Chemical Screening by Time-Resolved X-Ray Scattering To Discover Allosteric Probes

Drug discovery relies on efficient identification of small-molecule leads and their interactions with macromolecular targets. However, understanding how chemotypes impact mechanistically important conformational states often remains secondary among high-throughput discovery methods. Here, we present a conformational discovery pipeline integrating time-resolved, high-throughput small-angle X-ray scattering (TR-HT-SAXS) and classic fragment screening applied to allosteric states of the mitochondrial import oxidoreductase apoptosis-inducing factor (AIF). By monitoring oxidized and X-ray-reduced AIF states, TR-HT-SAXS leverages structure and kinetics to generate a multidimensional screening dataset that identifies fragment chemotypes allosterically stimulating AIF dimerization. Fragment-induced dimerization rates, quantified with time-resolved SAXS similarity analysis (

Chemical screening by time-resolved X-ray scattering to discover allosteric probes

Drug discovery relies on efficient identification of small-molecule leads and their interactions with macromolecular targets. However, understanding how chemotypes impact mechanistically important conformational states often remains secondary among high-throughput discovery methods. Here, we present a conformational discovery pipeline integrating time-resolved, high-throughput small-angle X-ray scattering (TR-HT-SAXS) and classic fragment screening applied to allosteric states of the mitochondrial import oxidoreductase apoptosis-inducing factor (AIF). By monitoring oxidized and X-ray-reduced AIF states, TR-HT-SAXS leverages structure and kinetics to generate a multidimensional screening dataset that identifies fragment chemotypes allosterically stimulating AIF dimerization. Fragment-induced dimerization rates, quantified with time-resolved SAXS similarity analysis (kVR), capture structure-activity relationships (SAR) across the top-ranked 4-aminoquinoline chemotype. Crystallized AIF-aminoquinoline complexes validate TR-SAXS-guided SAR, supporting this conformational chemotype for optimization. AIF-aminoquinoline structures and mutational analysis reveal active site F482 as an underappreciated allosteric stabilizer of AIF dimerization. This conformational discovery pipeline illustrates TR-HT-SAXS as an effective technology for targeting chemical leads to important macromolecular states

A new structural framework for integrating replication protein A into DNA processing machinery

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA’s DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA’s DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways

Evolving SAXS versatility: solution X-ray scattering for macromolecular architecture, functional landscapes, and integrative structural biology.

Small-angle X-ray scattering (SAXS) has emerged as an enabling integrative technique for comprehensive analyses of macromolecular structures and interactions in solution. Over the past two decades, SAXS has become a mainstay of the structural biologist's toolbox, supplying multiplexed measurements of molecular shape and dynamics that unveil biological function. Here, we discuss evolving SAXS theory, methods, and applications that extend the field of small-angle scattering beyond simple shape characterization. SAXS, coupled with size-exclusion chromatography (SEC-SAXS) and time-resolved (TR-SAXS) methods, is now providing high-resolution insight into macromolecular flexibility and ensembles, delineating biophysical landscapes, and facilitating high-throughput library screening to assess macromolecular properties and to create opportunities for drug discovery. Looking forward, we consider SAXS in the integrative era of hybrid structural biology methods, its potential for illuminating cellular supramolecular and mesoscale structures, and its capacity to complement high-throughput bioinformatics sequencing data. As advances in the field continue, we look forward to proliferating uses of SAXS based upon its abilities to robustly produce mechanistic insights for biology and medicine

Evolving SAXS versatility: solution X-ray scattering for macromolecular architecture, functional landscapes, and integrative structural biology.

Small-angle X-ray scattering (SAXS) has emerged as an enabling integrative technique for comprehensive analyses of macromolecular structures and interactions in solution. Over the past two decades, SAXS has become a mainstay of the structural biologist's toolbox, supplying multiplexed measurements of molecular shape and dynamics that unveil biological function. Here, we discuss evolving SAXS theory, methods, and applications that extend the field of small-angle scattering beyond simple shape characterization. SAXS, coupled with size-exclusion chromatography (SEC-SAXS) and time-resolved (TR-SAXS) methods, is now providing high-resolution insight into macromolecular flexibility and ensembles, delineating biophysical landscapes, and facilitating high-throughput library screening to assess macromolecular properties and to create opportunities for drug discovery. Looking forward, we consider SAXS in the integrative era of hybrid structural biology methods, its potential for illuminating cellular supramolecular and mesoscale structures, and its capacity to complement high-throughput bioinformatics sequencing data. As advances in the field continue, we look forward to proliferating uses of SAXS based upon its abilities to robustly produce mechanistic insights for biology and medicine

Visualizing and accessing correlated SAXS data sets with Similarity Maps and Simple Scattering web resources.

Constructing a comprehensive understanding of macromolecular behavior from a set of correlated small angle scattering (SAS) data is aided by tools that analyze all scattering curves together. SAS experiments on biological systems can be performed on specimens that are more easily prepared, modified, and formatted relative to those of most other techniques. An X-ray SAS measurement (SAXS) can be performed in less than a milli-second in-line with treatment steps such as purification or exposure to modifiers. These capabilities are valuable since biological macromolecules (proteins, polynucleotides, lipids, and carbohydrates) change conformation or assembly under specific conditions that often define their biological role. Furthermore, mutation or post-translational modification change their behavior and provides an avenue to tailor their mechanics. Here, we describe tools to combine multiple correlated SAS measurements for analysis and review their application to biological systems. The SAXS Similarity Map (SSM) compares a set of scattering curves and quantifies the similarity between them for display as a color on a grid. Visualizing an entire correlated data set with SSMs helps identify patterns that reveal biological functions. The SSM analysis is available as a web-based tool at https://sibyls.als.lbl.gov/saxs-similarity/. To make data available and promote tool development, we have also deployed a repository of correlated SAS data sets called Simple Scattering (available at https://simplescattering.com). The correlated data sets used to demonstrate the SSM are available on the Simple Scattering website. We expect increased utilization of correlated SAS measurements to characterize the tightly controlled mechanistic properties of biological systems and fine-tune engineered macromolecules for nanotechnology-based applications

Targeting Allostery with Avatars to Design Inhibitors Assessed by Cell Activity: Dissecting MRE11 Endo- and Exonuclease Activities

For inhibitor design, as in most research, the best system is question dependent. We suggest structurally defined allostery to design specific inhibitors that target regions beyond active sites. We choose systems allowing efficient quality structures with conformational changes as optimal for structure-based design to optimize inhibitors. We maintain that evolutionarily related targets logically provide molecular avatars, where this Sanskrit term for descent includes ideas of functional relationships and of being a physical embodiment of the target's essential features without requiring high sequence identity. Appropriate biochemical and cell assays provide quantitative measurements, and for biomedical impacts, any inhibitor's activity should be validated in human cells. Specificity is effectively shown empirically by testing if mutations blocking target activity remove cellular inhibitor impact. We propose this approach to be superior to experiments testing for lack of cross-reactivity among possible related enzymes, which is a challenging negative experiment. As an exemplary avatar system for protein and DNA allosteric conformational controls, we focus here on developing separation-of-function inhibitors for meiotic recombination 11 nuclease activities. This was achieved not by targeting the active site but rather by geometrically impacting loop motifs analogously to ribosome antibiotics. These loops are neighboring the dimer interface and active site act in sculpting dsDNA and ssDNA into catalytically competent complexes. One of our design constraints is to preserve DNA substrate binding to geometrically block competing enzymes and pathways from the damaged site. We validate our allosteric approach to controlling outcomes in human cells by reversing the radiation sensitivity and genomic instability in BRCA mutant cells