Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography   

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Dec 2016


We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR complexes and further purification. The 3D structures of the complex trapped in the phosphotransferase and in the phosphatase reaction steps, were solved by X-ray crystallography using molecular replacement. The solution was not trivial, and we found that in silico-generated models used as search probes, were instrumental to succeeding in placing a large portion of the complex in the asymmetric unit. Electron density maps were then clear enough to allow for manual model building attaining complete atomic models. These methods contribute to tackling a major challenge in the bacterial signaling field, namely obtaining stable kinase:regulator complexes, in distinct conformational states, amenable for high-resolution crystallographic studies.

Keywords: Signaling proteins, Protein phosphorylation, Trapping conformational rearrangements, Structure-based mutagenesis, X-ray crystallography, Protein engineering


Structural information about bacterial signaling complexes, especially of two-component systems (TCSs), is still scarce (Casino et al., 2009; Gao and Stock, 2009). TCSs comprise a sensory histidine-kinase (HK) and a response regulator (RR) partner, present in almost all bacteria, they allow the cells to perceive the environment and to react accordingly through adaptive responses. Structural information is even more limited when it comes to TCS complexes adopting different functional states, despite the importance of such switching mechanism in signal transmission (Trajtenberg et al., 2016). We have studied the DesK-DesR pathway (de Mendoza, 2014), a TCS from Bacillus subtilis involved in regulating the cell membrane composition in adaptation to cues that reduce the bilayer’s fluidity, such as cold shock.

The protocols we have developed were aimed at overcoming major technical bottlenecks, encompassing complex purification, crystallization and X-ray structure determination. Most of these hurdles likely arise from the intrinsic flexibility and heterogeneity that characterize TCS proteins. With the purpose of trapping the DesK:DesR complex in defined signaling steps, it is useful to recall some details based on previous findings from our laboratory. The protocols have been developed to work with DesKC, a truncated DesK variant comprising the entire cytoplasmic region of DesK, without the trans-membrane sensory domain, which is catalytically competent to phosphotransfer to DesR, as well as to dephosphorylate P~DesR (Albanesi et al., 2004). As for the response regulator partner, DesR, we have chosen to use a truncated form, including the entire receiver domain (REC), competent for all DesK-mediated phosphotransfer reactions (Trajtenberg et al., 2014), but lacking the C-terminal DNA-binding domain, and thus minimizing potential inter-domain flexibility issues.

In order to trap the DesKC:DesR complex in the phosphotransfer step of the signaling pathway, we chose to use the phosphomimetic point mutant DesKC-His188Glu. This variant, when not bound to DesR, adopts a structural conformation very similar to the phosphorylated form of wild-type DesKC (Albanesi et al., 2009), hence an attractive template to mimic the phosphorylated HK just prior to the transfer reaction, also avoiding effective transfer to take place.

On the other hand, in order to trap the DesKC:DesR complex in the dephosphorylation step, previous work was instrumental by uncovering a switch mechanism of DesK, swapping between ‘active’ (kinase-on/phosphatase-off) and ‘inactive’ (phosphatase-on/kinase-off traits) states of the kinase (Albanesi et al., 2009). Briefly, the conformational transition of DesK from its kinase-active to the inhibited form, implicates the assembly of a coiled-coil structure within the central Dimerization and His-phosphotransfer (DHp) domain, a coiled-coil that is otherwise ‘broken’ when the kinase is active. The DHp, an all-helical domain, connects the trans-membrane sensor with the Catalytic ATP-binding (CA) domains, hence the identified DHp’s conformational switching plays a key role in signal transmission through long-range allosteric rearrangements. Such mechanistic insights later led to constructing a coiled-coil hyper-stabilized variant (DesKSTA) (Saita et al., 2015), harboring point-mutations at key positions (Ser150Ile, Ser153Leu and Arg157Ile) that stabilize a phosphatase-constitutive form (Saita et al., 2015). The corresponding soluble construct, with the trans-membrane domain truncated (DesKCSTAB), indeed displays a phosphatase-trapped 3D structure (Trajtenberg et al., 2016). DesKCSTAB was used to trap the DesKC:DesR complex in the dephosphorylation step, as described in this protocol.

Copyright Imelio et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Imelio, J. A., Larrieux, N., Mechaly, A. E., Trajtenberg, F. and Buschiazzo, A. A. (2017). Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography. Bio-protocol 7(16): e2510. DOI: 10.21769/BioProtoc.2510.
  2. Trajtenberg, F., Imelio, J. A., Machado, M. R., Larrieux, N., Marti, M. A., Obal, G., Mechaly, A. E. and Buschiazzo, A. (2016). Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action. Elife 5.

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