Crystal structure of the Escherichia coli regulator of σ⁷⁰, Rsd, in complex with σ⁷⁰ domain 4

Summary

The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) σ⁷⁰ factor. Rsd plays a role in alternative σ factor-dependent transcription by biasing the competition between σ⁷⁰ and alternative σ factors for the available core RNAP. Here, we determined the 2.6 Å-resolution X-ray crystal structure of Rsd bound to σ⁷⁰ domain 4 (σ⁷⁰₄), the primary determinant for Rsd binding within σ⁷⁰. The structure reveals that Rsd binding interferes with the two primary functions of σ⁷⁰₄, core RNAP binding and promoter –35 element binding. Interestingly, the most highly conserved Rsd residues form a network of interactions through the middle of the Rsd structure that connect the σ⁷⁰₄-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between σ⁷⁰₄ binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation.

Introduction

In bacteria, the 450 kDa RNA polymerase (RNAP) holoenzyme, comprising the evolutionarily conserved catalytic core (subunit composition α₂ββ′ω) combined with the initiation-specific σ subunit, directs transcription initiation ¹. The principal control point of gene expression in bacteria is transcription initiation, and a major mechanism by which bacteria regulate transcription initiation is through regulation of σ activity ². The activity of most σ factors is determined by their cellular level, their affinity for RNAP, and their interactions with regulatory proteins that bind and modulate σ factor function.

In Escherichia coli (Ec), the primary σ factor, σ⁷⁰, has the highest affinity for core RNAP and is also the most abundant σ factor throughout the growth cycle ^{3; 4}. Thus, it is not clear how alternative σ factors capture sufficient core RNAP to express genes under their control. The discovery of the σ⁷⁰-binding protein Rsd provided a partial solution to this problem ⁵. Biochemical and genetic studies demonstrated that Rsd can indirectly stimulate transcription from alternative σ factor-dependent promoters by binding to σ⁷⁰ with a stoichiometry of 1:1 ^5–10. Mapping of the primary Rsd-binding determinant on σ⁷⁰ was localized to σ⁷⁰ domain 4 (σ⁷⁰₄; ^10–12, a structural domain of σ⁷⁰ that binds the RNAP β-subunit flap-tip, recognizes the –35 promoter element, and is a target for many transcriptional activators) ^13–17.

The amino acid sequence of Rsd is 55% similar to the sequence of the Pseudomonas aeruginosa (Paer) AlgQ transcription regulator. Paer is an opportunistic pathogen and causative agent of microbial corrosion. AlgQ is important in regulating the production of alginate, neuraminidase, and pyoverdine; factors that are necessary for promoting Paer virulence ^18–22. Ec Rsd can complement AlgQ in the production of pyoverdine ¹⁸. Thus, it has been proposed that Paer AlgQ binds to and modulates the activity of Paer σ⁷⁰ in a manner similar to Ec Rsd ^{11; 18}.

Here, we describe the 2.6 Å-resolution X-ray crystal structure of the Ec σ⁷⁰₄/Rsd complex. The results provide a structural basis for future experiments addressing the role of both Rsd and AlgQ in regulating σ activity.

Results

Crystallization, structure determination, and overall structure of the σ⁷⁰₄/Rsd complex

Rsd appears to interact with Ec σ⁷⁰ primarily through determinants within σ⁷⁰₄ ^10–12. Although Ec σ⁷⁰₄ on its own is poorly behaved for structural studies ²³, co-expression of Ec σ⁷⁰₄ (Ec σ⁷⁰ residues 541–613) with Rsd resulted in a soluble complex that was purified to homogeneity with high yield (Materials and Methods). Alongside the Ec σ⁷⁰₄/Rsd complex, Paer AlgQ and Paer σ⁷⁰₄ were co-expressed and purification of the complex attempted. Although there is evidence that the proteins interact (Ref. ¹¹; L.F.W. and S.A.D., unpublished), the complex of Paer proteins was unstable and did not survive the purification procedure (data not shown).

