Protocol for in vitro phosphorylation of the MUS81-binding region of SLX4 using CDK1-cyclin B.

Phosphorylation is a post-translational modification that can alter protein structure and regulate protein-protein interactions. Here, we present a procedure for in vitro phosphorylation of the MUS81-binding region of SLX4 (SLX4MBR) using cyclin-dependent kinase 1-cyclin B. We describe steps for the dialysis and phosphorylation of target proteins followed by purification using size-exclusion chromatography. Finally, we detail a system to monitor phosphorylation effectiveness and identify phosphorylated residues. We anticipate this protocol to be readily adapted for other protein targets or kinases. For complete details on the use and execution of this protocol, please refer to Payliss et al. (2022).1.

Development of a yeast whole-cell biocatalyst for MHET conversion into terephthalic acid and ethylene glycol.

BACKGROUND: Over the 70 years since the introduction of plastic into everyday items, plastic waste has become an increasing problem. With over 360 million tonnes of plastics produced every year, solutions for plastic recycling and plastic waste reduction are sorely needed. Recently, multiple enzymes capable of degrading PET (polyethylene terephthalate) plastic have been identified and engineered. In particular, the enzymes PETase and MHETase from Ideonella sakaiensis depolymerize PET into the two building blocks used for its synthesis, ethylene glycol (EG) and terephthalic acid (TPA). Importantly, EG and TPA can be re-used for PET synthesis allowing complete and sustainable PET recycling. RESULTS: In this study we used Saccharomyces cerevisiae, a species utilized widely in bioindustrial fermentation processes, as a platform to develop a whole-cell catalyst expressing the MHETase enzyme, which converts monohydroxyethyl terephthalate (MHET) into TPA and EG. We assessed six expression architectures and identified those resulting in efficient MHETase expression on the yeast cell surface. We show that the MHETase whole-cell catalyst has activity comparable to recombinant MHETase purified from Escherichia coli. Finally, we demonstrate that surface displayed MHETase is active across a range of pHs, temperatures, and for at least 12 days at room temperature. CONCLUSIONS: We demonstrate the feasibility of using S. cerevisiae as a platform for the expression and surface display of PET degrading enzymes and predict that the whole-cell catalyst will be a viable alternative to protein purification-based approaches for plastic degradation.

Phosphorylation of the DNA repair scaffold SLX4 drives folding of the SAP domain and activation of the MUS81-EME1 endonuclease.

The DNA repair scaffold SLX4 has multifaceted roles in genome stability, many of which depend on structure-selective endonucleases. SLX4 coordinates the cell cycle-regulated assembly of SLX1, MUS81-EME1, and XPF-ERCC1 into a tri-nuclease complex called SMX. Mechanistically, how the mitotic kinase CDK1 regulates the interaction between SLX4 and MUS81-EME1 remains unclear. Here, we show that CDK1-cyclin B phosphorylates SLX4 residues T1544, T1561, and T1571 in the MUS81-binding region (SLX4MBR). Phosphorylated SLX4MBR relaxes the substrate specificity of MUS81-EME1 and stimulates cleavage of replication and recombination structures, providing a biochemical explanation for the chromosome pulverization that occurs when SLX4 binds MUS81 in S-phase. Remarkably, phosphorylation of SLX4MBR drives folding of an SAP domain, which underpins the high-affinity interaction with MUS81. We also report the structure of phosphorylated SLX4MBR and identify the MUS81-binding interface. Our work provides mechanistic insights into how cell cycle-regulated phosphorylation of SLX4 drives the recruitment and activation of MUS81-EME1.

Exploring the Structures and Functions of Macromolecular SLX4-Nuclease Complexes in Genome Stability.

All organisms depend on the ability of cells to accurately duplicate and segregate DNA into progeny. However, DNA is frequently damaged by factors in the environment and from within cells. One of the most dangerous lesions is a DNA double-strand break. Unrepaired breaks are a major driving force for genome instability. Cells contain sophisticated DNA repair networks to counteract the harmful effects of genotoxic agents, thus safeguarding genome integrity. Homologous recombination is a high-fidelity, template-dependent DNA repair pathway essential for the accurate repair of DNA nicks, gaps and double-strand breaks. Accurate homologous recombination depends on the ability of cells to remove branched DNA structures that form during repair, which is achieved through the opposing actions of helicases and structure-selective endonucleases. This review focuses on a structure-selective endonuclease called SLX1-SLX4 and the macromolecular endonuclease complexes that assemble on the SLX4 scaffold. First, we discuss recent developments that illuminate the structure and biochemical properties of this somewhat atypical structure-selective endonuclease. We then summarize the multifaceted roles that are fulfilled by human SLX1-SLX4 and its associated endonucleases in homologous recombination and genome stability. Finally, we discuss recent work on SLX4-binding proteins that may represent integral components of these macromolecular nuclease complexes, emphasizing the structure and function of a protein called SLX4IP.

