Mechanism of environmentally driven conformational changes that modulate H-NS DNA-bridging activity
Although the bacterial genome is compacted by a vast variety of factors , including DNA supercoiling , and macromolecular crowding , it owes much of its organization to nucleoid-associated proteins ( Dame , 2005; Dame et al . , 2011; Dillon and Dorman , 2010; Dorman , 2013; Rimsky and Travers , 2011; Travers and Muskhelishvili , 2005; Luijsterburg et al . , 2008; Dame and Tark-Dame , 2016 ) . A key protein in nucleoid organization of Gram-negative bacteria is the Histone-like Nucleoid Structuring protein ( H-NS ) . Genome-wide binding studies have revealed that H-NS binds along the genome in long patches ( Grainger et al . , 2006; Kahramanoglou et al . , 2011; Lucchini et al . , 2006; Navarre , 2006; Oshima et al . , 2006 ) , which have been proposed to mediate the formation of genomic loops ( Noom et al . , 2007; van der Valk et al . , 2014 ) . H-NS is also an important regulator of global gene expression , implied in mediating global transcriptional responses to environmental stimuli ( osmolarity , pH , temperature ) ( Atlung and Ingmer , 1997 ) , and operating as xenogeneic silencer , silencing horizontally integrated DNA ( Navarre et al . , 2006 ) . A large fraction of Escherichia coli and Salmonella genes ( 5–10% ) is thus regulated ( usually repressed ) by the action of H-NS . H-NS operation is modulated by environmental stimuli and through interplay with other proteins ( Atlung and Ingmer , 1997; Stoebel et al . , 2008 ) . In solution , the H-NS protein exists as a dimer , which oligomerizes at high concentrations ( Ceschini et al . , 2000; Spurio et al . , 1997 ) . H-NS consists of three structural domains: a C-terminal domain responsible for DNA binding ( Shindo et al . , 1995 ) , a N-terminal dimerization domain ( Bloch et al . , 2003; Cerdan et al . , 2003; Esposito et al . , 2002; Ueguchi et al . , 1996 ) and a central dimer-dimer interaction domain responsible for multimer formation ( Arold et al . , 2010; Leonard et al . , 2009 ) . These two interaction domains are connected by a long α-helix ( Arold et al . , 2010 ) ( helix α3 ) . H-NS exhibits two seemingly distinct DNA-binding modes: DNA bridging ( Dame et al . , 2006; Dame et al . , 2000; Dame et al . , 2001; Dame et al . , 2002; Schneider et al . , 2001 ) , the condensation of DNA by intra- and inter- molecular DNA binding by H-NS and DNA stiffening , the rigidification of DNA through the formation of a H-NS-DNA filament ( Amit et al . , 2003; Dame and Wuite , 2003; Liu et al . , 2010 ) . These modes have been attributed to the basic functional H-NS unit ( a dimer ) binding to DNA either in cis or in trans ( Wiggins et al . , 2009; Joyeux and Vreede , 2013 ) . H-NS paralogues StpA , Sfh , Hfp , and truncated derivatives such as H-NST , have been proposed to modulate H-NS function by forming heteromers with H-NS ( Baños et al . , 2008; Deighan et al . , 2003; Müller et al . , 2010; Williams et al . , 1996; Williamson and Free , 2005 ) , with DNA-binding properties different from homomeric H-NS . Members of the Hha/YmoA family of proteins are H-NS co-regulators with limited sequence homology to H-NS ( Madrid et al . , 2007 ) . At many targets along the genome , H-NS and Hha co-localize . Localization of Hha at these sites is strictly H-NS dependent , whereas the genome-wide binding pattern of H-NS is only mildly affected by Hha ( Ueda et al . , 2013 ) . Although evidence has been put forward that the concentration of divalent ions determines the binding mode of H-NS ( Liu et al . , 2010 ) , a mechanistic explanation is lacking . Moreover , the possible effect of co-regulators of H-NS , such as Hha , on these binding modes has remained unexplored . To obtain a better understanding of the molecular basis underlying the H-NS binding modes , it is crucial to determine the effects of ion valence and concentration , as well as the presence of helper proteins on both the stiffening and the bridging mode . Here , we investigate DNA stiffening on short DNA tethers using Tethered Particle Motion ( TPM ) . As intramolecular DNA bridging does not occur on short DNA tethers , DNA stiffening can be uncoupled from DNA bridging . In addition , to accurately determine intermolecular DNA-bridging efficiencies in solution , we developed a sensitive quantitative bulk assay . Using these two assays , we unravel the assembly pathway of bridged DNA-H-NS-DNA complexes and the roles of mono- and divalent ions , helper proteins Hha and YdgT , and truncated H-NS derivatives . Finally , Molecular Dynamics ( MD ) simulations reveal that ions and interacting proteins directly alter H-NS structure from a ‘closed’ bridging incapable to an ‘open’ bridging capable conformation , thus providing a molecular understanding of the modulation of H-NS function .