Rod-like crystals with approximate dimensions of 180 × 20 × 20 μm, space group p6₄ (a = b = 84.111 Å, c = 84.219 Å), were grown using vapor diffusion (Materials and Methods). The crystals contained one 26.7 kDa σ⁷⁰₄/Rsd complex per asymmetric unit, with a solvent content of 64%. The crystals diffracted isotropically to 2.6 Å-resolution. The structure of the complex was solved by Multiwavelength Anomalous Diffraction ²⁴ using data from selenomethionyl-substituted protein crystals (Table 1; Figure S1). Cycles of iterative model building and crystallographic refinement converged to an R/R_free of 0.239/0.267 at 2.6 Å-resolution (Table 1).

Table 1.

Crystallographic Analysis

Diffraction Data
Data Set	Beamline	Wavelength (Å)	Resolution (Å)	No. of Reflections (tot./unique)	Completeness (%) (tot./last shell)	I/σ(I) (tot./last shell)	Rsymc (%) (tot./last shell)	No. of Sites
Native	NSLSa X29	1.1	50–2.6 (2.7–2.6)	144,443/10,405	99.1 (98.8)	17.2 (2.2)	8 (48.6)
SeMet1(λ1)	APS SGXb 31-ID-D	0.9793 (peak)	20–2.9 (3.0-2.9)	117,509/7,527	98.2 (87.1)	8.5 (1.3)	11.7 (44.7)	4
SeMet1(λ1)	APS SGXb 31-ID-D	0.9641 (remote)	20–3.1 (3.21-3.1)	132,551/6,187	99.5 (97.5)	7.7 (1.4)	13.5 (52.1)	4
Crystal space group, P64; unit cell, a = b = 84.111 Å, c = 84.219 Å; Figure of Meritd (30 - 2.9 Å resolution), 0.35
Refinement (against Native data set)
Resolution (Å)	50 – 2.6
No. of solvent molecules	98
Rcryst/Rfreee (%)	24.0/27.3
Rmsd bond lengths	0.008 Å
Rmsd bond angles	1.328°

^aNational Synchrotron Light Source, Brookhaven, NY;

^bAdvanced Photon Source-Structural GenomiX, Argonne, IL;

^cR_sym =Σ|I − <I>|/ΣI, where I is observed intensity and <I> is average intensity obtained from multiple observations of symmetry related reflections;

^dFigure of merit calculated by SHARP (de la Fortelle et al. 1997);

^eR_cryst = Σ||F_observed| − |F_calculated||/Σ|F_observed|, R_free = R_cryst calculated using 5% random data omitted from the refinement.

The structure reveals a σ⁷⁰₄/Rsd complex with a stoichiometry of 1:1 (Figure 1). This is consistent with the gel filtration behavior of the σ⁷⁰₄/Rsd complex during purification (data not shown), and with previous biochemical experiments indicating a 1:1 stoichiometry of the full-length σ⁷⁰/Rsd complex in solution ¹⁰.

Figure 1. Structure of the σ⁷⁰₄/Rsd complex.

Ribbon diagrams showing two orthogonal views of the complex, color-coded as shown. The α-helices of Rsd, H1–H5, are labeled. The α-carbon of Gly68 at the position of the kink in H3, is shown as a CPK sphere and labeled. A 7-residue disordered segment connecting H2 to H3 is represented by green dots.

Rsd structure

The Rsd structure comprises four core α-helices (helices H2–H5; Figure 1) packed in an up-and-down bundle with a slight left-handed twist. An additional short N-terminal α-helix (H1) is tucked against the side of the bundle against H2 and H5. The Rsd fold is remarkably similar to the vinculin tail domain. Superposition of Rsd with the vinculin tail domain (chicken vinculin residues 901–1046; Ref. 25) over the entire length of Rsd (residues 1–153) but excluding exposed loops connecting the α-helices yields a root-mean-square deviation of 2.53 Å over 116 α-carbons (Figure S2). Rsd therefore belongs to the vinculin/α-catenin superfamily of proteins ²⁶. Vinculin and α-catenin are structural components of the eukaryotic cytoskeleton, while Rsd is a bacterial transcriptional regulator. At this point we do not believe there is any functional significance to the observed structural similarity.

