Functional roles of the pre-sensor I insertion sequence in an AAA+ bacterial enhancer binding protein

Abstract

Molecular machines belonging to the AAA+ superfamily of ATPases use NTP hydrolysis to remodel their versatile substrates. The presence of an insertion sequence defines the major phylogenetic pre-sensor I insertion (pre-SIi) AAA+ superclade. In the bacterial sigma(54)-dependent enhancer binding protein phage shock protein F (PspF) the pre-SIi loop adopts different conformations depending on the nucleotide-bound state. Single amino acid substitutions within the dynamic pre-SIi loop of PspF drastically change the ATP hydrolysis parameters, indicating a structural link to the distant hydrolysis site. We used a site-specific protein-DNA proximity assay to measure the contribution of the pre-SIi loop in sigma(54)-dependent transcription and demonstrate that the pre-SIi loop is a major structural feature mediating nucleotide state-dependent differential engagement with Esigma(54). We suggest that much, if not all, of the action of the pre-SIi loop is mediated through the L1 loop and relies on a conserved molecular switch, identified in a crystal structure of one pre-SIi variant and in accordance with the high covariance between some pre-SIi residues and distinct residues outside the pre-SIi sequence.

Fig. 1

Location and sequence of the pre-SIi loop in PspF₁₋₂₇₅. A. Crystal structure of the PspF₁₋₂₇₅ hexamer with the location of the L1 and pre-SIi loops, Helix 3 and Helix 4 highlighted (PDB 2BJW). The residues that comprise the PspF pre-SIi are indicated. B. Left panel: The consensus pre-SIi insertion sequence obtained using the 289 annotated Pfam (00158) bEBP sequences. Alignment of these sequences suggests that conservation of the pre-SIi loop varies, with the RVGG motif being the most conserved sequence. Right panel: The pre-SIi sequences of structurally characterized bEBPs PspF₁₋₂₇₅, NtrC1, ZraR and DctD.

Fig. 2

Specific pre-SIi variants demonstrate defects in transcription activation. A. Some pre-SIi variants fail to support Eσ⁵⁴ transcription from the super-coiled (SC) Sinorhizobium meliloti nifH promoter (10 min activation time). The full-length (FL) transcript is as indicated. The Q136A and P137A/T variants demonstrate significant levels of transcription activity. B. Top: Schematic and nucleotide sequence of the S. meliloti nifH duplex promoter probe with the consensus promoter elements GG (positions −26 and −25) and GC (positions −14 and −13) underlined. Bottom: Abortive transcription gel showing the pre-SIi variants ability to support Eσ⁵⁴ open complex formation on the linear duplex probe. The abortive transcript UpGGG is indicated. The relative numbers of complexes formed on the duplex probe in the presence of the pre-SIi variants are indicated in the graph below the gel. C. As in B but using the pre-opened promoter probe. The lowercase letters in bold type-face indicate the non-complementary residues in the pre-opened promoter probe.

Fig. 3

The pre-SIi variants defective for open complex formation fail to interact with σ⁵⁴. A. Top: Schematic and nucleotide sequence of the S. meliloti nifH mismatch promoter probe, the lowercase letters in bold type-face indicate non-complementary residues in the mismatch promoter probe. Bottom: SDS-PAGE gel showing the cross-linking profiles of σ⁵⁴–DNA complexes formed on the mismatch promoter probe in the presence of 4 mM dATP and either PspF₁₋₂₇₅WT or variants. The migration positions of the cross-linked σ⁵⁴–DNA and PspF₁₋₂₇₅–DNA species are indicated. Native-PAGE gel illustrating supershift complexes (σ⁵⁴ss–DNA) are formed in the presence of PspF₁₋₂₇₅WT (lane 2) and the S135A (lane 6), Q136A (lane 7) and P137A/T (lanes 8 and 9) variants. The migration positions of the supershift (σ⁵⁴ss–DNA) and binary σ⁵⁴–DNA (σ⁵⁴–DNA) complexes, free DNA and percentage DNA bound in each complex is indicated. B. SDS-PAGE gel as in A but in the presence of core RNAP. The migration positions of the cross-linked β/β′–DNA and σ⁵⁴–DNA species are indicated. Cross-linked β/β′–DNA species are only observed with the transcription competent PspF₁₋₂₇₅ variants (WT, Q136A and P137A/T).

Fig. 4

Gel filtration profiles of PspF₁₋₂₇₅ (WT and variants) in the absence and presence of ADP. Nucleotide-dependent apparent dimer/hexamer equilibrium of PspF₁₋₂₇₅ proteins as obtained by analytical HPLC gel-filtration chromatography using a Biosep 3000 column (Phenomenex), in the absence (A) and presence (B) of ADP. Chromatographs are overlaid and offset but not normalized. The PspF₁₋₂₇₅ variants are as indicated.

Fig. 5

The activities of the V132A and L138A variants are rescued by ADP–AlF. A. SDS-PAGE gel showing the cross-linking profiles of σ⁵⁴–DNA complexes formed on the mismatch promoter probe in the presence of ADP–AlF. The migration positions of the cross-linked σ⁵⁴–DNA and PspF₁₋₂₇₅–DNA species are indicated. A cross-linked PspF₁₋₂₇₅–DNA species is observed in reactions containing the V132A (lane 4), S135A (lane 6), Q136A (lane 7), P137A/T (lanes 8–9) and L138A (lane 10) variants. Native-PAGE gel illustrating that stable ADP–AlF trapped complexes are only observed in the presence of PspF₁₋₂₇₅ WT (lane 2) and the S135A (lane 6), Q136A (lane 7) and P137A/T (lanes 8–9) variants. The migration positions of the σ⁵⁴–DNA-PspF₁₋₂₇₅:ADP–AlF (trapped) and binary σ⁵⁴–DNA (σ⁵⁴–DNA) complexes, free DNA and percentage DNA bound in each complex are as indicated. B. SDS-PAGE gel as in A but on the duplex promoter probe in the presence of core RNAP. The migration positions of the cross-linked β/β′–DNA, σ⁵⁴–DNA and PspF₁₋₂₇₅–DNA species are indicated.

Fig. 6

A conserved switch between the pre-SIi and L1 loops exists within bEBPs. A. The covariance between the PspF pre-SIi sequence (RVGGSQPLQ; colour-coded as shown) and PspF residues 80–98 was calculated and depicted graphically. The strong covariance of residue E81 with pre-SIi residues R131, G134 and L138 (arrowed) and residue E97 and pre-SIi residue R131 (arrowed) is indicated by high covariance scores. B. The crystal structures of the apo-PspF₁₋₂₇₅R131A (yellow) and apo-PspF₁₋₂₇₅WT (blue; PDB 2BJW) demonstrate the effect of the R131A mutation on the pre-SIi loop conformation. The two structures were aligned on the main chain atoms of residues 35–42. The positions of residues E81 (L1 loop), E125 (Helix 4), R168 (putative R-finger), R131 and V132 (pre-SIi) and the R131A mutation (in the context of the PspF₁₋₂₇₅R131A structure) are indicated. Structural features relevant to bEBPs such as the L1 and pre-SIi loops, Helix 3 and Helix 4 are labelled. Clear local differences between the apo-PspF₁₋₂₇₅WT and apo-PspF₁₋₂₇₅R131A structures are apparent in the pre-SIi loop conformation, as well as a significant rotation of residue E81 (L1 loop) resulting in the disruption of the E81-R131 link.