In order to dissect the role of divalent ions in the formation of bridged and stiffened complexes , we applied a novel , sensitive , and quantitative DNA-bridging assay and carried out TPM experiments ( providing a quantitative and selective readout of DNA stiffening ) . The DNA-bridging assay relies on immobilization of bait DNA on magnetic microparticles and the capture and detection of 32P labeled prey DNA if DNA-DNA bridge formation occurs ( see Figure 1—figure supplement 4b for a schematic depiction of the assay ) . 80% of initial prey DNA is recovered at high H-NS concentrations ( see Figure 1a ) . In the absence of either the H-NS protein or bait DNA , no prey DNA is recovered under our experimental conditions . Next , we used this assay to quantify the DNA-bridging efficiency of H-NS as a function of the amount of Mg2+ ions ( see Figure 1b ) , reproducing the qualitative results of Liu et al . ( 2010 ) and providing independent confirmation of the previously observed effects . The concentration range from 0 to 10 mM Mg2+ is considered to be physiologically relevant ( Hurwitz and Rosano , 1967 ) . Importantly , the transition from no bridging to complete bridging is abrupt between 4–6 mM Mg2+ , indicating that changes in Mg2+ concentration might drive a binary switch . Earlier studies have shown that H-NS binding along a single DNA molecule results in DNA stiffening ( Amit et al . , 2003; Liu et al . , 2010 ) . However , due to the co-occurrence of DNA bridging , previous studies were incapable of measuring DNA stiffening in the presence of Mg2+ . Here , we used TPM to investigate DNA stiffening as a function of Mg2+ concentration . In TPM , the Root Mean Square displacement ( RMS ) of bead movement is a direct reflection of tether stiffness and length ( Figure 1—figure supplement 4a ) . DNA-binding proteins can affect both , but previous studies have shown that the DNA contour length is not affected by binding of H-NS ( Dame et al . , 2006; Dame et al . , 2001 ) . Thus , an increase in stiffness due to H-NS binding translates into a higher RMS value of a DNA tether ( Figure 1d ) . Here , we measured the effects of H-NS on DNA stiffness in the absence and presence of 10 mM Mg2+ and confirmed that H-NS stiffens DNA ( Amit et al . , 2003; Liu et al . , 2010 ) ; importantly , our experiments reveal that Mg2+ does not affect the stiffness of the fully formed H-NS-DNA complexes at saturation , as in both conditions the RMS is the same ( Figure 1d ) . Analysis of the binding characteristics using the McGhee-von Hippel equation revealed that the association constant of H-NS is somewhat reduced in the presence of Mg2+ , while cooperativity increases under these conditions ( Figure 3—figure supplement 1a , d ) . The reduction in DNA-binding affinity of H-NS may be attributed to shielding of the negatively charged phosphate backbone by Mg2+ . The cooperative binding of H-NS and DNA stiffening observed by TPM suggest that H-NS multimerizes along DNA , likely via the recently defined dimer-dimer interaction domain ( Arold et al . , 2010 ) . Multimerization along DNA has been previously suggested ( Esposito et al . , 2002; Williams et al . , 1996 ) but has never been conclusively demonstrated . To test this hypothesis , we generated a mutant , H-NSY61DM64D , predicted to have disrupted dimer-dimer interaction based on the H-NS1-83 crystal structure ( Arold et al . , 2010 ) . Size exclusion chromatography showed that this H-NS mutant indeed exists solely as a dimer in solution independent of protein concentration ( Figure 1—figure supplement 1b ) , whereas wild-type H-NS forms large multimeric structures ( Figure 1—figure supplements 1a and Arold et al . , 2010; Leonard et al . , 2009 ) . The multimerization behavior of both proteins was unaffected by the presence of Mg2+ . Electrophoretic Mobility Shift Assay confirmed that the DNA binding of the H-NS mutant is intact ( Figure 1—figure supplement 2 ) . TPM experiments reveal that H-NSY61DM64D binding does not lead to the formation of stiff H-NS-DNA filaments ( Figure 1e ) . The RMS is reduced compared to that of bare DNA , indicating not only that dimer-dimer interactions are disrupted , but also that individual H-NS dimers mildly distort DNA . DNA-bridging experiments reveal that H-NSY61DM64D is also incapable of forming DNA-H-NS-DNA complexes ( Figure 1—figure supplement 3 ) . This indicates that individual H-NSY61DM64D dimers do not form stable bridges and that dimer-mediated bridging , involving H-NS-dimer binding cooperativity due to high local DNA concentration adjacent to existing bridges ( Dame et al . , 2006; Dame et al . , 2000 ) , is insufficient to explain the formation of bridged DNA-H-NS-DNA complexes . The nature of the effect of Mg2+ on DNA-bridging efficiency is not understood . An increase in affinity would be expected if Mg2+ would only facilitate interactions between the DNA phosphate backbone and negatively charged residues on H-NS , but this is not observed ( Figure 3—figure supplement 1 ) . As Mg2+ does not affect the multimeric state of H-NS in solution ( Figure 1—figure supplement 1 ) , a model involving an effect on H-NS multimerization can be excluded . A structural effect of Mg2+ on individual units within H-NS filaments