A distinctive structural feature of Rsd is a pronounced kink (~30°) in H3. The kink occurs around a highly conserved Gly residue at position 68 (Figure 1). In an alignment of 113 Rsd orthologs (Supplemental Information), Gly occurs at this position in 110 of the sequences (97% identity, Figure 2). Moreover, the primary Rsd binding interface with σ⁷⁰₄ is centered at the kink (Figure 1). All of the absolutely conserved Rsd residues that contact σ⁷⁰₄ lie near the H3 kink (Asp63, Ser66, Phe70; Figures 2 and 3). These observations suggest that the kink is a conserved feature of the Rsd structure that is important for σ⁷⁰₄ binding.

Figure 2. Sequence conservation among Rsd orthologs.

The sequence on top shows the consensus sequence from an alignment of 113 Rsd orthologs (see supplemental information), while the histogram above it denotes the level of sequence conservation at each position (red bar, 100% conserved; dark blue bar, less than 20%). Shown are only two sequences from the full alignment, E. coli Rsd (top) and Paer AlgQ, represented in one-letter amino acid code and identified by the species at the right. The numbers at the beginning of each line indicate amino acid positions relative to the start of each protein sequence. The numbers at the top of the sequences indicate the amino acid position in E. coli Rsd. Amino acid identity with the consensus sequence is indicated by blue shading. The α-helices in the Rsd structure are indicated above the Rsd sequence as rectangles (labeled H1–H5), loops are indicated by a solid line. Rsd positions that contact σ⁷⁰₄ are denoted by black dots above the helices.

Figure 3. The σ⁷⁰₄/Rsd interactions.

A) Schematic diagram denoting molecular interactions between Rsd and σ⁷⁰₄. Van der Waals interactions (<4 Å) are listed on the left, polar interactions (hydrogen bonds, salt bridges) are listed on the right.

B) Stereo view of the σ⁷⁰₄/Rsd interface. Protein α-carbon backbones are shown as worms, color-coded as in Figure 1. The two α-helices of Rsd harboring amino acid residues that interact with σ⁷⁰₄ (H3 and H4) are labeled. Amino acid side chains that participate in interprotein interactions are shown, with carbon atoms color-coded as the backbone worm, nitrogen atoms colored blue, and oxygen atoms colored red. Interprotein polar interactions (hydrogen bonds or salt bridges) are indicated by dashed lines.

Interactions between σ⁷⁰₄ and Rsd

Binding of σ⁷⁰₄ to Rsd buries a modest 779 Ų of protein surface area. Nevertheless, the protein/protein complex is very stable, as it survives several purification steps without dissociating. All of the interactions between σ⁷⁰₄ and Rsd are schematically diagrammed in Figure 3A, and a stereo view of the interface with the involved amino acids is shown in Figure 3B.

The σ⁷⁰₄/Rsd interface, centered about the kink in Rsd-H3, involves hydrophobic interactions with a patch of highly conserved residues from H3 exposed on the surface of Rsd (Figure 3). Rsd residues Val62 and Leu65 are conserved as hydrophobic residues, while Ser66 (which makes van der Waals contacts through its β-carbon), Ala67, and Phe70 are absolutely conserved (Figure 2). The interface includes polar interactions involving mostly residues from Rsd-H4. The protein/protein interaction is also stabilized by two salt bridges between pairs of highly conserved, oppositely-charged residues (σ⁷⁰₄-Arg562/Rsd-Asp63; σ⁷⁰₄-Arg596/Rsd-Asp108).

The structural observations are completely consistent with biochemical and genetic studies on the σ⁷⁰₄/Rsd interaction. Previous studies implicated σ⁷⁰₄ region 4.2 as the primary determinant in the σ⁷⁰/Rsd interaction ^10–12, which is borne out by the structure (Figure 3A). Detailed genetic studies have implicated residues in σ⁷⁰₄ that the structure shows are involved in, or are near, the σ⁷⁰₄/Rsd interface (σ⁷⁰₄-590/591/593/595/596/598; ^10–12. A more recent study showed that mutation of Rsd-Asp63 (involved in a conserved salt bridge with σ⁷⁰₄-Arg562) to Ala caused a severe defect in Rsd binding to σ^{70 9}.

