Biology subjects

Isiorho, E. A.

Publications and source records attributed to Isiorho, E. A..

4 recordsLinked to original sources

bioRxiv2026

Structural Basis of a Novel Heme Binding Bacterial One-Component Switch

One-component systems (OCSs) integrate sensory and effector functions within a single protein, enabling rapid gene expression changes in response to environmental cues. Here, we characterized a novel heme binding OCS protein, FG214, from Fimbriimonas ginsengisoli, a redox-regulated helix-turn-helix transcription factor in which heme iron ligand state controls a monomer-to-dimer switch. Data supporting this included our observation of the FG214 PAS domain binding a hexacoordinate heme b in oxidized conditions and undergoing a slate of redox and ligand-dependent conformational changes, transitioning from a monomer to a homodimer. Spectroscopic and structural data revealed that oxidation stabilizes the likely HTH-PAS intramolecular domain interface, while reduction of the heme iron dissociates the HTH, freeing previously-sequestered homodimerization surfaces. Similar effects were seen by addition of a small molecule ferric heme ligand, as directly visualized with a 1.47 [A] crystal structure of an imidazole-bound truncated construct. Using in vitro DNA-binding assays, we identified an artificial promoter sequence and demonstrated ligand-enhanced protein-DNA binding. Finally, we performed in vivo proof of concept experiments establishing FG214 as a redox-sensitive scaffold for biosensor engineering. Together, these findings define FG214 as a novel heme-binding PAS DNA binding protein, complementing known heme-PAS two-component signaling switches.

biochemistry↗

bioRxiv2025

Context-Dependent Variability Of HIF Heterodimers Influences Interactions With Macromolecular And Small Molecule Partners

Hypoxia inducible factors (HIFs) are transcription factors that coordinate cellular responses to low oxygen levels, functioning as an /{beta} heterodimer which binds a short hypoxia response element (HRE) DNA sequence. Prior studies suggest HIF/HRE complexes are augmented by the binding of additional factors nearby, but those interactions are not well understood. Here, we integrated structural and biochemical approaches to investigate several functionally relevant HIF assemblies with other protein, small molecule, and DNA partners. First, we used cryo-electron microscopy (cryo-EM) to establish HIF-1 and HIF-2 self-assemble to form novel "dimer-of-heterodimers" (DoHD) complexes on extended human EPO enhancer sequences, with one heterodimer bound at a canonical HRE site and the second binding in an inverted fashion to an HRE-adjacent sequence (HAS) 8 bp away. Consistent with ARNT PAS-B domains predominating interactions within a DoHD, we found HIF-1 and HIF-2 co-assemble mixed DoHD complexes on the same DNA. Second, we saw that despite the increased complexities of the larger complexes, ligands for the isolated ARNT or HIF-2 PAS-B domains are still capable of binding and disrupting both the heterodimer and DoHD complexes, albeit with variable potencies depending on the ligand. Finally, we combined cryo-EM and hydrogen- deuterium exchange by mass spectrometry (HDX-MS) to show how HIF-1 and HIF-2 heterodimers engage the transforming acidic coiled-coil containing protein 3 (TACC3) coactivator via both ARNT and HIF- subunits, though this was unseen in the larger DoHD. Our findings highlight the importance of both molecular context and dynamics in biomolecular complex formation, adding to the complexities of potential regulation. Significance StatementHypoxia inducible factors (HIFs) are transcription factors that regulate oxygen-dependent cellular processes with implications in certain types of cancers. Current molecular structures of HIFs bound to short DNA fragments provide insights into their function, but leave open questions about how they bind longer natural DNA fragments and interact with small molecules and protein coactivators. Integrating structural and biochemical techniques, we discovered a novel assembly in which two HIFs bind together on a single extended DNA fragment, forming a "dimer-of-heterodimers", which exhibits some differences in ligand and coactivator binding than heterodimers or isolated PAS domains. Our studies highlight how functional contexts can shift structural paradigms and provide greater insight into the mechanisms by which HIFs and similar bHLH-PAS transcription factors operate.

biochemistry↗

bioRxiv2025

The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

Eukaryotic elongation factor-2 kinase (eEF-2K), a member of the -kinase family, modulates translational rates by phosphorylating eEF-2, a GTPase that facilitates the translocation of the nascent chain on the ribosome during the elongation phase of protein synthesis. eEF-2K is regulated by diverse cellular cues, many of which sensitize it to the Ca2+-effector protein calmodulin (CaM). CaM, which binds and allosterically activates eEF-2K in the presence of Ca2+, contains two structural "lobes," each with a pair of Ca2+-binding EF-hands. Using kinetic analysis, we demonstrate that the isolated C-terminal lobe of CaM (CaMC) is sufficient to engage and fully activate eEF-2K in a Ca2+-dependent fashion. Genetically fusing CaMC to the N-terminus of eEF-2K, upstream of its critical CaM-targeting motif (CTM) via a flexible 2-glycine linker, results in a chimeric species (C-LiNK) that is constitutively active independent of external CaM and Ca2+. A structure of the C-LiNK functional core reveals no significant deviation in the overall conformations of the interacting modules and orientations of key catalytic-site residues relative to the heterodimeric complex between full-length CaM and eEF-2K. These observations demonstrate that, in contrast to other CaM-regulated kinases, CaMC alone is sufficient to activate eEF-2K fully. The proximity effect of CaMC in the context of C-LiNK removes the requirement for external Ca2+, whose apparent role is to enhance the CaM-affinity of eEF-2K and drive kinase activation. The responsiveness of eEF-2K to regulatory stimuli in cells appears to be lost in C-LiNK, presumably due to its permanently "on" state.

biochemistry↗

bioRxiv2024

Variations in kinase and effector signaling logic in a two component signaling network

The general stress response (GSR) protects bacteria from a wide range of stressors. In Alphaproteobacteria, GSR activation is coordinated by HWE/HisKA2 family histidine kinases (HKs), which can exhibit non-canonical structure and function. For example, while most light-oxygen-voltage sensor-containing HKs are light activated dimers, the Rubellimicrobium thermophilum RT-HK has inverted "dark on, light off" signaling logic with a tunable monomer/dimer equilibrium. Here, we further investigate these atypical behaviors of RT-HK and characterize its downstream signaling network. Using hydrogen-deuterium exchange mass spectrometry, we find that RT-HK uses a signal transduction mechanism similar to light-activated systems, despite its inverted logic. Mutagenesis reveals that RT-HK autophosphorylates in trans, with changes to the J helix linking sensor and kinase domains affecting autophosphorylation levels. Exploring downstream effects of RT-HK, we identified two GSR genetic regions, each encoding a copy of the central regulator PhyR. In vitro measurements of phosphotransfer from RT-HK to the two putative PhyRs revealed that RT-HK signals only to one, and does so at an increased intensity in the dark, consistent with its reversed logic. X-ray crystal structures of both PhyRs revealed a substantial shift within the receiver domain of one, suggesting a basis for RT-HK specificity. We probed further down the pathway using nuclear magnetic resonance to determine that the single NepR homolog interacts with both unphosphorylated PhyRs, and this interaction is decoupled from activation in one PhyR. This work expands our understanding of HWE/HisKA2 family signal transduction, revealing marked variations from signaling mechanisms previously identified in other GSR networks.

biochemistry↗