How to Change the Oligomeric State of a Circular Protein Assembly: Switch from 11-Subunit to 12-Subunit TRAP Suggests a General Mechanism

Abstract

BackgroundMany critical cellular functions are performed by multisubunit circular protein oligomers whose internal geometry has evolved to meet functional requirements. The subunit number is arguably the most critical parameter of a circular protein assembly, affecting the internal and external diameters of the assembly and often impacting on the protein's function. Although accurate structural information has been obtained for several circular proteins, a lack of accurate information on alternative oligomeric states has prevented engineering such transitions. In this study we used the bacterial transcription regulator TRAP as a model system to investigate the features that define the oligomeric state of a circular protein and to question how the subunit number could be manipulated.Methodology/Principal FindingsWe find that while Bacillus subtilis and Bacillus stearothermophilus TRAP form 11-subunit oligomers, the Bacillus halodurans TRAP exclusively forms 12-subunit assemblies. Significantly, the two states of TRAP are related by a simple rigid body rotation of individual subunits around inter-subunit axes. We tested if such a rotation could be induced by insertion or deletion mutations at the subunit interface. Using wild type 11-subunit TRAP, we demonstrate that removal of five C-terminal residues at the outer side of the inter-subunit axis or extension of an amino acid side chain at the opposite, inner side, increased the subunit number from 11 to 12. Our findings are supported by crystal structures of TRAP oligomers and by native mass spectrometry data.Conclusions/SignificanceThe subunit number of the TRAP oligomer can be manipulated by introducing deletion or addition mutations at the subunit interface. An analysis of available and emerging structural data on alternative oligomeric states indicates that the same principles may also apply to the subunit number of other circular assemblies suggesting that the deletion/addition approach could be used generally to engineer transitions between different oligomeric states.

Trp RNA-Binding Attenuation Protein: Modifying Symmetry and Stability of a Circular Oligomer

“…The scenario found in the 12-mer B. halodurans TRAP, regarding the exclusion of the C-terminus, could logically be mimicked by truncating the 4 or 5 C-terminal residues, removing this segment from the subunit interface. This is observed in the previously determined structures of B. stearothermophilus E71stop TRAP and B. subtilis K71stop TRAP [9]. With respect to thermal stability accompanying the 11-mer to 12-mer transition, we find that this truncation results in an increase in melting temperature, so that at 50 µM L-tryptophan the increase in melting temperature between B. stearothermophilus wild type and E71stop is 6°C, from 81.4°C to 87.4°C.…”

“…The tryptophan RNA-binding attenuation protein, TRAP, possesses similar geometric features to other circular assemblies, being assembled of multiple subunits and containing a central tunnel. Typically composed of 11 or 12 identical subunits, with a molecular weight of 90.6 to 101.9 kDa [8] , [9] , TRAP regulates L-tryptophan biosynthesis genes by attenuation in many Bacilli [10] . When activated by increased levels of L-tryptophan – bound to receptor sites nestled between adjacent subunits – TRAP binds single stranded RNA at the leader region of the trpEDCFBA operon transcript.…”

“…The presence of L-tryptophan – and hence the bound holo-TRAP state – has been associated with a greater structural rigidity and integrity [14] . Prior to the recent determination of the B. stearothermophilus apo-TRAP crystal structure [15] TRAP was crystallized in the presence of L-tryptophan [8] , [9] . We find that L-tryptophan binds with a K d in the 2.8 µM to 6.9 µM range, with small variation between TRAP from different species.…”

Influence of Structural Symmetry on Protein Dynamics

“…The smaller population of the 12-mer TRAP compared with the 11-mer is primarily attributed to subtle differences in the inter-subunit interactions. Antson et al [32] recently found that B. halodurans TRAP exclusively forms a 12-mer ring. The crystal structure of the 12-mer B. halodurans TRAP showed the C-terminal residues with a conformation different from those of the 11-mer TRAP of B. subtilis or B. stearothermophilus , which forms different interactions with the adjacent subunit allowing an increase in the diameter of the ring [32].…”

“…Antson et al [32] recently found that B. halodurans TRAP exclusively forms a 12-mer ring. The crystal structure of the 12-mer B. halodurans TRAP showed the C-terminal residues with a conformation different from those of the 11-mer TRAP of B. subtilis or B. stearothermophilus , which forms different interactions with the adjacent subunit allowing an increase in the diameter of the ring [32]. However, the present study shows that symmetry significantly influences dynamics, and should be another important factor for not only stability but also biological function (for example, ligand binding) of ring proteins, especially for large ring structures like the present case of C 11 and C 12 .…”