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.

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

Scabin is a mono-ADP-ribosyltransferase toxin/enzyme and possible virulence factor produced by the agriculture pathogen, . 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 R-R-N-STT-W-W-(QxE) sequence motif was proposed as a catalytic signature of the Pierisin family of DNA-acting toxins.

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.

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 , and . Due to the rise in antibiotic resistance, new strategies must be developed to treat the growing spectre of drug resistant infections. Targeting the injectisome 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.

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.

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 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.

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.