X-ray crystallographic analyses of archaeal DNA binding proteins TrmBL2 and TrmB

In archaea, packaging of DNA into chromatin and transcriptional regulation are closely related processes and the proteins involved therein often exhibit overlapping roles. In case of extremophilic archaea, the challenge to protect the DNA from detrimental effects of the harsh environments has led many of these chromatin proteins to evolve the additional function of DNA protection. The multiple roles these proteins play in DNA metabolism makes them interesting candidates for structural studies.The first and major part of this work deals with the structural elucidation of TrmBL2, a member of the TrmB family of transcriptional regulators. Recent studies have to a great extent established the role of TrmBL2 in both chromatin shaping and transcriptional regulation. Upon association with DNA, TrmBL2 has been shown to form thick fibrous structures. Deletion of TrmBL2, in addition to the loss of fibrous structure, leads to upregulation of many unrelated genes thereby providing evidence for its role as a dual functional protein.For structure determination, TrmBL2 was heterologously expressed in E.coli, subjected to ion-exchange and size exclusion chromatographic purification and crystallized with 19 or 17 bp TGM (Thermococcales Glycolytic Motif) dsDNA. In the absence of a suitable molecular replacement model, phases were determined by the Selenium Single Wavelength Anomalous Dispersion (Se-SAD) method. The structure of DNA-free TrmBL2 expressed in Pyrococcus furiosus was subsequently determined by molecular replacement.TrmBL2 crystallizes as a tetramer in an asymmetric unit, both in the DNA-bound and DNA-free forms. The structure reveals an extended winged Helix Turn Helix (ewHTH) domain at the N-terminus followed by a coiled coil dimerization domain and a Phospholipase D (PLD) like domain at the C-terminus. While the electron density of the sugar-phosphate backbone of the bound TGM dsDNA is clearly distinguishable, the density for the nucleobases is averaged and represents a superposition of three binding modes with a 3 bp shift around the central 19 bp DNA at the 5’ and 3’ ends thereby explaining the observed 25 bp density. For the 17 bp DNA, the observed 21 bp density could be explained by a similar 2 bp shift at the 5’ and 3’ ends. During refinement, the occupancy of the nucleotides was adjusted so that the overall occupancy sums to the actual number of the base pairs used in crystallization. Given the non-specific binding of TrmBL2 to the DNA, the observed multiple binding modes and the resultant averaging out of the nucleobase density is not surprising.The structure of the DNA-free TrmBL2 does not show any major differences from the DNA-bound structure.The TrmBL2-DNA complex structure described in this work shows a hitherto unknown mode of tetramerization and DNA binding. The analyses of the crystal structures provide a basis for the reported non-specific binding of TrmBL2 to the DNA and also provides an explanation for its observed roles in chromatin structuring and transcriptional regulation.The second part of this work details the efforts to devise a protocol for overexpressing TrmB and to overcome the low solubility issues of this protein with the ultimate aim of solving the structure of TrmB in complex with TM and MD promoters. Towards this end several constructs were tried but the problem of TrmB proteolysis proved to be a major hindrance in the realization of these goals.publishe

Structural Insights into Nonspecific Binding of DNA by TrmBL2, an Archaeal Chromatin Protein

The crystal structure of TrmBL2 from the archaeon Pyrococcus furiosus shows an association of two pseudosymmetric dimers. The dimers follow the prototypical design of known bacterial repressors with two helix–turn–helix (HTH) domains binding to successive major grooves of the DNA. However, in TrmBL2, the two dimers are arranged at a mutual displacement of approximately 2 bp so that they associate with the DNA along the double-helical axis at an angle of approximately 80°.While the deoxyribose phosphate groups of the double-stranded DNA (dsDNA) used for co-crystallization are clearly seen in the electron density map, most of the nucleobases are averaged out. Refinement required to assume a superposition of at least three mutually displaced dsDNAs. The HTH domains interact primarily with the deoxyribose phosphate groups and polar interactions with the nucleobases are almost absent.This hitherto unseen mode of DNA binding by TrmBL2 seems to arise from nonoptimal protein–DNA contacts made by its four HTH domains resulting in a low-affinity, nonspecific binding to DNA

