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Insights into the Structure of Sulfolobus Nucleoid using Engineered Sac7d Dimers with Defined Orientation Gokul Turaga, Stephen P. Edmondson, Kelley Smith, and John William Shriver Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00810 • Publication Date (Web): 21 Oct 2016 Downloaded from http://pubs.acs.org on October 25, 2016

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Insights into the Structure of Sulfolobus Nucleoid using Engineered Sac7d Dimers with Defined Orientation

Gokul Turaga, Stephen P. Edmondson, Kelley Smith and John W. Shriver* Department of Chemistry University of Alabama in Huntsville, Huntsville, AL 35899

Running Title: Archaeal Nucleoid Structure Keywords: Archaea, Sulfolobus, Nucleoid, Chromatin, DNA binding

*Address for correspondence: John W. Shriver Department of Chemistry Materials Science Building John Wright Drive University of Alabama in Huntsville Huntsville, AL 35899

Phone: 256-824-2477; Fax: 256-824-6349 email: [email protected]

† This work was supported by grant GM49686 from the National Institutes of Health to JWS and SPE.

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Abbreviations:

SAXS: Small angle x-ray scattering; HSQC: heteronuclear single quantum coherence; K9C*: K9C lysine-9 to cysteine-9 Sac7d mutant protein with a His-tag on the C-terminus.


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Abstract The structure of Archaeal chromatin or nucleoid is believed to have characteristics similar to that found in both eukaryotes and bacteria. Recent comparative studies have suggested that DNA compaction in Archaea requires a bridging protein (e.g. Alba) along with either a wrapping protein (e.g. a histone) or a bending protein such as Sac7d. While x-ray crystal structures demonstrate that Sac7d binds as a monomer to create a significant kink in duplex DNA, the structure of a multiprotein-DNA complex has not been established. Using crosslinked dimers of Sac7d with defined orientation, we present evidence which indicates that Sac7d is able to largely coat duplex DNA in vivo by binding in alternating head-to-head and tail-to-tail orientations. Although each Sac7d monomer promotes a significant kink of nearly 70°, coated DNA is expected to be largely extended due to compensation of repetitive kinks with helical symmetry.


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Chromatin proteins function to compact genomic DNA in all domains of life (1-3). They also regulate defined regions of the genome for replication and transcription. While much is known about the structure and function of chromatin in eukaryotes, significantly less is known about bacterial chromatin (nucleoid) and the role of a number of proteins in defining a dynamic but yet compact structure (4-7). Archaea are especially intriguing since they display similarities to both eukaryotes and bacteria (4). For example, some archaea compact genomic DNA using true histones, while others lack proteins with any resemblance to histones and employ a wide variety of apparently unrelated proteins. It is thought that a better understanding of alternative solutions to the DNA packing problem may provide insight into the basic requirements and principles of DNA compaction and accessibility (8, 9).

Surprisingly, none of the many chromatin proteins used by Archaea are conserved across all species within the domain, and no single protein appears to be absolutely required.

The

reason for this may be provided by a recent characterization of chromatin proteins as “wrappers”, “bridgers”, and “benders”

(3)

. A comparison of the distributions of the three classes in Archaea

shows that a bridging protein, Alba, usually occurs along with either a wrapper or a bender (10-12). Benders such as Sul7 or Cren7 are proposed to function similarly to the bacterial protein HU and assist in packing loops created by a bridger. Notably, in this model Sul7 and Cren7 function as isolated monomers promoting local bends that presumably stabilize the loop

(3, 13)

. Supporting

evidence is provided by the x-ray crystal structure of Sul7-DNA complexes (14), as well as AFM and magnetic tweezers experiments

(15)

. The work presented here is designed to increase our

understanding of the packing of multiple Sul7 proteins on DNA in solution.


