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The majority of pathogenic mutations in the neurofibromatosis type I (NF1) gene reduce total neurofibromin protein expression through premature truncation or microdeletion, but it is less well understood how loss-of-function missense variants drive NF1 disease. We have found that patient variants in codons 844 to 848, which correlate with a severe phenotype, cause protein instability and exert an additional dominant-negative action whereby wild-type neurofibromin also becomes destabilized through protein dimerization. We have used our neurofibromin cryogenic electron microscopy structure to predict and validate other patient variants that act through a similar mechanism. This provides a foundation for understanding genotype-phenotype correlations and has important implications for patient counseling, disease management, and therapeutics.

RAF inhibitors unexpectedly induce ERK signaling in normal and tumor cells with elevated RAS activity. Paradoxical activation is believed to be RAS dependent. In this study, we showed that LY3009120, a pan-RAF inhibitor, can unexpectedly cause paradoxical ERK activation in KRASG12C-dependent lung cancer cell lines, when KRAS is inhibited by ARS1620, a KRASG12C inhibitor. Using H/N/KRAS-less mouse embryonic fibroblasts, we discovered that classical RAS proteins are not essential for RAF inhibitor-induced paradoxical ERK signaling. In their absence, RAF inhibitors can induce ERK phosphorylation, ERK target gene transcription, and cell proliferation. We further showed that the MRAS/SHOC2 complex is required for this process. This study highlights the complexity of the allosteric RAF regulation by RAF inhibitors, and the importance of other RAS-related proteins in this process.

Bundles of stiff filaments are ubiquitous in the living world, found both in the cytoskeleton and in the extracellular medium. These bundles are typically held together by smaller cross-linking molecules. We demonstrate, analytically, numerically, and experimentally, that such bundles can be kinked, that is, have localized regions of high curvature that are long-lived metastable states. We propose three possible mechanisms of kink stabilization: a difference in trapped length of the filament segments between two cross-links, a dislocation where the endpoint of a filament occurs within the bundle, and the braiding of the filaments in the bundle. At a high concentration of cross-links, the last two effects lead to the topologically protected kinked states. Finally, we explore, numerically and analytically, the transition of the metastable kinked state to the stable straight bundle.

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

Microbes face many physical, chemical, and biological insults from their environments. In response, cells adapt, but whether they do so cooperatively is poorly understood. Here, we use a model social bacterium, Myxococcus xanthus, to ask whether adapted traits are transferable to naïve kin. To do so we isolated cells adapted to detergent stresses and tested for trait transfer. In some cases, strain-mixing experiments increased sibling fitness by transferring adaptation traits. This cooperative behavior depended on a kin recognition system called outer membrane exchange (OME) because mutants defective in OME could not transfer adaptation traits. Strikingly, in mixed stressed populations, the transferred trait also benefited the adapted (actor) cells. This apparently occurred by alleviating a detergent-induced stress response in kin that otherwise killed actor cells. Additionally, this adaptation trait when transferred also conferred resistance against a lipoprotein toxin delivered to targeted kin. Based on these and other findings, we propose a model for stress adaptation and how OME in myxobacteria promotes cellular cooperation in response to environmental stresses. [ABSTRACT FROM AUTHOR]

Amyloid-β (Aβ) is present in humans as a 39- to 42-amino acid residue metabolic product of the amyloid precursor protein. Although the two predominant forms, Aβ(1-40) and Aβ(1-42), differ in only two residues, they display different biophysical, biological, and clinical behavior. Aβ(1-42) is the more neurotoxic species, aggregates much faster, and dominates in senile plaque of Alzheimer's disease (AD) patients. Although small Aβ oligomers are believed to be the neurotoxic species, Aβ amyloid fibrils are, because of their presence in plaques, a pathological hallmark of AD and appear to play an important role in disease progression through cell-to-cell transmissibility. Here, we solved the 3D structure of a disease-relevant Aβ(1-42) fibril polymorph, combining data from solid-state NMR spectroscopy and mass-per-length measurements from EM. The 3D structure is composed of two molecules per fibril layer, with residues 15-42 forming a double-horseshoe-like cross-β-sheet entity with maximally buried hydrophobic side chains. Residues 1-14 are partially ordered and in a β-strand conformation, but do not display unambiguous distance restraints to the remainder of the core structure.