TrmBL2 from Pyrococcus furiosus Interacts Both with Double-Stranded and Single-Stranded DNA

In many hyperthermophilic archaea the DNA binding protein TrmBL2 or one of its homologues is abundantly expressed. TrmBL2 is thought to play a significant role in modulating the chromatin architecture in combination with the archaeal histone proteins and Alba. However, its precise physiological role is poorly understood. It has been previously shown that upon binding TrmBL2 covers double-stranded DNA, which leads to the formation of a thick and fibrous filament. Here we investigated the filament formation process as well as the stabilization of DNA by TrmBL2 from Pyroccocus furiosus in detail. We used magnetic tweezers that allow to monitor changes of the DNA mechanical properties upon TrmBL2 binding on the single-molecule level. Extended filaments formed in a cooperative manner and were considerably stiffer than bare double-stranded DNA. Unlike Alba, TrmBL2 did not form DNA cross-bridges. The protein was found to bind double- and single-stranded DNA with similar affinities. In mechanical disruption experiments of DNA hairpins this led to stabilization of both, the double- (before disruption) and the single-stranded (after disruption) DNA forms. Combined, these findings suggest that the biological function of TrmBL2 is not limited to modulating genome architecture and acting as a global repressor but that the protein acts additionally as a stabilizer of DNA secondary structure.publishe

Kinetics of TrmBL2 binding to dsDNA at different concentrations of TrmBL2.

<p>The kinetics were measured by monitoring the change of the DNA length at a force of 0.4 pN, which linearly changes with the coverage of the DNA by the protein. The data shown were recorded from the same DNA molecule. At room temperature, the filament formation is completed (95%) within approximately 10 minutes and the final DNA length (dotted line) is observed.</p

A model for the TrmBL2 filament using the crystal structure of TrmBL2 with bound dsDNA.

<p>In the crystal structure of TrmBL2 from <i>Pyrococcus furiosus</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156098#pone.0156098.ref010" target="_blank">10</a>] four protein molecules bind to the 19 basepairs long TGM dsDNA. In the filament model shown in panel A, it comprises the first quarter with DNA (pink) and the protein tetramer (green). First an octameric complex was constructed from two copies of the crystal structure. In the uppermost panel it comprises the left halve with one pink, one red DNA piece, one green and one yellow tetramer. Two copies of the octameric complex were shifted in tandem to get a continuous dsDNA running through both as seen in panel A. The resulting dsDNA in the hexadecamer has zero overall curvature. The hexadecameric filament model is shown in ribbon representation in three orthogonal views (A-C). Note the almost full coverage of the DNA by TrmBL2 and that the DNA is in an almost perfectly linear conformation.</p

TrmBL2-filament formation on dsDNA.

<p><b>(A)</b> Schematic representation of dsDNA length change as result of TrmBL2 binding. In the absence of protein (left) the bare DNA is in a random coil configuration at low persistence length. Upon protein binding the stiffness of the DNA increases markedly, resulting at low forces in a more extended DNA conformation seen as an increase in the end-to-end distance by ΔL (right). <b>(B)</b> Force extension curves of dsDNA in the absence of TrmBL2 (black squares) and presence of 0.28 μM (green triangles) and 18 μM (red circles) TrmBL2 for a single bead. Fits of the data with the WLC model (shown as solid lines) provided, respectively, filament contour lengths of 3.36 ± 0.07 μm, 3.31 ± 0.06 μm and 3.28 ± 0.04 μm (blue dashed line) as well as persistence lengths of 36.4 ± 2.9 nm, 82.17 ± 14.0 nm and 126.13 ± 31.4 nm. ΔL indicates the length change expected at a force of 0.4 pN as applied in the kinetic measurements (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156098#pone.0156098.g002" target="_blank">Fig 2</a>). <b>(C)</b> Filament contour length (top panel), persistence length (black squares, bottom panel) and DNA coverage (blue circles, bottom panel) as function of the TrmBL2 concentration. The coverage was calculated using the persistence lengths obtained from force extension data. A fit to the data from four beads (solid grey and blue lines) provided a half-coverage concentration of 0.3 ± 0.2 μM and a Hill coefficient of 3.0 ± 0.33 for the DNA coverage. The error bars resulted from averaging apparent contour length, persistence length and coverage from four different beads.</p

