H-NS_ST site1 and site2 form homodimers to enable H-NS multimerization in a head-to-head/tail-to-tail fashion (Figure 1B; Arold et al., 2010). In concert with the DNA interaction of the individual domains, this homo-oligomerization is required for tight DNA binding and hence gene repression. In our previous study, we showed that only H-NS_ST site2 dimers unfold and dissociate within a biologically relevant temperature range, whereas site1 dimers remain unaffected (Shahul Hameed et al., 2019). The higher stability of the site1 dimer of H-NS_ST is explained by a substantially larger contact surface between the two monomers (ca. 3300 Ų compared to ca. 850 Ų for site1 and site2, respectively, according to PDBePISA [Krissinel and Henrick, 2007]).

To investigate whether this mechanism is conserved in other H-NS orthologs, we built homology models for H-NS_EA, H-NS_BA, and H-NS_IL using the crystal structure of the H-NS_ST site1–site2 fragment as a template (PDB ID: 3NR7) (Arold et al., 2010). Next, we constructed a tetrameric model as a minimal representation that conserves all features of the H-NS multimer. This tetramer contained two full-length H-NS monomers (residues 1–137, with templates PDB IDs: 3NR7 and 2L93) and two partial monomers, truncated before site2 (residues 1–52) (Figure 1C). To probe differences in environmental responses of the orthologs, we first used conventional all-atom MD. We simulated all four tetramers (~100,000 atoms in each system; see Materials and methods) for 200 ns at three different conditions (0.15 M NaCl, 293 K; 0.50 M NaCl, 293 K/20°C; or 0.15 M NaCl, 313 K/40 °C) (Supplementary file 1B).

The tetramer simulations at 0.15 M NaCl and 20°C produced a lower residue fluctuation level in site1 (local root-mean-square fluctuation [RMSF] 0.4–1.9 Å) than in site2 (local RMSF 0.5–4.4 Å) for all four orthologs (Supplementary file 1C). The higher stability of the site1 dimer is explained by the generally higher number of nonpolar contacts than in the site2 dimer (Figure 2—figure supplement 1). These contacts involved conserved hydrophobic amino acid residues, notably L5 (or I5) and L8 of α1, L14 of α2, and L23, L26, V36, and V37 (or I37) of α3 (Figure 2). Our in silico mutant stability prediction analysis corroborated qualitatively the importance of hydrophobic residues for stabilizing the site1 dimer, in particular of L5, L8, L23, and L26 (Supplementary file 1D and 1E).

These interactions remained formed in all site1 dimers in our tetramer simulations (at 0.15 M NaCl at 20°C) and tetramer simulations at higher salinity (at 0.50 M NaCl at 20°C) or higher temperature (at 0.15 M NaCl at 40°C). Hence, we found that the stability of the site1 dimers resulted mainly from strong and conserved nonpolar packing. Indeed, recombinantly expressed site1 fragments of all four orthologs formed ~15 kDa dimers in size exclusion chromatography–multi-angle light scattering. These dimers were stable within the temperature range relevant for environment-sensing (aggregation temperature, T_agg > 37 °C; Figure 2—figure supplement 2). We concluded that the mechanism observed for H-NS_ST – where site1 remains stable and the site2 stability is affected by the environment – is conserved in H-NS_EA, H-NS_BA, and H-NS_IL.

