Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Salmonella effector kinase SteC is activated by host-mediated phosphorylation

ABSTRACT: The pathogen Salmonella, which causes significant human morbidity and mortality, encodes an effector kinase, SteC, which mediates actin polymerisation and cell migration of the infected cell. Analysis of the sequence and predicted structure alongside canonical eukaryotic serine-threonine kinases raises the questions of how it is catalytically active and how this activity is regulated. Here, we reveal that SteC is activated following the phosphorylation of a highly conserved residue, S379, by a host kinase. This induces a dramatic increase in SteC nucleotide binding affinity, providing an explanation for S379’s requirement for substrate phosphorylation and actin polymerisation. Further mutational analysis revealed HD and DGD motifs within the depleted C lobe that are important for function, and may represent non-canonical mimics of HxD and DFG motifs in eukaryotic serine/threonine kinases. Meanwhile, the C-tail of SteC, encompassing amino acids 429-457, is essential for function following translocation from Salmonella, even though it is not required for catalysis in vitro. Overall, our findings uncover two previously unappreciated mechanisms that mediate the activity of the only Salmonella effector kinase within the host.

INSTRUMENT(S):

ORGANISM(S): Salmonella Enterica Subsp. Enterica Serovar Typhimurium Str. T000240

SUBMITTER: Sarah Maslen

LAB HEAD: Dr. Katrin Rittinger

PROVIDER: PXD061217 | Pride | 2026-06-12

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	SteC_State.csv	Csv
	Timesh_SteC.zip	Other

Items per page:

1 - 2 of 2

Similar Datasets

Bacterial effectors mediate kinase reprogramming through a series of evolutionary conserved amino acids that mimic eukaryotic motifs

Project description:Bacteria have evolved numerous biochemical processes that underpin their biology and pathogenesis. The small, non-enzymatic bacterial (Salmonella) effector SteE mediates kinase reprogramming, whereby the canonical serine/threonine host kinase GSK3 gains tyrosine-directed activity towards neosubstrates, promoting Salmonella virulence. Yet, both the mechanism behind the switch in GSK3’s activity and the diversity of this phenomenon remain to be determined. Here we show that kinase reprogramming of GSK3 is mediated by putative homologues from diverse Gram-negative pathogens. Next, we identify both the molecular basis of how SteE targets GSK3 and then uncover that the SteE-induced tyrosine activity conferred on GSK3 occurs by mimicry of an L/xGxP motif, previously shown to mediate GSK3 autophosphorylation on a tyrosine. Together, we demonstrate how a family of intrinsically disordered proteins mediate kinase reprogramming through the molecular mimicry of eukaryotic short linear motifs. With these advances comes the potential for the rationale design of synthetic reprogramming proteins

2025-04-25 | PXD058368 | Pride

Structural bases for integrase-RNA binding and retention of viral RNA within HIV-1 capsid cores

Project description:HIV-1 integrase (IN) promotes encapsulation of viral genomic RNA into mature viral cores, and this function is a target for ongoing antiretroviral drug development efforts. We determined a cryo-EM structure of a primate lentiviral IN complexed with RNA, which revealed a linear filament made of IN octamer repeat units, each comprising a pair of asymmetric homotetramers. The assembly is stabilized through IN-RNA interactions primarily involving the IN C-terminal domains and RNA backbone. Remarkably, the spacing and orientation of the IN filament repeat units closely matched those of consecutive capsid (CA) hexamers within the mature CA lattice. Using cryo-EM images of native purified HIV-1 cores, we refined the structure of the IN filament as it propagates along the luminal side of the CA lattice. Each IN tetramer within the filament nestled in a CA hexamer, engaging closely with the major homology regions. Substitutions of residues involved in IN-CA contacts yielded eccentric virions with RNA nucleoids located outside of the cores. Collectively, our results establish the structural basis for the HIV 1 IN-RNA interaction and reveal that IN forms an RNA-binding module on the luminal side of the mature CA lattice.

2025-12-04 | PXD070910 | Pride

Structural and mechanistic insights into the CAND1-mediated SCF substrate receptor exchange

Project description:Modular SCF (SKP1-CUL1-Fbox) ubiquitin E3 ligases orchestrate multiple cellular pathways in eukaryotes. Their variable SKP1-Fbox substrate receptor (SR) modules enable regulated substrate recruitment and subsequent proteasomal degradation. CAND proteins are essential for the efficient and timely exchange of SRs. To gain structural understanding of the underlying molecular mechanism, we reconstituted a CAND1-driven exchange reaction of substrate-bound SCF alongside its co-E3 ligase DCNL1 and visualised it by cryo-EM. We describe high-resolution structural intermediates including a ternary CAND1-SCF complex, as well as conformational and compositional intermediates representing SR- or CAND1-dissociation. We describe in molecular detail how CAND1-induced conformational changes in CUL1/RBX1 provide an optimised DCNL1 binding site and reveal an unexpected dual role for DCNL1 in CAND1-SCF dynamics. Moreover, a partially dissociated CAND1-SCF conformation accommodates cullin neddylation, leading to CAND1 displacement. Our structural findings, together with functional biochemical assays help formulate a detailed model for CAND-SCF regulation.

2023-09-18 | PXD042178 | Pride

Recruitment of trimeric eIF2 by phosphatase non-catalytic subunit PPP1R15B

Project description:HDX-MS of R15B and it's interactions with trimeric eIF2 substrate. Here we integrated diverse approaches to elucidate how the PP1 non-catalytic subu-nit PPP1R15B (R15B) captures its full trimeric eIF2 substrate. We found that the substrate recruitment module of R15B is largely disordered with three short helical elements, H1, H2 and H3. H1 and H2 form a clamp that grasps the substrate in a re-gion remote from the phosphorylated residue. A homozygous N423D variant, adja-cent to H1, reducing substrate binding and dephosphorylation was discovered in a ra-re syndrome with microcephaly, development delay and intellectual disability. These findings explain how R15B captures its 125 kDa substrate by binding the far end of the complex relative to the phospho-site to present it for dephosphorylation by PP1, a paradigm of broad relevance.

