The strand exchange domain of tumor suppressor PALB2 is intrinsically disordered and promotes oligomerization-dependent DNA compaction.

The partner and localizer of BRCA2 (PALB2) is a scaffold protein linking BRCA1 with BRCA2 and RAD51 during homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR in cells, while the PALB2 DNA-binding domain (PALB2-DBD) supports DNA strand exchange . We determined that PALB2-DBD is intrinsically disordered beyond a single N-terminal α-helix.

Selective deuteration of an RNA:RNA complex for structural analysis using small-angle scattering.

The structures of RNA:RNA complexes regulate many biological processes. Despite their importance, protein-free RNA:RNA complexes represent a tiny fraction of experimentally-determined structures. Here, we describe a joint small-angle X-ray and neutron scattering (SAXS/SANS) approach to structurally interrogate conformational changes in a model RNA:RNA complex.

Early Events in G-quadruplex Folding Captured by Time-Resolved Small-Angle X-Ray Scattering.

Time-resolved small-angle X-ray experiments (TR-SAXS) are reported here that capture and quantify a previously unknown rapid collapse of the unfolded oligonucleotide as an early step in G4 folding of hybrid 1 and hybrid 2 telomeric G-quadruplex structures. The rapid collapse, initiated by a pH jump, is characterized by an exponential decrease in the radius of gyration from 20.6 to 12.

Design of intrinsically disordered protein variants with diverse structural properties.

Intrinsically disordered proteins (IDPs) perform a broad range of functions in biology, suggesting that the ability to design IDPs could help expand the repertoire of proteins with novel functions. Computational design of IDPs with specific conformational properties has, however, been difficult because of their substantial dynamics and structural complexity. We describe a general algorithm for designing IDPs with specific structural properties.

A General Mechanism for the General Stress Response in Bacteria.

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains.

Structural insights into the formation of repulsive netrin guidance complexes.

Netrins dictate attractive and repulsive responses during axon growth and cell migration, where the presence of the receptor Uncoordinated-5 (UNC-5) on target cells results in repulsion. Here, we showed that UNC-5 is a heparin-binding protein, determined its structure bound to a heparin fragment, and could modulate UNC-5-heparin affinity using a directed evolution platform or structure-based rational design. We demonstrated that UNC-5 and UNC-6/netrin form a large, stable, and rigid complex in the presence of heparin, and heparin and UNC-5 exclude the attractive UNC-40/DCC receptor from binding to UNC-6/netrin to a large extent.

: new developments for a free open-source program for small-angle scattering data reduction and analysis.

is a free open-source program for reduction, analysis and modelling of biological small-angle scattering data. Here, the new developments in version 2 are described. These include improved data reduction using ; updated automated Guinier fitting and finding algorithms; automated series ( size-exclusion chromatography coupled small-angle X-ray scattering or SEC-SAXS) buffer- and sample-region finding algorithms; linear and integral baseline correction for series; deconvolution of series data using regularized alternating least squares (); creation of electron-density reconstructions using electron density via solution scattering (); a comparison window showing residuals, ratios and statistical comparisons between profiles; and generation of PDF reports with summary plots and tables for all analysis.

The Ruminococcus bromii amylosome protein Sas6 binds single and double helical α-glucan structures in starch.

Resistant starch is a prebiotic accessed by gut bacteria with specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses Sas6 (Starch Adherence System member 6), which consists of two starch-specific carbohydrate-binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). Here, we present the crystal structures of Sas6 and of RbCBM74 bound with a double helical dimer of maltodecaose.

Design of intrinsically disordered protein variants with diverse structural properties.

Intrinsically disordered proteins (IDPs) perform a wide range of functions in biology, suggesting that the ability to design IDPs could help expand the repertoire of proteins with novel functions. Designing IDPs with specific structural or functional properties has, however, been difficult, in part because determining accurate conformational ensembles of IDPs generally requires a combination of computational modelling and experiments. Motivated by recent advancements in efficient physics-based models for simulations of IDPs, we have developed a general algorithm for designing IDPs with specific structural properties.

Structural and functional studies reveal the molecular basis of substrate promiscuity of a glycosyltransferase originating from a major agricultural pest.

The two-spotted spider mite, Tetranychus urticae, is a major cosmopolitan pest that feeds on more than 1100 plant species. Its genome contains an unprecedentedly large number of genes involved in detoxifying and transporting xenobiotics, including 80 genes that code for UDP glycosyltransferases (UGTs). These enzymes were acquired via horizontal gene transfer from bacteria after loss in the Chelicerata lineage.

BioXTAS RAW 2: new developments for a free open-source program for small angle scattering data reduction and analysis.

BioXTAS RAW is a free, open-source program for reduction, analysis and modelling of biological small angle scattering data. Here, the new developments in RAW version 2 are described. These include: improved data reduction using pyFAI; updated automated Guinier fitting and finding algorithms; automated series (e.

