Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold.

Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å. We apply this system to the key cancer signaling protein KRAS (19 kDa in size), obtaining four structures of oncogenic mutational variants by cryo-EM. Importantly, a structure for the key G12C mutant bound to an inhibitor drug (AMG510) reveals significant conformational differences compared to prior data in the crystalline state. The findings highlight the promise of cryo-EM scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases.

Mapping the DNA-Binding Motif of Scabin Toxin, a Guanine Modifying Enzyme from Streptomyces scabies.

Scabin is a mono-ADP-ribosyltransferase toxin/enzyme and possible virulence factor produced by the agriculture pathogen, Streptomyces scabies. Recently, molecular dynamic approaches and MD simulations revealed its interaction with both NAD+ and DNA substrates. An Essential Dynamics Analysis identified a crab-claw-like mechanism, including coupled changes in the exposed motifs, and the Rß1-RLa-NLc-STTß2-WPN-WARTT-(QxE)ARTT sequence motif was proposed as a catalytic signature of the Pierisin family of DNA-acting toxins. A new fluorescence assay was devised to measure the kinetics for both RNA and DNA substrates. Several protein variants were prepared to probe the Scabin-NAD-DNA molecular model and to reveal the reaction mechanism for the transfer of ADP-ribose to the guanine base in the DNA substrate. The results revealed that there are several lysine and arginine residues in Scabin that are important for binding the DNA substrate; also, key residues such as Asn110 in the mechanism of ADP-ribose transfer to the guanine base were identified. The DNA-binding residues are shared with ScARP from Streptomyces coelicolor but are not conserved with Pierisin-1, suggesting that the modification of guanine bases by ADP-ribosyltransferases is divergent even in the Pierisin family.

Cryo-EM structure of the EspA filament from enteropathogenic Escherichia coli: Revealing the mechanism of effector translocation in the T3SS.

The type III secretion system (T3SS) is a virulence mechanism employed by Gram-negative pathogens. The T3SS forms a proteinaceous channel that projects a needle into the extracellular medium where it interacts with the host cell to deliver virulence factors. Enteropathogenic Escherichia coli (EPEC) is unique in adopting a needle extension to the T3SS-a filament formed by EspA-which is absolutely required for efficient colonization of the gut. Here, we describe the cryoelectron microscopy structure of native EspA filaments from EPEC at 3.6-Å resolution. Within the filament, positively charged residues adjacent to a hydrophobic groove line the lumen of the filament in a spiral manner, suggesting a mechanism of substrate translocation mediated via electrostatics. Using structure-guided mutagenesis, in vivo studies corroborate the role of these residues in secretion and translocation function. The high-resolution structure of the EspA filament could aid in structure-guided drug design of antivirulence therapeutics.

Cryo-EM analysis of the SctV cytosolic domain from the enteropathogenic E. coli T3SS injectisome.

The bacterial injectisome and flagella both rely on type III secretion systems for their assembly. The syringe-like injectisome creates a continuous channel between the bacterium and the host cell, through which signal-modulating effector proteins are secreted. The inner membrane pore protein SctV controls the hierarchy of substrate selection and may also be involved in energizing secretion. We present the 4.7 Å cryo-EM structure of the SctV cytosolic domain (SctVC) from the enteropathogenic Escherichia coli injectisome. SctVC forms a nonameric ring with primarily electrostatic interactions between its subunits. Molecular dynamics simulations show that monomeric SctVC maintains a closed conformation, in contrast with previous studies on flagellar homologue FlhA. Comparison with substrate-bound homologues suggest that a conformational change would be required to accommodate binding partners.

On the road to structure-based development of anti-virulence therapeutics targeting the type III secretion system injectisome.

The type III secretion system injectisome is a syringe-like multimembrane spanning nanomachine that is essential to the pathogenicity but not viability of many clinically relevant Gram-negative bacteria, such as enteropathogenic Escherichia coli, Salmonella enterica and Pseudomonas aeruginosa. Due to the rise in antibiotic resistance, new strategies must be developed to treat the growing spectre of drug resistant infections. Targeting the injectisome via an 'anti-virulence strategy' is a promising avenue to pursue as an alternative to the more commonly used bactericidal therapeutics, which have a high propensity for resulting resistance development and often more broad killing profile, including unwanted side effects in eliminating favourable members of the microbiome. Building on more than a decade of crystallographic work of truncated or isolated forms of the more than two dozen components of the secretion apparatus, recent advances in the field of single-particle cryo-electron microscopy have allowed for the elucidation of atomic resolution structures for many of the type III secretion system components in their assembled, oligomerized state including the needle complex, export apparatus and ATPase. Cryo-electron tomography studies have also advanced our understanding of the direct pathogen-host interaction between the type III secretion system translocon and host cell membrane. These new structural works that further our understanding of the myriad of protein-protein interactions that promote injectisome function will be highlighted in this review, with a focus on those that yield promise for future anti-virulence drug discovery and design. Recently developed inhibitors, including both synthetic, natural product and peptide inhibitors, as well as promising new developments of immunotherapeutics will be discussed. As our understanding of this intricate molecular machinery advances, the development of anti-virulence inhibitors can be enhanced through structure-guided drug design.

