sspB structure and its conservation among C. crescentus, E. coli and H. influenzae ^[42.1]

The sspBα dimeric structure is stabilized by two α-helices in interaction, as part B of the figure above shows, each of them located at the N-terminus of either sspBα molecule. The subsequent parts of the protein form a domain consisting of two β-sheet structures, together building up the ssrA binding site. An unstructured area at the C-terminus being referred to as the XB module forms the ClpX binding part of the protein. It is connected to the rest of the molecule via a linker domain. ^[42.2]
Chien et al. ^[42.1] compared crystal structures of C. crescentus sspBα and its E. coli and H. influenzae sspB orthologs, discovering that in sspBα the α-helices are significantly longer, more twisted and cover a larger cross section area than the other two sspB orthologs. Also considering that β-sheets are rotated by around 20° in comparison to E. coli and H. influenzae orthologs, this leads to an antiparallel orientation of the two ssrA tagged protein bound to the ssrA binding sites of an sspBα dimer in C. crescentus, while they are parallel in γ-protobacterial sspB.

By measuring GFP fluorescence intensity, decrease of GFP-^CCssrA concentration (1) without sspBα added, (2) with mutated sspBα(Q74A) added , (3) with wildtype sspBα added can be visualized. ^[42.1]

Chien et al. point out that although there are the remarkable differences in protein structure between sspBα and its γ-protobacterial ortholog, they show up with similar effectiveness in binding proteins tagged with the related ssrA peptide. But it turned out in their research that effectiveness of sspBα binding to the protein which needs to be tethered to the ClpXP protease strongly depends on which ssrA ortholog the protein is tagged with. sspBα binds firmly to ^CCssrA, with an affinity being 175 times as large as for binding to ^ECssrA (i.e. the E. coli ortholog). By comparing the crystal structures of both sspBα and the compound of sspBα and ^CCssrA, Chien et al. further proved that binding of sspBα to ^CCssrA does not lead to significant changes of its 3D conformation.

References

[42.1] Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918
[42.2] Bivalent tethering of sspB to ClpXP is required for efficient substrate delivery: a protein design study, Bolon DN et al., Mol Cell, 2004, PMID: 14967151

Template:Team:Bonn:NetworkData

From 2013.igem.org