of the different fimbrial types needs to be regulated. The control mechanisms are complex and act at multiple levels. Environmental factors may affect the on–off switching of the fimbrial genes indirectly by influencing the expression patterns of certain proteins. In addition, there is cross-regulation between the different fimbrial gene clusters [3–5]. Many types of fimbriae expressed by UPEC strains are structurally similar and their genetic organizations show high resemblance. The P fimbriae, commonly associated with pyelonephritis [6], are the most studied and have been described extensively. The pap gene cluster, sufficient for the production of P fimbriae, consists of nine structural and two regulatory genes. The structural genes for P fimbriae encode both minor and major subunits, building up the fimbrial rod, as well as proteins important for the transport and assem- bly of the fimbriae at the bacterial surface (Fig. 1). Recent studies have revealed additional genes in the promoter distal region of the main fimbrial operons, and their role also appears to be at the regulatory level [7,8]. The pap genes are transcribed from two divergent promoters, P B and P I , separated by an intercistronic region containing binding sites for the PapB protein, which is a key factor in the regulation of fimbriae [9,10]. Other important regulators that operate in this region include cAMP receptor protein (CRP), Lrp, PapI, DNA adenine methylase and histone-like nucle- oid-structuring protein [11]. UPEC commonly express F1C fimbriae, a fimbrial type often found in strains also carrying genes for type 1 or P-fimbrial expression [12]. The receptors for F1C fimbriae have been identified as glycosphingolipids [13,14] and their specific interaction allows the F1C fimbriae to adhere to the collecting ducts and distal tubules of the kidney [15]. The organization of the genetic determinant for F1C fimbriae, the foc operon, is very similar to that of the pap operon, with only a few transposed genes. The foc gene cluster includes seven structural and two pro- pap J96 Usher AdhesinChaperone Minor subunits I B A E GF K D J H C Y X Regulation (?) Regulators Major prs J96 I B A E GFKD J H C X subunit prf 536 I B A E GFKD J H C X sfa IHE3034 C B A S HGED F Y X foc 536 I B A F HG C I D Y X HNS CRP Lrp H-NS focB focI P B P I + – Site 2Site 1 B + – Fig. 1. Genetic organization of different fimbrial gene clusters: pap (P fimbriae), prs (Prs fimbriae), prf (P-related fimbriae), sfa (S fimbriae) and foc (F1C fimbriae). A representative E. coli strain carrying the gene cluster is indicated. The transcription factor FocB is acting in the int- ercistronic region between the focI and focB genes, together with a number of other regulatory proteins. FocB has also been described to have a repressive effect on fim expression (encoding type 1 fimbriae) [5]. Boxes with the same color represent genes with similar function. Recent findings suggest that fimbrial gene clusters also include the previously unrecognized novel regulatory genes, X and Y [8]. Modified from Sjo ¨ stro ¨ m et al. [8]. U. W. Hultdin et al. Structure of FocB FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS 3369 moter-proximal regulatory genes [16,17] (Fig. 1). One of these regulatory genes encodes the FocB protein, a member of the PapB family of fimbrial transcriptional factors. The members of the PapB family identified so far exist in both the E. coli and Salmonella species and are all involved in the regulation of fimbrial expression. At the amino acid level, FocB is 81% identical to PapB and 100% identical to SfaB, the protein regulating the expression of S-fimbriae [17], both of which are associ- ated with E. coli newborn meningitis strains [18]. FocB is also 47% identical to the PefB protein of Salmo- nella typhimurium [19], and 34% and 28% identical to the FaeB and FanB proteins, respectively. The latter two proteins are regulators of the K88 and K99 fimbri- al types that are expressed by enterotoxigenic E. coli and cause diarrhea in domestic animals. In many respects, the roles of the FaeB and FanB proteins in transcriptional regulation are similar to that of PapB in the regulation of the pap operon [20,21]. UPEC strains