Structural basis for transthiolation intermediates in the ubiquitin pathway.

Transthiolation (also known as transthioesterification) reactions are used in the biosynthesis of acetyl coenzyme A, fatty acids and polyketides, and for post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins1-3. For the Ub pathway, E1 enzymes catalyse transthiolation from an E1~Ub thioester to an E2~Ub thioester. Transthiolation is also required for transfer of Ub from an E2~Ub thioester to HECT (homologous to E6AP C terminus) and RBR (ring-between-ring) E3 ligases to form E3~Ub thioesters4-6. How isoenergetic transfer of thioester bonds is driven forward by enzymes in the Ub pathway remains unclear. Here we isolate mimics of transient transthiolation intermediates for E1-Ub(T)-E2 and E2-Ub(T)-E3HECT complexes (where T denotes Ub in a thioester or Ub undergoing transthiolation) using a chemical strategy with native enzymes and near-native Ub to capture and visualize a continuum of structures determined by single-particle cryo-electron microscopy. These structures and accompanying biochemical experiments illuminate conformational changes in Ub, E1, E2 and E3 that are coordinated with the chemical reactions to facilitate directional transfer of Ub from each enzyme to the next.

DNA asymmetry promotes SUMO modification of the single-stranded DNA-binding protein RPA.

Repair of DNA double-stranded breaks by homologous recombination (HR) is dependent on DNA end resection and on post-translational modification of repair factors. In budding yeast, single-stranded DNA is coated by replication protein A (RPA) following DNA end resection, and DNA-RPA complexes are then SUMO-modified by the E3 ligase Siz2 to promote repair. Here, we show using enzymatic assays that DNA duplexes containing 3' single-stranded DNA overhangs increase the rate of RPA SUMO modification by Siz2. The SAP domain of Siz2 binds DNA duplexes and makes a key contribution to this process as highlighted by models and a crystal structure of Siz2 and by assays performed using protein mutants. Enzymatic assays performed using DNA that can accommodate multiple RPA proteins suggest a model in which the SUMO-RPA signal is amplified by successive rounds of Siz2-dependent SUMO modification of RPA and dissociation of SUMO-RPA at the junction between single- and double-stranded DNA. Our results provide insights on how DNA architecture scaffolds a substrate and E3 ligase to promote SUMO modification in the context of DNA repair.

Chemical tools for probing the Ub/Ubl conjugation cascades.

Conjugation of ubiquitin (Ub) and structurally related ubiquitin-like proteins (Ubl's), essential for many cellular processes, employs muti-step reactions orchestrated by specific E1, E2 and E3 enzymes. The E1 enzyme activates the Ub/Ubl C-terminus in an ATP-dependent process that results in the formation of a thioester linkage with the E1 active site cysteine. The thioester activated Ub/Ubl is transferred to the active site of an E2 enzyme which then interacts with an E3 enzyme to promote conjugation to the target substrate. The E1-E2-E3 enzymatic cascades utilize labile intermediates, extensive conformational changes, and vast combinatorial diversity of short-lived protein-protein complexes to conjugate Ub/Ubl to various substrates in a regulated manner. In this review, we discuss various chemical tools and methods used to study the consecutive steps of Ub/Ubl activation and conjugation, which are often too elusive for direct studies. We focus on methods developed to probe enzymatic activities and capture and characterize stable mimics of the transient intermediates and transition states thereby providing insights into fundamental mechanisms in the Ub/Ubl conjugation pathways.

Indazole to 2-cyanoindole scaffold progression for mycobacterial lipoamide dehydrogenase inhibitors achieves extended target residence time and improved antibacterial activity.

Tuberculosis remains a leading cause of death from a single infection worldwide. Drug resistance to existing and even new antimycobacterials calls for research into novel targets and unexplored mechanisms of action. Recently we reported on the development of tight-binding inhibitors of Mycobacterium tuberculosis (Mtb) lipoamide dehydrogenase (Lpd), which selectively inhibit the bacterial but not the human enzyme based on a differential modality of inhibitor interaction with these targets. Here we report on the striking improvement in inhibitor residence time on the Mtb enzyme associated with scaffold progression from an indazole to 2-cyanoindole. Cryo-EM of Lpd with the bound 2-cyanoindole inhibitor 19 confirmed displacement of the buried water molecule deep in the binding channel with a cyano group. The ensuing hours-long  improvement in on-target residence time is associated with enhanced antibacterial activity in axenic culture and in primary mouse macrophages. Resistance to 2-cyanoindole inhibitors involves mutations within the inhibitor binding site that have little effect on inhibitor affinity but change the modality of inhibitor-target interaction, resulting in fast dissociation from Lpd. These findings underscore that on-target residence time is a major determinant of antibacterial activity and in vivo efficacy.