Dynamics of Scabin toxin. A proposal for the binding mode of the DNA substrate.

Scabin is a mono-ADP-ribosyltransferase enzyme and is a putative virulence factor produced by the plant pathogen, Streptomyces scabies. Previously, crystal structures of Scabin were solved in the presence and absence of substrate analogues and inhibitors. Herein, experimental (hydrogen-deuterium exchange), simulated (molecular dynamics), and theoretical (Gaussian Network Modeling) approaches were systematically applied to study the dynamics of apo-Scabin in the context of a Scabin·NAD+·DNA model. MD simulations revealed that the apo-Scabin solution conformation correlates well with the X-ray crystal structure, beyond the conformation of the exposed, mobile regions. In turn, the MD fluctuations correspond with the crystallographic B-factors, with the fluctuations derived from a Gaussian network model, and with the experimental H/D exchange rates. An Essential Dynamics Analysis identified the dynamic aspects of the toxin as a crab-claw-like mechanism of two topological domains, along with coupled deformations of exposed motifs. The "crab-claw" movement resembles the motion of C3-like toxins and emerges as a property of the central ß scaffold of catalytic single domain toxins. The exposure and high mobility of the cis side motifs in the Scabin ß-core suggest involvement in DNA substrate binding. A ternary Scabin·NAD+·DNA model was produced via an independent docking methodology, where the intermolecular interactions correspond to the region of high mobility identified by dynamics analyses and agree with binding and kinetic data reported for wild-type and Scabin variants. Based on data for the Pierisin-like toxin group, the sequence motif Rß1-RLa-NLc-STTß2-WPN-WARTT-(QxE)ARTT emerges as a catalytic signature involved in the enzymatic activity of these DNA-acting toxins. However, these results also show that Scabin possesses a unique DNA-binding motif within the Pierisin-like toxin group.

Characterization of the catalytic signature of Scabin toxin, a DNA-targeting ADP-ribosyltransferase.

Scabin was previously identified as a novel DNA-targeting mono-ADP-ribosyltransferase (mART) toxin from the plant pathogen 87.22 strain of Streptomyces scabies Scabin is a member of the Pierisin-like subgroup of mART toxins, since it targets DNA. An in-depth characterization of both the glycohydrolase and transferase enzymatic activities of Scabin was conducted. Several protein variants were developed based on an initial Scabin·DNA molecular model. Consequently, three residues were deemed important for DNA-binding and transferase activity. Trp128 and Trp155 are important for binding the DNA substrate and participate in the reaction mechanism, whereas Tyr129 was shown to be important only for DNA binding, but was not involved in the reaction mechanism. Trp128 and Trp155 are both conserved within the Pierisin-like toxins, whereas Tyr129 is a unique substitution within the group. Scabin showed substrate specificity toward double-stranded DNA containing a single-base overhang, as a model for single-stranded nicked DNA. The crystal structure of Scabin bound to NADH - a competitive inhibitor of Scabin - was determined, providing important insights into the active-site structure and Michaelis-Menten complex of the enzyme. Based on these results, a novel DNA-binding motif is proposed for Scabin with substrate and the key residues that may participate in the Scabin·NAD(+) complex are highlighted.

Scabin, a Novel DNA-acting ADP-ribosyltransferase from Streptomyces scabies.

A bioinformatics strategy was used to identify Scabin, a novel DNA-targeting enzyme from the plant pathogen 87.22 strain of Streptomyces scabies Scabin shares nearly 40% sequence identity with the Pierisin family of mono-ADP-ribosyltransferase toxins. Scabin was purified to homogeneity as a 22-kDa single-domain enzyme and was shown to possess high NAD(+)-glycohydrolase (Km (NAD) = 68 ± 3 µm; kcat = 94 ± 2 min(-1)) activity with an RSQXE motif; it was also shown to target deoxyguanosine and showed sigmoidal enzyme kinetics (K0.5(deoxyguanosine) = 302 ± 12 µm; kcat = 14 min(-1)). Mass spectrometry analysis revealed that Scabin labels the exocyclic amino group on guanine bases in either single-stranded or double-stranded DNA. Several small molecule inhibitors were identified, and the most potent compounds were found to inhibit the enzyme activity with Ki values ranging from 3 to 24 µm PJ34, a well known inhibitor of poly-ADP-ribosyltransferases, was shown to be the most potent inhibitor of Scabin. Scabin was crystallized, representing the first structure of a DNA-targeting mono-ADP-ribosyltransferase enzyme; the structures of the apo-form (1.45 Å) and with two inhibitors (P6-E, 1.4 Å; PJ34, 1.6 Å) were solved. These x-ray structures are also the first high resolution structures of the Pierisin subgroup of the mono-ADP-ribosyltransferase toxin family. A model of Scabin with its DNA substrate is also proposed.