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Sul7 refers to a class of small (~7.6 kDa) monomeric, DNA binding proteins found in the hyperthermophile Sulfolobus

(16-18)

. Cren7 is structurally homologous to Sul7 and is thought to

function similarly (19). The best characterized Sul7 examples are Sac7d and Sso7d from S. acidocaldarius and S. solfataricus, respectively (17). These are some of the most highly expressed proteins in Sulfolobus, contributing about 5% of total cellular protein(20) with a cellular concentration of approximately 1 mM, or roughly 3,000,000 Sul7 molecules per cell. Fluorescence titrations indicate a DNA binding site size of 4 bp with a dissociation constant on the order of 10-5 M at physiological salt concentrations(21-23). Since the total protein concentration is approximately two orders of magnitude greater than the Kd, formation of a DNA complex is highly favored in vivo. With a genome of 3 x 106 bp, the number of binding sites is significantly less than the number of Sul7 molecules in the cell (essentially ¼). Given the high concentration of Sul7, the genome is expected to be largely coated with Sul7 in vivo, as claimed by Mai et al. (20), and isolated single copies of Sul7 on genomic DNA should be rare.

The goal of the work presented here is to determine how Sul7 proteins coat DNA. Crystal structures show that binding is associated with intercalation of two amino acid side chains (V26 and M29) to promote a sharp kink at a single base pair step of about 66°, one of the largest observed for any DNA-binding protein (14, 24, 25). Since the intercalating side chains lie on one side of the DNA interface, the binding orientation of the adjacent Sac7d molecules dictates the distance between successive intercalation sites and kinks in the DNA (Figure 1). The intercalating residues V26 and M29 occur at an edge of a three-stranded sheet in a region we refer to as the “head”. At the opposite end of the binding site is the “tail” defined by the two


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strands of an N-terminal β-ribbon. Because of the asymmetry in the DNA binding site, head-totail packing leads to a separation between kinks of 4 bp, but with head-to-head (and tail-to-tail) binding the separation alternates between 2 and 6 bp.

The distortion introduced by Sac7d may favor a non-random orientation for adjacent proteins resulting in a DNA-induced preferred binding orientation of Sac7d, i.e. indirect readout may define a preferred orientation of adjacent monomers even if there are no cooperative interactions between proteins (26-28). There are two published reports indicating preferential orientations of adjacent Sac7d molecules on DNA. SAXS data collected on duplex DNA saturated with Sac7d were fit with a model containing adjacent, head-to-tail monomers with a regular kink repeat of 4 base pairs (29). In contrast, the NMR solution structure of the homologous Sso7d-DNA complex showed two-fold symmetry with two monomers oriented head-to-head at the center of the 12-mer (30). So far all attempts to obtain a crystal structure of more than a single Sul7 bound to DNA have proven unsuccessful (31).

Although fluorescence titrations do not show evidence of cooperative binding, there is some evidence for indirect interactions between adjacent Sac7d monomers on DNA (22). Forward titrations (Sac7d into DNA) followed by circular dichroism of DNA demonstrates that with increasing protein binding density there is cooperative unwinding of the DNA (22). At low binding density, the protein binds but negligible unwinding is observed. Above a threshold dictated by the DNA sequence, adjacent Sac7d are close enough to act cooperatively to unwind the DNA. Cooperativity due to this effect is presumably not observed in fluorescence titrations


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because the latter are performed as reverse titrations, DNA into protein, with saturating protein at the beginning of the titration.

We present here experiments designed to investigate the existence of preferential binding orientation of adjacent Sac7d molecules. We use disulfide crosslinking to study binding preferences for engineered dimers in head-to-tail, head-to-head and tail-to-tail orientations. Disulfide crosslinking also enables us to investigate preferential orientations of adjacent Sac7d monomers in DNA complexes. Native Sac7d contains no cysteine, allowing the introduction of single cysteines at specific sites for chemical crosslinking of two monomers using either oxidation or bi-functional crosslinking reagents.


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MATERIALS AND METHODS Preparation of Sac7d and single cysteine mutants Sac7d was prepared and purified using methods described previously

(21)

. Single amino

acid substitutions (K9C, G10C, K28C, and G27C) were created in the Sac7d expression vector using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene / Agilent). Mutations were confirmed by DNA sequencing, and the proteins were expressed in E. coli BL21(DE3)pLysS using procedures described previously for Sac7d without change

(21)

.

