Ferric reduction by a CYBDOM protein counteracts increased iron availability in root meristems induced by phosphorus deficiency.

To mobilize sparingly available phosphorus (P) in the rhizosphere, many plant species secrete malate to release P sorbed onto (hydr)oxides of aluminum and iron (Fe). In the presence of Fe, malate can provoke Fe over-accumulation in the root apoplast, triggering a series of events that inhibit root growth. Here, we identified HYPERSENSITIVE TO LOW P1 (HYP1), a CYBDOM protein constituted of a DOMON and a cytochrome b561 domain, as critical to maintain cell elongation and meristem integrity under low P. We demonstrate that HYP1 mediates ascorbate-dependent trans-plasma membrane electron transport and can reduce ferric and cupric substrates in Xenopus laevis oocytes and in planta. HYP1 expression is up-regulated in response to P deficiency in the proximal zone of the root apical meristem. Disruption of HYP1 leads to increased Fe and callose accumulation in the root meristem and causes significant transcriptional changes in roots. We further demonstrate that HYP1 activity overcomes malate-induced Fe accumulation, thereby preventing Fe-dependent root growth arrest in response to low P. Collectively, our results uncover an ascorbate-dependent metalloreductase that is critical to protect root meristems of P-deficient plants from increased Fe availability and provide insights into the physiological function of the yet poorly characterized but ubiquitous CYBDOM proteins.

DNA facilitates heterodimerization between human transcription factors FoxP1 and FoxP2 by increasing their conformational flexibility.

Transcription factors regulate gene expression by binding to DNA. They have disordered regions and specific DNA-binding domains. Binding to DNA causes structural changes, including folding and interactions with other molecules. The FoxP subfamily of transcription factors in humans is unique because they can form heterotypic interactions without DNA. However, it is unclear how they form heterodimers and how DNA binding affects their function. We used computational and experimental methods to study the structural changes in FoxP1's DNA-binding domain when it forms a heterodimer with FoxP2. We found that FoxP1 has complex and diverse conformational dynamics, transitioning between compact and extended states. Surprisingly, DNA binding increases the flexibility of FoxP1, contrary to the typical folding-upon-binding mechanism. In addition, we observed a 3-fold increase in the rate of heterodimerization after FoxP1 binds to DNA. These findings emphasize the importance of structural flexibility in promoting heterodimerization to form transcriptional complexes.

Effect of temperature and nucleotide on the binding of BiP chaperone to a protein substrate.

BiP (immunoglobulin heavy-chain binding protein) is a Hsp70 monomeric ATPase motor that plays broad and crucial roles in maintaining proteostasis inside the cell. Structurally, BiP is formed by two domains, a nucleotide-binding domain (NBD) with ATPase activity connected by a flexible hydrophobic linker to the substrate-binding domain. While the ATPase and substrate binding activities of BiP are allosterically coupled, the latter is also dependent on nucleotide binding. Recent structural studies have provided new insights into BiP's allostery; however, the influence of temperature on the coupling between substrate and nucleotide binding to BiP remains unexplored. Here, we study BiP's binding to its substrate at the single molecule level using thermo-regulated optical tweezers which allows us to mechanically unfold the client protein and explore the effect of temperature and different nucleotides on BiP binding. Our results confirm that the affinity of BiP for its protein substrate relies on nucleotide binding, by mainly regulating the binding kinetics between BiP and its substrate. Interestingly, our findings also showed that the apparent affinity of BiP for its protein substrate in the presence of nucleotides remains invariable over a wide range of temperatures, suggesting that BiP may interact with its client proteins with similar affinities even when the temperature is not optimal. Thus, BiP could play a role as a "thermal buffer" in proteostasis.

Guiding protein design choices by per-residue energy breakdown analysis with an interactive web application.

Recent developments in machine learning have greatly facilitated the design of proteins with improved properties. However, accurately assessing the contributions of an individual or multiple amino acid mutations to overall protein stability to select the most promising mutants remains a challenge. Knowing the specific types of amino acid interactions that improve energetic stability is crucial for finding favorable combinations of mutations and deciding which mutants to test experimentally. In this work, we present an interactive workflow for assessing the energetic contributions of single and multi-mutant designs of proteins. The energy breakdown guided protein design (ENDURE) workflow includes several key algorithms, including per-residue energy analysis and the sum of interaction energies calculations, which are performed using the Rosetta energy function, as well as a residue depth analysis, which enables tracking the energetic contributions of mutations occurring in different spatial layers of the protein structure. ENDURE is available as a web application that integrates easy-to-read summary reports and interactive visualizations of the automated energy calculations and helps users selecting protein mutants for further experimental characterization. We demonstrate the effectiveness of the tool in identifying the mutations in a designed polyethylene terephthalate (PET)-degrading enzyme that add up to an improved thermodynamic stability. We expect that ENDURE can be a valuable resource for researchers and practitioners working in the field of protein design and optimization. ENDURE is freely available for academic use at: http://endure.kuenzelab.org.