Gel shift assay of TrmBL2 binding to ssDNA and dsDNA.

<p>The lanes contained TrmBL2 at increasing concentration (solid black ramp) and DNA. <b>(A)</b> Gel of ssDNA incubated with TrmBL2 at different concentrations. Marked with arrows are the ssDNA/TrmBL2 complex (top) and the ssDNA alone (bottom). <b>(B)</b> Gel of dsDNA incubated with TrmBL2 at different concentrations. Marked with arrows are the dsDNA/TrmBL2 complex (top) and the ssDNA alone (bottom). Note that unhybridized ssDNA runs at lower molecular weight. The bare dsDNA sample shows 2 bands, one annealed dsDNA band and one ssDNA band which resulted from unhybridised residual 60mer. The unhybridised band made up approximately 5% of the total fluorescence. <b>(C)</b> Titration curve using measured intensities of the bands indicate a K_D of 2.0 μM for ssDNA. Data were fitted using a logistic binding model. <b>(D)</b> Titration curve after analysis of band intensities for TrmBL2 binding to dsDNA indicate a K_D of 1.0 μM. Data were fitted using a Hill equation.</p

Hairpin unzipping at constant force reveals discrete protein dissociation events.

<p><b>(A)</b> Time trajectory of DNA hairpin unzipping and rezipping in absence of TrmBL2. The force was alternated between 20.6 pN and 1.6 pN to induce the conformational changes of the hairpin. Hairpin unzipping occurred under these conditions in a single fast step. <b>(B)</b> Time trajectory of DNA hairpin unzipping and rezipping in presence of 30 μM TrmBL2. Different forces from 20.6 pN to 26.5 pN were used for unzipping (see force axis). Hairpin opening occurred in small steps obviously due to displacement of bound protein (see detail in the right panel). <b>(C)</b> Number of base pairs unzipped in single steps for the traces shown under (B). The heights of the steps were determined manually and converted into the number of opened base pairs with a force-dependent algorithm (unpublished).</p

DNA unzipping in absence and presence of TrmBL2.

<p>A DNA hairpin is anchored through a dsDNA spacer at one end to the surface and at the other end to the magnetic bead (top). At sufficient force the hairpin is disrupted. When lowering the force, the hairpin spontaneously closes. <b>(B-E)</b> Unzipping (cyan) and rezipping (blue) cycles of the hairpin (see arrows for direction) in absence and presence of TrmBL2 as indicated in the figures. Hairpin rezipping is already affected at low TrmBL2 concentrations (19 nM) while hairpin unzipping is affected only when significant fractions of the DNA are covered by the protein (from 187 nM on). This shows that bound TrmBL2 shifts the unzipping to higher force and the rezipping to lower force indicating that the protein binds both, dsDNA and ssDNA.</p

Altered supercoiling response of dsDNA in presence of TrmBL2 for different forces.

<p>Arrows indicate the sense of rotation whereas the colour indicates the applied force. <b>(A)</b> Supercoiling curves in absence of TrmBL2 at different forces. The lighter and darker shade of a given color show curves that were obtained when inducing negative and positive supercoiling, respectively. Small cartoons of the plectoneme formation are shown. <b>(B)</b> Supercoiling curves taken at the same conditions in presence of 4.6 μM TrmBL2. The presented curves were all recorded using the same tether.</p