Protein purity was

demonstrated by SDS gel electrophoresis using Tris-Tricine, 16% acrylamide, 1.5 mm precast gels (Jule). Protein concentration was measured using an extinction coefficient ε280 of 1.1 ml mg-1 cm

(21)

. Cysteine mutants were reduced with 10 mM dithiothreitol followed by separation

on a PD-10 desalting column (GE Healthcare) prior to oxidation or chemical crosslinking.

Dimer synthesis by oxidation Sac7d with single cysteine substitutions were dimerized by addition of diamide (0.1 mM) with essentially complete conversion in 1 hour (10 mM NaH2PO4, pH 7.2, 25 °C). Dimers were separated from monomer by cation exchange chromatography with HiTrap SP Sepharose (5 ml column,10 mM NaH2PO4, pH 7.0, 0 – 1 M NaCl gradient) on an Akta Purifier. Dimer purity was demonstrated by non-reducing PAGE.

Production of the head-to-tail Sac7d dimer A His6 tag was incorporated into the K9C mutant by inserting the gene for Sac7d-K9C into pET-28(b) to add the sequence NSSSVDKLAAALEHHHHHH to the C-terminus. This


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provided a His tag “handle” separated from the Sac7d K9C C-terminus by a 13 residue spacer arm. The expressed protein was found largely in inclusion bodies which were dissolved by adding 8 M urea to the cell lysis buffer. Purification on a HisTrap HP (GE Healthcare) with a 0.0 – 0.5 M imidazole gradient (50 mM NaH2PO4, 0.25 M NaCl, pH 7.5) produced the cysteine tail mutant with a His tag, which we refer to as K9C*.

Oxidation of an equimolar mixture of K9C* (tail cysteine) and K28C (head cysteine) resulted in three dimers: K28C-K28C (head-to-head, no His-tag), K28C-K9C* (head-to-tail, one His-tag) and K9C*-K9C* (tail-to-tail, two His-tags). The three species were readily separated on a HisTrap HP Ni-column (GE Healthcare) with a 0.0 to 0.5 M imidazole gradient.

Crosslinking of proteins in DNA complexes Equimolar head (K28C) and tail (K9C*) single cysteine mutant proteins were mixed to give a final concentration of 21 µM total protein and 15 µM (bp) DNA in 10 mM sodium phosphate (pH 7.2) at room temperature. The effect of three different DNA sequences on crosslinking were tested in these experiments: 12-mer DNA duplex with the sequence used for the 1bbx NMR solution structure, 100-mer duplex poly(dGdC), and 100-mer duplex poly(dIdC). The protein-DNA complex was incubated with 25 mM DTT in a total volume of 2.5 ml for 30 minutes at 25 °C. DTT was quickly removed by gel exclusion chromatography on PD-10 columns and the reduced complex was immediately treated with either diamide (as described above) or with pPDM (40 µM) for one hour. The identity of the crosslinked species was determined by gel electrophoresis under non-reducing conditions.


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Circular Dichroism. Circular dichroism spectra were collected on an Olis 1500 Spectrometer in 10 mM KH2PO4 (pH 7.2 at 200 C) as described (21). Protein spectra were collected with a concentration of 0.015 mg/ml in the 1 mm path length cells. The CD of DNA (8 mer and 100 mer) was monitored using 1cm cells by titrating protein (0.8 mM stock solutions) into DNA (6 µM, nucleotides). Secondary structure content was calculated using the dichroweb server employing SELCON3 (32, 33)

, CDSSTR (32, 34, 35) and CONTINLL (36, 37) algorithms.