could explain the observed effects of Mg2+ on the bridging efficiency of H-NS . To investigate the role of Mg2+ on individual H-NS dimers we carried out MD simulations of an H-NS dimer in both the absence and presence of Mg2+ , using our previously established model of a full-length H-NS dimer ( van der Valk et al . , 2014 ) . Visual inspection of the H-NS dimer simulations at 50 mM KCl reveals that H-NS changes from an ‘open’ extended conformation into more compact ‘closed’ shapes ( see Figure 2a for snapshots from these simulations , Figure 2—figure supplement 1 for examples of the ‘closed’ conformation , or Figure 2—video 1 for a movie of one such simulation ) . The three domains in H-NS interact , and these inter-domain interactions are facilitated by partial unfolding and buckling of the long central α helix ( helix α3 ) connecting the dimerization and dimer-dimer interaction domains . The average distance between the donor and acceptor of all helical hydrogen bonds ( O-H distance ) in helix α3 indicates that the buckle forms in region Glu42-Ala49 ( Figure 2b ) . By analyzing the O-H distance between residues Ser45 and Ala49 in time , key residues at the site of buckle formation ( see Figure 2—figure supplement 3 ) , we found that buckles can be reversible and irreversible , within the simulation time scale of 50 ns . Reversible buckles , caused by thermal fluctuations , typically last a few nanoseconds and occur several times during a single simulation run ( see green line in Figure 2—figure supplement 3 for an example ) . Irreversible buckles , stabilized by inter-domain interactions , do not return to a helical conformation during our simulations ( see red line in Figure 2—figure supplement 3 for an example ) . To characterize the interactions that occur during the simulations , we generated contact maps that show the probability of finding interactions between residues , with a contact defined as the minimum distance between two residues being 0 . 6 nm or less , see Materials and methods for details of this analysis ( Figure 2—figure supplement 2a ) . These maps reveal that the DNA-binding domain interacts with other parts of the protein complex . In absence of Mg2+ , the DNA-binding domain interacts with the dimerization domain , rendering the DNA-binding QGR motif ( residues 112–114 ) ( Gordon et al . , 2011 ) of one DNA-binding domain inaccessible ( see the snapshots in Figure 2a ) . Although the simulations were performed in absence of DNA , these observations suggest that DNA bridging is not possible in such a conformation , as the H-NS dimer can bind DNA only through its remaining/second DNA-binding domain . In this ‘closed’ conformation , interactions occur between the IRT residues at position 10–12 and the AMDEQGK residues at position 122–128 . These interactions are hydrophilic in nature , supplemented by a salt bridge between R11 and D124 or E125 . In the presence of Mg2+ interactions between the DNA-binding domain and the dimerization domain no longer occur ( see the snapshots in Figure 2a or the movie in Figure 2—video 2 ) , The absence of such interactions is further illustrated by the contact map in Figure 2—figure supplement 2b and in higher detail in Figure 2—figure supplement 5 . The likelihood of finding Mg2+ interacting with ( i . e . being within 0 . 6 nm of ) H-NS residues , indicated by PMg2+ , revealed that Mg2+ has a preference for glutamate residues in region 22–35 ( Figure 2c , Figure 2—figure supplement 8 ) , where the ions shield this region from interacting with the DNA-binding domains . The Mg2+ ions transiently interact with the glutamate residues , with residence times in the order of a few ns ( as seen in Figure 2—video 2 ) . Furthermore , Mg2+ ions interact with region 98–105 ( sequence DENGE ) , right next to the DNA-binding QGR motif ( Figure 2c ) . The presence of Mg2+ stabilizes the ‘open’ conformation of H-NS , ensuring that DNA bridging can occur . Furthermore , we noted that Mg2+ is also located close to the buckle and may directly stabilize helix α3 through interactions with Glu42 , Glu43 , Glu44 , and Ser45 ( Figure 2—figure supplement 7 ) , resulting in an ‘open’ , bridging capable , H-NS conformation ( see Figure 2—videos 1 and 2 ) . These data suggest that Mg2+ modulates H-NS by shielding interactions between the DNA-binding domain and dimerization domain , and by influencing the conformation of helix α3 . Based on these observations , we designed an H-NS mutant predicted to bridge DNA independent of Mg2+ . We therefore generated a mutant in which several of the amino acids involved in buckle formation ( E43 , E44 , S45 ) were substituted with alanines . In our DNA-bridging assay , H-NSE43A , E44A , S45A retains its ability to bridge DNA , but indeed achieves high DNA recovery ( ±50% ) in the absence of Mg2+ ( Figure 2—figure supplement 9a ) . Low concentrations of Mg2+ are sufficient to reach saturated DNA bridging ( up to 80% DNA recovery ) ( Figure 2—figure supplement 9a ) . This observation independently confirms our model that the