X-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations, must be measured. Until now, measurement of diffuse scattering from protein crystals has been scarce because of the extra effort of collecting diffuse data. Here, we present 3D measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practices for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested translation-libration-screw (TLS), liquidlike motions (LLM), and coarse-grained normal-modes (NM) models of protein motions. The LLM model provides a global picture of motions and was refined against the diffuse data, whereas the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. These results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that dynamics-inspired NM structural models can simultaneously agree with both Bragg and diffuse scattering.

The unique inheritance pattern of the X chromosome exposes it to natural selection in a way that is different from that of the autosomes, potentially resulting in accelerated evolution. We perform a comparative analysis of X chromosome polymorphism in 10 great ape species, including humans. In most species, we identify striking megabase-wide regions, where nucleotide diversity is less than 20% of the chromosomal average. Such regions are found exclusively on the X chromosome. The regions overlap partially among species, suggesting that the underlying targets are partly shared among species. The regions have higher proportions of singleton SNPs, higher levels of population differentiation, and a higher nonsynonymous-to-synonymous substitution ratio than the rest of the X chromosome. We show that the extent to which diversity is reduced is incompatible with direct selection or the action of background selection and soft selective sweeps alone, and therefore, we suggest that very strong selective sweeps have independently targeted these specific regions in several species. The only genomic feature that we can identify as strongly associated with loss of diversity is the location of testis-expressed ampliconic genes, which also have reduced diversity around them. We hypothesize that these genes may be responsible for selective sweeps in the form of meiotic drive caused by an intragenomic conflict in male meiosis.

X-ray diffraction from protein crystals includes both sharply peaked Bragg reflections and diffuse intensity between the peaks. The information in Bragg scattering is limited to what is available in the mean electron density. The diffuse scattering arises from correlations in the electron density variations and therefore contains information about collective motions in proteins. Previous studies using molecular-dynamics (MD) simulations to model diffuse scattering have been hindered by insufficient sampling of the conformational ensemble. To overcome this issue, we have performed a 1.1-μs MD simulation of crystalline staphylococcal nuclease, providing 100-fold more sampling than previous studies. This simulation enables reproducible calculations of the diffuse intensity and predicts functionally important motions, including transitions among at least eight metastable states with different active-site geometries. The total diffuse intensity calculated using the MD model is highly correlated with the experimental data. In particular, there is excellent agreement for the isotropic component of the diffuse intensity, and substantial but weaker agreement for the anisotropic component. Decomposition of the MD model into protein and solvent components indicates that protein-solvent interactions contribute substantially to the overall diffuse intensity. We conclude that diffuse scattering can be used to validate predictions from MD simulations and can provide information to improve MD models of protein motions.

The soil bacterium Bacillus subtilis is a model organism to investigate the formation of biofilms, the predominant form of microbial life. The secreted protein BslA self-assembles at the surface of the biofilm to give the B. subtilis biofilm its characteristic hydrophobicity. To understand the mechanism of BslA self-assembly at interfaces, here we built a molecular model based on the previous BslA crystal structure and the crystal structure of the BslA paralogue YweA that we determined. Our analysis revealed two conserved protein-protein interaction interfaces supporting BslA self-assembly into an infinite 2-dimensional lattice that fits previously determined transmission microscopy images. Molecular dynamics simulations and in vitro protein assays further support our model of BslA elastic film formation, while mutagenesis experiments highlight the importance of the identified interactions for biofilm structure. Based on this knowledge, YweA was engineered to form more stable elastic films and rescue biofilm structure in bslA deficient strains. These findings shed light on protein film assembly and will inform the development of BslA technologies which range from surface coatings to emulsions in fast-moving consumer goods. [ABSTRACT FROM AUTHOR]