Differential scanning calorimetry Differential scanning calorimetry (DSC) data were collected on a MicroCal extended range VP-DSC instrument as described previously (38). Protein samples were dialyzed overnight against the indicated buffer. The sample and reference cells were loaded with protein and dialysis buffer, respectively, approximately 30 psi pressure was applied to prevent cavitation during heating, and scans were conducted at a rate of 1 °C/min. Repetitive scans were performed on the same sample to demonstrate reversibility. Nonlinear least squares fitting of the data was performed using in-house software as previously described to obtain the Tm and change in enthalpy for monomer and dimer unfolding (38). .

1

H-15N HSQC nuclear magnetic resonance NMR data was collected on a Varian INOVA 800 MHz NMR spectrometer. 15N-labelled

protein was obtained by expression in M9 minimal media supplemented with

15

NH4Cl and

purified as described above for unlabeled protein. Samples were prepared to a final volume of 0.6 ml with 90% H2O and 10% D2O, and the pH was adjusted to 4.5 with HCl without correction


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for the deuterium isotope effect. 1H chemical shifts were referenced to internal DSS and

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N

shifts were referenced to liquid ammonia using relative frequencies (39). Assignments were made as previously described (40, 41).

Quantification of binding by fluorescence quenching Intrinsic protein (tryptophan) fluorescence intensity was measured on a Fluoromax-3 fluorimeter with excitation at 295 nm (5 nm slit) and emission at 355 nm (5 nm slit). Aliquots (e.g. 5 µl) of double stranded DNA (0.3 M in 10 mM KH2PO4, pH 7.2, 20 °C) were titrated into 3.0 ml protein samples (0.15 µM in 10 mM phosphate buffer) in a 4 ml quartz fluorescence cell (1 cm path) with continuous stirring and temperature control. Concentrations of nucleic acid and protein stock solutions were determined spectrophotometrically using the protein extinction coefficient reported above, and an extinction coefficient of poly[dGdC]•poly[dGdC] at 260 nm

(42).

8,400 L/cm.mole for

All experiments are performed at 25 °C using a water

circulating bath coupled to the cell holder. The fluorescence intensity decreased with increasing DNA, and reached a plateau at saturating DNA concentrations. No correction was needed for an inner filter effect. Photobleaching of the Sac7d was not observed during the titrations.

Binding parameters were obtained by fitting the dependence of the observed intrinsic tryptophan fluorescence quenching on the total protein and DNA concentrations after correction for dilution and light scattering. Titration data were fit by nonlinear regression to a binding model that incorporated the McGhee-von Hippel treatment of non-specific protein binding to an infinite lattice (22, 43).


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The observed fluorescence, Fobs, was converted to fluorescence quenching, Qobs, based on the initial fluorescence, Fi (i.e. that measured prior to adding DNA) (44):

Qobs = (Fi − Fobs) / Fi

(1)

A binding isotherm was obtained by plotting the observed quenching as a function of total DNA added. The curve increased from 0 to Qmax, the plateau observed at saturating DNA. A direct correlation between the observed quenching and the fraction of protein bound to DNA allowed the concentration of bound protein to be calculated from the fractional change in quenching:

Lb = (Qobs / Qmax ) * Lt

(2)

where Lt is the total protein concentration (22). The free protein concentration is given by

Q  Lf = Lt − Lb = Lt −  obs  Lt  Q max 

(3)

Non-specific, non-cooperative binding of protein to an infinite lattice leads to an apparent negative cooperativity due to exclusion of binding sites as proteins can bind with less than one binding site between them. The effect was described by the McGhee-von Hippel model (43) such that


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Lf =

ν

K(1− nν )(

1− nν ) n−1 1 − (n − 1)ν

(4)

where K is the binding affinity, n the binding site size (in basepairs), ν is the binding density given by

ν = Lb / Dt

(5)

and Dt is the total DNA concentration. Using this treatment, Lf (and therefore Qobs) was defined by n, K, and Qmax as a function of Lt, and Dt. Non-linear regression using Igor (Wavemetrics, Oregon) was used to obtain optimized values of n, K, and Qmax for each titration data set (Qobs values as a function of Lt and Dt).