Structure and function of the metagenomic plastic-degrading polyester hydrolase PHL7 bound to its product.

The recently discovered metagenomic-derived polyester hydrolase PHL7 is able to efficiently degrade amorphous polyethylene terephthalate (PET) in post-consumer plastic waste. We present the cocrystal structure of this hydrolase with its hydrolysis product terephthalic acid and elucidate the influence of 17 single mutations on the PET-hydrolytic activity and thermal stability of PHL7. The substrate-binding mode of terephthalic acid is similar to that of the thermophilic polyester hydrolase LCC and deviates from the mesophilic IsPETase. The subsite I modifications L93F and Q95Y, derived from LCC, increased the thermal stability, while exchange of H185S, derived from IsPETase, reduced the stability of PHL7. The subsite II residue H130 is suggested to represent an adaptation for high thermal stability, whereas L210 emerged as the main contributor to the observed high PET-hydrolytic activity. Variant L210T showed significantly higher activity, achieving a degradation rate of 20 µm h-1 with amorphous PET films.

Structural basis of the activation of PPARγ by the plasticizer metabolites MEHP and MINCH.

Di-2-ethylhexyl phthalate (DEHP) and its substitute 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) are widely used as plasticizers but may have adverse health effects. Via hydrolysis of one of the two ester bonds in the human body, DEHP and DINCH form the monoesters MEHP and MINCH, respectively. Previous studies demonstrated binding of these metabolites to PPARγ and the induction of adipogenesis via this pathway. Detailed structural understanding of how these metabolites interact with PPARγ and thereby affect human health is lacking until now. We therefore characterized the binding modes of MINCH and MEHP to the ligand binding domain of PPARγ by X-ray crystallography and molecular dynamics (MD) simulations. Both compounds bind to the activating function-2 (AF-2) binding site via an interaction of the free carboxylates with the histidines 323 and 449, tyrosine 473 and serine 289. The alkyl chains form hydrophobic interactions with the tunnel next to cysteine 285. These binding modes are generally stable as demonstrated by the MD simulations and they resemble the complexation of fatty acids and their metabolites to the AF-2 site of PPARγ. Similar to the situation for these natural PPARγ agonists, the interaction of the free carboxylate groups of MEHP and MINCH with tyrosine 473 and surrounding residues stabilizes the AF-2 helix in the upward conformation. This state promotes binding of coactivator proteins and thus formation of the active complex for transcription of the specific target genes. Moreover, a comparison of the residues involved in binding of the plasticizer metabolites in vertebrate PPARγ orthologs shows that these compounds likely have similar effects in other species.

Antarctic Polyester Hydrolases Degrade Aliphatic and Aromatic Polyesters at Moderate Temperatures.

Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics in the packaging industry, and consequently has become one of the main components of plastic waste found in the environment. However, several microorganisms have been described to encode enzymes that catalyze the depolymerization of PET. While most known PET hydrolases are thermophilic and require reaction temperatures between 60°C and 70°C for an efficient hydrolysis of PET, a partial hydrolysis of amorphous PET at lower temperatures by the polyester hydrolase IsPETase from the mesophilic bacterium Ideonella sakaiensis has also been reported. We show that polyester hydrolases from the Antarctic bacteria Moraxella sp. strain TA144 (Mors1) and Oleispira antarctica RB-8 (OaCut) were able to hydrolyze the aliphatic polyester polycaprolactone as well as the aromatic polyester PET at a reaction temperature of 25°C. Mors1 caused a weight loss of amorphous PET films and thus constitutes a PET-degrading psychrophilic enzyme. Comparative modeling of Mors1 showed that the amino acid composition of its active site resembled both thermophilic and mesophilic PET hydrolases. Lastly, bioinformatic analysis of Antarctic metagenomic samples demonstrated that members of the Moraxellaceae family carry candidate genes coding for further potential psychrophilic PET hydrolases. IMPORTANCE A myriad of consumer products contains polyethylene terephthalate (PET), a plastic that has accumulated as waste in the environment due to its long-term stability and poor waste management. One promising solution is the enzymatic biodegradation of PET, with most known enzymes only catalyzing this process at high temperatures. Here, we bioinformatically identified and biochemically characterized an enzyme from an Antarctic organism that degrades PET at 25°C with similar efficiency to the few PET-degrading enzymes active at moderate temperatures. Reasoning that Antarctica harbors other PET-degrading enzymes, we analyzed available data from Antarctic metagenomic samples and successfully identified other potential enzymes. Our findings contribute to increasing the repertoire of known PET-degrading enzymes that are currently being considered as biocatalysts for the biological recycling of plastic waste.