Compared to site1 dimers, site2 dimers harbor fewer nonpolar contacts, only involving residues L58 (or I58), Y61 (or F61), M64 (or A64), I70, and L75 (or I75) (Figure 2). Hence, while site1 dimerization was largely maintained by nonpolar packing, site2 dimerization was strongly driven by electrostatic interactions from selective salt bridges. MD simulations revealed that these salt bridges were in a dynamic equilibrium between forming, breaking, and rearranging. These salt bridges were either formed in cis, within the site2 monomer, (e.g. E52-R56 and R62-E63 in H-NS_ST) or in trans, between two monomers in the site2 dimer (e.g. R54-D71’, R54-E74’, and K57-D68’ in H-NS_ST, where the apostrophe denotes residues from the second chain; illustrated in Figure 3). In addition to substitutions that delete (E52A in H-NS_BA; E63S and D68A in H-NS_IL) or weaken (E63Q in H-NS_EA; D71N in H-NS_BA) these salt bridges, our simulations showed different levels of site2 salt bridge stability among orthologs (Figure 3, Supplementary file 1F): (1) The inter-monomer salt bridge R/K54-E/D74’ was stable in all our simulations at 20°C and 0.15 M NaCl, but less likely to form at an increased temperature (40°C) or salinity (0.50 M NaCl), suggesting that this salt bridge is involved in environmental sensing (Figure 3A). (2) Absent in H-NS_IL, the inter-monomer salt bridge K57-D68’ remained formed during all our simulations of H-NS_ST, H-NS_EA, and H-NS_BA, indicating a ‘housekeeping’ role for the stability of the site2 dimer in all orthologs except for H-NS_IL (Figure 3B).

Our simulations show how specific protein dynamics might modulate the ortholog’s response to salinity or temperature. For example, we observed increased bending of the α3 backbone (annotated by the black arrow in Figure 3C) at high temperature (40°C) or high salinity (0.50 M NaCl) (Figure 3—figure supplement 1). Although α3 bending occurred in all orthologs, it only significantly affected the site2 dimer of H-NS_ST by separating R54 from E74’ or D71’, suggesting that this mechanism contributed to the salt and temperature sensitivity of H-NS_ST site2, whereas it was not strong enough to significantly affect site2 stability in other orthologs.

Another example was given by H-NS_EA, where an alternative R54-D71’ salt bridge formed whenever the R54-E74’ contact was broken at 40°C. This alternative R54-D71’ salt bridge stabilized the H-NS_EA site2 dimer at the higher temperature, suggesting that this compensatory mechanism resulted in a decreased sensitivity to temperature (Figure 3C). H-NS_IL provided a final example for a specific response. Compared with the R54-E74’ salt bridge (Figure 3A), the K57-D68’ salt bridge only varied slightly in all our simulations (Figure 3B). However, the substitution D68A in H-NS_IL supplanted the electrostatic interaction with a nonpolar interaction, which was broken at 40°C in our simulations (Figure 3D). This effect suggested that H-NS_IL had a reduced sensitivity to salinity, while remaining sensitive to temperature.

To complement the dynamics of H-NS orthologs from our conventional MD simulations, we used extensive simulations with umbrella sampling (US) to quantitate the overall site2 stability. We calculated the potential of mean force (PMF) for site2 dimer dissociation (residues 50–82, ~46,000 atoms) of the four H-NS orthologs for three different conditions (low salinity/low temperature, high salinity, or high temperature). The site2 monomers were not constrained and remained structurally flexible during the dissociation process. To ensure convergence in the PMFs, we employed long windows (54 ns) in simulations totaling 52 µs (details provided in the SI; see Figure 3—figure supplement 2 for resulting histograms and PMFs along the dissociation coordinate). According to the free-energy difference between the dimerization and dissociation states (∆G = G_dimer − G_dissociation), we estimated the energetic impact from increased salinity and temperature as follows: ∆∆G = ∆G_{high salinity or temperature}− ∆G_{293K, 0.15M NaCl} (Figure 3E). Notably, high salinity (0.50 M NaCl) or temperature (40°C) decreased the stability of the H-NS_ST site2 dimer by 2.2 kcal/mol. H-NS_BA displayed a similar sensitivity to temperature but a lower sensitivity to salinity, which destabilized the dimer by 1.5 kcal/mol. Interestingly, our data indicated that H-NS_EA was only sensitive to salinity, whereas raising the temperature had little impact on the stability of the H-NS_EA site2 dimer. Conversely, H-NS_IL only responded to temperature, whereas the increased salinity did not affect the stability of its site2 dimer (∆G ~ 0 kcal/mol). Collectively, our conventional MD simulations and PMF calculations suggested how, on the atomic level, changes in the site2 sequence may alter the sensitivity of the H-NS orthologs to different environmental changes.