stretch of glutamates at the buckle is responsible for Mg2+ sensing and Mg2+-dependent bridging by H-NS . Additionally , we note that the DNA-binding affinity of H-NE43A , E44A , S45A is not significantly altered and that its DNA binding cooperativity is similar to that of wildtype H-NS in the absence of Mg2+ ( Figure 2—figure supplement 9b , Figure 3—figure supplement 1d ) . Yet , in the presence of Mg2+ , wild-type H-NS exhibits a far more cooperative DNA binding ( Figure 3—figure supplement 1d ) . This suggests that the transition between the ‘open’ and ‘closed’ conformation of H-NS promotes lateral filament formation by H-NS . Although it has long been known that the expression of some H-NS controlled genes ( such as the proU operon ) is modulated by the osmolarity of the medium ( Cairney et al . , 1985 ) , the underlying mechanism remains undetermined . Previous studies have revealed that the H-NS DNA stiffening mode is mildly sensitive to the KCl concentration ( Amit et al . , 2003; Liu et al . , 2010 ) . Using TPM , we were able to confirm these observations . The reduction in DNA stiffening is gradual ( Dame and Wuite , 2003 ) and modest ( Figure 1f ) . It is thus questionable whether the multimerization of H-NS along DNA alone is sufficient to explain its role in repression of transcription ( and modulation thereof ) . Could the modulation of gene repression be due to ionic effects on DNA-bridging efficiency ? Using our DNA-bridging assay , we observed complete abolishment of H-NS DNA bridging by KCl at concentrations exceeding 120 mM ( Figure 1c ) , a binary response , similar to what we observed for the Mg2+ titration . This in vitro observation mirrors the in vivo response of the ProU operon , at which KCl concentrations exceeding 100 mM are required to alleviate H-NS-mediated repression ( Cairney et al . , 1985 ) . Control experiments using K-glutamate ( Figure 1—figure supplement 5 ) confirm that K+ , and not the counter-ion , is responsible for the observed effects , even though the counter ion may affect the DNA-binding affinity of the protein ( Figure 1—figure supplements 5c and Leirmo et al . , 1987 ) This strong and abrupt effect on DNA bridging , while leaving DNA stiffening essentially unaffected , might indicate that H-NS reverts to the ‘closed’ conformation by the addition of K+ . To investigate this effect at a structural level we performed MD simulations at high KCl concentrations . The presence of 130 mM KCl alters interactions between the various domains in H-NS ( see contact maps in Figure 2—figure supplement 2c and d ) . In addition to interactions between the DNA-binding domain and the dimerization domain , the two DNA-binding domains in the dimer interact with each other . Moreover , the DNA-binding domains interact with helix α3 . In particular regions 98–105 ( KYSYVDENGE ) and 123–129 ( EQGKS ) are involved in these interactions . The average distance between the donors and acceptors in the hydrogen bonds within helix α3 ( Figure 2b ) indicates that that buckles in helix α3 also occur at a high KCl concentration , at the same location as determined by low-salt conditions . These observations indicate that the ‘closed’ state can have multiple forms ( see Figure 2—figure supplement 1 for snapshots ) , but that all these conformations block the DNA-binding motif QGR from interacting with DNA . The presence of Mg2+ does not significantly alter the occurrence of buckles ( Figure 2b ) . Instead , Mg2+ is capable of deterring interactions between the DNA-binding domain and the dimerization domain by shielding residues in the dimerization domain involved in these interactions ( Figure 2c , Figure 2—figure supplement 8 ) . However , the probability of interactions occurring between the DNA-binding domain and other parts of the protein is reduced significantly in the presence of Mg2+ ( Figure 2—figure supplement 2 and Figure 2—figure supplement 5 ) . High K+ concentrations therefore inhibit H-NS bridging by promoting the ‘closed’ conformation of H-NS even in the presence of Mg2+ and elucidates the modulatory and regulatory effects of KCl and osmolarity . In addition to environmental factors such as osmolarity , H-NS activity is affected by interactions with other proteins in vivo . Members of the Hha/YmoA protein family , such as Hha and YdgT , are known to cooperate with H-NS in repression of genes ( Baños et al . , 2008 ) , while other proteins such as H-NST are capable of inhibiting H-NS function ( Liu et al . , 2010 ) , likely by hampering H-NS multimerization . To systematically investigate the latter mechanism , we designed and synthesized truncated H-NS derivatives , targeting the H-NS dimerization domain ( H-NS1-58 ) or dimer-dimer interaction domain ( H-NS56-83 ) . Interfering with H-NS dimerization , through the addition of H-NS1-58 in DNA-bridging experiments , we observed a reduction in DNA recovery from 75% to 20% at ratios higher than 1: 3 H-NS1-58/ H-NS ( Figure 3a ) . Similarly , targeting the dimer-dimer interaction domain ( through H-NS56-82 ) resulted