often carry several different fimbrial operons within their genome [12]. For example, the UPEC strain J96 carries at least the operons fim, pap, prs and foc [22–24]. However, less than 10% of the bacteria display more than one fimbrial type on their surfaces simultaneously. Thus, fimbrial expression involves cross-regulatory interactions between the dif- ferent operons. Recent experiments suggest that an intricate hierarchy exists with respect to the the cross- regulation of the pap and foc operons because FocB could stimulate the expression of pap, whereas PapB is insufficient for stimulating the expression of foc by itself [5]. In concordance, the PapB family members share a common core structure, as revealed by multiple sequence alignments. These homologous proteins share almost completely conserved regions that are known to be important for oligomerization and DNA-binding [25]. In the present study, we describe the structure of FocB, which is the first reported structure of a member of the PapB protein family. FocB crystallizes as dimers, in which each subunit contains one helix-turn- helix (HTH) motif. Crystal packing interactions suggest that there are two dimerization interfaces of interest for the in vivo function of FocB. Amino acids known to be important for DNA-binding are exposed on the protein surface, although they are not located in the recognition helix of the HTH motif. The results obtained from DNA-shift assays suggest that FocB binds to the DNA minor groove, thus indicating a DNA-binding pattern different from that of classical HTH motifs. Results Structure determination The E. coli FocB structure (109 amino acid residues) was determined at a resolution of 1.4 A ˚ by multiwave- length anomalous diffraction [26] from a single crystal of the selenomethionine labeled protein. The crystal comprised two molecules per asymmetric unit. Apart from several residues at the N- and C-terminal ends, all protein residues could be modeled into the electron density. The final model contains residues 10–99 of chain A and residues 10–97 of chain B. The final R-values, R work = 0.196 and R free = 0.222, are higher than expected for this resolution. This is probably a result of the additional electron density ascribed to the N- and C-terminal residues of the protein, which were not modeled in the structure because of disorder. Table 1 summarizes the statistics of X-ray data collec- tion and the results for the structural refinement of FocB. The coordinates and structure factors are depos- ited in the Protein Data Bank (PDB) (accession code 3M8J). The structure of FocB As anticipated from CD measurements and secondary structure prediction software [27] (Fig. 2), the amino acid chain of FocB forms an all a-helical structure that comprises five a-helices: a1 (Asp12-Ser21), a2 (30Glu-Ser40), a3 (Asp45-Gly58), a4 (Arg61-Tyr68), and a5 (Asn71-Tyr95) (Fig. 3). Of these, helices a4 and a5 and the connecting turn (Gln69-Asn71) comprise an HTH motif, which appears to be quite different from the canonical HTH motifs found in many bacterial transcription factors [30–32]. In particular, the second helix of the HTH motif, the putative recognition helix, is much longer in the FocB structure than in a typical HTH motif. Structural similarities with the helix- loop-helix motif in homeodomains also strengthen the view that FocB harbors an atypical HTH motif. The helix a5, at the center of the domain, can be divided into two parts based on its amino acid compo- sition. The N-terminal part, comprising residues Asn72 to Asn86, is amphiphatic, whereas the C-terminal part, starting at residue Val87 and extending to the last mod- eled residue Tyr95, is predominantly hydrophobic in nature, with the exception of one residue, Arg91. The hydrophobic surface of the amphiphatic N-terminal part of the helix is shielded from the solvent by three short anti-parallel helices a2–a4 situated perpendicular to a5. The interactions between these four helices form Structure of FocB U. W. Hultdin et al. 3370 FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS Table 1. Data collection and refinement statistics. Data collection Native Peak Inflection Remote Wavelength (A ˚ ) 0.9792 0.9792 0.9795 0.9754 