In a previous study (Shahul Hameed et al., 2019), we had shown that melting and dissociation of site2 dimers allow H-NS_ST to adapt a closed conformation in which the linker-DNAbd fragment interacts with a negatively charged region on site1 α3 (Figure 4—figure supplement 1A) and that this autoinhibitory interaction is incompatible with DNA interactions. However, due to extensive signal broadening of mainly linker amides exchanging with water, our conventional proton-detected NMR analysis based on exchangeable amide H/N-observed correlations did not allow confident mapping of the binding site on the C-terminal region (Shahul Hameed et al., 2019; Figure 4—figure supplement 1B). Herein, we overcame this limitation by using proton-less ¹³C-detected NMR analysis to complete the resonance assignment of the linker-DNAbd fragment (Figure 4 and Figure 4—figure supplement 1C). These complete carbon chemical shifts allowed us to elucidate the structural mechanism of H-NS_ST autoinhibition fully and, in a second step, to use this understanding to investigate the existence of this closed conformation in the orthologs.

We first titrated unlabeled H-NS_ST site1 (residues 1–57) onto the ¹³C,¹⁵N-labeled H-NS_ST C-terminal region (Ct_ST, residues 84–137), comprising the linker (residues 84–93) and DNAbd (residues 94–137) (Figure 4A, Figure 4—figure supplement 1A). Motif identification from chemical shifts (MICS) (Shen and Bax, 2013) revealed that the interaction promoted the formation of a short type VIII β-turn in residues 89–92 (MICS confidence coefficient was 0.69). No significantly stable other motif besides the residual random coil was identified (Figure 4B,C). This sharp turn brings the positively charged linker side chains K87, K89, R90, R93 closer to each other than in the free state, presumably as a result of pairing them with opposite charges on site1 (Shahul Hameed et al., 2019; Figure 4B,C).

To further probe the local dynamics of the polypeptide chain, we determined the random-coil-index order parameter RCI-S² based on the fully assigned ¹³C-resonances for each residue for the ligand-free and site1-saturated Ct_ST. The dynamics of the well-ordered DNAbd domain remained unchanged with or without site1 present, in agreement with its only minor involvement in the autoassociation (Figure 4D). Conversely, the linker residues 84–95 were disordered without regular secondary motifs in the absence of site1 (RCI-S² < 0.35). Upon addition of site1, the local dynamics decreased, particularly within the stretch of four amino acids K89-R90-A91-A92 (RCI-S² > 0.6) that predominantly form the type VIII β-turn according to MICS. Nonetheless, the overall RCI-S² of the linker remained low, although experimental conditions resulted in >99% of ligand saturation of the labeled Ct_ST, demonstrating that the association with site1 did not substantially restrict the linker’s movements (Figure 4D).

Collectively, our analysis established that the autoinhibitory site1:Ct_ST association was driven by oppositely charged residues located on site1 and the linker, and involved only a small region of the DNAbd. The center of this linker region, residues 89–92, rigidified upon binding and predominantly formed a β-turn conformation. However, the resulting intramolecular interaction was maintained through ‘fuzzy’ charge-pairing that did not fix the partners into a structurally stable complex.

Having established the detailed autoinhibitory interactions between site1 and the Ct region in H-NS_ST, we next examined the H-NS orthologs. Based on our structural models (initial homology models and models from conventional MD), the electrostatic surface of the Ct was well conserved across all H-NS orthologs (Figure 5A). This level of conservation was expected, given that this region is also required for DNA association (Gao et al., 2017) – a role that needs to be conserved in all H-NS. Conversely, the site1 surface that binds to Ct was not conserved across all orthologs. While H-NS_EA was similar to H-NS_ST in the overall charge distribution, α3 of H-NS_BA showed a distinctly basic surface. H-NS_IL displayed an intermediate electrostatic character, with features closer to H-NS_ST/H-NS_EA (Figure 5A). These findings suggested that the stability of the closed conformation varies across orthologs. To test this prediction, we carried out in vitro binding experiments using microscale thermophoresis (MST).

