Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action
- Institut Pasteur de Montevideo, Uruguay
- Universidad de Buenos Aires, Argentina
Abstract
Two-component systems (TCS) are protein machineries that enable cells to respond to input signals. Histidine kinases (HK) are the sensory component, transferring information toward downstream response regulators (RR). HKs transfer phosphoryl groups to their specific RRs, but also dephosphorylate them, overall ensuring proper signaling. The mechanisms by which HKs discriminate between such disparate directions, are yet unknown. We now disclose crystal structures of the HK:RR complex DesK:DesR from Bacillus subtilis, comprising snapshots of the phosphotransfer and the dephosphorylation reactions. The HK dictates the reactional outcome through conformational rearrangements that include the reactive histidine. The phosphotransfer center is asymmetric, poised for dissociative nucleophilic substitution. The structural bases of HK phosphatase/phosphotransferase control are uncovered, and the unexpected discovery of a dissociative reactional center, sheds light on the evolution of TCS phosphotransfer reversibility. Our findings should be applicable to a broad range of signaling systems and instrumental in synthetic TCS rewiring.
Data availability
-
Crystal structure of phosphorylated DesKCPublicly available at the RCSB Protein Data Bank (accession no: 5IUM).
-
Crystal structure of the DesK-DesR complex in the phosphatase statePublicly available at the RCSB Protein Data Bank (accession no: 5IUN).
-
Crystal structure of the DesK-DesR complex in the phosphotransfer state with low Mg2+ (20 mM)Publicly available at the RCSB Protein Data Bank (accession no: 5IUJ).
-
Crystal structure of the DesK-DesR complex in the phosphotransfer state with high Mg2+ (150 mM)Publicly available at the RCSB Protein Data Bank (accession no: 5IUK).
-
Crystal structure of the DesK-DesR complex in the phosphotransfer state with high Mg2+ (150 mM) and BeF3Publicly available at the RCSB Protein Data Bank (accession no: 5IUL).
Article and author information
Author details
Alejandro Buschiazzo
Funding
Agencia Nacional de Investigación e Innovación (FCE2009_1_2679)
- Felipe Trajtenberg
- Alejandro Buschiazzo
Agence Nationale de la Recherche (PCV06_138918)
- Alejandro Buschiazzo
FOCEM (COF 03/11)
- Alejandro Buschiazzo
Centro de Biologia Estructural del Mercosur
- Alejandro Buschiazzo
Agencia Nacional de Investigación e Innovación (FCE2007_219)
- Felipe Trajtenberg
- Alejandro Buschiazzo
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2016, Trajtenberg et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action
eLife 5:e21422.
https://doi.org/10.7554/eLife.21422
Further reading
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Liquid-liquid phase separation (LLPS) involving intrinsically disordered protein regions (IDRs) is a major physical mechanism for biological membraneless compartmentalization. The multifaceted electrostatic effects in these biomolecular condensates are exemplified here by experimental and theoretical investigations of the different salt- and ATP-dependent LLPSs of an IDR of messenger RNA-regulating protein Caprin1 and its phosphorylated variant pY-Caprin1, exhibiting, for example, reentrant behaviors in some instances but not others. Experimental data are rationalized by physical modeling using analytical theory, molecular dynamics, and polymer field-theoretic simulations, indicating that interchain ion bridges enhance LLPS of polyelectrolytes such as Caprin1 and the high valency of ATP-magnesium is a significant factor for its colocalization with the condensed phases, as similar trends are observed for other IDRs. The electrostatic nature of these features complements ATP’s involvement in π-related interactions and as an amphiphilic hydrotrope, underscoring a general role of biomolecular condensates in modulating ion concentrations and its functional ramifications.
-
- Structural Biology and Molecular Biophysics
PROteolysis TArgeting Chimeras (PROTACs) are small molecules that induce target protein degradation via the ubiquitin-proteasome system. PROTACs recruit the target protein and E3 ligase; a critical first step is forming a ternary complex. However, while the formation of a ternary complex is crucial, it may not always guarantee successful protein degradation. The dynamics of the PROTAC-induced degradation complex play a key role in ubiquitination and subsequent degradation. In this study, we computationally modelled protein complex structures and dynamics associated with a series of PROTACs featuring different linkers to investigate why these PROTACs, all of which formed ternary complexes with Cereblon (CRBN) E3 ligase and the target protein bromodomain-containing protein 4 (BRD4BD1), exhibited varying degrees of degradation potency. We constructed the degradation machinery complexes with Culling-Ring Ligase 4A (CRL4A) E3 ligase scaffolds. Through atomistic molecular dynamics simulations, we illustrated how PROTAC-dependent protein dynamics facilitating the arrangement of surface lysine residues of BRD4BD1 into the catalytic pocket of E2/ubiquitin cascade for ubiquitination. Despite featuring identical warheads in this PROTAC series, the linkers were found to affect the residue-interaction networks, and thus governing the essential motions of the entire degradation machine for ubiquitination. These findings offer a structural dynamic perspective on ligand-induced protein degradation, providing insights to guide future PROTAC design endeavors.