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Results The NMR solution structure of free Sac7d (1sap) (45) and crystal structures of Sac7dDNA complexes (1azp) (14) indicate solvent exposed regions in the protein sequence where a single cysteine might be introduced to serve as a crosslinking site (Figure 1). One region is composed of the second and third residues in a tight turn (V26-G27-K28-M29) beginning and ending with the intercalating valine and methionine residues . We refer to this region of the DNA binding site as the “head”. At the opposite end of the protein, the N-terminal β-ribbon “tail” extending away from the globular body of the protein contains another tight turn (Y8-K9G10-E11) with the second and third residues well positioned for placement of a crosslinking cysteine. Model building indicates that the side chain of a cysteine placed at one of these four positions (viz. G27, K28, K9, and G10) would extend away from the protein to enable crosslinking and dimer formation.

Sac7d with single cysteine substitutions at the above four candidate positions was expressed in E. coli and purified using ion exchange chromatography. The mutant proteins consistently showed the presence of nearly equal amounts of monomer and dimer (detected by non-reducing PAGE), with the higher molecular weight dimer eluting from a cation exchange column at higher salt concentration. PAGE under reducing conditions (DTT) showed only monomeric Sac7d. Essentially complete conversion of monomer to dimer was promoted with 0.1 mM diamide

(46)

, followed by purification with cation exchange chromatography. Sac7d

dimers produced in this manner were stable for months after removing the oxidizing agent, with


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essentially no reversion to monomer under normal storage conditions (10 mM NaH2PO4, pH 7.0, 0.3 M NaCl) as detected by non-reducing PAGE.

Diamide oxidation of single Sac7d cysteine mutants was used to produce head-to-head (G27C-G27C and K28C-K28C) and tail-to-tail (K9C-K9C and G10C-G10C) dimers. Oxidation of an equimolar mixture of both head and tail mutant protein (e.g. K9C with G27C) gave a mixture of head-to-head, tail-to-tail, and head-to-tail dimers. We were unable to separate this mixture of dimers by cation exchange chromatography, making it difficult to obtain a pure preparation of head-to-tail dimer using this approach.

The introduction of a C-terminal His tag into Sac7d provided a convenient “handle” at the end of the C-terminal α-helix extending away from the DNA binding interface. Addition of the His tag to K9C (referred to as K9C*) allowed for purification of the K9C mutant from a mixture of cysteine mutants. Oxidation of reduced K9C* with equimolar, reduced G27C gave G27C-G27C head-to-head dimer (no histags), K9C*-G27C head-to-tail dimer (one histag), and K9C*-K9C* tail-to-tail dimer (two histags). Chromatography of the mixture on a Ni column permitted excellent separation of the three species: G27C-G27C with no His tags was not retained by the column and the head-to-tail dimer with a single His tag had a lower affinity than the double His tag mutant (Figure 2).

Circular dichroism spectra of the mutant proteins in monomer and dimer forms showed little difference relative to native Sac7d. Comparison of calculated secondary structures for the


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mutant dimers of Sac7d with those of recombinant Sac7d indicated that cysteine mutations did not significantly alter the secondary structure of the protein (Table 1).

A more comprehensive indicator of structural integrity was provided by 1H-15N-HSQC spectra. Overlays of HSQC spectra of native Sac7d and Sac7d dimers showed negligible differences in NH chemical shifts except at the site of the single residue substitution, indicating that the cysteine mutations and dimerization did not affect protein folding (Figure 3).

The positions of the cysteine substitutions as well as the high positive charge on the surface of the protein (pI 9.7; 13 lysines, 4 arginines, 5 aspartates, and 7 glutamates) was expected to lead to two independent globular domains connected by a flexible linker. Differential scanning calorimetry (DSC) was used to investigate potential domain size changes and domain-domain interactions in the dimers (Figure 4). As expected, dimerization by crosslinking led to negligible change in Tm and ∆H of unfolding. The essentially identical ∆H values indicates that the Sac7d subunits in the dimer behave as independent domains. As observed for the Sac7d monomer (38), thermal unfolding of the dimer was reversible.

