Serine-linked PARP1 auto-modification controls PARP inhibitor response.

Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are recruited and activated by DNA damage, resulting in ADP-ribosylation at numerous sites, both within PARP1 itself and in other proteins. Several PARP1 and PARP2 inhibitors are currently employed in the clinic or undergoing trials for treatment of various cancers. These drugs act primarily by trapping PARP1 on damaged chromatin, which can lead to cell death, especially in cells with DNA repair defects. Although PARP1 trapping is thought to be caused primarily by the catalytic inhibition of PARP-dependent modification, implying that ADP-ribosylation (ADPr) can counteract trapping, it is not known which exact sites are important for this process. Following recent findings that PARP1- or PARP2-mediated modification is predominantly serine-linked, we demonstrate here that serine ADPr plays a vital role in cellular responses to PARP1/PARP2 inhibitors. Specifically, we identify three serine residues within PARP1 (499, 507, and 519) as key sites whose efficient HPF1-dependent modification counters PARP1 trapping and contributes to inhibitor tolerance. Our data implicate genes that encode serine-specific ADPr regulators, HPF1 and ARH3, as potential PARP1/PARP2 inhibitor therapy biomarkers.

Mechanistic insights into the three steps of poly(ADP-ribosylation) reversal.

Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.

Viral macrodomains: a structural and evolutionary assessment of the pharmacological potential.

Viral macrodomains possess the ability to counteract host ADP-ribosylation, a post-translational modification implicated in the creation of an antiviral environment via immune response regulation. This brought them into focus as promising therapeutic targets, albeit the close homology to some of the human macrodomains raised concerns regarding potential cross-reactivity and adverse effects for the host. Here, we evaluate the structure and function of the macrodomain of SARS-CoV-2, the causative agent of COVID-19. We show that it can antagonize ADP-ribosylation by PARP14, a cellular (ADP-ribosyl)transferase necessary for the restriction of coronaviral infections. Furthermore, our structural studies together with ligand modelling revealed the structural basis for poly(ADP-ribose) binding and hydrolysis, an emerging new aspect of viral macrodomain biology. These new insights were used in an extensive evolutionary analysis aimed at evaluating the druggability of viral macrodomains not only from the Coronaviridae but also Togaviridae and Iridoviridae genera (causing diseases such as Chikungunya and infectious spleen and kidney necrosis virus disease, respectively). We found that they contain conserved features, distinct from their human counterparts, which may be exploited during drug design.

The structural basis for an on-off switch controlling Gßγ-mediated inhibition of TRPM3 channels.

TRPM3 channels play important roles in the detection of noxious heat and in inflammatory thermal hyperalgesia. The activity of these ion channels in somatosensory neurons is tightly regulated by µ-opioid receptors through the signaling of Gßγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gßγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gßγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gß1 that are individually necessary for TRPM3 inhibition by Gßγ. The 10-amino-acid Gßγ-interacting domain in TRPM3 is subject to alternative splicing. Its inclusion in or exclusion from TRPM3 channel proteins therefore provides a mechanism for switching on or off the inhibitory action that Gßγ proteins exert on TRPM3 channels.

Structural Insights into the Binding of Natural Pyrimidine-Based Inhibitors of Class II Aminoacyl-tRNA Synthetases.

The pyrimidine-containing Trojan horse antibiotics albomycin and a recently discovered cytidine-containing microcin C analog target the class II seryl- and aspartyl-tRNA synthetases (serRS and aspRS), respectively. The active components of these compounds are competitive inhibitors that mimic the aminoacyl-adenylate intermediate. How they effectively substitute for the interactions mediated by the canonical purine group is unknown. Employing nonhydrolyzable aminoacyl-sulfamoyl nucleosides substituting the base with cytosine, uracil, and N3-methyluracil the structure-activity relationship of the natural compounds was evaluated. In vitro using E. coli serRS and aspRS, the best compounds demonstrated IC50 values in the low nanomolar range, with a clear preference for cytosine or N3-methyluracil over uracil. X-ray crystallographic structures of K. pneumoniae serRS and T. thermophilus aspRS in complex with the compounds showed the contribution of structured waters and residues in the conserved motif-2 loop in defining base preference. Utilizing the N3-methyluracil bound serRS structure, MD simulations of the fully modified albomycin base were performed to identify the interacting network that drives stable association. This analysis pointed to key interactions with a methionine in the motif-2 loop. Interestingly, this residue is mutated to a glycine in a second serRS (serRS2) found in albomycin-producing actinobacteria possessing self-immunity to this antibiotic. A comparative study demonstrated that serRS2 is poorly inhibited by the pyrimidine-containing intermediate analogs, and an equivalent mutation in E. coli serRS significantly decreased the affinity of the cytosine congener. These findings highlight the crucial role of dynamics and solvation of the motif-2 loop in modulating the binding of the natural antibiotics.