Interdependence of the rad50 hook and globular domain functions.

Rad50 contains a conserved Zn(2+) coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here, we focused on rad50 mutations flanking the Zn(2+)-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double-strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end joining, and DNA double-strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled-coil and globular ATPase domains, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions.

Design and Semisynthesis of Biselectrophile-Functionalized Ubiquitin Probes To Investigate Transthioesterification Reactions.

Ubiquitin (Ub) regulates a wide array of cellular processes through post-translational modification of protein substrates. Ub is conjugated at its C-terminus to target proteins via an enzymatic cascade in which covalently bound Ub thioesters are transferred from E1 activating enzymes to E2 conjugating enzymes, and then to certain E3 protein ligases. These transthioesterification reactions proceed via transient tetrahedral intermediates. A variety of chemical strategies have been used to capture E1-Ub-E2 and E2-Ub-E3 mimics, but these introduce modifications that disrupt atomic spacing at the linkage point relative to the native tetrahedral intermediate. We have developed a biselectrophilic PSAN warhead that can be installed in place of the conserved C-terminal glycine in Ub and used to form ternary protein complexes linked via cyanomethyldithioacetals that closely mimic the native tetrahedral intermediates. Investigation of the reactivity of the warhead and substituted analogues led to an effective semisynthetic route to Ub-1-PSAN, which was used to form a ternary E1-Ub*-E2 complex as a mimic of the transthioesterification intermediate.

Method for accurate determination of dissociation constants of optical ratiometric systems: chemical probes, genetically encoded sensors, and interacting molecules.

Ratiometric chemical probes and genetically encoded sensors are of high interest for both analytical chemists and molecular biologists. Their high sensitivity toward the target ligand and ability to obtain quantitative results without a known sensor concentration have made them a very useful tool in both in vitro and in vivo assays. Although ratiometric sensors are widely used in many applications, their successful and accurate usage depends on how they are characterized in terms of sensing target molecules. The most important feature of probes and sensors besides their optical parameters is an affinity constant toward analyzed molecules. The literature shows that different analytical approaches are used to determine the stability constants, with the ratio approach being most popular. However, oversimplification and lack of attention to detail results in inaccurate determination of stability constants, which in turn affects the results obtained using these sensors. Here, we present a new method where ratio signal is calibrated for borderline values of intensities of both wavelengths, instead of borderline ratio values that generate errors in many studies. At the same time, the equation takes into account the cooperativity factor or fluorescence artifacts and therefore can be used to characterize systems with various stoichiometries and experimental conditions. Accurate determination of stability constants is demonstrated utilizing four known optical ratiometric probes and sensors, together with a discussion regarding other, currently used methods.

Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly.

The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small ß-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.

Relationship between the architecture of zinc coordination and zinc binding affinity in proteins--insights into zinc regulation.

Zinc proteins are an integral component of the proteome of all domains of life. Zn(II), one of the most widespread transition elements, serves multiple functions in proteins, such as a catalytic co-factor, a structural center and a signaling component. The mechanism by which proteins associate with and dissociate from Zn(II) and the factors that modulate their affinity and stability remain incompletely understood. In this article, we aim to address how zinc binding sites present in proteins differ in their architecture and how their structural arrangement is associated with protein function, thermodynamic and kinetic stability, reactivity, as well as zinc-dependent regulation. Here, we emphasize that the concentration-dependent functionality of the interprotein zinc binding site may serve as another factor regulating the relationship between cellular Zn(II) availability and protein function.

Femtomolar Zn(II) affinity of minimal zinc hook peptides--a promising small tag for protein engineering.

The minimal zinc hook peptide of Rad50 and its alanine mutants form highly stable Zn(II) complexes. These peptides were successfully used as a small, efficient tag for reversible Zn(II)-mediated protein homodimerization. The high stability, its biological consequences and potential applications in protein engineering are discussed.