Protein-DNA binding affinity was quantitatively described by analyzing reverse titrations (DNA into protein) monitored with DNA-induced quenching of the intrinsic fluorescence of Sac7d. Previous work has demonstrated that the fractional quenching of Sac7d tryptophan (W24) fluorescence is a direct indicator of the fraction of DNA-bound protein

(22)

. The DNA

affinities of head-to-head, tail-to-tail, and head-to-tail dimers for synthetic 100-mer poly(dGdC) were measured in relatively low ionic strength under conditions previously used for native and


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recombinant Sac7d (Table 2). The binding affinities for all of the dimers were identical, within experimental error, to that of monomeric Sac7d. The binding site size for the dimers was similar to that of the monomer. A slightly lower site size for the dimer may be due to some species binding with only a single domain (the other dangling in solution and thereby effectively reducing the site size). However the magnitude in maximal quenching observed for the dimers is almost identical to that observed for monomeric Sac7d, 0.80 ± 0.1 compared to 0.84 ± 0.1.

Since the NMR solution structure of a Sso7d-DNA complex indicated a preference for head-to-head binding of two monomers (30), we also determined the binding parameters for headto-head, head-to-tail and tail-to-tail dimers binding to the same 12-mer DNA sequence used for the solution NMR studies (1bbx). The results were similar to those observed for poly(dGdC) with no obvious preference observed for any dimer (Table 3).

We also created dimers by crosslinking with bifunctional crosslinking agents to increase the distance between monomers and as well as the flexibility of the connection. The reagents were all bifunctional maleimides separated by ethylene glycol, butane, hydroxybutane, hexane, and benzene: BMB (1,4-bismaleimidobutane), BMH (1,6-bismaleimidohexane), BM(PEG)2 (2,8 bismaleimidodiethyeneglycol), BMBD (1,4-bismaleimido-2,3-dihydrdoxybutane), and pPDM (p-phenylenedimaleimide). None of the crosslinked proteins showed a binding affinity significantly different from that observed by oxidation with diamide (data not shown).

Finally, we investigated the existence of preferred binding orientation in protein-DNA complexes by crosslinking a mixture of single cysteine mutant proteins with different DNA


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sequences. With a binding affinity on the order of 106 M-1 we might expect a dissociation rate constant of 0.01 sec-1 (assuming a diffusion-limited second order on-rate of 108 M-1 sec-1), or a lifetime on the order of 100 sec. Thus during the time of a crosslinking experiment (e.g. 60 min), the complex is likely to dissociate and re-associate hundreds of times, the DNA should provide a bias in crosslinking orientations reflecting preferred orientations if they exist. As shown in Figure 5, complexing Sac7d with 12-mer (1bbx sequence), 100-mer duplex poly(dGdC) and poly(dIdC) resulted in predominantly head-to-head and tail-to-tail dimers with significantly less head-to-tail dimer. The relative amounts of dimers observed in the absence of DNA was consistent with that observed by ion exchange chromatography (Figure 3). Notably, crosslinking with pPDM showed a significant amount of head-to-tail dimer, presumably due to the need for greater separation between monomers to accommodate the crosslinker. These results indicate that adjacent monomers pack onto DNA preferentially in an alternating head-to-head, tail-to-tail pattern.


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DISCUSSION We have used selective incorporation of single cysteines to allow the engineering of dimers of Sac7d with defined subunit orientations, viz. head-to-head, tail-to-tail, and head-to-tail dimers. Similar to previous work, we have found that the stability of this small, stable protein was not affected by the modification and the protein remains functional. In other work, Sac7d and Sso7d have been fused or crosslinked to different proteins to create dimers with nonsequence specific DNA binding capability. Sac7d has been fused to a DNA polymerase to enhance processivity (47). It has also been fused to HIV integrase to create a highly soluble, hyperactive integrase (48). A Sac7d dimer created by disulfide crosslinking through engineered C-terminal cysteines proved to have significantly different binding modes from that observed for the monomer (49). This is perhaps not too surprising given the fact that the C-terminus projects from the side of the protein opposite to the binding interface so that such a dimer would have DNA binding sites facing away from each other.