in complete abolishment of DNA bridging ( Figure 3a ) . Next , we investigated the effects of H-NS derivatives on H-NS DNA stiffening using TPM . Only at very high H-NS derivative concentrations ( 30-fold excess ) reduction of DNA stiffening was observed ( Figure 3b ) . These experiments reveal that both H-NS dimerization and dimer-dimer interactions can be effectively targeted for inhibition of H-NS activity and that the respective domains are crucial to the formation of bridged filaments . This suggests that natural H-NS inhibitors such as H-NST operate by disrupting DNA bridging and provides clues for rational design of artificial peptide inhibitors of H-NS . Gene regulation by H-NS often occurs in conjunction with other proteins; these co-regulators are known to interact with H-NS at specific loci along the genome . Two such proteins , Hha , and YdgT , are members of the Hha/YmoA ( Madrid et al . , 2007 ) family of proteins . In order to understand the modulation of H-NS function by these proteins we investigated their influence on the H-NS DNA binding modes . We observed that Hha , when added at equimolar concentrations , enhances DNA bridging by H-NS at low Mg2+ concentrations ( Figure 3c ) . A similar enhancement of H-NS-mediated DNA bridging was observed with the Hha paralogue , YdgT . While Hha and YdgT promote DNA bridging to a similar extent , YdgT promotes DNA bridging at significantly lower concentrations , likely due to a higher affinity for H-NS . At higher concentrations of YdgT the effect on H-NS-mediated bridging closely resembles the bridging profile obtained for H-NSE43A , E44A , S45A ( Figure 3—figure supplement 2 ) . To determine whether enhanced DNA bridging is due to structural changes in H-NS-DNA filaments , we investigated the effects of Hha and YdgT on DNA stiffening ( Figure 3d ) . TPM experiments show a mild increase in H-NS mediated DNA stiffening in the presence of Hha . We observe a negative offset in RMS in the presence of YdgT , indicating a more compact conformation . Specifically , this is evident in TPM experiments containing H-NS , YdgT , and Mg2+ , where in the presence of Mg2+ and YdgT , H-NS causes ‘DNA collapse’ in TPM ( data not shown ) , similar to earlier observations for H-NS in the presence of Mg2+ without ( Liu et al . , 2010; Wang et al . , 2014 ) or with Hha added ( Wang et al . , 2014 ) . This ‘DNA collapse’ is attributed to H-NS-mediated DNA bridge formation . One possible explanation for the effects of Hha and YdgT , is that they effectively increase the DNA-binding affinity of H-NS ( Ali et al . , 2013 ) . To investigate whether Hha affects H-NS conformation , we performed MD simulations , incorporating structural information from the recently resolved H-NS1-43-Hha co-crystal structure ( Ali et al . , 2013 ) . Our MD simulations reveal that Hha does not prevent buckles in helix α3 ( see Figure 2b ) . Instead Hha alters the interactions between the dimerization domain and the DNA-binding domain ( Figure 2—figure supplement 2 ) by blocking access to parts of dimerization domain . This hypothesis is further supported by interactions between Hha and other parts of H-NS , including the DNA-binding domain and helix α3 ( Figure 2—figure supplement 6 ) . In the presence of Mg2+ and Hha , the contacts between the DNA-binding domain and dimerization domain are reduced even further . This shows that Hha modulates H-NS function by stabilizing the ‘open’ -bridging capable- conformation of H-NS .
It has been known for many years that H-NS binding induces gene silencing . H-NS activity in vivo is modulated by physico-chemical factors ( osmolarity , pH , temperature ) and interaction partners . These findings support the hypothesis that H-NS plays a role in environmental adaptation . However , mechanistically it is unclear how functional modulation of H-NS by such factors is achieved . Based on our findings , we conclude that H-NS is incapable of bridging or stiffening DNA as dimers . H-NS dimers bind DNA in cis ( Dame et al . , 2006; Dame et al . , 2000 ) and associate side-by-side along DNA , likely via the recently identified dimer-dimer interaction domain ( Arold et al . , 2010 ) , resulting in DNA stiffening . This process is cooperative as H-NS dimers interact with neighbors , as well as with DNA . Our studies reveal that H-NS-DNA filaments are structurally very similar , independent of the presence of Mg2+ ( Figure 1d ) . But functionally , these H-NS-DNA filaments are distinct . H-NS can be ‘activated’ by Mg2+ , which promotes a conformational change , rendering both DNA-binding domains of H-NS dimers accessible for DNA bridging . In our model , the assembly of bridged complexes proceeds in distinct steps: ( 1 ) nucleation ( Lang et al . , 2007 ) ( binding of an H-NS dimer at a high affinity site ) , ( 2 ) lateral filament growth by H-NS dimer-dimer interactions ( leading to DNA stiffening ) and ( 3 ) bridging of the assembled filament to bare DNA provided in trans ( Figure 4 ) . Each step can potentially be modulated by osmolarity and protein