Unit cell P2 1 2 1 2 1 P2 1 2 1 2 1 P2 1 2 1 2 1 P2 1 2 1 2 1 Unit cell parameters (A ˚ ) a = 47.39 a = 47.8 a = 47.8 a = 47.8 b = 58.82 b = 59.3 b = 59.3 b = 59.3 c = 65.84 c = 66.1 c = 66.2 c = 66.2 Range of resolution (A ˚ ) 47.40–1.40 ⁄ (1.47–1.40) 47.77–1.90 ⁄ (2.00–1.90) 47.80–2.00 ⁄ (2.11–2.00) 47.77–2.05 ⁄ (2.16–2.05) R merge a 0.036 ⁄ (0.314) 0.058 ⁄ (0.381) 0.055 ⁄ 0.335) 0.056 ⁄ (0.428) Number of observations 230628 213993 187663 174385 Number of unique reflections 36163 15361 13252 12331 completeness (%) 97.8 ⁄ (97.4) 100.0 ⁄ (100.0) 100.0 ⁄ (100.0) 99.9 ⁄ (100.0) I ⁄ rI 28.6 ⁄ (6.1) 29.7 ⁄ (6.6) 32.6 ⁄ (8.3) 33.0 ⁄ (6.7) Redundancy 6.4 ⁄ (6.4) 13.9 ⁄ (12.5) 14.2 ⁄ (14.5) 14.1 ⁄ (14.4) Anomalous completeness (%) 99.9 ⁄ (99.9) 99.9 ⁄ (100.0) 99.9 ⁄ (100.0) Anomalous multiplicity 7.5 ⁄ (6.5) 7.6 ⁄ (7.6) 7.6 ⁄ (7.6) Refinement Number of reflections 34198 R-factor b 0.196 R free c 0.222 Number of atoms 1539 Number of water molecules 148 Overall mean B (A ˚ 2 ) 17.3 Protein atoms (A ˚ 2 ) 15.8 Water molecules (A ˚ 2 ) 28.1 rmsd bond length (A ˚ ) 0.017 rmsd bond angles (°) 1.61 Ramachandran plot favoured (%) 100 Ramachandran plot accepted (%) 0 Ramachandran plot outliers (%) 0 PDB code 3M8J a For replicate reflections, R = RI hi ) <I h >| ⁄ R<I h >; I hi = intensity measured for reflection h in data set i,<I h > = average intensity for reflection h calculated from replicate data. b R-factor = R||F o | ) |F c || ⁄ R|F o |; F o and F c are the observed and calculated structure factors, respectively. c R free is based upon 5% of the data randomly culled and not used in the refinement. Fig. 2. Sequence alignment (BLAST) [28] of FocB and PapB from E. coli. The sequence identity is 81%. Residues not identical are highlighted in yellow. Secondary structural elements from the current structure of FocB are shown in bold with the HTH motif highlighted in red. The first and last residue visible in the electron density of the FocB structure is highlighted in blue. Residues Arg61 and Cys65, which were previ- ously found to be important for DNA-binding, are boxed in pink, whereas some residues previously found to be important for oligomerization are boxed in green [25]. The secondary structure elements predicted with JPRED3 [29] are shown in blue (H, helices; E, b-strands). Interest- ingly, the a1 helix in FocB was predicted to be a b-strand in PapB. U. W. Hultdin et al. Structure of FocB FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS 3371 a stable hydrophobic core. The first helix a1 is posi- tioned perpendicular to a5, but situated on the oppo- site side of helix a5 with respect to helices a2–a4. Only a few contacts are formed between a1 and a5, and include one hydrogen bond between the main chain nitrogen atom of Leu23 and the side chain Oc1 atom of Thr83, and one hydrophobic interaction between the side chain of Leu18 and the Cc2 atom of Thr83. FocB dimerization Cross-linking and size exclusion chromatography stud- ies showed that FocB forms predominantly dimers in solution [27]. Furthermore, the packing of molecules in the crystal structure suggests that FocB is dimeric. The asymmetric unit comprises two molecules of FocB. Crystal packing contacts provide two alternative possi- bilities for homodimeric FocB interactions. At the first dimer interface, the two monomers, chains A and B in the asymmetric unit, form extensive contacts between their helices a2 and a5(Fig. 4A, subunits colored in light and dark blue). These helices are positioned per- pendicular with respect to each other in a four-helix arrangement. The two monomers are related by a non- crystallographic two-fold symmetry operation, and we refer to this interface as dimer Interface-I. The inter- Fig. 3. Ribbon representation of the structure of FocB. Helices a1–a5 are displayed, with the HTH motif highlighted in blue. The conserved DNA-binding residues Arg61 and Cys65 are shown as ball and sticks. A BC Fig. 4. Crystal packing of the FocB struc- ture. (A) Subunits forming Interface-I are colored in light and dark blue; subunits of Interface-II are colored in light and dark coral. (B) Interactions in Interface-I. (C) Interactions in Interface-II. For clarity, only selected residues are shown as