Melissa Ann Graewert

Despite its essential role in pre-mRNA splicing, the structure and functional significance of the intrinsically disordered N-terminal domain (NTD) of SF3B1, a core component of the SF3b spliceosome complex, remain poorly understood. Here, we combine nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), biochemistry, bioinformatics approaches, with molecular dynamics simulations to characterize the conformational landscape of the SF3B1-NTD and its phosphorylation-dependent remodelling. Specifically, we focus on the 186–341 region ( ^186–341 SF3B1), which contains at least four regulatory phosphorylation sites. We reveal that ^186–341 SF3B1 is highly dynamic, lacking a stable secondary structure, and that phosphorylation of T211, T235, T313, and T328 induces a shift toward more compact states driven by the formation of intramolecular interactions. Additionally, we unveil that ^186–341 SF3B1 interacts with RNA primarily via the arginine residues flaking one of the conserved tryptophan-centred motifs present within SF3B1-NTD. These interactions are transient and heterogeneous, reflecting a nonspecific yet sequence-dependent recognition mechanism. Collectively, this study provides a comprehensive description of the conformational landscape and functional features of the SF3B1-NTD, and expands the portfolio of its possible interactors, thus broadening our understanding of how intrinsic disorder, post-translational modifications, and sequence-encoded motifs cooperate to regulate pre-mRNA splicing. Highlights: SAXS and NMR experiments reveal that SF3B1-NTD is intrinsically disordered Phosphorylation compacts the SF3B1 intrinsically disordered region SF3B1 intrinsically disordered region interacts with RNA at W200-centred motif

Rho-associated protein kinases (ROCK1/2) are critical regulators of fundamental cellular processes and represent promising therapeutic targets for various pathologies, particularly cardiovascular disease. While the terminal globular kinase domain and the PH-C1 tandem (PHn-C1-PHc) are well characterized, the sequence-structure-function relationship of the central amphipathic α-helical segment remains poorly understood. Utilizing an integrative biophysical approach—including multi-angle light scattering (MALS), analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), negative-stain electron microscopy (EM), X-ray crystallography, and 3D molecular modeling—we elucidate the structural organization of the central coiled-coil region of ROCK1. Notably, the 2.2 Å resolution crystal structure of the homology region 1 (HR1) of ROCK1 reveals a canonical parallel coiled-coil dimer. This structural arrangement diverges significantly from the antiparallel coiled-coil HR1 module found in other Rho effector kinases. Combined with the structural reconstitution of full-length ROCK1, our data demonstrates that the central segment of ROCK1 forms a highly elongated coiled-coil dimer. Furthermore, EM analysis of the isolated full coiled-coil domain uncovers a dynamic oligomerization state, transitioning between a dimer and a tetramer. Together, these biophysical insights establish a foundation that opens avenues for future investigation into ROCK1 regulation, activation, and downstream signaling mechanisms.

Rho-associated protein kinases (ROCK1/2) play a key role in many fundamental cellular processes and are promising therapeutic targets for various diseases, particularly cardiovascular disease. However, unlike the terminal globular kinase domain and the PH-C1 tandem (PHn-C1-PHc), the sequence-structure-function relationship of the central amphipathic α-helical segment of ROCK proteins is not well understood. This study employed multiple biophysical techniques, including multi-angle light scattering (MALS), analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), negative-stain electron microscopy (EM), X-ray crystallography, and 3D molecular modeling to elucidate the structural basis of the entire ROCK1 segment. The study yielded significant new insights into the structural organization of ROCK proteins. Notably, the crystal structure of the homology region 1 (HR1) of ROCK1 (ROCK ^HR1 ), located at the N-terminus of the amphipathic segment and determined at 2.2 Å resolution, provided the first experimental evidence of a canonical parallel coiled-coil dimer. This structure differs significantly from the antiparallel coiled-coil HR1 module found in other Rho effector kinases. Along with the structural reconstitution of full-length ROCK1, this evidence demonstrates that the central segment of ROCK1 forms a highly elongated coiled-coil dimer. EM analysis using purified ROCK ^FCC revealed that this dimer switches between a dimer and a tetramer.

Biological small-angle X-ray scattering (SAXS) is a versatile and powerful technique for investigating the structural and biophysical properties of biologically and pharmaceutically relevant macromolecules and nanoparticles. SAXS offers detailed insights into macromolecular composition, size, shape and internal structure, while addressing key aspects such as oligomeric state, stability, molecular interactions, and conformational flexibility. Recently, asymmetrical-flow field-flow fractionation (AF4) was successfully coupled to SAXS, enabling online size-based fractionation and analysis of polydisperse samples. This approach allows precise, size-dependent characterization, offering significant advancements in the study of polydisperse systems. We have integrated an AF4 device at the P12 beamline at the European Molecular Biology Laboratory and implemented technical adaptations allowing full automation to make the system suitable for routine user access. We provide streamlined workflows and troubleshooting resources for both novice and advanced SAXS users thereby equipping them with clear guidance on performing AF4–SAXS measurements. The general principles of our set-up are easily adaptable to other beamlines which have integrated (or are planning to integrate) a similar system.

Small-angle X-ray and neutron scattering (SAXS and SANS) are versatile techniques for studying the structure of various materials and particles, enabling investigations of structures from a few to hundreds of nanometres. However, interpreting SAXS or SANS data can be challenging, hence the need for effective training. Here, we present the website SAStutorials.org, which offers tutorials on small-angle scattering (SAS) data analysis. The website serves as a learning platform that supports active learning both in the classroom and through self-study. The tutorials cover basic concepts, advanced modelling and theoretical aspects of SAS. Each tutorial follows a structure based on the principle of SOLO taxonomy, guiding learners from minimal or no knowledge in a given area to the ability to tackle real-world problems. SAStutorials.org has been developed as a community tool, providing tutorials that encompass a broad range of data analysis types, leveraging various programs and investigating different materials. Moreover, the website and all its data are open source, encouraging contributions from the community.

Cerebral dopamine neurotrophic factor (CDNF) is an unconventional neurotrophic factor that is a disease-modifying drug candidate for Parkinson’s disease. CDNF has pleiotropic protective effects on stressed cells, but its mechanism of action remains incompletely understood. Here, we use state-of-the-art advanced structural techniques to resolve the structural basis of CDNF interaction with GRP78, the master regulator of the unfolded protein response (UPR) pathway. Subsequent binding studies confirm the obtained structural model of the complex, eventually revealing the interaction site of CDNF and GRP78. Finally, mutating the key residues of CDNF mediating its interaction with GRP78 not only results in impaired binding of CDNF but also abolishes the neuroprotective activity of CDNF-derived peptides in mesencephalic neuron cultures. These results suggest that the molecular interaction with GRP78 mediates the neuroprotective actions of CDNF and provide a structural basis for development of next generation CDNF-based therapeutic compounds against neurodegenerative diseases.

Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment. In solution, chemical and biophysical parameters that have an influence on the structure and dynamics of molecules can be varied and their effect on conformational changes can be monitored in time-resolved XFEL and SAXS experiments. We report here the collection of scattering form factors of proteins in solution using FEL X-rays. The form factors correspond to the scattering signal of the protein ensemble alone; the scattering contributions from the solvent and the instrument are separately measured and accurately subtracted. The experiment was done using a liquid jet for sample delivery. These results pave the way for time-resolved studies and measurements from dilute samples, capitalizing on the intense and short FEL X-ray pulses.

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0–1 Å⁻¹ are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to 2.2 Å⁻¹ showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set.