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5494–5502 Nucleic Acids Research, 2005, Vol. 33, No. 17 doi:10.1093/nar/gki868 Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis Thirumananseri Kumarevel, Hiroshi Mizuno1 and Penmetcha K. R. Kumar* Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, Tsukuba, Ibaraki 305-8566, Japan and 1NEC Soft Ltd, 1-18-6, Shinkiba, Koto-ku, Tokyo 106-8608, Japan Received July 10, 2005; Revised August 22, 2005; Accepted September 9, 2005 PDB ID: 1WRN, 1WRO and 1WPT ABSTRACT terminator structure (+459 to +571 nt, Figure 1) in between the hutP and hutH genes, and regulates the transcription of the HutP is an RNA-binding protein that regulates the hut operon by an anti-termination mechanism (1–3). Several expression of the histidine utilization (hut) operon bacterial proteins that regulate anti-terminator/terminator in Bacillus subtilis, by binding to cis-acting regulatory structures have been described, including TRAP, PyrR, sequences on hut mRNA. It requires L-histidine and LacT, BglG, SacT and GlpP (4–10). One such protein is an Mg21 ion for binding to the specific sequence the hutP gene product, HutP, of B.subtilis. HutP is a 16.2 within the hut mRNA. In the present study, we show kDa protein that shares 60% sequence identity with that several divalent cations can mediate the HutP– the HutP proteins found in other Bacillus species, such as RNA interactions. The best divalent cations were Bacillus halodurans (11), Bacillus cereus (12) and Bacillus Mn21,Zn21 and Cd21, followed by Mg21,Co21 and anthracis (13). Ni21, while Cu21,Yb21 and Hg21 were ineffective. In HutP is a positive regulatory protein that interacts specif- ically with the terminator region (3) of hut mRNA. Similar to the HutP–RNA interactions, divalent cations cannot the other transcriptional regulatory proteins mentioned above, be replaced by monovalent cations, suggesting that it requires a ligand for its activation; HutP requires L-histidine a divalent metal ion is required for mediating the as a ligand for binding to the terminator RNA (3). For com- protein–RNA interactions. To clarify their importance, plete activation of HutP, a concentration of 10 mM L- we have crystallized HutP in the presence of three histidine was sufficient for binding to the cognate RNA different metal ions (Mg21,Mn21 and Ba21), which (14). Using various analogs of L-histidine, we determined revealed the importance of the metal ion binding that the imidazole ring and the backbone of L-histidine site. Furthermore, these analyses clearly demon- are important for supporting RNA binding in vitro (14,15). strated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition. Terminator/anti-terminator region INTRODUCTION Genes responsible for the degradation and utilization of hutP hutH hutU hutI hutG hutM L-histidine, as a carbon and nitrogen source under nutrient- limiting conditions, are located within the hut operon in Bacillus subtilis. The operon consists of six open reading frames; the hutP gene is located near the promoter, and five Histidine Utilization Genes other genes, hutH, hutU, hutI, hutG and hutM, are located Figure 1. Histidine utilizing (hut) operon of B.subtilis. Schematic representa- far downstream from the promoter. Previous biochemical tion of the hut operon, showing the arrangement of the hutP, terminator/anti- and genetic studies suggested that the hut mRNA forms a terminator (+459 to +571 nt), and structural genes. *To whom correspondence should be addressed. Tel: +81 298 61 6085; Fax: +81 298 61 6095; Email: [email protected] Present address: Thirumananseri Kumarevel, Structural and Molecular Biology Laboratory, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan Ó The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2005, Vol. 33, No. 17 5495 Obviously, it is intriguing how the ligand (L-histidine) modu- (SRP)–Ffh complex (24), the C5 protein–M1 RNA complex lates HutP for binding to the terminator. In order to obtain (25) and the E.coli L11–23S rRNA ribosomal protein complex insights into these interactions, we recently solved the (26), have been evaluated. Interestingly, these RNA–protein crystal structure of the HutP–HBN (L-histidine analog, complexes involve longer RNAs (S4–16S, SPR–Ffh, C5 L-histidine b-naphthylamide) complex (14). The complex protein–M1 RNA and L11–23S rRNA complexes) within structure revealed that HutP forms a hexamer, which consists the complex, and they specifically require divalent metal of three dimers of HutP. The L-histidine was located in ions for