Small-molecule modulators of INAVA cytosolic condensate and cell-cell junction assemblies.

Epithelial cells lining mucosal surfaces distinctively express the inflammatory bowel disease risk gene INAVA. We previously found that INAVA has dual and competing functions: one at lateral membranes where it affects mucosal barrier function and the other in the cytosol where INAVA enhances IL-1ß signal transduction and protein ubiquitination and forms puncta. We now find that IL-1ß-induced INAVA puncta are biomolecular condensates that rapidly assemble and physiologically resolve. The condensates contain ubiquitin and the E3 ligase ßTrCP2, and their formation correlates with amplified ubiquitination, suggesting function in regulation of cellular proteostasis. Accordingly, a small-molecule screen identified ROS inducers, proteasome inhibitors, and inhibitors of the protein folding chaperone HSP90 as potent agonists for INAVA condensate formation. Notably, inhibitors of the p38α and mTOR pathways enhanced resolution of the condensates, and inhibitors of the Rho-ROCK pathway induced INAVA's competing function by recruiting INAVA to newly assembled intercellular junctions in cells where none existed before.

Highly Hydrophilic Segments Attached to Hydrophobic Peptides Translocate Rapidly across Membranes.

Hydrophilic segments attached to transmembrane helices often cross membranes. In an increasing number of cases, it has become apparent that this occurs in a biologically relevant post-translational event. In this study, we investigate whether juxta-membrane (JM) hydrophilic sequences attached to hydrophobic helices are able to rapidly cross lipid bilayers via their ability or inability to block hydrophobic helix interconversion between a transmembrane (TM) and non-TM membrane-associated state. Interconversion was triggered by changing the protonation state of an Asp residue in the hydrophobic core of the peptides, and peptide configuration was monitored by the fluorescence of a Trp residue at the center of the hydrophobic sequence. In POPC vesicles, conversion of the TM to non-TM state at high pH and the non-TM to TM state at low pH was rapid (seconds or less) for KK, KKNN, and the KKNNNNNN flanking sequences on both N- and C-termini and the KLFAGHQ sequence that flanks the spontaneously TM-inserting 3A protein of polio virus. In vesicles composed of 6:4 (mol/mol) POPC/cholesterol, interconversion was still rapid, with the exception of the peptide flanked by KKNNNNNN sequences, for which the half time of interconversion slowed to minutes. This behavior suggests that, at least in membranes with low levels of cholesterol, movement of hydrophilic JM segments (and analogous hydrophobic loops in multipass TM proteins) across membranes may be more facile than previously thought. This may have important biological implications.

Effect of lipid composition and amino acid sequence upon transmembrane peptide-accelerated lipid transleaflet diffusion (flip-flop).

We examined how hydrophobic peptide-accelerated transleaflet lipid movement (flip-flop) was affected by peptide sequence and vesicle composition and properties. A peptide with a completely hydrophobic sequence had little if any effect upon flip-flop. While peptides with a somewhat less hydrophobic sequence accelerated flip-flop, the half-time remained slow (hours) with substantial (0.5mol%) peptide in the membranes. It appears that peptide-accelerated lipid flip-flop involves a rare event that may reflect a rare state of the peptide or lipid bilayer. There was no simple relationship between peptide overall hydrophobicity and flip-flop. In addition, flip-flop was not closely linked to whether the peptides were in a transmembrane or non-transmembrane (interfacial) inserted state. Flip-flop was also not associated with peptide-induced pore formation. We found that peptide-accelerated flip-flop is initially faster in small (highly curved) unilamellar vesicles relative to that in large unilamellar vesicles. Peptide-accelerated flip-flop was also affected by lipid composition, being slowed in vesicles with thick bilayers or those containing 30% cholesterol. Interestingly, these factors also slow spontaneous lipid flip-flop in the absence of peptide. Combined with previous studies, the results are most consistent with acceleration of lipid flip-flop by peptide-induced thinning of bilayer width.

Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism.

