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.
Highlights
Multisubunit circular proteins play key roles in a variety of biological mechanisms
For example for the viral portal proteins, a single functional oligomeric state is found in vivo and it is not known if the alternative oligomeric states are active
We examined the factors that define the oligomeric state in a circular protein oligomer by studying the Bacillus trp RNA-binding attenuation protein (TRAP)
Summary
A number of proteins including bacterial toxins [1], viral portal proteins [2], flagellar motor proteins [3,4], Sm-like proteins [5], components of the type III secretion system [6] and protective antigen of anthrax toxin [7,8] can form circular oligomers with alternative oligomeric states. We examined the factors that define the oligomeric state in a circular protein oligomer by studying the Bacillus trp RNA-binding attenuation protein (TRAP). 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. 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
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