Figure 1.
Structure of B. halodurans TRAP.
(A) Sequence alignment of TRAP proteins from different species produced by ClustalW [18]. The extent of sequence conservation is depicted in grey with identical residues in black and non-conserved in white. Residues important for the conformation of the C-terminus are highlighted by red stars and residues that interact with RNA are highlighted by yellow stars. (B) Ribbon diagrams viewed along the 12-fold axis (two opposite views). Each subunit is shown in a different color. The C-terminus of each subunit, starting from residue 71, is shown by a thick yellow ribbon. L-Tryptophan molecules, bound in deep pockets between adjacent subunits, are shown as van der Waals models with carboxyl oxygen atoms in red, nitrogen atoms in blue and carbon atoms in yellow.
Table 1.
Data collection and refinement statistics.
Table 2.
Summary of native mass spectrometry analysis.
Table 3.
Average distance (Å) between Cα atoms of RNA-binding residues belonging to adjacent subunits.
Figure 2.
Comparison of 11-mer and 12-mer TRAP assemblies.
(A) Dimers of B. halodurans TRAP (red and yellow) and wild-type B. stearothermophilus TRAP (both subunits in blue) were least-square fitted using main chain atoms of single subunit (shown on the right). Cα-models are shown with the segment 72–75 highlighted by wide traces. View is from outside the TRAP ring toward its center with the central rotation axis vertical. The inter-subunit rotational axis relating the 11-mer and 12-mer oligomers is shown by dashed line. (B, C, and D) Comparison of the C-terminus conformation in 11-mer and 12-mer TRAP. C-terminal residues starting from 71 and residues stabilizing the conformation of the C-terminus are shown by sticks, the rest of the subunit interface is shown by ribbons.
Figure 3.
Individual TRAP subunits are shown as ball-and-stick models in different shades of grey. Inter-subunit rotation axes are roughly parallel to the central oligomer axis and are depicted by black crosses shown in yellow circles. (A) Removal of the C-terminal segment (green) at one side of the axis or (B) introduction of methylene group through Val-11-Leu mutation (blue) at the other side of the axis, allows subunits to roll around the inter-subunit rotation axis to form a 12-mer.
Figure 4.
Native mass spectrometry analysis.
Nanoflow electrospray mass spectra for (A) B. halodurans TRAP, (B) B. stearothermophilus E71stop TRAP and (C) B. subtilis V11L TRAP. A stable 12-mer species was identified in each sample; peaks corresponding to different charge states and different numbers of bound tryptophan molecules are labeled.
Figure 5.
General model for transition between alternative oligomeric states.
Transition from n-subunit to (n+1)-subunit state could be accomplished either by deletion (green triangles) at the outer side of the inter-subunit rotation axis (A) or insertion (light blue triangles) at the inner side of the axis (B). Inserts with three-dimensional representation show two adjacent subunits of each oligomer, with the intersubunit axis relating rotation of adjacent subunits during transition between the two states shown in yellow.
Table 4.
Increase in the size of the central tunnel.