Identification and characterization of the Hfq bacterial amyloid region DNA interactions

Abstract

Nucleic acid amyloid proteins interactions have been observed in the past few years. These interactions often promote protein aggregation. Nevertheless, molecular basis and physiological consequences of these interactions are still poorly understood. Additionally, it is unknown whether the nucleic acid promotes the formation of self-assembly due to direct interactions or indirectly via sequences surrounding the amyloid region. Here we focus our attention on a bacterial amyloid, Hfq. This protein is a pleiotropic bacterial regulator that mediates many aspects of nucleic acids metabolism. The protein notably mediates mRNA stability and translation efficiency by using stress-related small non coding regulatory RNA. In addition, Hfq, thanks to its amyloid C-terminal region, binds and compacts DNA. A combination of experimental methodologies, including synchrotron radiation circular dichroism (SRCD), gel shift assay and infrared (FTIR) spectroscopy have been used to probe the interaction of Hfq C-terminal region with DNA. We clearly identify important amino acids in this region involved in DNA binding and polymerization properties. This allows to understand better how this bacterial amyloid interacts with DNA. Possible functional consequence to answer to stresses are discussed.

Keywords: Bacterial adaptation; Bacterial amyloid; DNA induced protein fibrillation; DNA:protein fibrils; Functional amyloid.

Fig. 1

EMSA analysis of Hfq-CTR wild type (WT) and mutants in the presence of DNA. Lane 1: Control, Hfq-CTR WT (this control was reprinted with permission from Biomacromolecules 2020, 21, 3668–3677. Copyright 2020 American Chemical Society); lane 2: Hfq-CTR S87A,S88A; lane 3: Hfq-CTR E99A,E100A,E102A; lane 4: Hfq-CTR Y83A. In these case a complex is formed with the peptide. Note that the complex, when it forms, migrates on the top of the gel (but not in the well that is not visible here), indicating that numerous peptides are bound to DNA. Sometimes 2 complexes of different size are present, probably corresponding to different numbers of CTRs bound to DNA . Taking into account the bridging properties of the CTR , we suspect these two bands could be (CTR_n:AT₅₉) and (CTR_n:AT₅₉)₂, where 2 (CTR_n:AT₅₉) are bridged. Lane 5: Hfq-CTR S80A,S81A; Lane 6: Hfq-CTR R66A; Lane 7: Hfq-CTR S65A,S69A,S72A; Lane 8: Hfq-CTR S93A,S98A; Lane 9: Hfq-CTR H70A,H71A,H84A,H85A; Lane 10: Hfq-CTR G76A, G77A, G78A. In these cases no complex is formed.

Fig. 2

K_D measurements of WT and mutated Hfq-CTR:dsDNA complexes using fluorescence anisotropy. In this case a dA:dT₂₀ dsDNA was used. WT Hfq-CTR (black) has an equilibrium dissociation constant K_D = 260 ± 10 nM; mutant Hfq-CTR S87A,S88A (blue) has a K_D = 460 ± 30 nM. The mutants Hfq-CTR Y83A (red) and Hfq-CTR E99A,E100A,E102A (green) have lower affinities with K_D = 1000 ± 75 nM and 890 ± 80 nM, respectively. For other mutants, namely Hfq-CTR S80A,S81A; Hfq-CTR R66A; Hfq-CTR S65A,S69A,S72A; Hfq-CTR S93A,S98A; Hfq-CTR H70A,H71A,H84A,H85A and Hfq-CTR G76A, G77A, G78A no complex is formed, titration curve was flat and not shown, in agreement with EMSA result.

Fig. 3

SRCD spectra of the CTR/DNA complex (blue), DNA (red) and CTR (green). Spectra of the individual components are measured with equivalent DNA and CTR concentrations. The dotted spectrum represents the relevant combination of the spectra pertaining to the individual components. (++), (+), (±) and (-) indicate the four main behaviors observed. An example of each behavior is presented. For instance for Hfq-CTR E99A,E100A,E102A/DNA spectra (top left corner, (++), a clear different spectrum is measured (blue) compared to the theoretical addition of the peptide and DNA spectra (dotted line). The same analysis for all other Hfq-CTR mutants with (-) behaviors are presented in Sup. Fig. S5. The qualitative comparison considering amplitude and peak position shifts between theoretical and measured spectra always correlates with EMSA and FTIR results.

Fig. 4

FTIR spectra of Hfq-CTR in the presence or absence of DNA. (a) peptides alone. (b) difference spectrum obtained by subtracting the (dA:dT)₅₉ contribution from the complex spectrum. We clearly observe in the Amide I band a contribution at ∼ 1620 cm⁻¹, indicative of the formation of the amyloid structure in the presence of DNA.

Fig. 5

Multiple sequence alignment of various bacterial Hfq CTRs. E. coli Hfq CTR aa presumably involved in DNA binding are indicated in red, in amyloid assembly in yellow and in DNA structural change in cyan (see 4.3). For GRAVY index hydrophobic uncharged residues are in blue, hydrophilic basic in red, hydrophilic acidic in purple and hydrophilic uncharged in dark green. The CTR regions are variable in length among bacteria and only those with a CTR comparable to that of E. coli Hfq are shown. In the alignment, strictly conserved R66 is indicated in hot pink; aa conserved in most Hfqs CTR are indicated in light pink. Other aa are indicated in yellow and green.