Low Carbon Footprint Recycling of Post-Consumer PET Plastic with a Metagenomic Polyester Hydrolase.

Earth is flooded with plastics and the need for sustainable recycling strategies for polymers has become increasingly urgent. Enzyme-based hydrolysis of post-consumer plastic is an emerging strategy for closed-loop recycling of polyethylene terephthalate (PET). The polyester hydrolase PHL7, isolated from a compost metagenome, completely hydrolyzes amorphous PET films, releasing 91 mg of terephthalic acid per hour and mg of enzyme. Vertical scanning interferometry shows degradation rates of the PET film of 6.8 µm h-1 . Structural analysis indicates the importance of leucine at position 210 for the extraordinarily high PET-hydrolyzing activity of PHL7. Within 24 h, 0.6 mgenzyme gPET -1 completely degrades post-consumer thermoform PET packaging in an aqueous buffer at 70 °C without any energy-intensive pretreatments. Terephthalic acid recovered from the enzymatic hydrolysate is then used to synthesize virgin PET, demonstrating the potential of polyester hydrolases as catalysts in sustainable PET recycling processes with a low carbon footprint.

Polycystin-1 Assembles With Kv Channels to Govern Cardiomyocyte Repolarization and Contractility.

BACKGROUND: Polycystin-1 (PC1) is a transmembrane protein originally identified in autosomal dominant polycystic kidney disease where it regulates the calcium-permeant cation channel polycystin-2. Autosomal dominant polycystic kidney disease patients develop renal failure, hypertension, left ventricular hypertrophy, and diastolic dysfunction, among other cardiovascular disorders. These individuals harbor PC1 loss-of-function mutations in their cardiomyocytes, but the functional consequences are unknown. PC1 is ubiquitously expressed, and its experimental ablation in cardiomyocyte-specific knockout mice reduces contractile function. Here, we set out to determine the pathophysiological role of PC1 in cardiomyocytes. METHODS: Wild-type and cardiomyocyte-specific PC1 knockout mice were analyzed by echocardiography. Excitation-contraction coupling was assessed in isolated cardiomyocytes and human embryonic stem cell-derived cardiomyocytes, and functional consequences were explored in heterologous expression systems. Protein-protein interactions were analyzed biochemically and by means of ab initio calculations. RESULTS: PC1 ablation reduced action potential duration in cardiomyocytes, decreased Ca2+ transients, and myocyte contractility. PC1-deficient cardiomyocytes manifested a reduction in sarcoendoplasmic reticulum Ca2+ stores attributable to a reduced action potential duration and sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) activity. An increase in outward K+ currents decreased action potential duration in cardiomyocytes lacking PC1. Overexpression of full-length PC1 in HEK293 cells significantly reduced the current density of heterologously expressed Kv4.3, Kv1.5 and Kv2.1 potassium channels. PC1 C terminus inhibited Kv4.3 currents to the same degree as full-length PC1. Additionally, PC1 coimmunoprecipitated with Kv4.3, and a modeled PC1 C-terminal structure suggested the existence of 2 docking sites for PC1 within the N terminus of Kv4.3, supporting a physical interaction. Finally, a naturally occurring human mutant PC1R4228X manifested no suppressive effects on Kv4.3 channel activity. CONCLUSIONS: Our findings uncover a role for PC1 in regulating multiple Kv channels, governing membrane repolarization and alterations in SERCA activity that reduce cardiomyocyte contractility.

Active Site Flexibility as a Hallmark for Efficient PET Degradation by I. sakaiensis PETase.

Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 Å resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic degradation.