sticks. Selected hydrogen bonds are shown in green (dotted line). Structure of FocB U. W. Hultdin et al. 3372 FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS face involves predominantly residues positioned at the hydrophobic C-terminal part of a5. An extensive hydrophobic core is formed over this interface, includ- ing four residues from a5 (AB-Leu85, AB-Leu88, AB-Val89 and AB-Leu92) and two residues from a2 (AB-Leu35 and AB-Ile39) (Fig. 4B). In addition, the side chains of AB-Tyr95 and AB-Gln32 stack and contribute to the hydrophobic core. Side chains of seven polar or charged residues positioned on helices a2 and a5 are involved in hydrogen bond formation over Interface-I (Table 2). The crystal packing interactions revealed a second putative dimer interface that we call Interface-II (Fig. 4A, subunits colored in light and dark coral). This interface is also formed by a four-helix arrangement comprising symmetry-related a1 and a5 helices. How- ever, at this interface, it is the N-terminal part of a5 that is involved. The polar sides of the two symmetry- related N-terminal parts of helix a5 are facing each other, and hydrogen bonds and two salt bridges are formed across the dimer interface (Table 2). Hydropho- bic contacts also exist between symmetry-related resi- dues AB¢-Phe14, AB¢-Leu15 and AB¢-Leu18, where B’ is a crystallographic symmetry-related copy of subunit B (symmetry transformation: )x, y +1⁄ 2, )z +1⁄ 2) (Fig. 4C). In this dimer, the hydrophobic residues bur- ied in the core of Interface-I are surface exposed. pisa is an interactive tool that can be used for explo- ration of protein interfaces [33]. pisa analysis of the two interfaces identified in FocB crystals suggested that both dimer alternatives are stable in solution. The solvation energy effects, D i G, of Interface-I and II were calculated to )16.1 kcalÆmol )1 and )8.1 kcalÆmol )1 , respectively. Furthermore, DG diss , which indicates the free energy of assembly dissociation, was calculated to 8.3 kcalÆmol )1 for Interface-I, and 1.5 kcalÆmol )1 for Interface-II. Contact areas of Interface-I and -II were estimated to 907 A ˚ 2 and 851 A ˚ 2 , respectively. Com- bined, the output data from pisa suggest that dimer Interface-I is significantly more stable than dimer Interface-II. FocB and PapB bind in the minor groove of double-stranded DNA In a previous study, Xia et al. [34] obtained evidence that PapB binds in the minor groove of DNA. This minor groove-binding property makes the protein unusual in the perspective of transcriptional activa- tors. To assess how the FocB protein interacts with DNA, we performed a electrophoretic DNA gel mobility shift test as a competition assay between FocB and distamycin, a minor groove-binding drug [35] and methyl green, a major groove-binding drug [36]. That distamycin could bind to DNA under the conditions used was evident from the mobility shift observed when ‡ 4 lm of distamycin was added to the DNA in the absence of protein (Fig . 5A, lane 4). At lower concentrations, partial occupancy of distamycin did not shift DNA (lanes 2 and 3). The DNA mobil- ity shift caused by 125 nm of FocB (lane 5) was com- pletely abolished when distamycin was present at concentration of 4 lm or higher (lanes 9–10). Addition of 1, 2, or 3 lm distamycin resulted in a gradually reduced amount of FocB bound to the DNA (lanes 6–8). Combined, the results suggest that distamycin competes with FocB for DNA-binding, and that the FocB-binding site on the DNA was increasingly occu- pied by distamycin at the tested concentrations. When challenging the FocB–DNA complex formation with methyl green at concentrations up to 100 lm,we observed no apparent inhibition of the DNA-binding, suggesting that FocB does not interact with the major groove (Fig. 5B). When methyl green was added at concentrations up to 100 lm in the absence of the protein, we did not observe any shift in mobility of the DNA but, at a very high concentration (1 mm), the DNA did not enter the gel (data not shown). That methyl green could bind to DNA under the conditions used was evident from DNA-binding tests with CRP (i.e. the cAMP receptor protein). CRP is a well char- acterized, major groove-interacting, DNA-binding