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

Abstract

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

“…TrmBL2 from Pyrococcus furiosus was cloned and expressed in E . coli BL21 (DE3) cells (New England Biolabs) which were cultivated in LB medium at 37°C as described previously [ 10 ]. Protein expression was induced by adding 1 mM IPTG to the cell culture at an OD600 of 0.6.…”

“…The protein containing fractions were identified by SDS-PAGE, pooled, concentrated and subjected to gel-filtration on a 60 ml Superdex 200 column equilibrated with 40 mM HEPES pH 7.5 and 150 mM NaCl. TrmBL2 eluted as a single peak corresponding to its dimeric form, in contrast to the tetramers obtained from Pyrococcus furiosus cells [ 10 ]. While this may be caused by differences in conditions in the bacterial and the archaeal cell, the crystal structure of dimeric TrmBL2 from E .…”

“…Another TrmB-like protein, VNG1451C from the halophilic archaeon Halobacterium salinarum NRC-1, controls genes encoding glycolytic and gluconeogenic pathways, purine biosynthesis, cobalamin biosynthesis, the tricarboxylic acid cycle, and glutamate synthesis [ 7 ]. Sequence similarities [ 8 ] as well as the crystal structures of TrmB [ 9 ] and TrmBL2 [ 10 ] show that both proteins share the same succession of homologous domains, a N-terminal DNA binding helix-turn-helix domain, a dimerization helix and a C-terminal domain. TrmB and TrmBL1 possess an additional domain at the C-terminus which is involved in binding of the effector sugar molecules.…”

“…Surprisingly, when bound to the ssDNA being produced during hairpin disruption, TrmBL2 inhibited double-helix formation similarly as single-strand binding proteins. Furthermore, the disruption experiments in combination with the recently determined atomic structure [ 10 ] provide evidence that the smallest broken protein-DNA interaction unit is a pair of DNA binding helix-turn-helix domains within the tetramer which bind to the same major groove. In summary, our tweezers data suggests that TrmBL2 stabilizes both dsDNA and ssDNA.…”

Protocols for Molecular Modeling with Rosetta3 and RosettaScripts

“…Phenix.Rosetta refinement has been successfully adapted to determine structures of the flavin binding center of the NqrC subunit of sodium-translocating NADH:quinone oxidoreductase, 81 the full-length protein and regulatory domain of Pseudomonas aeruginosa OxyR, 82 the apo-TrmBL2 structure to understand nonspecific binding of DNA by TrmBL2, 83 and the αβ T cell antigen receptor (TCR)–CD1a complex. 84 …”