Crystal structure of TrmB

The threedimensional structure of TrmB was determined to a resolution of 3.1Å with crystal-lographic R_work and R_{f ree} factors of 24.2% and 27.9%, respectively. As mentioned in section 3.3.3.2, the asymmetric unit of the crystal contained only one molecule of TrmB corresponding to a very high solvent content of more than 82%. Due to this high solvent content, the over-all B-factors are very high and parts of the electron density, especiover-ally in the solvent exposed

TrmB

Spacegroup P3221

Unit cell 156.8, 156.8, 79.1

Wavelength 1.0162

Resolution 30-3.1 (3.2-3.1) Observed reflections 80323 (7698) Completeness 97.9 (99.7)

I/σ_I 18.26 (1.28)

R_meas 5.4 (103.8)

R_mrgd−F 11.9 (157)

Table 3.8:Statistics for the best dataset of TrmB. The crystals frozen in liquid propane led to slightly better R_meas values than those frozen in liquid nitrogen.

3.3 Structure of TrmB with bound sucrose

Figure 3.10:Cutout of the packing of a TrmB crystal. The helix-turn-helix motif of the yellow colored DNA binding domain of TrmB extends into the solvent.

DNA-binding part of the protein are very poor. There is no electron density corresponding to the first two amino acids of TrmB as well as for the N terminal His tag. (see also figure3.10).

TrmB consists of the sugar binding domain as described in section3.2, a short linker and the N-terminal DNA-binding domain which consists of fiveα-helices (H0-H4) and threeβ-strands (S1-S2-S3), with an H0-H1-S1-H2-H3-S2-S3-H4 topology. The N-terminal helix H0 (residues 3-20) is followed by the H1 helix (23-35), the H2 helix (39-42), the H3 helix (47-57), a β hairpin (60-74) and the H4 helix (77-102) (The electron density for parts of helix H0 and H2 is very poor). The overall fold of the monomer can be divided into three structural domains:

An N-terminal H0 helix, a central winged helix-turn-helix domain (see section1.3.1.4) and the C-terminal H4 helix. Figure3.11gives an overview of the TrmB structure.

Sucrose binding In the interstice between the two domains of the sugar binding domain of TrmB, electron density corresponding to sucrose was visible. The sucrose forms hydrogen bonds with the same residues as the nonreducing part of maltose did (see section3.2.4.4), but there is no hydrogen bond between the sucrose and Ser²²⁹of the sugar binding helix of the sugar binding domain (see figure3.12). This is consistent with the idea of the binding modes for the

3 Crystal Structure of the archaeal transcriptional regulator TrmB

Figure 3.11:Structure of TrmB with bound sucrose. The N-terminal DNA-binding domain consists of a winged helix-turn-helix motif with a preceding and successive helix. The wing and the recognition helix of the helix-turn-helix motif are colored yellow. The DNA-binding domain is connected to the sugar binding domain via a short linker. The electron density for helix H2 and helix H0 was very poor.

3.3 Structure of TrmB with bound sucrose

Figure 3.12:Superposition of TrmB_∆2−109with bound maltose (colored slate) (see section3.2) and TrmB with bound sucrose (colored grey, with the sucrose colored yellow/red). The superposition of the struc-tures was done with the software THESEUS [235] using a maximum likelihood approach. As ex-pected (see section3.2.4.4), there is no interaction between sucrose and Ser²²⁹of the sugar binding helix. Minor shifts of theβ-sheet above and the loop region below the sugar can be seen.

different sugars bound by TrmB and the results of the sugar binding assays with mutants as described in section3.2.4.4. A comparison of the sugar binding domains of TrmB with bound sucrose and of TrmB_∆2−109 with bound maltose shows minor movements of the loop region below and of theβ-strand above the sugar. The overall structures of the sugar binding domains with bound maltose and sucrose show no further differences.

Dimerization One of the symmetry mates of the crystal reveals the most probable dimeriza-tion mode of TrmB (that differs from the dimerizadimeriza-tion mode proposed in secdimeriza-tion3.2.5.3in the light of the structure of the sugar binding domain of TrmB). The H4 helices of two monomers form a short antiparallel coiled coil with one knobs-into-holes packing interaction (see figure 3.13). The length of the coiled coil rod in the TrmB dimer leads to a separation of its two H3

recognition helices by about 30Å.

3.3.4 Discussion

The architecture of the DNA-binding part of the TrmB dimer resembles those of the puta-tive DNA-binding protein Sto12a of the thermoacidophilic archaeonSulfolobus tokodaii[236],

3 Crystal Structure of the archaeal transcriptional regulator TrmB

Figure 3.13:The H4 helices of the two TrmB monomers form a coiled coil. One knobs-into-holes interaction occurs in the middle of the short coiled coil where in each case a phenylalanine is opposed by a leucine. The arrows indicate the direction from N to C terminus.

