Biology subjects

Hunte, C.

Publications and source records attributed to Hunte, C..

6 recordsLinked to original sources

bioRxiv2026

Modular assembly of mitochondrial complex III from a dynamic matrix domain pool

Mitochondrial complex III is a central component of the cellular energy metabolism. It is an obligate dimer of catalytic and accessory subunits that are encoded by two genomes. How its large matrix domain, formed by the core proteins Qcr1 and Qcr2, is built and joined to the membrane part and during assembly is not known. Here we show that Qcr1 and Qcr2 preassemble independently of complex III into matrix domain modules. Cryo-EM structures from a cardiolipin-deficient yeast strain and of recombinant protein resolved single, double and triple matrix domains, which are linked by the C-terminal tail of Qcr1. In the mature complex III, the same tail binds cytochrome b and cardiolipin. The modules form a dynamic pool, in which assembly species interconvert and exchange subunits. Mutations at the module interfaces, including mimics of human disease variants, reshape this pool and deletion of the Qcr1 tail impairs respiratory growth. Substitution of a tyrosine residue linked to parkinsonism exposes a conserved cysteine residue to oxidation. A cryo-EM structure of an assembly intermediate shows a cap of two matrix domains bound to a monomeric membrane module of cytochrome b, Qcr7, Qcr8 and cytochrome c1. We propose that complex III receives its matrix domain from a dynamic pool of prebuilt modules, which organizes the dimerization of the complex.

biochemistry↗

bioRxiv2026

Human NHE1 binds full-length calcineurin with high affinity and inhibits substrate dephosphorylation

Calcineurin (Cn) interacts with the Na+/H+-exchanger NHE1 through its calcineurin-binding domain (NHE1-CnBD). How activation of full-length Cn influences this interaction and its functional consequences has remained unclear. Here, we quantified the interaction between full-length human Cn and NHE1-CnBD in distinct activation states using fluorescence anisotropy and quantitative size-exclusion chromatography. Ca2+/calmodulin activation increased Cn affinity for NHE1-CnBD from submicromolar to tens of nanomolar affinity and stabilized the complex. NHE1-CnBD outcompeted the immunosuppressive cyclosporin A/cyclophilin A complex for Cn binding and selectively inhibited dephosphorylation of a phosphopeptide substrate while enhancing hydrolysis of p-nitrophenyl phosphate. These findings identify NHE1 as a high-affinity, substrate-competitive Cn regulator that blocks substrate recognition rather than catalysis, providing a mechanistic framework for reciprocal regulation between Cn and NHE1.

biochemistry↗

bioRxiv2026

Biomolecular condensate architecture of an autophagic cargo at molecular resolution in situ

Biomolecular condensates organise cellular biochemistry, yet their molecular architecture in situ remains poorly understood. During selective autophagy, macromolecules frequently accumulate into biomolecular condensates, forming discrete entities for autophagic engulfment and degradation - ideal systems for structural analysis. We employed in situ cryo-electron tomography to determine the near-atomic resolution structure of Aminopeptidase 1 condensates within cells. These condensates form densely packed, spherical assemblies with amorphous organisation and liquid-like properties, elucidating the requirements of a selective autophagic cargo for exclusive targeting. Structural analysis and multiscale simulations reveal that the short, transient -helical structures in the disordered N-terminus of Aminopeptidase 1 enable site-specific, coiled-coil-like interactions required for condensate formation and properties. A single point mutation that increases -helical propensity directly modulates condensate viscosity and dynamics from a liquid-like to a glass-like state, while preserving local molecular packing. Our results demonstrate that disordered regions encode both specificity and material properties through transiently structured motifs, linking sequence specificity to phase behaviour in cells and expanding the molecular logic of phase separation in cells.

cell biology↗

bioRxiv2025

Molecular determinants of selective and high-affinity binding of the scaffold protein PDZK1 to the transporter URAT1