The results presented here show no significant difference in the binding of the head-tohead, head-to-tail, and tail-to-tail Sac7d dimers to 100-mer poly(dGdC) duplex DNA or the 12 mer DNA duplex used for the NMR solution structure of Agback et al. (30). However, crosslinking of the protein complexed with DNA shows a bias for head-to-head and tail-to-tail dimers with little head-to-tail orientation. Given this clear preference for two of the orientations, it is surprising that there is no evidence of increased binding affinities for the corresponding dimers relative to head-to-tail. As demonstrated with Lac repressor, a dimer that is cross-linked in a putative preferred orientation should exhibit greater affinity relative to a monomer or those


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in less than optimal orientations (50, 51). The lack of enhanced binding may be attributed to possible structural constraints that prevent optimal complex formation. Presumably the proximity of reactive cysteines in the preferred head-to-head and tail-to-tail orientations is sufficient to bias the crosslinking reaction, even though the product will have an affinity less than expected and comparable to the head-to-tail dimer.

We conclude that Sac7d is likely to coat DNA in vivo with alternating head-to-head and tail-to-tail orientations of the monomers. As we have shown previously, DNA fully saturated with Sac7d is largely extended due to the repetitive counteracting kinks (29). A model showing the effect of alternating head-to-head and tail-to-tail binding is shown in Figure 6. Therefore, the available solution data indicates that under conditions approaching saturation, Sac7d is more appropriately viewed as a “stiffening” protein, rather than a “bending” protein. Although the xray structure demonstrated that the protein induces a remarkably large kink into duplex DNA, given the dissociation constant and the concentrations expected in vivo we expect that Sac7d does not function as a “bender” unless there are sufficient concentrations of competing proteins or interactions with other proteins that promote the binding of isolated Sac7d monomers. It remains to be seen how Sac7d affects the structure of DNA in conjunction with other nucleoid proteins under in vivo conditions.

Acknowledgements: This work was supported by grant GM49686 from the National Institutes of Health to JWS and SPE.


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Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Tables: Table 1. Calculated secondary structure in Sac7d monomer and engineered dimers. Fractions of α-helix, β-sheet, β-turn and other were calculated from the CD spectra for each protein. CD spectra were collected in 0.01 M phosphate buffer, pH 7.2 at 20 ºC.

Protein Sac7d K9C dimer K28C dimer K9C*K28C

α-helix 24 32

β-strand 33 36

β-turn 16 13

Others 27 19

25

35

16

24

28

27

21

24


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Table 2. Monomer and Dimer Sac7d Binding to poly(dGdC) Comparison of DNA binding of monomer and dimer Sac7d to 100 bp duplex poly(dGdC), i.e. essentially infinite duplex DNA. Binding affinity, site size and maximal quenching were obtained by nonlinear least squares fitting of fluorescence titrations as described in the text.

Ka

n

Protein

(107 M-1)

(bp)

Qmax

Sac7d

2.4

3.6

0.84

K9C dimer (TT#)

3.0

3.2

0.89

G10C dimer (TT)

1.5

2.6

0.86

G27C dimer (HH)

1.5

3.5

0.79

K28C dimer (HH)

1.3

3.3

0.83

K9C*-G27C (HT)

1.7

2.7

0.79

K9C*-K28C (HT)

2.3

2.3

0.60

* Estimated error: Ka: 0.6 x 107; n: 0.5; Qmax: 0.1, based on repeated measurements. #

TT: Tail-to-tail; HH: head-to-head; HT: head-to-tail.


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Table 3. Monomer and Dimer Sac7d Binding to 12 bp Duplex DNA* Comparison of DNA binding of monomer and dimer Sac7d to 12mer DNA. The DNA sequence was that used for the NMR solution structure of the protein-DNA complex reported by Agback et al. (30). Binding affinity, site size and maximal quenching were obtained by nonlinear least squares fitting of fluorescence titrations as described in the text.