the interactions. The metal ions in these complexes between the two monomers of HutP and participates in an are primarily needed for folding/stabilizing alternative RNA extensive hydrogen bond network within the hydrophobic conformations for recognition by the cognate protein, rather pocket (14). Furthermore, we mapped the HutP binding than directly mediating the interactions. On the other hand, sites within the terminator region, as the UAGNNNNUAG- RNA-binding proteins that bind to single-stranded RNA, such NNNNUAG recognition motif, where N indicates any base, as Sex-lethal (27), Poly(A) binding protein (28), transcription and also identified the important chemical groups of the bases termination factor Rho (29) and TRAP (30), recognize and within the core region (UAG) (16). bind their RNAs even in the absence of metal ions. Thus, based To analyze the ability of HutP to bind to mRNA, the reac- on these examples, it appears that the divalent metal ions are tions are carried out in the presence of L-histidine (10 mM) primarily required in the RNA–protein complexes when and Mg2+ ions (5 mM). However, we do not know what func- longer RNAs (>55 nt) are involved, to obtain a specific sec- tion the metal ions play in anti-termination complex forma- ondary structure for complex formation [e.g. the L11–RNA tion. In order to understand the role of metal ions in the complex (31)]. Alternatively, it is possible in some cases that formation of this complex, we used a gel mobility shift the metal ions may be required to mediate specific interactions assay, and these studies suggested that Mg2+ ions are important between the RNA and the protein, without interacting with for the HutP–RNA interactions (17). Furthermore, our qua- the RNA. ternary complex analyses revealed that the Mg2+ ions were In the present study, we show that a single-stranded RNA- simultaneously coordinated within the histidine cluster of binding protein, HutP, is an L-histidine and divalent metal HutP and the L-histidine ligand. Upon these interactions, ion-dependent anti-termination protein that binds to the the Mg2+ ion was found to facilitate appropriate structural RNA sequences within the terminator region of hut mRNA. rearrangements, especially at the L-histidine binding site Among the 15 divalent metal ions tested, only Cu2+,Yb2+ and and the loop regions of HutP, thus leading to the recognition Hg2+ failed to support the HutP–RNA interactions, while Mn2+, of the cognate RNA sequence (17). All of the structural Zn2+ and Cd2+ were the best metal ions. Monovalent rearrangements caused by the divalent metal ions are essential metal ions, such as, Na+ and K+, could not substitute for for the RNA recognition. The L-histidine movement only the divalent metal ions in the HutP–RNA interaction. These occurred in the presence of the metal ion. The side chain of analyses suggest that a metal ion binding pocket exists within Arg88 moved in the opposite direction of the L-histidine and the HutP protein that specifically coordinates with its formed a salt bridge, leading to the disruption of the hydro- counterparts. To determine whether specific divalent metal phobic pocket formed by the salt bridge between Arg88 and ion interactions occur with HutP, we solved the crystal Glu81 in the HutP–HBN complex, and formed a new salt structures of HutP in the presence of three metal ions, bridge with Arg98. In order to establish this new salt bridge Mn2+,Ba2+ and Mg2+. Interestingly, all three divalent ions interaction with the L-histidine ligand, the conformation of the reside in a precise position and interact specifically with the residue Arg98 changes drastically, and this is accompanied by HutP residues His73, His77 and His138, and the L-histidine a large shift in the Ca position of the next residue, Thr99. ligand. A comparison of the complex structures of HutP in Owing to these structural rearrangements caused by the dival- the presence and absence of divalent metal ions suggests that ent metal ion, the Thr99 side chain forms two hydrogen bonds the metal ions cause structural rearrangements, especially at with the N3 and 20-OH of the A base in the UAG motif. The the loop regions, by interacting with the histidine cluster and involvement of the metal ions in the histidine cluster causes L-histidine ligand. These structural rearrangements of the the His138 residue to reorient its imidazole ring to coordinate HutP protein might be required for recognizing the specific with the divalent metal ion.

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