Structural Basis of the Subcellular Topology Landscape of Escherichia coli.

Cellular proteomes are distributed in multiple compartments: on DNA, ribosomes, on and inside membranes, or they become secreted. Structural properties that allow polypeptides to occupy subcellular niches, particularly to after crossing membranes, remain unclear. We compared intrinsic and extrinsic features in cytoplasmic and secreted polypeptides of the Escherichia coli K-12 proteome. Structural features between the cytoplasmome and secretome are sharply distinct, such that a signal peptide-agnostic machine learning tool distinguishes cytoplasmic from secreted proteins with 95.5% success. Cytoplasmic polypeptides are enriched in aliphatic, aromatic, charged and hydrophobic residues, unique folds and higher early folding propensities. Secretory polypeptides are enriched in polar/small amino acids, ß folds, have higher backbone dynamics, higher disorder and contact order and are more often intrinsically disordered. These non-random distributions and experimental evidence imply that evolutionary pressure selected enhanced secretome flexibility, slow folding and looser structures, placing the secretome in a distinct protein class. These adaptations protect the secretome from premature folding during its cytoplasmic transit, optimize its lipid bilayer crossing and allowed it to acquire cell envelope specific chemistries. The latter may favor promiscuous multi-ligand binding, sensing of stress and cell envelope structure changes. In conclusion, enhanced flexibility, slow folding, looser structures and unique folds differentiate the secretome from the cytoplasmome. These findings have wide implications on the structural diversity and evolution of modern proteomes and the protein folding problem.

The Preprotein Binding Domain of SecA Displays Intrinsic Rotational Dynamics.

SecA converts ATP energy to protein translocation work. Together with the membrane-embedded SecY channel it forms the bacterial protein translocase. How secretory proteins bind to SecA and drive conformational cascades to promote their secretion remains unknown. To address this, we focus on the preprotein binding domain (PBD) of SecA. PBD crystalizes in three distinct states, swiveling around its narrow stem. Here, we examined whether PBD displays intrinsic dynamics in solution using single-molecule Förster resonance energy transfer (smFRET). Unique cysteinyl pairs on PBD and apposed domains were labeled with donor/acceptor dyes. Derivatives were analyzed using pulsed interleaved excitation and multi-parameter fluorescence detection. The PBD undergoes significant rotational motions, occupying at least three distinct states in dimeric and four in monomeric soluble SecA. Nucleotides do not affect smFRET-detectable PBD dynamics. These findings lay the foundations for single-molecule dissection of translocase mechanics and suggest models for possible PBD involvement during catalysis.

Protein folding in the cell envelope of Escherichia coli.

While the entire proteome is synthesized on cytoplasmic ribosomes, almost half associates with, localizes in or crosses the bacterial cell envelope. In Escherichia coli a variety of mechanisms are important for taking these polypeptides into or across the plasma membrane, maintaining them in soluble form, trafficking them to their correct cell envelope locations and then folding them into the right structures. The fidelity of these processes must be maintained under various environmental conditions including during stress; if this fails, proteases are called in to degrade mislocalized or aggregated proteins. Various soluble, diffusible chaperones (acting as holdases, foldases or pilotins) and folding catalysts are also utilized to restore proteostasis. These responses can be general, dealing with multiple polypeptides, with functional overlaps and operating within redundant networks. Other chaperones are specialized factors, dealing only with a few exported proteins. Several complex machineries have evolved to deal with binding to, integration in and crossing of the outer membrane. This complex protein network is responsible for fundamental cellular processes such as cell wall biogenesis; cell division; the export, uptake and degradation of molecules; and resistance against exogenous toxic factors. The underlying processes, contributing to our fundamental understanding of proteostasis, are a treasure trove for the development of novel antibiotics, biopharmaceuticals and vaccines.

Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF.

Escherichia coli MazF (EcMazF) is the archetype of a large family of ribonucleases involved in bacterial stress response. The crystal structure of EcMazF in complex with a 7-nucleotide substrate mimic explains the relaxed substrate specificity of the E. coli enzyme relative to its Bacillus subtilis counterpart and provides a framework for rationalizing specificity in this enzyme family. In contrast to a conserved mode of substrate recognition and a conserved active site, regulation of enzymatic activity by the antitoxin EcMazE diverges from its B. subtilis homolog. Central in this regulation is an EcMazE-induced double conformational change as follows: a rearrangement of a crucial active site loop and a relative rotation of the two monomers in the EcMazF dimer. Both are induced by the C-terminal residues Asp-78-Trp-82 of EcMazE, which are also responsible for strong negative cooperativity in EcMazE-EcMazF binding. This situation shows unexpected parallels to the regulation of the F-plasmid CcdB activity by CcdA and further supports a common ancestor despite the different activities of the MazF and CcdB toxins. In addition, we pinpoint the origin of the lack of activity of the E24A point mutant of EcMazF in its inability to support the substrate binding-competent conformation of EcMazF.