Ka

n

Protein

(107 M-1)

(bp)

Qmax

Sac7d

1.4

3.6

0.62

K9C dimer (TT)

1.9

2.5

0.68

G10C dimer (TT)

1.0

2.6

0.88

G27C dimer (HH)

1.7

3.5

0.83

K9C*-G27C (HT)

2.3

3.3

0.72

K9C*-K28C (HT)

1.8

2.6

0.64

*12mer sequence was that used for the 1bbx NMR solution structure

(30)

: CTAGCGCGCTAG.

Errors and abbreviations as in Table 2.


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Figure Legends

Figure 1. An X-ray crystal structure of Sac7d (blue) binding to an eight base pair DNA duplex (grey) (PDB 1azp). The DNA binding interface is defined by the three stranded β-sheet and the tight turn in the N-terminal β-ribbon. Both the N-terminus (at the right) and the C-terminus (at the end of the helix, top right) extend away from the DNA binding site. The intercalating residues V26 and M29 (both shown in cyan) are shown in the tight turn at the top with G27 (red) and K28 (green) intervening, while the β-ribbon turn at the bottom contains K9 (red) and G10 (green). The region containing the intercalating residues is referred to as the “head”, while the opposite end is the “tail”. Single cysteine substitutions at K9, G10, G27 or K28 permitted crosslinking by oxidation or bifunctional crosslinking agents to enable head-to-head, head-to-tail, and tail-to-tail dimers.

Figure 2. Separation of head-to-tail dimer using a his-tag attached to the C-terminus of one of the monomers. In this experiment K9C* with his-tag was mixed with an equimolar amount of G27C and oxidized with diamide. The effluent prior to initiation of an imidazole concentration gradient contained G27dimers, while the K9C*-G27C dimer with only one his-tag was eluted at lower imidazole than the K9C* dimer with two his-tags. The imidazole gradient is indicated in red with the scale on the right.


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Biochemistry

Figure 3. Comparison of the changes 1H,15N HSQC peak positions in Sac7d with formation of the K28C dimer through oxidation with diamide. Peak positions are reported as weighted average of the 1H and

N chemical shifts using + . The only difference was

15

observed at K28, the site of the substitution.

Figure 4. Differential scanning calorimetry of recombinant Sac7d (blue) compared to the K9C dimer obtained by diamide oxidation (red). Solid curves show the results of a nonlinear least squares fit of the heat capacity as a function of temperature with Sac7d giving a Tm of 90.8 ± 0.1 °C and a ∆H of 78 kcal/mol, while the dimer showed a Tm of 91.4 ± 0.1 °C and a ∆H of 75 kcal/mol.

Figure 5. SDS-gel electrophoresis of Sac7d dimers obtained by crosslinking in the presence and absence of DNA. Sac7d runs at 10kDa in a 16.0% Tris-Tricene under non-reducing conditions. Lane 1: molecular weight ladder, lane 2: K28C and K9C* single cysteine mutant proteins, followed by K28C and K9C* crosslinked by oxidation with diamide in the presence of 12-mer DNA (1bbx, lane 3), poly(dIdC), lane 4 and poly(dGdC), lane 5), and lane 6 : crosslinking with pPDM.

Figure 6. A model of a Sac7d DNA complex showing the effect of fully coating DNA with Sac7d oriented in head-to-head, tail-to-tail orientations with kinks separated by 2 and 6 base pairs, respectively.

The model was created in Chimera by imposing the coordinates for the

crystal structure of Sac7d-DNA complex (1AZP) onto 32-mer poly(dGdC) with 7 Sac7d monomers.


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Figure 1.


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Figure 2.


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Figure 3.


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Figure 4.


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Figure 5.

F


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Figure 6.


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For Table of Contents Use Only:


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Figure 1.


Biochemistry

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Figure 2.


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Figure 3.


Biochemistry

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Figure 4.


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Figure 5.

F


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Figure 6.


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Insights into the Structure of Sulfolobus Nucleoid ... - ACS Publications

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