pro- tein, used here as a control. The addition of 100 lm methyl green abolished all of the CRP binding to Table 2. Hydrogen bonds formed at the two plausible dimer inter- faces of FocB. Interface-I Chain A Distance (A ˚ ) Chain B Glu 31 (Oe2) 2.5 Tyr 95 (Og) Gln 32 (Ne2) 3.0 Tyr 96 (Og ) Gly 38 (O) 2.6 Arg 84 (Ng2) Arg 84 (Ng2) 2.9 Gly 38 (O) Asn 86 (Nd2) 2.6 Tyr 96 (Og) Tyr 96 (Og) 3.0 Gln 32 (Ne 2) Tyr 96 (Og) 2.6 Asn 86 (Nd2) Interface-II Chain A Distance (A ˚ ) Chain B¢ Ser 76 (Oc) 3.2 Ser 76 (Oc) Ser 76 (Oc) 3.3 Asn 72 (Nd2) Asp 12 (Od1) 2.9 Arg 81 (Ne) Asp 12 (Od2) 3.0 Arg 81 (Ng2) Arg 81 (Ne) 2.8 Asp 12 (Od1) Arg 81 (Ng2) 2.9 Asp 12 (Od2) U. W. Hultdin et al. Structure of FocB FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS 3373 DNA (data not shown). Taken together, our results strongly suggest that FocB binds DNA by minor groove interactions. The FocB structure shows a fold similar to DNA-binding HTH proteins Structural similarity searches using the dali server [37,38] identified a number of structures similar to FocB. The top seven dali hits are presented in Table 3. Generally, the protein structures identified by dali comprise DNA-binding proteins with HTH motifs and, for many of the hits, structures in complex with DNA or RNA are available in the PDB. Struc- tural comparisons of FocB with some of the top DALI hits are shown in Fig.6. The dali server rates structural similarity by the z-score, where values above 2 are considered to be significant hits. KorA, with a z-score above 7 and a sequence identity of 18%, can tentatively be considered relevant [38]. The secondary structure organization of KorA with a recognition helix of a HTH motif flanked by three short helices is also strikingly similar to the FocB structure (Fig. 6A). Furthermore, KorA is a homodimeric repressor pro- tein with two HTH motifs that bind in the major groove on opposite sides of the DNA [39]. With the N-terminal end of its recognition helix, KorA recog- nizes and binds a 12 bp symmetric operator. The side chains of Gln37 in the first helix of the HTH motif, and Arg48, Gln53 and Arg57 in its second helix, are particularly important for specific interaction with the DNA [39]. In FocB, these residues correspond to Arg61, positioned on helix a4, and Asn72, Thr77 and Arg81, positioned on a5. Interestingly, substitution of Arg61 and Arg81 impair DNA-binding in PapB, indi- cating that these residues are critical for DNA-binding also in FocB [25]. The DNA-recognition domain of RNA polymerase r E -factor, another of the FocB structurally similar proteins, shows a DNA interaction similar to that of KorA. The r E -factor is also a homodimer, with one HTH motif per subunit, interacting exclusively with the major grooves of two different DNA strands [40] (Fig. 6B). Hepatocyte nuclear factor-1 (HNF-1b) also shares structural similarity to FocB (Fig. 6C). Differ- ent from KorA and the RNAP r E -factor, this protein binds to the major groove using both helices of its HTH motif [41]. Also, DNA bound to HNF-1b is ori- ented  90° with respect to the position of the DNA bound to KorA or RNAP r E -factor. Among the top DALI-hits, we also found the struc- ture of the G1 ⁄ S specific cyclin-D1 protein. In this protein, the two helices that share structural similarity to a HTH motif do not have DNA-binding function A B Fig. 5. Gel mobility shift assay of the FocB protein binding to DNA in the absence or presence of the DNA-binding compounds distamy- cin (minor groove-binding) and methyl green (major groove-binding), respectively. A 401 bp DNA fragment containing four repeats of the 9 bp long sequence constituting the primary FocB-binding sequence [5] was used as target DNA. The shifted bands in the gel represent- ing different DNA complexes with protein or the tested compounds are indicated along the left side. (A) Effect of distamycin on FocB- binding. (B) Lack of effect of methyl green on FocB-binding. Table 3. DALI search results. Protein Chain PDB code z-score rmsd Alignment length % sequence identity KorA, transcriptional repressor protein B 2W7N 7.7 2.0 68 18 Hel308, helicase A 2P6U 5.8 2.6 73 15 G1 ⁄ S specific cyclin-D1 A 2W9Z 5.4 3.0 72 10 Hypothetical UPF0122 A 1XSV 5.1 2.8 64 