Interaction hot spots for phase separation revealed by NMR studies of a CAPRIN1 condensed phase.

The role of biomolecular condensates in regulating biological function and the importance of dynamic interactions involving intrinsically disordered protein regions (IDRs) in their assembly are increasingly appreciated. While computational and theoretical approaches have provided significant insights into IDR phase behavior, establishing the critical interactions that govern condensation with atomic resolution through experiment is more difficult, given the lack of applicability of standard structural biological tools to study these highly dynamic large-scale associated states. NMR can be a valuable method, but the dynamic and viscous nature of condensed IDRs presents challenges. Using the C-terminal IDR (607 to 709) of CAPRIN1, an RNA-binding protein found in stress granules, P bodies, and messenger RNA transport granules, we have developed and applied a variety of NMR methods for studies of condensed IDR states to provide insights into interactions driving and modulating phase separation. We identify ATP interactions with CAPRIN1 that can enhance or reduce phase separation. We also quantify specific side-chain and backbone interactions within condensed CAPRIN1 that define critical sequences for phase separation and that are reduced by O-GlcNAcylation known to occur during cell cycle and stress. This expanded NMR toolkit that has been developed for characterizing IDR condensates has generated detailed interaction information relevant for understanding CAPRIN1 biology and informing general models of phase separation, with significant potential future applications to illuminate dynamic structure-function relationships in other biological condensates.

Side chain electrostatic interactions and pH-dependent expansion of the intrinsically disordered, highly acidic carboxyl-terminus of γ-tubulin.

Intramolecular electrostatic attraction and repulsion strongly influence the conformational sampling of intrinsically disordered proteins and domains (IDPs). In order to better understand this complex relationship, we have used nuclear magnetic resonance to measure side chain pKa values and pH-dependent translational diffusion coefficients for the unstructured and highly acidic carboxyl-terminus of γ-tubulin (γ-CT), providing insight into how the net charge of an IDP relates to overall expansion or collapse of the conformational ensemble. Many of the pKa values in the γ-CT are shifted upward by 0.3-0.4 units and exhibit negatively cooperative ionization pH profiles, likely due to the large net negative charge that accumulates on the molecule as the pH is raised. pKa shifts of this magnitude correspond to electrostatic interaction energies between the affected residues and the rest of the charged molecule that are each on the order of 1 kcal mol-1 . Diffusion of the γ-CT slowed with increasing net charge, indicative of an expanding hydrodynamic radius (rH ). The degree of expansion agreed quantitatively with what has been seen from comparisons of IDPs with different charge content, yielding the general trend that every 0.1 increase in relative charge (|Q|/res) produces a roughly 5% increase in rH . While γ-CT pH titration data followed this trend nearly perfectly, there were substantially larger deviations for the database of different IDP sequences. This suggests that other aspects of an IDP's primary amino acid sequence beyond net charge influence the sensitivity of rH to electrostatic interactions.

The structural and functional effects of Hg(II) and Cd(II) on lipid model systems and human erythrocytes: A review.

The anthropogenic mobilization of mercury and cadmium into the biosphere has led to an increased and ineludible entry of these metals into biological systems. Here we discuss the impact of Hg(II) and Cd(II) on lipid model systems and human erythrocytes from a biophysical perspective. After a brief introduction to their implications on human health, studies that have investigated the effects of Hg(II) and Cd(II) on lipid model systems and human erythrocytes are discussed. In terms of lipids as toxicological target sites, predominantly variations in lipid head groups have been the source of investigation. However, as research in this field progresses, the effects of Hg(II) and Cd(II) on other structural features, such as acyl chain length and unsaturation, and other important lipid components and complex biomimetic lipid mixtures, will require further examinations. This review provides an analysis of what has been learned collectively from the diverse methodologies and experimental conditions used thus far. Consequently, there is a need for more comprehensive and thorough investigations into the effects of Hg(II) and Cd(II) on lipid membranes under consistent experimental conditions such as pH, ionic strength, temperature, and choice of lipid model system.