Biology subjects

Itzen, A.

Publications and source records attributed to Itzen, A..

6 recordsLinked to original sources

bioRxiv2026

The Shewanella oneidensis Fic enzyme SoFic targets the switch-Iregion of EF-Tu for AMPylation

Fic enzymes mediate diverse post-translational modifications, including adenosine monophosphate (AMP) transfer and removal, referred to as AMPylation and deAMPylation, respectively. We identified the prokaryotic translation elongation factor Tu (EF-Tu) as an AMPylation target of the Fic enzyme SoFic. SoFic can constitutively reverse EF-Tu modification via deAMPylation whereas AMPylation depends on SoFic homodimerization. The complex crystal structure between SoFic and EF-Tu confirms a conserved target binding mode across evolutionary distant Fic enzymes. AMPylation disrupts EF-Tus regulatory switch-I region, causing translational inhibition. SoFic furthermore binds to its promotor DNA, suggesting a dual function as transcriptional and translational regulator in bacterial cells. Together, our structural and biochemical data provide valuable insights into the functional and regulatory diversity of Fic enzymes.

biochemistry↗

bioRxiv2026

The Legionella phosphocholinase AnkX modulates IMPDH2 regulation and cytoophidia dynamics

Legionella pneumophila, the causative agent of Legionnaires disease, secretes more than 300 different effector proteins into the host cell to modulate processes such as signal transduction, membrane dynamics, and metabolism. A key regulator of cellular metabolism and proliferation is inosine 5-monophosphate dehydrogenase type II (IMPDH2), which catalyzes the rate-limiting step of de novo GTP biosynthesis. IMPDH2 assembles into filaments and larger structures known as cytoophidia, which enable fine-tuned regulation of enzymatic activity in response to changing nucleotide demands. Here, we identify human IMPDH2 as a previously unrecognized host target of the Legionella effector AnkX. We show that AnkX post-translationally modifies IMPDH2 within its regulatory domain by covalently attaching a phosphocholine moiety. While this post-translational modification does not alter the catalytic activity of IMPDH2, it disrupts filament formation, thereby impairing nucleotide-dependent regulation of enzyme activity. Consequently, AnkX-mediated phosphocholination affects IMPDH2 filament assembly, cytoophidia formation, and subcellular localization to the specific Legionella-containing vacuole (LCV). Thus, L. pneumophila subverts host GTP metabolism by posttranslationally modifying a central enzyme of the GTP biosynthetic pathway. Graphical abstract description O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/701698v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@18f7724org.highwire.dtl.DTLVardef@1d70e86org.highwire.dtl.DTLVardef@1fa8890org.highwire.dtl.DTLVardef@11bc5ba_HPS_FORMAT_FIGEXP M_FIG C_FIG The Legionella effector AnkX post-translationally modifies the human host enzyme IMPDH2 by transferring a phosphocholine group using CDP-choline as co-substrate. We identified the modification site and discovered that this post-translational modification disrupts the ability of IMPDH2 to assemble into filaments and cytoophidia. By shifting the equilibrium toward free octamers, AnkX-mediated PCylation perturbs the structural regulation of IMPDH2 and alters GTP metabolism in L. pneumophila-infected host cells. This figure was inspired by (https://www.biorxiv.org/content/10.1101/2024.07.29.605679v2).

microbiology↗

bioRxiv2024

Resolving the Dynamic Interplay of the Conformational States in the TPR Domain of Human AMPylase FicD

The human Fic-enzyme FicD plays an important role in regulating the Hsp70 homolog BiP in the endoplasmic reticulum (ER): FicD reversibly modulates BiPs activity through attaching an adenosine monophosphate (AMP) to the substrate binding domain. This reduces BIPs chaperone activity by shifting it into a conformation with reduced substrate affinity. Crystal structures of FicD in the apo, ATP-bound, and BiP-bound states revealed significant conformational variability in the tetratricopeptide repeat (TPR) motifs. In this study, we investigate the conformational dynamics of FicDs TPR motifs using two-color single-molecule Forster resonance energy transfer (smFRET). We demonstrate that the TPR motifs exhibit conformational dynamics between the TPR-out and TPR-in conformations on timescales ranging from milliseconds to microseconds. In addition, we extend our investigation on multiple labeling positions within FicD, revealing how conformational dynamics vary depending on the location within the TPR motif. We quantify the motions with dynamic photon distribution analysis (PDA) for the FRET constructs and propose a conformational landscape model for FicD where the TPR-in/out states exist in an equilibrium that is altered due to the presence of ATP and BiP.

biophysics↗

bioRxiv2020

Monoclonal anti-AMP-antibodies reveal broad and diverse AMPylation patterns in cancer cells

AMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recent progress in the field reveals an emerging role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis, however, generic tools to study AMPylation are required. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse which are capable of protein backbone independent recognition of AMPylation, in denatured (Western Blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, the anti-AMP mABs will aid the advancement of understanding AMPylation and the spectrum of modified targets.

biochemistry↗

bioRxiv2019

PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2 mediated phosphorylation at Threonine72

Loss of function mutations in the PINK1 kinase are causal for autosomal recessive Parkinson disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2. Strikingly we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and NMR structure of Ser111-phosphorylated Rab1B that does not demonstrate any major changes suggesting that the phosphorylated SF3 motif may disrupt effector-Switch II interactions. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A suggesting that small molecule activators of PINK1 may have therapeutic potential in patients harbouring LRRK2 mutations.

biochemistry↗

bioRxiv2019

Phosphorylation of Ser111 in Rab8a modulates Rabin8 dependent activation by perturbation of side chain interaction networks

GTPases are key-players in cellular signaling processes. Phosphorylation of Rab proteins, which belong to the Ras superfamily of small GTPases regulating intracellular transport, has recently been implicated in the pathogenesis of Parkinson Disease (PD). For Rab8a, it was shown that serine 111 phosphorylation (pS111) is dependent on the protein kinase PINK1, and that mimicking the phosphorylation at S111 by a serine/glutamate substitution (S111E) impaired Rab8a activation by its cognate nucleotide exchange factor (GEF) Rabin8. Here, we performed comparative Molecular Dynamics and free energy simulations on Rab8a and Rab8a:Rabin8 complexes to elucidate the molecular details on how pS111 and S111E may influence the interaction with Rabin8. The simulations indicate that S111E and pS111 establish an intramolecular interaction with arginine 79 (R79). In the complex, this interaction persists, and therefore perturbs a favorable intermolecular salt-bridge contact between R79 in Rab8a and the acidic aspartate 187 (D187) in Rabin8. Binding free analysis reveals that S111E and pS111, as well as the mutation R79A, in Rab8a drastically reduce the binding affinity to Rabin8. Combining the R79A mutation with S111E or pS111, respectively, nearly diminishes Rab8a-Rabin8 binding. In vitro experiments confirm our computational results showing that the nucleotide exchange rates of the respective Rab8a mutants are decreased by >80% in the presence of Rabin8 compared to wild type. In addition to specific insights into how S111 phosphorylation of Rab8a can influence GEF-mediated activation, the simulations demonstrate how side chain modifications in general can allosterically influence the network of surface side chain interactions between binding partners.

biophysics↗