interaction partners . Here , we show that these factors most effectively target DNA bridging . What are the implications of our observations ? Our observations add to the large body of evidence showing that regulation of transcription via H-NS is complex , and that it does not proceed via a single , simple , well-defined mechanism . The most straightforward form of repression by H-NS is via occlusion of RNA polymerase from the promoter ( Lim et al . , 2012; Prosseda et al . , 2004; Göransson et al . , 1990 ) . Whether this mechanism of repression involves lateral filament formation or bridging is unclear . It is expected that both types of complexes assembled at a promoter site can in principle occlude RNA polymerase . A second mechanism of repression is to trap RNA polymerase at the promoter , preventing promoter escape ( Schröder and Wagner , 2000; Shin et al . , 2005 ) . RNA polymerase trapping likely involves DNA bridging of promoter upstream and downstream elements ( Dame et al . , 2002; Shin et al . , 2005 ) . A third mechanism of repression by H-NS is to interfere with RNA polymerase progression during active transcription by intragenic binding ( Dole et al . , 2004 ) . In this model , both modes could interfere with transcription . However , it was suggested recently that only bridged filaments are capable of interfering with transcription ( Kotlajich et al . , 2015 ) , with lateral filaments likely being disassembled as RNA polymerase encounters them . Generally , the type of complex formed by H-NS is expected to depend on the type and number of ions , combined with local DNA conformation ( dependent on DNA sequence or DNA topology ) , and the presence of modulating proteins . The interplay between these factors will determine the strength of the complex , and degree of repression . Thus , different H-NS repressed genes are expected to be subject to different types of modulation , providing a key to a coordinated response in gene expression to altered conditions and selectivity for the interplay with specific co-regulators at specific target regions .
H-NS/H-NSY61DM64D/H-NSE43A , E44A , S45A . BL21 ( DE3 ) ( RRID: WB-STRAIN: HT115 ( DE3 ) ) Δhns::kan/frt pLysE ( NT201 , our lab ) cells transformed with plasmids expressing H-NS/H-NS mutants were grown to an OD600 of 0 . 4 , and induced for 2 hr using IPTG ( 500 μM ) . For the H-NE43A , E44A , S45A protein , the cells were co-transformed with pRD252 , coding for LacI to help suppress leaky expression of H-NE43A , E44A , S45A . The cells were pelleted and lysed by sonication in 100 mM NH4Cl , 20 mM Tris pH 7 . 2 , 10% glycerol , 8 mM β-mercaptoethanol , 3 mM benzamidine ) . The soluble fraction was loaded onto a P11 column and eluted using a 100 mM-1 M NH4Cl gradient , the protein eluted at 280 mM NH4Cl . The peak fractions were dialysed to buffer B ( identical to buffer A , but containing 130 mM NaCl instead of NH4Cl ) by overnight dialysis . The dialysate was loaded onto a heparin column ( GE Healthcare ) and eluted using a 130 mM-1 M NaCl gradient , the protein eluted at 350 mM NaCl . The pooled peak fractions were dialysed to buffer B and concentrated using a 1 ml Resource-Q column ( GE Healthcare ) . The purity of the protein was verified on an SDS-PAGE gel . The protein concentration was determined using a Bicinchoninic Acid assay ( Pierce BCA protein assay kit , Thermo Scientific ) . Size exclusion chromatography was done using a Superose-12 column with a flow of 0 . 3 ml/min , pre-equilibrated with 10 mM Tris-HCl , 50 mM KCl , 5% glycerol containing or lacking 10 mM MgCl2 . The absorbance of the eluting fractions was measured at 215 nm . These experiments were performed in triplicate . Streptavidin-coated paramagnetic Dynabeads M280 ( Invitrogen ) were washed once with 100 μL of 1xPBS and twice with Coupling Buffer ( CB: 20 mM Tris-HCl pH 8 . 0 , 2 mM EDTA , 2 M NaCl , 2 mg/mL BSA ( ac ) , 0 . 04% Tween20 ) according to manufacturer instructions . After washing , the beads were resuspended in 200 µL CB containing 100 nM biotinylated DNA . Next , the bead suspensions were incubated for 30 min on a rotary shaker ( 1000 rpm ) at 25°C . After incubation , the beads were washed twice with Incubation buffer ( IB: 10 mM Tris-HCl pH 8 . 0 , 50 mM KCl , 10* mM MgCl2 , 5% v/v Glycerol , 1 mM DTT and 1 mM Spermidine ) before resuspension in IB and addition of ±8000 cpm of radioactively labeled 32P 685 bp DNA . Radioactive DNA was supplemented with unlabeled 685 bp DNA to maintain a constant ( 20 nM ) DNA concentration . The DNA-bridging protein H-NS ( concentrations indicated in the text ) , and if applicable Hha or YdgT were added and the mixture was incubated for 30 min on a shaker ( 1000 rpm ) at 25°C . To remove unbridged prey DNA , the beads were washed with IB , before resuspension in 12 μL stop buffer ( 10 mM Tris pH 8 . 0 , 1 mM EDTA , 200 mM NaCl , 0 . 