The renal solute carrier URAT1 (SLC22A12) is essential for urate homeostasis, with loss-of-function linked to renal hypouricemia, nephrolithiasis and lower gout risk. URAT1 function depends on binding the multi-PDZ domain scaffold protein PDZK1 (NHERF3), with a similar role suggested for the related NHERF1. The molecular basis of these interactions remains poorly understood. Using fluorescence anisotropy, we show that full-length human PDZK1 binds the C-terminal peptide of URAT1 with high affinity (KD 170 nM), unlike NHERF1 (KD >70 {micro}M). The PDZ1 domain of PDZK1 alone is sufficient for high-affinity binding (KD 160 nM), while PDZ4 provides a secondary site (KD 1.35 {micro}M), with both interactions characterized by rapid kinetics. Gel filtration shows that PDZK1 can bind two URAT1 peptides. X-ray structures of individual PDZ domains from PDZK1 and NHERF1 complexed with the URAT1 peptide reveal the molecular determinants for PDZK1s higher affinity and selectivity. Murine Pdzk1 and Nherf1 bind Urat1 with high affinity indicating species-specific interactions. These data provide insights into URAT1 regulation by PDZ scaffold proteins with relevance for understanding urate homeostasis regulation and related disorders.

biochemistry↗

bioRxiv2022

Conformational dynamics and target-dependent myristoyl switch of calcineurin B homologous protein 3

Calcineurin B homologous protein 3 (CHP3) is an EF-hand Ca2+-binding protein involved in regulation of cancerogenesis, cardiac hypertrophy and neuronal development via interactions with sodium/proton exchangers (NHEs) and signalling proteins. CHP3 binds Ca2+ with micromolar affinity providing the basis to respond to intracellular Ca2+ signals. Ca2+ binding and myristoylation are important for CHP3 function but the underlying molecular mechanism remained elusive. Here, we show that Ca2+ binding and myristoylation independently affect conformational dynamics and functions of human CHP3. Ca2+ binding increased flexibility and hydrophobicity of CHP3 indicative of an open conformation. CHP3 in open Ca2+-bound conformation had higher affinity for NHE1 and associated stronger with lipid membranes compared to the closed Mg2+-bound conformation. Myristoylation enhanced flexibility of CHP3 and decreased its affinity to NHE1 independently of the bound ion, but did not affect its binding to lipid membranes. The data exclude the proposed Ca2+-myristoyl switch for CHP3. Instead, they document a Ca2+-independent exposure of the myristoyl moiety induced by binding of the target peptide to CHP3 enhancing its association to lipid membranes. We name this novel regulatory mechanism "target-dependent myristoyl switch". Taken together, the interplay of Ca2+ binding, myristoylation and target binding allows for a context-specific regulation of CHP3 functions.

biochemistry↗

bioRxiv2022

Remorin proteins serve as membrane topology scaffolds in plants

Organization of membrane topologies in plants has so far been mainly attributed to the cell wall and the cytoskeleton. Taking rhizobial infections of legume root cells, where plasma membranes undergo dynamic and large-scale topology changes, as an initial model, we challenged this paradigm and tested whether additional scaffolds such as plant-specific remorins that accumulate on highly curved and often wall-less plasma membrane domains, control local membrane dynamics. Indeed, loss-of-function mutants of the remorin protein SYMREM1 failed to develop stabilized membrane tubes as found in colonized cells in wild-type plants, but released empty membrane spheres instead. Expression of this and other remorins in wall-less protoplasts allowed engineering different membrane topologies ranging from membrane blebs to long membrane tubes. Reciprocally, mechanically induced membrane indentations were equally stabilized by SYMREM1. This function is likely supported by remorin oligomerization into antiparallel dimers and the formation of higher order membrane scaffolding structures. Taken together we describe an evolutionary confined mechanism that allows the stabilization of large-scale membrane conformations and curvatures in plants. One-sentence summaryThe remorin SYMREM1 evolved as structural membrane scaffold that stabilizes membrane tubulation and curvature during symbiotic intracellular infections.

plant biology↗