SOLEIL II - Come to SOLEIL
- Phonebook & CONTACTS
In experiments carried out at the PROXIMA 2A and SWING beamlines at SOLEIL scientists from the Institut de Biology et Chemistry des Protéines (IBCP) and the International Center for Infectiology Research in Lyon in collaboration with researchers from the University of Leicester in the UK, have unravelled one of the strategies Legionella use to successfully invade host cells.
Legionnaires' disease (also known as Legion Fever) is an aggressive form of pneumonia caused by Legionella bacteria. These bacteria are water-borne and often found in urban aquatic systems or water storage tanks. During infection the bacterium invades lung epithelial cells and macrophages, and reproduces rapidly within infected cells.
Eukaryotic cells use protein phosphorylation cascades as switches to control cellular processes, and as sensing, or relay « signalling » pathways. Recently, bacterial pathogens have been shown to subvert these signalling pathways to promote invasion and replication by injecting proteins that phosphorylate and modify the activity of host cell proteins. A group led by Laurent Terradot (IBCP, Lyon) solved the crystal structure of an active form of one such protein LegK4 using data collected at the microfocus beamline Proxima-2A.
A novel dimeric form stabilizes essential catalytic elements
The structure reveals an atypical eukaryotic-like kinase domain with some remarkable adaptations. The protein has several unique features, and self-assembles to generate a dimeric interface not previously observed in the protein kinase super-family. This novel dimerization mode is stabilized by a conserved alpha helical segment (αG), which usually plays an important role in anchoring the activation-loop during protein substrate binding in kinases. The dimerization model was confirmed by small angle X-ray scattering (SAXS) at SWING. Complementary enzymatic studies demonstrate that LegK4 is a constitutively active enzyme and the structural data suggests that the dimer assembly stabilizes the active conformation in the absence of phosphorylation.
Impact
Bacterial kinase effectors such as LegK4, represent a common strategy used by pathogens to subvert host cell defences. While related proteins identified so far contained minimal kinase catalytic domains, the structure of LegK4 published in Scientific Reports is more similar to classic eukaryotic kinases albeit with some remarkable adaptations. Notably, nucleotide binding does not involve the canonical glycine-rich activation loop, but is instead mediated by unusual residues in nearby structural elements.
The newly described dimer and atypical activation loop suggest a structural explanation for the constitutive activity of LegK4. Sequence comparisons indicate that some of these elements may be shared with LegK1 and other members of this protein family.
The structure also points toward specific mechanisms of kinase regulation that could be exploited by the bacteria to re-route different host pathways. While a number of these questions remain open, this study paves the way for further structural and mechanistic studies aiming at understanding the role of eukaryotic-like kinases during bacterial infection.
Figure : LegK41–445 dimer organisation.
(a) Side (upper panel) and top (lower panel) views of the dimer with chain A coloured in grey and shown as a surface and chain B coloured in blue and shown as ribbon. The cap and FHB domains of the two chains are coloured in pink and yellow, respectively.
(b) Comparison of the experimental SAXS curve of apo-LegK41–445 (black) with theoretical SAXS curves of apo-LegK41–445 monomer (orange), dimer (magenta) and AMP-PNP•LegK41–445 dimer (blue). χ2 obtained with FOXS server51 are indicated.
(c) Detailed view of the dimer interface coloured as in (a) with participating residues shown as ball and stick.