2% SDS ) . All samples were quantified through liquid scintillation counting over 10 min . All values recovered from the DNA-bridging assay were corrected for background signal ( using a sample lacking H-NS ) , and normalized to a reference sample containing the amount of labeled 32P 685 bp DNA used in the assay . The samples were then run on a 5% 0 . 5x TBE gel to ensure DNA integrity . DNA bridging was calculated based on a reference sample containing 2 µL of prey DNA . All DNA-bridging experiments were performed in triplicate . Unless indicated otherwise all DNA-bridging experiments were performed in the presence of 10 mM of MgCl2 and 3 , 3 µM of H-NS ( 10% more H-NS than is required for saturation - see Figure 1A ) . Each experiment contains 50 mM KCl , to which additional KCl or K-glutamate are added depending on the experimental condition tested . Tethered particle motion experiments were performed as reported previously ( Driessen et al . , 2014; van der Valk et al . , 2017 ) . Flow cells were prepared as described with minor modifications ( Driessen et al . , 2014; van der Valk et al . , 2017 ) . Here , before flowing in protein diluted in the experimental buffer ( 10 mM Tris-HCl pH 8 . 0 , 50 mM KCl , 10 mM MgCl2/EDTA , 5% v/v Glycerol , 1 mM DTT ) the flow cell was washed using 4 flow cell volumes with the experimental buffer . Next , the flow cell was incubated for 10 min with protein solution before sealing the flow cell . The flow cell was maintained at a constant temperature of 25°C . Measurements were started 10 min after the introduction of protein solution . TPM experiments were done at least in duplicate . The data were analyzed as previously described ( Driessen et al . , 2014; van der Valk et al . , 2017 ) . The RMS was first converted to persistence length ( Lp ) using Equation 1 ( Göransson et al . , 1990 ) : ( 1 ) RMS=233- 156 ( 1+0 . 08Lp ) 0 . 45 Lp values were then used to calculate the fractional coverage ( Equation 2 ) ( Göransson et al . , 1990 ) : ( 2 ) nϑ=1Lp , measured-1Lp , naked1Lp , saturated-1Lp , naked The fractional coverage ( d ) was fit using the McGhee-von Hippel model for cooperative lattice binding ( Equation 3-5 ) ( McGhee and von Hippel , 1974 ) : ( 3 ) ϑc=K∙1-d∙2ω+11-d+ϑ-R2ω-11-dn-1∙1-n+1ϑ+R2 ( 1-d ) 2 where ( 4 ) R= ( 1-n+1ϑ ) 2+4ωϑ ( 1-d ) andd=nϑ Here , the association constant ( K ) is described as a function of the protein concentration ( c ) and a cooperativity parameter ( ω ) . To this end , weighted orthogonal distance regression ( ODR ) was performed to estimate the parameters of the nonlinear implicit equation describing the cooperative ligand binding . The binding site size ( n ) of H-NS was fixed to a value of 30 bp during regression , which corresponds to values determined previously ( Dame et al . , 2006; Amit et al . , 2003 ) . The association constant ( K ) and the cooperativity parameter ( ω ) were assumed to be positive real numbers . A custom fitting routine was implemented in Fortran and makes use of the ODRPACK library ( Boggs et al . , 1992 ) . The starting conformation of the full-length H-NS dimer was constructed as described previously ( van der Valk et al . , 2014 ) . The system was placed in a periodic dodecahedron box with a distance of at least 0 . 8 nm between the box edge and the most extended atom of the protein dimer , followed by the addition of water and ions . With this system we performed Molecular Dynamics ( MD ) simulations of full length H-NS at different concentrations of KCl , and MgCl2 and with the addition of Hha , summing up to a total of six different systems . Hha was added by aligning the crystal structure containing the H-NS – Hha complex ( PDB code 4ICG [Ali et al . , 2013] ) with the full-length structural model and copying the Hha molecules . System size ranged from 513457 atoms for the Hha systems to around 1 , 135 , 000 atoms for the other four systems . Interactions between atoms were described by the AMBER99-SB-ILDN force field ( Lindorff-Larsen et al . , 2010 ) , in combination with the TIP3P water model ( Jorgensen et al . , 1983 ) . Long-range electrostatic interactions were treated via the Particle Mesh Ewald method ( Darden et al . , 1993; Essmann et al . , 1995 ) with a short-range electrostatic cutoff distance at 1 . 1 nm . Van der Waals interactions were cut off at 1 . 1 nm . Preparation of the systems consisted of energy minimization equilibration of the solvent . Energy minimization was performed by the conjugate gradient method . After energy minimization , the positions of water molecules and ions were equilibrated by a 1 ns molecular dynamics run at a temperature of 298 K and a pressure of 1 bar in which the heavy atoms in the protein were position-restrained with a force constant in each direction of 1000 kJ/mol nm . After preparation , we performed 16 50 ns runs for each system , varying initial conditions by assigning new random starting velocities drawn from the Maxwell-Boltzmann distribution at 298 K . All simulations were performed with GROMACS v . 4 . 6 . 