Structure Determination of TrmB and TrmB-like Transcriptional Regulators from the hyperthermophilic Archaeon Pyrococcus furiosus
Zusammenfassung der Projektergebnisse
We report here about the funding period 2009 – 2011, the extension of the project to 2013, granted in 2010, and the continuation by us without funding till 2016. This work continued a similar precursor project which aimed at the TrmB class of transcriptional regulators in thermophilic archaea. At the beginning, work by one of our groups led to the discovery of TrmB as a dual-function transcriptional regulator of maltose, glucose and sucrose via the TM and MD operators. Sequence homologies revealed two further homologs, TrmBL1 and TrmBL2. During the previous project we were able to determine the crystal structure of the TrmB effector binding domain (EBD) in complex with maltose and got crystals of the full-length protein with bound sucrose. In the follow-up project summarized here we performed the crystal structure analysis of the latter and published the result. The TrmB structure suggests a dimeric DNA binding conformation. We identified the N-terminal 'extended winged-helix-turn-helix' DNA binding domain (DBD), the dimerization domain (CC) and compared the residues involved in maltose and sucrose binding. The proposed dimer model exhibits the expected distance of the recognition helices of the two DBDs to fit into two adjacent major grooves along the DNA. Moreover, the coiled-coil of two CC domains forms a '11 residues over 3 turns' right-handed coiled-coil. In the discussion we compared the structure of TrmB with crystal structures of other DNA binding proteins. Moreover, we propose a model how TrmB can repress transcription of both, pseudo-palindromic TM and non-palindromic MD operator sequences in different protein conformations dependent on the type of bound ligand and on the operator sequence. TrmBL1 crystallization which was planned in our work schedule failed in spite of time-consuming and extensive attempts. TrmBL2 crystallization, however, succeeded in absence and in presence of bound DNA. TrmBL2, in contrast to TrmB and TrmBL1, lacks the C-terminal part of the EBD. It exhibits some affinity for the MD operator sequence but no clear hint to transcriptional regulation had been shown at the onset of the project. During our crystal structure analysis we studied a homolog of TrmB from T. kodakarensis with Electron microscopy and other techniques and showed that the protein binds non-specifically and cooperatively in large numbers so that the DNA-protein complex assumes the appearance of a thick filament. Our crystal structure showed that two TrmBL2 dimers bound to a single double helical DNA segment. The base pairs with which the recognition helices of the ewHTH domains in the two TrmBL2 dimers interact, are shifted by two along the DNA. This causes a rotation by about 80° of the two dimers with respect to an approximated DNA axis. In the view along the DNA axis it is seen that TrmBL2 covers half of the DNA surface. The protein bends the DNA upon binding which is a common feature of many protein-DNA complexes. In addition we studied the interaction of TrmBL2 with DNA by magnetic tweezer experiments. This revealed further effects of TrmB binding on the double helix formation and the length of the DNA. We also demonstrated TrmBL2 binding to single DNA strands. In short, the binding of TrmBL2 to a ~3.5 micrometer long DNA causes an increased bending rigidity of the filament and a shortening of the DNA contour length by 2%. Hairpin unzipping experiments showed stabilization of the double helix by TrmBL2 binding and unzipping occurring in discrete steps which are not seen in absence of protein. Ten was the smallest number of basepairs which opened simultaneously. The crystal structure of DNA with bound TrmBL2 and the data from literature as well as the magnetic tweezer data led us to propose a model for the DNA with bound protein which is in accord with the observations. In the discussion we suggest that within the TrmB-sheath, the thermal movements of DNA may be reduced so that the chemical structure is protected. The stiff coating thus may function to stabilize the DNA against a variety of possible damages.
Projektbezogene Publikationen (Auswahl)
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The three-dimensional structure of TrmB, a transcriptional regulator of dual function in the hyperthermophilic archaeon Pyrococcus furiosus in complex with sucrose. Protein Science, Vol. 22. 2013, Issue 6, pp. 800–808.
Krug, M., Lee, S.-J., Boos, W., Diederichs, K., Welte, W.
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Structural Insights into Nonspecific Binding of DNA by TrmBL2, an Archaeal Chromatin Protein. Journal of Molecular Biology, Vol. 427. 2015, Issue 20, pp. 3216-3229.
Ahmad, M., Waege, I., Hausner, W., Thomm, M., Boos, W., Diederichs, K., Welte, W.
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TrmBL2 from Pyrococcus furiosus Interacts Both with Double-Stranded and Single-Stranded DNA. Plos One, Vol. 11. 2016, Issue 5: e0156098.
Wierer, S., Daldrop, P., Ahmad M. Boos, W., Drescher, M. Welte, W., Seidel, R.