These binding experiments between site1 and Ct confirmed that the strength of the autoassociation was similar for H-NS_ST and H-NS_EA (Figure 5B). The autoassociation was 10-fold stronger in H-NS_IL than in H-NS_ST, despite a less acidic site1. Hence, autoinhibition in H-NS_IL might include additional and/or different interactions. Conversely, H-NS_BA did not show a significant capacity for autoassociation, as expected from its markedly more basic site1 surface. In agreement, the double H-NS_ST site1 mutant E34K/E42K (designed to make the electrostatic site1 surface of H-NS_ST B. aphidicola like) dramatically lowered its affinity for H-NS_STCt (Figure 5—figure supplement 1). In addition to the reduced electrostatic complementarity, H-NS_BACt also has a proline residue (P91) in position 3 of the β-turn region, which is highly unfavorable for this secondary structure element (Creighton, 1990). Indeed, at room temperature, H-NS_BACt associated only very weakly with H-NS_ST site1 (K_d > mM), whereas the Ct of H-NS_EA and H-NS_IL bound to H-NS_ST site1 with a similar affinity than H-NS_STCt (K_ds were 31.2 ± 3 µM and 18.2 ± 2 µM, respectively Figure 5—figure supplement 1A,B). Given that all four Cts display comparable electrostatic surfaces, the loss of affinity for H-NS_BACt supported the importance of the β-turn. Increasing the temperature decreased the self-association strength 2- to 3-fold in H-NS_ST and H-NS_IL and more than 10-fold in H-NS_EA (Figure 5B). It also decreased the K_d for H-NS_BA to values beyond the measurement range.

We concluded that the strength of the autoinhibitory conformation is mostly modulated by the electrostatic surface characteristics of site1. The Ct is constrained by the requirement to preserve the overlapping DNA binding surface, but can decrease autoinhibition by disrupting the β-turn conformation.

We next experimentally assessed the response of the H-NS orthologs to physicochemical changes using dynamic light scattering (DLS). DLS provides the average hydration radius R_H of the particles in solution, and hence gives a proxy for the tendency of H-NS molecules to form site2-mediated multimers or (still site1-linked) dimers. Thus, the R_H is a convoluted signal of both effects, that is the relative strength of site2 multimerization and of the autoinhibitory conformation (if it exists). We measured the R_H under different salt concentrations and temperatures. As reported previously, H-NS_ST showed a clear drop in R_H from 10°C to 40 °C (Figure 6A; Shahul Hameed et al., 2019). The marked decrease of R_H for curves at 0.15, 0.25, and 0.50 M NaCl indicated a strong inverse correlation between salinity and site2 stability, in agreement with a key role of salt bridges in stabilizing the site2 dimers.

All three H-NS orthologs displayed a similar behavior overall, further supporting that the general mechanism of site2-mediated multimerization and environment-sensing was preserved. However, we noted important differences in the orthologs’ response characteristics (Figure 6A): (1) Of the four orthologs, H-NS_ST responded most strongly to salinity and temperature, consistent with the broken R54-E74’ salt bridge and large site2 RMSF in our high-salinity or high-temperature simulations. (2) H-NS_EA was less temperature sensitive and showed weaker multimerization than the other orthologs. Indeed, our simulations suggested that H-NS_EA site2 can rearrange the inter-dimer salt bridge and form either R54-E74’ or R54-D71’ to maintain site2 stability at higher temperatures. (3) H-NS_BA had the highest tendency to multimerize among all the orthologs tested, which might partly be explained by the absence of the autoinhibitory conformation. Compared to H-NS_ST, our PMF calculations showed a slightly higher sensitivity to temperature and a slightly reduced sensitivity to salinity. Although these tendencies were apparent in our DLS data, these data were also affected by the fact that H-NS_BA required more than 150 mM NaCl to stay in solution, but H-NS_BA nonetheless aggregated at 30°C. (4) H-NS_IL showed a decreased sensitivity to salinity compared to H-NS_ST, as suggested by our computational analysis (i.e. the lack of the site2 K57-D68’ salt bridge, the lack of salt-promoted free-energy changes, and the attenuated electrostatic site1 surface).