3 ( Pronk et al . , 2013 ) at the Dutch National Supercomputer with the leap-frog integration scheme and a time step of 2 fs , using LINCS ( Hess et al . , 1997 ) to constrain all bonds . All simulations were performed in the isothermal-isobaric ensemble at a pressure of 1 bar , using the v-rescale thermostat ( Bussi et al . , 2007 ) and the Parrinello-Rahman barostat ( Parrinello and Rahman , 1981 ) . Frames were stored every 10 ps . The first 10 ns of each simulation are excluded from analysis , unless stated otherwise . Analysis focused on determining contacts between domains , between H-NS and Hha , between H-NS and ions , and helical hydrogen bonds . Contact maps of interactions between residues in the H-NS dimer system were obtained by first calculating the minimum distance between each residue pair in the system . A residue pair is counted to be in contact if they are at a minimum distance of 0 . 6 nm or less . The probability of a contact is then calculated as the average over all 16 simulations ( excluding the first 10 ns ) and displayed as a contact probability matrix . We used a modified version of the g_mdmat tool in GROMACS ( Pronk et al . , 2013 ) in combination with Perl scripts to generate contact maps . To determine the location of ions with respect to the H-NS system , we calculated the minimum distance between each residue in the H-NS dimer and the ions and counted a contact if the distance between an H-NS residue and an ion is 0 . 6 nm or less . These contact probabilities ( PMg2+ , PK+ and PCl- ) are averaged over all 16 simulations and the two monomers . A similar procedure was performed to determine the probability of contacts between H-NS and Hha , PHha , and between H-NS domains , with P1-40 indicating the probability of finding the DNA-binding domain ( residues 96–137 ) close to the dimerization domain ( residues 1–40 ) . P96-137 indicates the probability of finding the dimerization domain close to the DNA-binding domain . To determine the location of the buckle in helix α3 , we calculated the helical hydrogen bond distances dO-N for residues 22–67 in each monomer between the backbone carbonyl oxygen O of residue i and the backbone amide nitrogen N of residue i + 4 . A hydrogen bond is counted to be in contact if they are at a minimum distance of 0 . 35 nm or less . These probabilities are averaged over all 16 simulations ( excluding the first 10 ns ) and the two monomers . Snapshots and movies were generated with PyMOL .
Bacteria frequently need to adapt to altered environmental conditions . Adaptation requires changes in gene expression , often mediated by global regulators of transcription . The nucleoid-associated protein H-NS is a key global regulator in Gram-negative bacteria and is believed to be a crucial player in bacterial chromatin organization via its DNA-bridging activity . H-NS activity in vivo is modulated by physico-chemical factors ( osmolarity , pH , temperature ) and interaction partners . Mechanistically , it is unclear how functional modulation of H-NS by such factors is achieved . Here , we show that a diverse spectrum of H-NS modulators alter the DNA-bridging activity of H-NS . Changes in monovalent and divalent ion concentrations drive an abrupt switch between a bridging and non-bridging DNA-binding mode . Similarly , synergistic and antagonistic co-regulators modulate the DNA-bridging efficiency . Structural studies suggest a conserved mechanism: H-NS switches between a ‘closed’ and an ‘open’ , bridging competent , conformation driven by environmental cues and interaction partners .
The genetic information every cell needs to work properly is encoded in molecules of DNA that are much longer than the cell itself . A key challenge in biology is to understand how DNA is organized to fit inside each cell , whilst still providing access to the information that it contains . Since the way DNA is organized can influence which genes are active , rearranging DNA plays an important role in controlling how cells behave . In Escherichia coli and many other bacteria , a protein called H-NS contributes to DNA reorganization by forming or rupturing loops in the DNA in response to changes in temperature , the levels of salt and other aspects of the cell’s surroundings . In controlling loop formation , it dictates whether specific genes are switched on or off . However , it remains unclear how H-NS detects the environmental changes . To address this question , van der Valk et al . used biochemical techniques to study the activity of H-NS from E . coli under different environmental conditions . The experiments show that changes in the environment cause structural changes to H-NS , altering its ability to form DNA loops . A previously unnoticed region of the protein acts as a switch to control these structural changes , and ultimately affects which genes are active in the cell . These findings shed new light on how bacteria organize their DNA and the strategies they have developed to adapt to different environments . The new protein region identified in H-NS may also be present in similar proteins found in other organisms . In the future , this knowledge may ultimately help to develop new antibiotic drugs that target H-NS proteins in bacteria .