2024-05-22 | PXD047538 | Pride

HDX-MS Analysis of CHK2 Incubated with Calmodulin

Project description:To determine the conformational dynamics upon binding of calmodulin to serine/threonine kinase CHK2.

2025-10-15 | PXD048473 | Pride

Droplet microfluidic hydrogen/deuterium exchange for investigating protein dynamics with millisecond precision exchange

Project description:Hydrogen/deuterium exchange (HDX) methods for studying protein dynamics would benefit from millisecond-scale incubations to probe intrinsically disordered proteins, highly dynamic regions and conformation changes. Here we investigate droplet microfluidics for rapid mixing to trigger D2O labelling, uniform incubations and rapid droplet merging for acid quenching in advance of mass spectrometry. A surfactant-free merging approach combining expansion elements for synchronised droplet collision proved robust. The high diffusive flux of D2O and protons enable microsecond mixing to trigger and arrest D2O labelling, respectively, affording the possibility of single millisecond incubations. Droplet HDX processors were used to measure the fast uptake characteristics of a model peptide. Forward exchange measurements demonstrate D2O labelling to be the rate-limiting step, in essence defining 10 milliseconds as the minimum practical incubation time for proteins in typical physiological conditions. With the ability to access millisecond time scales the fast dynamics of calmodulin, a model of calcium-triggered allostery with rapid conformational switching, was investigated. At 10 milliseconds, we could observe significant deuterium uptake within the well-defined EF-hand motifs (Ca2+ binding sites). These findings demonstrate millisecond HDX enabled by droplet microfluidics allows areas of heightened plasticity to be detected within a stably folded protein. Associated dataset: PXD077479

2025-06-23 | PXD063880 | Pride

HDX-MS on secondary transporters

Project description:Secondary transporters undergo structural rearrangements to catalyze substrate translocation across the cell membrane – yet how such conformational changes happen within a lipid environment remains poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS) with molecular dynamics (MD) simulations to understand how lipids regulate the conformational dynamics of secondary transporters at the molecular level. Using the homologous transporters XylE, LacY and GlpT from Escherichia coli as model systems, we discover that conserved networks of charged residues act as molecular switches that drive the conformational transition between different states. We reveal that these molecular switches are regulated by interactions with surrounding phospholipids and show that phosphatidylethanolamine interferes with the formation of the conserved networks and favors an inward-facing state. Overall, this work provides insights into the importance of lipids in shaping the conformational landscape of an important class of transporters.

2018-10-16 | PXD011060 | Pride

Droplet microfluidic hydrogen/deuterium exchange for investigating protein dynamics with millisecond precision exchange

2026-04-22 | PXD077479 | Pride

Activation loop phosphorylation tunes conformational dynamics underlying Pyk2 tyrosine kinase activation

Project description:Pyk2 is a multidomain non-receptor tyrosine kinase that undergoes a complex, multi-stage activation process. Ca2+-flux induces conformational rearrangements that relieve autoinhibitory FERM domain interactions. The kinase domain phosphorylates a key linker residue to recruit Src kinase. Pyk2 and Src mutually phosphorylate activation loop residues to confer full activation. While the mechanisms of autoinhibition are established, the conformational dynamics associated with autophosphorylation and Src recruitment remain unclear. Here, we employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map the conformational changes associated with Src-mediated activation segment phosphorylation in a Pyk2 construct encompassing FERM and kinase domains (residues 20-692). Results reveal increased dynamics at regulatory interfaces spanning FERM and kinase domains. Phosphorylation of the activation segment stabilizes two antiparallel beta strands linking activation and catalytic loops. Increased dynamics of the C-terminal end of the activation loop propagate to the F-helix, explaining how phosphorylation prevents reversion to the autoinhibitory FERM interaction. HDX-guided site-directed mutagenesis and kinase activity profiling establish a mechanism for phosphorylation-induced active site sculpting to confer high activity.

2023-03-08 | PXD034028 | Pride

Nanobody-mediated modulation of long RSH enzymes Rel and RelA catalysis by restriction of their conformational landscape

Project description:Long RSH enzymes, Rel and RelA, are the master regulators of (p)ppGpp alarmone levels in bacteria. Their catalytic activity is governed by transitions between a compact, hydrolysis-competent (HDON) state and an elongated, synthesis-competent (SYNTHON) state. The equilibrium between these states is modulated by factors such as “starved” ribosomes and regulatory proteins DarB, EIIANTR, ACP and YtfK. Here, we identify and characterize camelid nanobodies that act as selective allosteric modulators by trapping Rel/RelA enzymes in distinct conformational states. Nanobodies that lock the TGS domain of RelA and prevent its activation by deacylated tRNA on starved ribosomes, strongly inhibit (p)ppGpp synthesis and suppress the virulence of E. coli in an animal model. Nb898 stabilizes Rel in the open SYNTHON state, enhancing synthesis activity while suppressing hydrolysis. Conversely, Nb585 traps Rel in a HDON conformation, strongly inhibiting alarmone synthesis while promoting (p)ppGpp hydrolysis. Structural and biochemical analyses reveal that nanobodies, like natural allosteric regulators, act by restricting the RSH enzyme’s conformational landscape. These findings establish nanobodies as powerful tools for dissecting RSH function and provide potential leads for developing protein-based RSH modulators.

2026-04-28 | PXD067451 | Pride