To corroborate these conclusions, we designed several site2 mutants and tested their effect on protein multimerization using fluorescence anisotropy. To eliminate the influence of the autoinhibitory site1:Ct interaction, we used H-NS constructs that lacked the Ct (H-NS 1–83). The normalized fluorescence polarization (NFP) of H-NS_ST declined between 20°C and ~40°C to a value of 0.2, indicating that the average particle size decreased with temperature, as observed in DLS. And as in DLS, increases in salt lowered the NFP, and hence, the propensity of the particles to form site2-linked multimers (Figure 6B). The H-NS_ST R54M mutant, disrupting the ‘housekeeping’ salt bridges R54-E74’ and R54-D71’, displayed an NFP of 0.2 at all temperatures and salt concentrations. Thus, this mutant supported the key roles of the R54-mediated salt bridges in H-NS multimerization and environment-sensing. To experimentally assess the role of the K57-D68’ salt bridge in conveying salt sensitivity, we introduced the D68A ‘IL-like’ mutation in H-NS_ST, and the A68D ‘ST-like’ mutation in H-NS_IL. These mutations abrogated salt sensitivity in H-NS_ST and introduced salt sensitivity in H-NS_IL, as predicted by our computational analysis (Figure 6B).

Collectively, our experimental observations revealed significant differences in response to physicochemical parameters, which were in agreement with our predictions based on the molecular features of the H-NS orthologs.

Environment-sensing through the pleiotropic gene regulator H-NS helps S. typhimurium to adapt when it is present inside its host mammal. In a previous study, we had shown that an increase in temperature, and to some extent salinity, dissociates the second dimerization element (site2). Melting of site2 produces two effects: first, it impedes synergistic DNA binding of H-NS multimers, and second, it allows H-NS to adopt an autoinhibitory conformation where DNA binding residues on the C-terminal linker-DNAbd fragment (herein abbreviated as the Ct) associate with the N-terminal site1 dimerization domain (Shahul Hameed et al., 2019). In our current study, we confirmed that site2 is the element that senses changes in physicochemical parameters, and we uncovered additional aspects of this process. In particular, proton-less NMR fully revealed the position and dynamics of the Ct residues involved in the autoinhibitory association with site1. We also showed that the formation of a β-turn in the linker residues 89–91 is associated with the autoinhibited conformation. The Ct residues critical for autoinhibition cannot reach site1 without site2 dissociation (Figure 3—figure supplement 2B), confirming that the closed autoinhibited conformation is mutually exclusive with H-NS multimerization. Our NMR analysis also demonstrated that this autoinhibition is achieved at a low entropic cost, maintaining a high flexibility with respect to the exact distribution of the interacting charges on both site1 and the linker-DNAbd fragment. On the one hand, avoiding the entropic penalty helps the autoinhibitory interaction to prevail against the competing DNA association. (Of note, the covalent link between site1 and the Ct will enhance their local concentration and hence their apparent affinity compared to our measurement based on separate domains in Figure 5B.) On the other hand, the fuzziness of the charge–charge interactions facilitates preserving the capacity for autoinhibition during bacterial evolution and adaptation.

Based on our refined molecular understanding of S. typhimurium H-NS, we then investigated environment-sensing of H-NS orthologs from bacteria that infect plants, bacteria that are endosymbionts of insects, and bacteria that are presumably free-living in or close to a hydrothermal vent. Across all four orthologs, we observed a conceptually similar response to temperature and salt, both overall and on an atomic level, where salt bridges play key roles. This similarity suggests that environment-sensing in H-NS evolved by co-opting an ancestral feature, namely the relative instability of the simple site2 helix-turn-helix dimerization motif. However, marked idiosyncrasies in the response of H-NS orthologs suggest that this ancestral feature was then adapted to fit the current habitat and lifestyle. Thus, our analysis suggests that environment-sensing by H-NS originated from an exaptation followed by adaptation. Our combined computational and experimental structural analysis allowed us to relate the observed in vitro features of this adaptation to events on a residual level: in particular, the salt bridge disposition and stability of site2, and the strength of the autoinhibition governed mostly by the electrostatics of site1 helix α3.

Although other factors inside bacteria can modify the in vitro behavior of the isolated protein, it is interesting to consider these idiosyncrasies with respect to the bacteria’s habitats (Figure 7):

Our integrative approach provided atomistic insights on how residue-level substitutions on a protein support adaptation of organisms to different lifestyles.