6 RNA polymerase r E -factor D 2H27 4.9 2.6 60 12 HNF-1b B 2H8R 4.7 3.5 67 6 Signal recognition particle, M-domain A 1HQ1 4.7 2.6 60 5 Structure of FocB U. W. Hultdin et al. 3374 FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS [42]. Other structures, such as domain 4 of the helicase Hel308 (Fig. 6D) and the RNA-binding M-domain of Ffh (Fig. 6E), gave examples of nucleic acid interac- tions different from classical HTH–major groove inter- actions. Domain 4 of Hel308 consists of a seven-helix bundle and together with other domains it forms a ring around the 3¢ tail of the unwound DNA oligonu- cleotide [43]. In this interaction, the central helix of domain 4 (corresponding to a5 in FocB) provides a ratchet for directional transport of the product DNA tail across the protein. Arg592 and Trp599 in the ratchet helix, corresponding to Thr77 and Arg84 in FocB, stack on base moieties of the single-stranded DNA. Amino acids that are positioned in the helix corresponding to helix a4 in FocB are not involved in DNA interaction. Ffh is a protein constituent of the signal recognition particle [44] and its M-domain con- tains a HTH motif that binds to the minor groove of signal recognition particle RNA with the small first helix of the motif (analogous to a4 in the FocB struc- ture) (Fig. 6E). Residues important for oligomerization and DNA-binding Alanine substitutions in PapB, made at positions con- served throughout the PapB family, have revealed a number of specific amino acids that appear to be par- ticularly important with respect to the ability of pro- teins to bind to their target DNA, and for their ability to form oligomeric complexes [25,34]. Two residues found to be important for DNA-binding in PapB (i.e. Arg61 and Cys65) are conserved and located on helix a4 in FocB (i.e. the helix preceding the presumed rec- ognition helix, a5) (Fig. 3). PapB binds DNA in an oligomeric fashion, probably in the form of dimers or tetramers [34]. Conserved residues shown to be impor- tant for oligomerization in PapB are spread out over the FocB structure. Some of these residues (i.e. Asp53, A B C D E Fig. 6. Protein–DNA and –RNA complex structures of the top five structural homologs of FocB identified by DALI [38]. Ribbon repre- sentations are shown to the left and overlays of Ca-traces of the structurally similar proteins (light blue) and FocB (dark red) are shown to the right. DNA strands are shown in yellow and orange, and the RNA strand is shown in coral. Parts of the structures superimposing on the HTH motif of FocB are highlighted in blue in the ribbon representation. To visualize the variety of DNA ⁄ RNA- binding sites, all proteins are shown with the same orientation of their HTH motif. (A) KorA (PDB code 2W7N), chain B, residues 3– 97 (38–67 in blue). (B) RNA polymerase r E -factor (PDB code 2H27), chain D, residues 122–190 (185–196 in blue). (C) HNF-1b (PDB code 2H8R), chain B, residues 90–185 (157–184 in blue). (D) Hel308 (PDB code 2P6R), chain A, residues 508–612 (576–612 in blue). (E) Ffh M-domain (PDB code 1HQ1), chain A, residues 13– 83, (48–82 in blue). U. W. Hultdin et al. Structure of FocB FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS 3375 Tyr54, Leu55 and Val56) are located in helix a3. Because this helix appears necessary for stabilizing the HTH motif, which is in itself apparently insufficient for independent folding, these mutations most likely affect the conformation of the whole protein structure. Other residues important for oligomerization include residues Leu35 and Leu36, which are buried in the dimer Interface-I. In this dimer constellation, the side chains of residues Tyr74, Phe75 and Ser76, also previ- ously shown to be important for oligomerization, are exposed on the surface of the protein. These residues, however, are buried at Interface-II. Discussion The FocB protein is a transcription factor involved in the regulation of genes for production of F1C fimbriae that are commonly expressed by UPEC. FocB belongs to the PapB family of adhesin regulators. Within this family, PapB is currently the most thoroughly charac- terized member and shares 81% sequence identity with FocB. The most significant difference in their sequences is located