Conserved residues in Rsd form an interacting network

The alignment of Rsd orthologs (Supplementary information) reveals 13 absolutely conserved residues distributed throughout helices H2–H5 (4 conserved residues in H2, 5 in H3, 1 in H4, 3 in H5; red bars in histogram of Figure 2). Only three of the absolutely conserved residues, all on H3, contact σ⁷⁰₄ (Rsd Asp63, Ser66, and Phe70; Figure 2). Remarkably, all 13 absolutely conserved residues form an interacting network that extends through the middle of the Rsd structure (Figure 4). The interacting network includes both polar (green lines in Figure 4B) and van der Waals (orange lines, Figure 4B) interactions. The network links the σ⁷⁰₄ binding surface with three exposed cavities on the Rsd surface (labelled I, II, and III in Figure 4).

Figure 4. Interacting network of conserved residues in Rsd.

A) Two orthogonal views of the σ⁷⁰₄/Rsd complex (similar views as Figure 1). The α-carbon backbones are shown as worms, with σ⁷⁰₄ colored orange, and Rsd colored grey. The side chains of the 13 absolutely conserved Rsd residues are shown (nitrogen, blue; oxygen, red), with carbon atoms colored magenta, except the three residues that interact directly with σ⁷⁰₄ (Asp63, Ser66, Phe70) are red. In the left view, transparent van der Waals surfaces for the conserved residues (color-coded as the atoms) is also shown, showing the continuous network through the middle of the Rsd structure. In the right view, the Rsd structure is shown as a cross-section, sliced at the level of the dashed line in the left view. On the right, the transparent molecular surface of Rsd is shown, colored grey except where the conserved residues are surface exposed. The Rsd helices (H1–H5), viewed mostly end-on, are labeled, as are the three cavities containing surface-exposed conserved residues (I, II, and III).

B) Schematic diagram showing the 13 conserved Rsd residues (bonds color-coded as in A, carbon atoms colored black) and illustrating the polar (hydrogen bonds or salt bridges, green lines) and van der Waals (< 4 Å, orange lines) interactions. The relative locations of σ⁷⁰₄, and cavities I, II, and II are indicated.

Taking the σ⁷⁰₄ binding surface of Rsd as the front, Cavity I is located on the side of the molecule and is lined with conserved residues Asp20 and Leu23 (Figure 4). Cavity II is located on the back of Rsd and is lined with conserved residues Arg26 and Asp142. Most interesting is Cavity III, a deep (approximately 8 Å), narrow (approximately 3 Å in diameter) ‘hole’ penetrating into the structure between H2 and H3. Cavity III is large enough to accommodate a water molecule. Cavity III is lined by conserved residues Trp22, Tyr64, Arg137, and Glu141. Most of the surface of Cavity III is lined with polar atoms (Trp22 NE1; Tyr64 O; Leu65 O; Gly68 N; Tyr73 OH; Arg137 NH1; Glu141 OE2), but one surface comprises the hydrophobic face of the aromatic ring of Tyr64. Note that conserved residues Arg137 and Glu141, which form a salt bridge (Arg137 NH1–Glu141 OE2 distance = 3.62 Å), are completely buried in the interior of the Rsd structure except for the exposure of Arg137 NH1 and Glu141 OE2 on the solvent-accessible surface of Cavity III.

Ecσ⁷⁰₄ structure and the relationship of Rsd with T4 AsiA

Structures of σ₄ from σ⁷⁰-family members have been observed individually (Thermus aquaticus [Taq] σ^A₄; Ref. 13), in complex with –35 element promoter DNA (Taq σA₄; Ref. 13), in complex with anti-σ factors (Ec σ^E₄; Ref. 27; Aquifex aeolicus σ²⁸₄; Ref. 28), and in complex with core RNAP (Taq σ^A₄; Ref. 16; Thermus thermophilus σ^A₄; Ref. 17). In all of these cases mentioned above, the σ₄ domain comprises a compact structural core of three α-helices (corresponding to residues 551–599 of Ec σ⁷⁰₄) that is essentially identical in all of the structures. The last two α-helices of the structural core make up the helix-turn-helix motif that is responsible for recognition of the promoter –35 element ¹³. In one case, however, the interaction of Ec σ⁷⁰₄ with the anti-σ and appropriator AsiA from bacteriophage T4 induces a dramatic structural rearrangement of σ⁷⁰₄ ²⁹.

