Studying Conformational Changes of the Yersinia Type-III-Secretion Effector YopO in Solution by Integrative Structural Biology.

The type-III secretion effector YopO helps pathogenic Yersinia to outmaneuver the human immune system. Injected into host cells, it functions as a Ser/Thr kinase after activation by actin binding. This activation process is thought to involve large conformational changes. We use PELDOR spectroscopy and small-angle X-ray scattering in combination with available crystal structures to study these conformational transitions. Low-resolution hybrid models of the YopO/actin structure in solution were constructed, where the kinase domain of YopO is tilted "backward" compared with the crystal structure, thus shortening the distance between actin and the kinase active site, potentially affecting the substrate specificity of YopO. Furthermore, the GDI domain of the hybrid models resembles a conformation that was previously observed in a crystal structure of the isolated GDI domain. We investigate possible structural reasons for the inactivity of the apo state, analyze its flexibility and discuss the biological implications.

Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems.

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central ß-sheet and two α-helical bundles. In addition, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3, and EspG5 chaperones using small-angle X-ray scattering reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3-PE5-PPE4 complex based on the small-angle X-ray scattering analysis.

Progress in small-angle scattering from biological solutions at high-brilliance synchrotrons.

Small-angle X-ray scattering (SAXS) is an established technique that provides low-resolution structural information on macromolecular solutions. Recent decades have witnessed significant progress in both experimental facilities and in novel data-analysis approaches, making SAXS a mainstream method for structural biology. The technique is routinely applied to directly reconstruct low-resolution shapes of proteins and to generate atomistic models of macromolecular assemblies using hybrid approaches. Very importantly, SAXS is capable of yielding structural information on systems with size and conformational polydispersity, including highly flexible objects. In addition, utilizing high-flux synchrotron facilities, time-resolved SAXS allows analysis of kinetic processes over time ranges from microseconds to hours. Dedicated bioSAXS beamlines now offer fully automated data-collection and analysis pipelines, where analysis and modelling is conducted on the fly. This enables SAXS to be employed as a high-throughput method to rapidly screen various sample conditions and additives. The growing SAXS user community is supported by developments in data and model archiving and quality criteria. This review illustrates the latest developments in SAXS, in particular highlighting time-resolved applications aimed at flexible and evolving systems.

Resolution of ab initio shapes determined from small-angle scattering.

Spatial resolution is an important characteristic of structural models, and the authors of structures determined by X-ray crystallography or electron cryo-microscopy always provide the resolution upon publication and deposition. Small-angle scattering of X-rays or neutrons (SAS) has recently become a mainstream structural method providing the overall three-dimensional structures of proteins, nucleic acids and complexes in solution. However, no quantitative resolution measure is available for SAS-derived models, which significantly hampers their validation and further use. Here, a method is derived for resolution assessment for ab initio shape reconstruction from scattering data. The inherent variability of the ab initio shapes is utilized and it is demonstrated how their average Fourier shell correlation function is related to the model resolution. The method is validated against simulated data for proteins with known high-resolution structures and its efficiency is demonstrated in applications to experimental data. It is proposed that henceforth the resolution be reported in publications and depositions of ab initio SAS models.

Weak protein-ligand interactions studied by small-angle X-ray scattering.

Small-angle X-ray scattering (SAXS) is a powerful technique for studying weak interactions between proteins and their ligands (other proteins, DNA/RNA or small molecules) in solution. SAXS provides knowledge about the equilibrium state, the stoichiometry of binding and association-dissociation processes. The measurements are conducted in a solution environment that allows easy monitoring of modifications in protein-ligand association state upon environmental changes. Model-free parameters such as the molecular mass of a system and the radius of gyration can be obtained directly from the SAXS data and give indications about the association state. SAXS is also widely employed to build models of biological assemblies at a resolution of approximately 10-20 Å. Low-resolution shapes can be generated ab initio, although more detailed and biologically interpretable information can be obtained by hybrid modelling. In the latter approach, composite structures of protein-ligand complexes are constructed using atomic models of individual molecules. These may be predicted homology models or experimental structures from X-ray crystallography or NMR. This review focuses on using SAXS data to model structures of protein-ligand complexes and to study their dynamics. The combination of SAXS with other methods such as size exclusion chromatography and dynamic light scattering is discussed.