at the N-terminal ends of the pro- teins, a region that is predicted to form a b-strand in the PapB protein (Fig. 2). In the present study, we have structurally characterized residues 10–99 of the 109 amino acid residue protein FocB at 1.4 A ˚ resolu- tion. The structure comprises five a-helices, including a HTH motif that is quite common for many DNA- binding proteins. From the amino acid sequence alone, no obvious recognition motifs similar to other DNA- binding proteins could be identified in FocB. Compar- ing the PapB sequence with the FocB structure shows that differences in their sequences are predominantly located in the a1-loop-a2 region of the N-terminus as well as in the C-terminal part of the two proteins. Thus, the core structure of PapB is very likely identical to that of FocB. Results from DNA-shift assays suggest that FocB, similar to PapB, is a minor groove-binding protein. In general, minor groove-binding proteins show various degrees of sequence specificity when binding to DNA. The lack of unique chemical features present within the minor groove requires various strategies for recog- nition. For example, the TATA box-binding protein finds and interacts with the minor groove of the TATA element, onto which it binds analgous to a saddle on a horse [45,46]. The integration host factor, on the other hand, uses a so-called winged helix motif to bind to regions of unusually narrow minor grooves [47]. A common feature of minor groove-binding proteins is their ability to bend DNA. For TATA box-binding protein and integration host factor, intercalation of hydrophobic residues between base pair steps in the DNA is the main cause of the extreme DNA-bending ability of these proteins [48]. Also, LacI [49,50] and PurR [51], members of the LacI family, bend their operator DNA. Both LacI and PurR form dimers attached tail-to-tail, where each dimer consists of an N-terminal DNA-binding domain and a C-terminal oligomerization domain. The DNA-binding headpieces of the N-terminal domains contain conventional major groove-binding HTH motifs, but symmetric hinge a-helices immediately adjacent to the headpieces bind deep in the minor groove, and intercalate leucine resi- dues into the central base pair step, which cause the DNA to bend. The structure of the FocB subunit contains one HTH motif. Generally, this motif is in its smallest functional form part of a core of three helices that form a right-handed helical bundle with a partly open configuration. The HTH motif, as it is known from transcription factors in both prokaryotes and eukary- otes, recognizes a specific DNA-sequence and is mostly associated with major groove-binding. Through inter- action between the second helix of the HTH motif (i.e. the recognition helix) and the DNA major groove, the sequence information is accessible for the protein [32,52,53]. Recently, proteins with minor groove-bind- ing HTH motifs have been identified. One example is the human DNA repair protein O6-alkylguanine- DNA-alkyltransferase [54,55]. Because DNA damage is not sequence dependent, it is favorable for DNA repair, as well as other types of nucleotide-flipping proteins, not to bind specifically to DNA. In O6-alkyl- guanine-DNA-alkyltransferase, one arginine residue, which is centrally positioned on the recognition helix, stacks between DNA bases in the minor groove, caus- ing the DNA to bend. In addition, small hydrophobic residues of the recognition helix are presented to the minor groove of the DNA, providing a nonsequence- specific interaction [54]. FocB and PapB bind to the minor groove of their target DNA. This is in agreement with their ability to bind A ⁄ T-triplets occurring at 9 bp intervals [34], where a classical, direct major groove interaction is less expected. We found that the conserved residues Arg61 and Cys65, known to be important for DNA-binding, are surface exposed and located in the helix preceding the putative recognition helix (Fig. 3). Also in the major groove-binding protein KorB, side chains that are not located in the recognition helix have been shown to be essential for the binding specificity [56]. In that case, the specific Thr211 and Arg240 are situated outside of the HTH motif, whereas the HTH