In the σ⁷⁰₄/Rsd complex, the structure of Ec σ⁷⁰₄ is nearly identical to the undistorted σ₄ structures that have been observed previously. For instance, superimposing the 49-residue structural core of Ec σ⁷⁰₄ (residues 551–599) from the σ⁷⁰₄/Rsd complex with Taq σ^A₄ (PDB ID 1KU3, residues 376–424; Ref. 13) results in an rmsd in α-carbon positions of 1.6 Å (Figure S3). Regions to the N- and C-terminus of the structural core deviate in structure, but these regions are known to be structurally variable depending on the functional context ¹³.

Pineda et al.³⁰ proposed that the T4-type phage AsiA orthologs and Rsd orthologs are members of a related family that interact similarly with their cognate σ factors. Although Jishage & Ishihama ⁵ concluded in their analysis that there was no significant sequence similarity between Rsd and T4 AsiA, Pineda et al. ³⁰ present an alignment between T4-type phage AsiA orthologs and the N-terminal 82 residues of Rsd and suggest that it indicates significant similiarity. However, the Pinda et al. ³⁰ alignment between T4-phage AsiA and Rsd[1–82] contains many gaps for such a short segment, and despite this the sequence identity between T4-phage AsiA and Rsd is only about 7% (6 out of 82 Rsd residues), which is similar to the sequence identity expected for the alignment of completely random sequences (6%). Indeed, structural comparison of T4 AsiA ³¹ and Rsd does not support the idea that these proteins belong to a related family of proteins (Figure S4). Moreover, T4 AsiA dramatically distorts the σ⁷⁰₄ structure ²⁹, while Rsd does not.

Rsd binding to σ⁷⁰₄ occludes both RNAP and promoter DNA binding by σ⁷⁰₄

In the RNAP holoenzyme, σ₄ interacts with the core RNAP by binding to the RNAP β-subunit flap-tip-helix ^14–17. Analysis of the σ⁷⁰₄/Rsd complex predicts that Rsd would sterically interfere with the binding of σ⁷⁰₄ to the β-flap-tip-helix (Figure 5). Rsd also directly interacts with several σ⁷⁰₄ residues that interact with the β-flap-tip in the RNAP holoenzyme (σ⁷⁰₄-Phe563, Leu595, and Arg599). Indeed, a core RNAP binding defect results from mutation of σ⁷⁰₄-Phe563 ^{32; 33}.

Figure 5. Rsd sterically occludes core RNAP and –35 element promoter DNA binding by σ⁷⁰₄.

The σ⁷⁰₄/Rsd complex is shown with the α-carbon backbones as worms (Rsd, green; σ⁷⁰₄, orange). Superimposed is the position of the β-flap-tip (T. aquaticus RNAP β-subunit residues 759–788, corresponding to E. coli RNAP β-subunit residues 887–916), shown as a cyan backbone worm as it would be interacting with σ₄ in the context of the RNAP holoenzyme ¹⁷. Also superimposed is the –35 element DNA as it would be interacting with σ4 13. Both the β-flap-tip and the –35 element sterically clash with Rsd. Below is a diagram schematically illustrating amino acid residues of σ4 that are involved in interactions with Rsd, the RNAP σ-subunit flap (Murakami et al., 2002; Vassylyev et al., 2002), and –35 element promoter DNA (Campbell et al., 2002). Shown in single-letter amino acid code is the sequence of Ecσ⁷⁰₄. Conserved regions 4.1 and 4.2 are indicated above the sequence. Dots below the sequence mark σ4 residues involved in interactions with Rsd (green dots), the RNAP β-flap (cyan dots), or –35 element promoter DNA (grey dots, DNA phosphate backbone; magenta dots, sequence-specific interactions). Residues that interact with Rsd and are also involved in interactions with the β-flap or –35 element DNA are boxed.