Type III Secretion: Building and Operating a Remarkable Nanomachine.

The Type III secretion system (T3SS) is a protein export pathway that is widespread in Gram-negative bacteria and delivers effector proteins directly into eukaryotic cells. At its core lie the injectisome (a sophisticated transmembrane secretion apparatus) and a complex network of specialized chaperones that target secretory proteins to the antechamber of the injectisome. The assembly of the system, and the subsequent secretion of proteins through it, undergo fine-tuned, hierarchical regulation. Here, we present the current understanding of the injectisome assembly process, secretion hierarchy, and the role of chaperones. We discuss these events in light of available structural and biochemical dissection and propose future directions essential to revealing mechanistic insight into this fascinating nanomachine.

An efficient method for the purification of proteins from four distinct toxin-antitoxin modules.

Toxin-antitoxin (TA) modules are stress response elements that are ubiquitous in the genomes of bacteria and archaea. Production and subsequent purification of individual TA proteins is anything but straightforward as over-expression of the toxin gene is lethal to bacterial and eukaryotic cells and over-production of the antitoxin leads to its proteolytic degradation because of its inherently unstructured nature. Here we describe an effective production and purification strategy centered on an on-column denaturant-induced dissociation of the toxin-antitoxin complex. The success of the method is demonstrated by its application on four different TA families, encoding proteins with distinct activities and folds. A series of biophysical and in vitro activity tests show that the purified proteins are of high quality and suitable for structural studies.

Escherichia coli antitoxin MazE as transcription factor: insights into MazE-DNA binding.

Toxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environmental stresses. The mazEF module encodes the MazF toxin and its cognate MazE antitoxin. The highly dynamic MazE possesses an N-terminal DNA binding domain through which it can negatively regulate its own promoter. Despite being one of the first TA systems studied, transcriptional regulation of Escherichia coli mazEF remains poorly understood. This paper presents the solution structure of C-terminal truncated E. coli MazE and a MazE-DNA model with a DNA palindrome sequence ∼ 10 bp upstream of the mazEF promoter. The work has led to a transcription regulator-DNA model, which has remained elusive thus far in the E. coli toxin-antitoxin family. Multiple complementary techniques including NMR, SAXS and ITC show that the long intrinsically disordered C-termini in MazE, required for MazF neutralization, does not affect the interactions between the antitoxin and its operator. Rather, the MazE C-terminus plays an important role in the MazF binding, which was found to increase the MazE affinity for the palindromic single site operator.

Structural and biophysical characterization of Staphylococcus aureus SaMazF shows conservation of functional dynamics.

The Staphylococcus aureus genome contains three toxin-antitoxin modules, including one mazEF module, SamazEF. Using an on-column separation protocol we are able to obtain large amounts of wild-type SaMazF toxin. The protein is well-folded and highly resistant against thermal unfolding but aggregates at elevated temperatures. Crystallographic and nuclear magnetic resonance (NMR) solution studies show a well-defined dimer. Differences in structure and dynamics between the X-ray and NMR structural ensembles are found in three loop regions, two of which undergo motions that are of functional relevance. The same segments also show functionally relevant dynamics in the distantly related CcdB family despite divergence of function. NMR chemical shift mapping and analysis of residue conservation in the MazF family suggests a conserved mode for the inhibition of MazF by MazE.

Crystallization of the Staphylococcus aureus MazF mRNA interferase.

mazEF modules encode toxin-antitoxin pairs that are involved in the bacterial stress response through controlled and specific degradation of mRNA. Staphylococcus aureus MazF and MazE constitute a unique toxin-antitoxin module under regulation of the sigB operon. A MazF-type mRNA interferase is combined with an antitoxin of unknown fold. Crystals of S. aureus MazF (SaMazF) were grown in space group P2(1)2(1)2(1). The crystals diffracted to 2.1 Šresolution and are likely to contain two SaMazF dimers in the asymmetric unit.

1H, 13C, and 15N backbone and side-chain chemical shift assignment of the staphylococcal MazF mRNA interferase.

MazF proteins are ribonucleases that cleave mRNA with high sequence-specificity as part of bacterial stress response and that are neutralized by the action of the corresponding antitoxin MazE. Prolonged activation of the toxin MazF leads to cell death. Several mazEF modules from gram-negative bacteria have been characterized in terms of catalytic activity, auto-regulation mechanism and structure, but less is known about their distant relatives found in gram-positive organisms. Currently, no solution NMR structure is available for any wild-type MazF toxin. Here we report the (1)H, (15)N and (13)C backbone and side-chain chemical shift assignments of this toxin from the pathogen bacterium Staphylococcus aureus. The BMRB accession number is 17288.