itself is typically placed with the recognition helix running Structure of FocB U. W. Hultdin et al. 3376 FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS along the major groove. Taken together, our findings suggest that the HTH motif in FocB is involved in DNA interactions that very likely are different from the classical HTH–major groove contacts. From dali searches, several proteins with similar structures to FocB were identified. We tried to identify the DNA-binding site in FocB based on the proteins identified by dali; however, the latter proteins dis- played very different types of DNA or RNA interac- tions (Fig. 6). Therefore, at this point, we refrain from speculating on where the exact DNA-binding site of FocB. FocB and PapB bind to DNA in an oligomeric fash- ion [34]. From the crystal structure of FocB, two pos- sible dimeric arrangements were identified. Of these, Interface-I is significantly more hydrophobic. Further- more, one of the dali hits, the hypothetical DNA-binding UPF0122 protein SAV1236 from Staphylococcus aureus (PDB code 1XSV; unpublished) (Table 3), is a dimer with a packing interaction resembling that of Interface-I in FocB (Fig. 7). Mutants that impair PapB oligomerization [25] are localized both at Interface-I and -II. We therefore con- sider that Interface-I represents the most stable dimeric form of FocB, but that Interface-II might play a role in the formation of larger oligomeric structures neces- sary for DNA-binding [34]. We hypothesize that several FocB dimers can bind to DNA side by side. The crystal structure of the FocB transcription factor provides an important starting point for further analyses aiming to understand the mechanisms of fimbrial gene regulation at the molecular level not just for FocB, but also for the entire family of related proteins. Experimental procedures Protein expression and purification of native and Se-Met FocB The overexpression and purification of native FocB (109 amino acid residues) has been described previously [27]. The full-length FocB protein was cloned into pETM11 and overexpressed in E. coli strain BL21 (DE3). The protein was purified on a Ni-NTA (Qiagen, Valencia, CA, USA) column followed by a Superdex 75 column (GE Healthcare, Milwaukee, WI, USA). During purification, the 6-His tag was removed with tobacco etch virus protease, leaving two extra residues, Gly and Ala, followed by Met1 correspond- ing to the native N-terminus. Pure fractions of the protein in 10 mm Hepes (pH 7.9), 500 mm NaCl, 5 mm EDTA and 0.1% b-mercaptoethanol were pooled and concentrated to 16 mgÆmL )1 , filtered through a 0.2 lm filter and stored at 4 °C. Selenomethionyl labeling of FocB (Se-Met FocB) was performed using the protocol as described previously [57]. FocB Se-Met was overexpressed in E. coli BL21 (DE3) cells grown in minimal medium (48 mm Na 2 HPO 4 ,22mm KH 2 PO 4 ,9mm NaCl, 19 mm NH 4 Cl, 2 mm MgSO 4 , 0.1 mm CaCl 2 ,4gÆL )1 glucose) with the addition of kanamycin (0.1 mgÆmL )1 )at37°C until D 600 = 0.6. The temperature AB Fig. 7. Structural similarity between (A) the dimer Interface-I of FocB and (B) the dimer interface of protein UPF0122 (PDB code 1XSV, unpublished). Residues Leu13-Leu76 of the protein matched residues Ser27- Ala93 of FocB with a z-score of 5.1 (Table 2). The structures are shown in two orientations. The visual alignment is based on the position of the HTH motif. U. W. Hultdin et al. Structure of FocB FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS 3377 [...]... Comparison of the genetic determinant coding for the S -fimbrial adhesin (sfa) of Escherichia coli to other chromosomally encoded fimbrial determinants Infect Immun 55, 194 0–1 943 Ott M, Hoschutzky H, Jann K, Van Die I & Hacker J (1988) Gene clusters for S fimbrial adhesin (sfa) and F1C fimbriae (foc) of Escherichia coli: comparative aspects of structure and function J Bacteriol 170, 398 3– 3990 Korhonen TK, Valtonen... 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Structure of FocB – a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli Ulrika W 3379 preliminary data analysis of FocB, a transcription fac- tor regulating fimbrial adhesin expression in uropatho- genic Escherichia coli. Acta Crystallogr