In the RNAP holoenzyme open promoter complex, in addition to the β-flap-tip helix, σ⁷⁰₄ also binds the promoter DNA –35 element. Analysis of the σ⁷⁰₄/Rsd complex predicts that Rsd would sterically interfere with the binding of σ⁷⁰₄ to the –35 element DNA (Figure 5). Rsd also directly interacts with several σ⁷⁰₄ residues that interact with the promoter DNA (σ⁷⁰₄-Arg562, Leu573, Arg584, and Arg588; Ref. 13). Both σ⁷⁰₄-Arg584 and Arg588 have been shown to be important for sequence-specific recognition of the –35 element ^{34; 35; 36}. We conclude that σ⁷⁰₄ bound to Rsd would not be able to interact with the RNAP β-flap-tip nor would it be able to interact with the promoter –35 element.

Conclusions

Rsd was identified on the basis of its tight binding to Ec σ^{70 5}. The binding is specific for σ⁷⁰, as Rsd does not associate with alternative σ factors ⁵. Data are consistent with Rsd playing a role in assisting alternative σ factor-dependent transcription by biasing the competition between σ⁷⁰ and alternative σ factors for the available core RNAP through its interaction with σ^{70 6–9}.

The structure of Rsd bound to σ⁷⁰₄, the primary determinant for Rsd binding within σ^{70 10–12}, reveals that Rsd binding interferes with the two primary functions of σ₄, core RNAP binding and promoter –35 element binding (Figure 5). Interestingly, the most highly conserved residues of Rsd form a network of interactions through the middle of the Rsd structure that connect the σ⁷⁰₄-binding surface with three cavities exposed on distant surfaces of Rsd. This suggests functional coupling between σ⁷⁰₄ binding and other binding surfaces of Rsd, either for other proteins or for as yet unkown small molecule effectors. Rsd is known to participate in protein/protein interactions in addition to the one with σ⁷⁰₄, including with the core RNAP ^{37; 38}, and with other σ⁷⁰ domains (L.F.W. and S.A.D., unpublished). The possibility that Rsd may interact with small molecule effectors, particularly through Cavity III (Figure 4), which does not seem appropriate for a protein/protein interaction, has not, to our knowledge, been considered previously. The identification of a putative small molecule ligand for Rsd could greatly facilitate the understanding of Rsd’s role in the regulation of transcription. Finally, due to the high sequence similarity between σ⁷⁰₄ from Ec and Paer, and between Ec Rsd and Paer AlgQ, the structure of the Ec σ⁷⁰₄/Rsd complex provides a structural basis for understanding the mechanism of action of AlgQ, a global regulator of virulence in the pathogen Paer.

Materials and Methods

Construction of the Ecσ⁷⁰₄/Rsd co-expression cassette

The Ec σ⁷⁰₄/Rsd co-expression plasmid was constructed in three steps based upon the procedure of ³⁹. First, DNA encoding full-length Rsd (residues 1–158) was amplified by the polymerase chain reaction (PCR), digested with NdeI and BamHI restriction endonucleases, and cloned between the NdeI and BamHI sites of pET21a (Novagen) generating pET21aRsd. Second, DNA encoding Ec σ⁷⁰₄ (Ec σ⁷⁰ residues 541–613) was amplified by PCR, cleaved with NdeI and HindIII, and cloned between the NdeI and HindIII sites of a pET28a-based plasmid, creating pSKB2Ecσ⁷⁰₄. Finally, using pSKB2Ecσ⁷⁰₄ as a template, the DNA encoding the pET28a-based translation initiation regions and the coding sequence of Ec σ⁷⁰₄ was amplified by PCR, cleaved with BamHI and HindIII, and cloned between the BamHI and HindIII sites of pET21aRsd, creating pET21aRsd/Ecσ⁷⁰₄.

