Sm-like proteins in Eubacteria: the crystal structure of the Hfq protein from Escherichia coli

Abstract

The Hfq protein was discovered in Escherichia coli in the early seventies as a host factor for the Qbeta phage RNA replication. During the last decade, it was shown to be involved in many RNA processing events and remote sequence homology indicated a link to spliceosomal Sm proteins. We report the crystal structure of the E.coli Hfq protein showing that its monomer displays a characteristic Sm-fold and forms a homo-hexamer, in agreement with former biochemical data. Overall, the structure of the E.coli Hfq ring is similar to the one recently described for Staphylococcus aureus. This confirms that bacteria contain a hexameric Sm-like protein which is likely to be an ancient and less specialized form characterized by a relaxed RNA binding specificity. In addition, we identified an Hfq ortholog in the archaeon Methanococcus jannaschii which lacks a classical Sm/Lsm gene. Finally, a detailed structural comparison shows that the Sm-fold is remarkably well conserved in bacteria, Archaea and Eukarya, and represents a universal and modular building unit for oligomeric RNA binding proteins.

Figure 1

The Hfq family. The organisms corresponding to the sequences are indicated on the left from top to bottom with entry names or access numbers in parenthesis. Proteobacteria: E.coli (HFQ_ECOLI), Shigella flexneri (HFQ_SHIFL), Salmonella typhimurium (HFQ_SALTY), Yersinia enterocolitica (HFQ_YEREN), Yersinia pestis (HFQ_YERPE), Erwinia carotovora (HFQ_ERWCA), Haemophilus influenzae (HFQ_HAEIN), Pasteurella multocida (HFQ_PASMU), Vibrio cholerae (HFQ_VIBCH), Pseudomonas aeruginosa (HFQ_PSEAE), Xanthomonas axonopodis (HFQ_XANAC), Xanthomonas campestris (HFQ_XANCP), Xylella fastidiosa (HFQ_XYLFA), Neisseria meningitidis (HFQ_NEIMA), Ralstonia solanacearum (HFQ_RALSO), Agrobacterium tumefaciens (HFQ_AGRT5), Brucella melitensis (HFQ_BRUME), Rhizobium loti (HFQ_RHILO), Azorhizobium caulinodans (HFQ_AZOCA), Caulobacter crescentus (HFQ_CAUCR). Aquificae: Aquifex aeolicus (HFQ_AQUAE). Thermotogae: Thermotoga maritima (HFQ_THEMA). Firmicutes: Clostridium acetobutylicum (HFQ_CLOAB), Clostridium perfringens (HFQ_CLOPE), Bacillus halodurans (HFQ_BACHD), Bacillus subtilis (HFQ_BACSU), Thermoanaerobacter tengcongensis (HFQ_THETN), S.aureus (Q99UG9). Archaea: M.jannaschii (Q58830). The numbering at the top corresponds to the E.coli sequence and the black arrow to the C-terminus of the short Hfq form. Conserved polar, basic and acidic residues appear in green, pink and violet, respectively, Gly and Pro in yellow, and a star indicates those involved in RNA binding in S.aureus (23). Blue boxes are conserved patches of hydrophobic residues. The 2D structure prediction from Jpred is indicated at the bottom as well as the 2D features seen in 3D structures [nomenclature according to Kambach et al. (18)].

Figure 2

Structure of the Hfq protein from E.coli. (A) Top and side views of the Hfq hexameric doughnut. Secondary structure elements are highlighted in one monomer with the N-terminal α-helix in pink and the five β-strands in blue. N- and C-termini pointing toward the top of the hexamer are indicated. (B) The dimer interface and H-bond interactions between strands β4′ and β5 of adjacent subunits. The 2Fo–Fc composite omit map (level 1.6σ) is shown in the region indicated by a square in (A). This figure was prepared using PyMol (Delano Scientific, San Carlos, CA).

Figure 3

Sequence and structure comparison of the Sm-fold in Sm/Lsm and Hfq monomers. (A) This stereoview shows the superposition of central monomers (see Materials and Methods) from each available structure using the following color code: Hfq monomers from E.coli and S.aureus without and with RNA (23), respectively called Hfq-EC, Hfq-SA and Hfq-SAr in (B), are colored in green; archaeal Lsm1 proteins from A.fulgidus and P.abyssi alone and with RNA (21,22,35), Pyrobaculum aerophilum (42) and M.thermoautotrophicum (43), respectively called Lsm1-PY, Lsm1-PYr, Lsm1-AF, Lsm1-Afr, Lsm1-PA, Lsm1-MT, are colored in blue; and Lsm2 from A.fulgidus, or Lsm2-AF, (35) is represented in cyan; human Sm momomers D1, D2, B and D3 (18), respectively called hSm-D1, hSm-D2, hSm-D3 and hSm-B, are shown in magenta. (B) This structure-based sequence alignment is restricted to the regions revealed by crystallographic studies (first and last observed residues are indicated on the left- and right-hand sides of the corresponding sequences). Gray boxes highlight the common backbone regions defining a minimal Sm-fold. These regions were used to superimpose the monomers (A) and to calculate RMSD values (Table 2). They mainly fit to the secondary structure features shown on top [nomenclature according to Kambach et al. (18)]. Overall conserved residues appear in orange, those specific to the Hfq family in green and those characteristic to Archaea and eukaryotes in magenta. Blue boxes indicate conserved patches of aliphatic or aromatic residues. Residues in loops L3 and L5 that form the NBP (A) in the structures of Lsm–RNA complexes are indicated by stars. Residues of the hSm-D2 variable region which are not seen in the structure are indicated by lower case letters. The residues belonging to loop L4 are separated from adjacent β-strands by gaps to highlight the variability of this region. Panel (A) was generated using ViewerLite (Accelrys Inc.).