4d7q
From Proteopedia
Crystal structure of a chimeric protein with the Sec7 domain of Legionella pneumophila RalF and the capping domain of Rickettsia prowazekii RalF
Structural highlights
FunctionPublication Abstract from PubMedA convenient strategy to interrogate the biology of regulatory proteins is to replace individual domains by an equivalent domain from a related protein of the same species or from an ortholog of another species. It is generally assumed that the overall properties of the native protein are retained in the chimera, and that functional differences reflect only the specific determinants contained in the swapped domains. Here we used this strategy to circumvent the difficulty in obtaining crystals of Rickettsia prowazekii RalF, a bacterial protein that functions as a guanine nucleotide exchange factor for eukaryotic Arf GTPases. A RalF homolog is encoded by Legionella pneumophila, in which a C-terminal capping domain auto-inhibits the catalytic Sec7 domain and localizes the protein to the Legionella-containing vacuole. The crystal structures of domain-swapped chimeras were determined and used to construct a model of Legionella RalF with a RMSD of less than 1A with the crystal structure, which validated the use of this approach to build a model of Rickettsia RalF. In the Rickettsia RalF model, sequence differences in the capping domain that target it to specific membranes are accommodated by a shift of the entire domain with respect to the Sec7 domain. However, local sequence changes also give rise to an artifactual salt bridge in one of the chimeras, which likely explains why this chimera is recalcitrant to activation. These findings highlight the structural plasticity whereby chimeras can be engineered, but also underline that unpredictable differences can modify their biochemical responses. On the use of Legionella/Rickettsia chimeras to investigate the structure and regulation of Rickettsia effector RalF.,Folly-Klan M, Sancerne B, Alix E, Roy CR, Cherfils J, Campanacci V J Struct Biol. 2014 Dec 9. pii: S1047-8477(14)00264-0. doi:, 10.1016/j.jsb.2014.12.001. PMID:25498244[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. Loading citation details.. Citations 4 reviews cite this structure No citations found References
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