Expression and Purification of the Ecσ⁷⁰₄/Rsd complex

The expression plasmid pET21aRsd/Ecσ⁷⁰₄ was transformed into BL21 (DE3) Ec cells and transformants were selected in the presence of the appropriate antibiotic. Cultures were grown at 37 °C to an A600 nm ~0.6 and induced with 1 mM IPTG for 3 hours at 30°C. Cells containing overexpressed proteins were harvested by centrifugation and stored at minus 80°C.

The Ec σ⁷⁰₄/Rsd complex (Ec σ⁷⁰₄ contains an N-terminal His₆-tag and PreScission cleavage site derived from the vector) was purified by HiTrap Ni²⁺-charged affinity chromatography (GE Healthcare), and the N-terminal His₆-tag was removed using PreScission protease (GE Healthcare). The sample was further purified by a second, subtractive HiTrap Ni²⁺-charged affinity chromatography step to remove uncleaved His₆-σ⁷⁰₄ protein and the His₆-tag, ion exchange chromatography (HiTrap Q Sepharose; GE Healthcare) and gel filtration chromatography (Superdex 75; GE Healthcare). Purified σ⁷⁰₄/Rsd complex was concentrated to ~16 mg/ml using centrifugal filtration units (Vivascience) and exchanged into buffer containing 10 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 5 mM DTT. Selenomethionyl-substituted complex was prepared for MAD analysis by suppression of methionine biosynthesis ⁴⁰, and purified using similar procedures.

Crystallization and Structure Determination

Rod-like crystals were initially obtained by vapour diffusion using a sitting drop setup in 96-well plates using a sparse matrix screen (Hampton Research) at 4°C by mixing 1 μL of protein solution with 1 μL of well solution (0.2 M KCl, 0.05 M MgCl₂, 0.05 M Tris-HCl, pH 7.5, 10% PEG4000) and equilibrating against 100 μL of well solution. Attempts to reproduce the original crystallization condition in a larger 24-well plate using a hanging drop vapour diffusion setup and a grid screen around the original condition yielded needle-like crystals. Crystal growth was optimized using a combination of grid screening and microseeding using Seed Beads (Hampton Research).

Crystals were prepared for cryocrystallography by incubating in cryosolution (0.2 M KCl, 0.05 M MgCl₂, 0.05 M Tris-HCl, pH 7.5, 10% PEG4000, 25% glycerol) for 1 minute. The crystals were then flash frozen in a vial of liquid nitrogen and stored at liquid nitrogen temperature. MAD data were collected from the crystals of selnomethioyl-substituted complex at two wavelengths corresponding to the peak and one remote value of the X-ray absorption spectrum (λ1 and λ2, respectively, Table 1).

Four Se sites (out of a possible six Se sites) were located using SnB ⁴¹ with the anomalous signal from the SeMet(λ1) dataset. Phases were calculated using SHARP ⁴². The experimental electron density map, after density modification using SOLOMON ⁴³, was excellent (Figure S1). Model building was performed manually using O ⁴⁴. Iterative model building and refinement against the native amplitudes were performed using CNS ⁴⁵. The final model contains Rsd residues 1–42 and 50–158 (the loop region 43–49 is disordered), Ec σ⁷⁰₄ residues 546–613, one Mg²⁺-ion, and 98 water molecules. Analysis of the structure using PROCHECK ⁴⁶ showed 91% of the residues in the most favored regions of the Ramachandran plot, and 9% in additional allowed regions (no residues in generously allowed or disallowed regions), and an overall G factor of 0.3.

Sequence alignment

The sequence alignment of 113 Rsd orthologs (Supplemental information) was generated by using the Ec K-12 Rsd sequence to BLAST the NCBI non-redundant database, followed by sequence retrieval. The downloaded sequences, which included both Rsd and Paer AlgQ, were then aligned using PCMA (ftp://iole.swmed.edu/pub/PCMA/) (PMID: 12584134).

Accession codes

The coordinates for the refined σ⁷⁰₄/Rsd structure have been deposited in the Protein Data Bank (accession code 2P7V).