Figure 3.14:Structure of a TrmB dimer as found within the TrmB crystals. One monomer is colored grey, the other slate. The recognition helix and the wing of the winged helix-turn-helix motif are colored yellow. The protein dimerizes via the formation of a coiled coil arrangement of the H4 helices of two monomers. The N and C termini of one monomer are indicated.

3.3 Structure of TrmB with bound sucrose

Figure 3.15:Sequence alignment of the DNA binding domain of TrmB (residues 1-102), Sto12a (PDB accession code 2D1H), Sso10a (PDB accession code 1R7J) and the protein fromA. fulgidus(PDB accession code 1SFX). There is a highly conserved leucine at the C terminus of the recognition helix (H3). The alignment was done using Kalign [238]. Secondary structure elements of the DNA binding domain of TrmB are indicated.

Sso10a, a member of a group of DNA-binding proteins thought to be important in chromatin structure and regulation in the hyperthermophilic archaeonsulfolobus solfataricus[237] and a putative helix-turn-helix transcription regulator fromArchaeoglobus fulgidus (no publication, PDB accession code:1SFX) (see figure3.16).

Sto12a as well as the putative transcription regulator fromA. fulgidus consist of 109 amino acids, Sso10a consists of 95 amino acids and the DNA binding domain of TrmB consists of 102 amino acids. The TrmB DNA binding domain shares 25% identical residues with theA.

fulgidusprotein, 20% with Sto12a and 19% with Sso10a. All three proteins dimerize and form an antiparallel coiled coil with C-terminal H4 helices. In the case of the Sso10a protein, the antiparallel coiled coil is packed through several knobs-into-holes packing interactions, while there is only one such interaction in the coiled coil of TrmB and Sto12a. Sso10a lacks the N-terminal helix corresponding to H0 in TrmB and Sto12a and the H4 helix is longer than that in TrmB and Sto12a. Although the protein fromArchaeoglobus fulgidus has the highest sequence identity with the DNA binding domain of TrmB, the structure of Sto12a resembles

3 Crystal Structure of the archaeal transcriptional regulator TrmB

Figure 3.16:Proteins exhibiting a similar architecture as the dimeric DNA-binding part of TrmB. All proteins differ in the length of the coiled coil and thus in the distance between their recognition helices. They also differ in the arrangement of the helix-turn-helix motif with respect to the coiled coil: Whereas the helix-turn-helix motif in Sso10a is oriented in a way, that its recognition helices face up, those of the three other proteins face to mutually opposite directions. The arrows point in direction of the N termini of the H4 helices of the proteins.

most the DNA-binding domain of TrmB. But interestingly the structure is “fixed” by disulfide bridges: There is one intermolecular disulfide bridge between the H0 and the H4 helices of each monomer and one intermolecular disulfide bridge between the two H0-H0’ helices of the dimer [237]. The proteins also differ in the topology of their dimer formation. If the proteins are oriented so that the winged helix-turn-helix domains are in front of the coiled coil plane, the coiled coil interfaces between the proteins differ completely. In the Sso10a dimer, when the structure is viewed from the N-terminus of the H4 helix with the winged helix-turn-helix above the H4 helix, the other H4 helix (H4’) is packed on the left side, in contrast to the right hand side packing in the case of Sto12a and TrmB and the protein fromA. fulgidus(see figure3.16). The distance between the H3 recognition helices in the Sso10a protein is approximately 55Å, in Sto12a about 34Å , 38Å for the A. fulgidus protein and in TrmB approximately 30Å. Both recognition helices of Sso10a face up front, whereas those of the three other proteins face to mutually opposite directions (one to the left and the other to the right, see figure3.16).

The fact that there are only minor differences between the sugar binding motif of TrmB with bound sucrose and TrmB_∆2−109with bound maltose was very suprising because according to the different effects of the two sugars on TrmB one should expect specific structural rear-rangements of the sugar binding domain upon binding of the different sugars. These structural rearrangements would then be transferred to the helix-turn-helix motif resulting in different

3.3 Structure of TrmB with bound sucrose

Figure 3.17:Structure of BmrR with bound DNA (PDB accession code: 1R8E). The wings and the recognition helices are colored yellow. One monomer is colored grey, the other is colored slate. The DNA is rotated approximately 65^◦counterclockwise with respect to the axes of the coiled coil helices. Ad-ditional DNA contacts are provided by residues within the orange colored helices.

DNA-binding abilities of TrmB. This lack of structural differences could be due to a mutation from valine to alanine in residue 161 that the crystallized TrmB accidentally exhibited com-pared with the wildtype protein (see section3.3.4.1). Nevertheless, the TrmB construct having this mutation in residue 161 only crystallized if sucrose was added to the protein solution. The fact that it could not be crystallized in presence of maltose indicates that there must be some structural differences between TrmB with bound maltose and TrmB with bound sucrose, even in presence of the V161A mutation.