A major question remaining in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xyloside α-1,3-xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly, the epidermal growth factor-like repeat acceptor substrate undergoes a large conformational change upon binding to the active site, providing a structural basis for substrate specificity. Our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations in which Notch dysregulation is known to cause cancer or developmental disorders.

Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis.

In translation, elongation factor Tu (EF-Tu) molecules deliver aminoacyl-tRNAs to the mRNA-programmed ribosome. The GTPase activity of EF-Tu is triggered by ribosome-induced conformational changes of the factor that play a pivotal role in the selection of the cognate aminoacyl-tRNAs. We present a 6.7-A cryo-electron microscopy map of the aminoacyl-tRNA x EF-Tu x GDP x kirromycin-bound Escherichia coli ribosome, together with an atomic model of the complex obtained through molecular dynamics flexible fitting. The model reveals the conformational changes in the conserved GTPase switch regions of EF-Tu that trigger hydrolysis of GTP, along with key interactions, including those between the sarcin-ricin loop and the P loop of EF-Tu, and between the effector loop of EF-Tu and a conserved region of the 16S rRNA. Our data suggest that GTP hydrolysis on EF-Tu is controlled through a hydrophobic gate mechanism.

Particle-verification for single-particle, reference-based reconstruction using multivariate data analysis and classification.

As collection of electron microscopy data for single-particle reconstruction becomes more efficient, due to electronic image capture, one of the principal limiting steps in a reconstruction remains particle-verification, which is especially costly in terms of user input. Recently, some algorithms have been developed to window particles automatically, but the resulting particle sets typically need to be verified manually. Here we describe a procedure to speed up verification of windowed particles using multivariate data analysis and classification. In this procedure, the particle set is subjected to multi-reference alignment before the verification. The aligned particles are first binned according to orientation and are binned further by K-means classification. Rather than selection of particles individually, an entire class of particles can be selected, with an option to remove outliers. Since particles in the same class present the same view, distinction between good and bad images becomes more straightforward. We have also developed a graphical interface, written in Python/Tkinter, to facilitate this implementation of particle-verification. For the demonstration of the particle-verification scheme presented here, electron micrographs of ribosomes are used.

Exploration of parameters in cryo-EM leading to an improved density map of the E. coli ribosome.

A number of image processing parameters in the 3D reconstruction of a ribosome complex from a cryo-EM data set were varied to test their effects on the final resolution. The parameters examined were pixel size, window size, and mode of Fourier amplitude enhancement at high spatial frequencies. In addition, the strategy of switching from large to small pixel size during angular refinement was explored. The relationship between resolution (in Fourier space) and the number of particles was observed to follow a lin-log dependence, a relationship that appears to hold for other data, as well. By optimizing the above parameters, and using a lin-log extrapolation to the full data set in the estimation of resolution from half-sets, we obtained a 3D map from 131,599 ribosome particles at 6.7A resolution (FSC=0.5).

Displaying 3D data on RNA secondary structures: coloRNA.

RNA performs a variety of diverse functions and therefore must adopt many different three-dimensional conformations. The number and complexity of RNA structures that are currently available are steadily increasing, necessitating the generation of versatile structure visualization tools. Here, we describe a new RNA secondary and tertiary structure visualization tool, the display program coloRNA. This program colors each nucleotide in a secondary structure schematic according to the value of an assigned property of the corresponding backbone phosphate group, such as the distance between corresponding residues in two atomic models of the same RNA molecule. To assist in analyzing tertiary structure, coloRNA also colors nucleotides based on the three-dimensional distances between a user-selected nucleotide and all others. Minimum and maximum thresholds can be used to focus in on, or eliminate, a particular value range. coloRNA can display a user-specified group of nucleotides by outlining the structure in an automatically assigned, but user-changeable color. As an example, we have used coloRNA to analyze a pair of recently published structures of the Escherichia coli 70S ribosome. When coloRNA is used to display the conformational difference between the two structures, the large movement of the small subunit head stands visually out from the background changes in the remaining domains of the small subunit.