Homomeric interactions of the MPZ Ig domain and their relation to Charcot-Marie-Tooth disease.

Mutations in MPZ (myelin protein zero) can cause demyelinating early-onset Charcot-Marie-Tooth type 1B disease or later onset type 2I/J disease characterized by axonal degeneration, reflecting the diverse roles of MPZ in Schwann cells. MPZ holds apposing membranes of the myelin sheath together, with the adhesion role fulfilled by its extracellular immunoglobulin-like domain (IgMPZ), which oligomerizes. Models for how the IgMPZ might form oligomeric assemblies has been extrapolated from a protein crystal structure in which individual rat IgMPZ subunits are packed together under artificial conditions, forming three weak interfaces.

The strand exchange domain of tumor suppressor PALB2 is intrinsically disordered and promotes oligomerization-dependent DNA compaction.

The Partner and Localizer of BRCA2 (PALB2) is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency in cells. The PALB2 DNA-binding domain (PALB2-DBD) supports strand exchange, a complex multistep reaction conducted by only a few proteins such as RecA-like recombinases and Rad52.

GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production.

GRB2 is an adaptor protein required for facilitating cytoplasmic signaling complexes from a wide array of binding partners. GRB2 has been reported to exist in either a monomeric or dimeric state in crystal and solution. GRB2 dimers are formed by the exchange of protein segments between domains, otherwise known as "domain-swapping".

Structure of a 28.5 kDa duplex-embedded G-quadruplex system resolved to 7.4 Å resolution with cryo-EM.

Genomic regions with high guanine content can fold into non-B form DNA four-stranded structures known as G-quadruplexes (G4s). Extensive in vivo investigations have revealed that promoter G4s are transcriptional regulators. Little structural information exists for these G4s embedded within duplexes, their presumed genomic environment.

Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro.

SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic. Its genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro). Polyprotein processing is essential yet incompletely understood.

Solution Structural Studies of Pre-amyloid Oligomer States of the Biofilm Protein Aap.

Staphylococcus epidermidis is a commensal bacterium on human skin that is also the leading cause of medical device-related infections. The accumulation-associated protein (Aap) from S. epidermidis is a critical factor for infection via its ability to mediate biofilm formation.

Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains.

Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 comprises self-associating regions that drive its homotypic liquid-liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS.

The Disordered Amino Terminus of the Circadian Enzyme Nocturnin Modulates Its NADP(H) Phosphatase Activity by Changing Protein Dynamics.

Endogenous circadian clocks control the rhythmicity of a broad range of behavioral and physiological processes, and this is entrained by the daily fluctuations in light and dark. Nocturnin () is a rhythmically expressed gene regulated by the circadian clock that belongs to the CCR4 family of endonuclease-exonuclease-phosphatase (EEP) enzymes, and the NOCT protein exhibits phosphatase activity, catalyzing the removal of the 2'-phosphate from NADP(H). In addition to its daily nighttime peak of expression, it is also induced by acute stimuli.

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome.

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS.

Long promoter sequences form higher-order G-quadruplexes: an integrative structural biology study of c-Myc, k-Ras and c-Kit promoter sequences.

We report on higher-order G-quadruplex structures adopted by long promoter sequences obtained by an iterative integrated structural biology approach. Our approach uses quantitative biophysical tools (analytical ultracentrifugation, small-angle X-ray scattering, and circular dichroism spectroscopy) combined with modeling and molecular dynamics simulations, to derive self-consistent structural models. The formal resolution of our approach is 18 angstroms, but in some cases structural features of only a few nucleotides can be discerned.

The Two Non-Visual Arrestins Engage ERK2 Differently.

Arrestin binding to active phosphorylated G protein-coupled receptors terminates G protein coupling and initiates another wave of signaling. Among the effectors that bind directly to receptor-associated arrestins are extracellular signal-regulated kinases 1/2 (ERK1/2), which promote cellular proliferation and survival. Arrestins may also engage ERK1/2 in isolation in a pre- or post-signaling complex that is likely in equilibrium with the full signal initiation complex.

A multi-step nucleation process determines the kinetics of prion-like domain phase separation.

Compartmentalization by liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mechanism underlying the organization of biomolecules in space and time. Here, we combine rapid-mixing time-resolved small-angle X-ray scattering (SAXS) approaches to characterize the assembly kinetics of a prototypical prion-like domain with equilibrium techniques that characterize its phase boundaries and the size distribution of clusters prior to phase separation. We find two kinetic regimes on the micro- to millisecond timescale that are distinguished by the size distribution of clusters.

Small-angle X-ray scattering experiments of monodisperse intrinsically disordered protein samples close to the solubility limit.

The condensation of biomolecules into biomolecular condensates via liquid-liquid phase separation (LLPS) is a ubiquitous mechanism that drives cellular organization. To enable these functions, biomolecules have evolved to drive LLPS and facilitate partitioning into biomolecular condensates. Determining the molecular features of proteins that encode LLPS will provide critical insights into a plethora of biological processes.