Journal of Molecular Biology

Synonymous codons encode the same amino acid but can differ in their usage and translational properties. In previous work we reported statistical differences in backbone dihedral angle distributions associated with synonymous codons in the Escherichia coli proteome. This finding has been questioned due to concerns regarding the statistical methodology used. Here we revisit the dataset using corrected statistical procedures and alternative statistical tests. Across multiple frameworks, the real dataset consistently shows an excess of small p-values relative to randomized controls, indicating detectable codon-associated differences in backbone conformation.

Pre-mRNA splicing is an essential step in eukaryotic gene expression during which spliceosomes remove introns from nascent RNAs while ligating the adjacent exons. Spliceosomes are cellular nanomachines composed of five small nuclear (snRNA) components and dozens of proteins, most of which are highly conserved. Despite the high conservation of many splicing factors between S. cerevisiae and H. sapiens, several protein components of the S. cerevisiae spliceosome are not essential for growth under normal laboratory conditions. This is particularly surprising for nonessential factors whose conserved domains contact the spliceosomes catalytic core. Uncovering a function for these splicing factors can be challenging since they are not required for viability, may engage in functionally redundant interactions, and may display only weak phenotypes in the absence of secondary mutations in other spliceosome components. One such nonessential factor is the Cwc15 protein. Cwc15s highly conserved N-terminus directly contacts the U2/U6 di-snRNA within the spliceosome catalytic core; yet its precise role in splicing has not been defined in any organism. In this work, we use molecular genetics in S. cerevisiae combined with splicing reporter assays to study Cwc15p function. We propose that Cwc15p not only promotes active site stability during 5 splice site cleavage but also impacts structural transitions into and out of this spliceosome conformation. This function may be critical for splicing in S. cerevisiae under nonoptimal conditions, facilitating use of weak or alternate splice sites, and could have implications for proofreading of spliceosome active site formation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/713263v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@b296c5org.highwire.dtl.DTLVardef@c87b91org.highwire.dtl.DTLVardef@287011org.highwire.dtl.DTLVardef@d59741_HPS_FORMAT_FIGEXP M_FIG C_FIG Article SummaryPre-mRNA splicing is carried out by large macromolecular machines called spliceosomes which are composed of several snRNAs and dozens of proteins. Despite decades of study, the functions of many splicing factors such as S. cerevisiae Cwc15p remain unknown. Cwc15p is highly conserved among eukaryotes and directly contacts the spliceosome catalytic core. Here, we have used genetic and splicing reporter assays to study the function of Cwc15p during splicing in vivo. We propose that Cwc15p both stabilizes the spliceosome active site during 5 splice site cleavage and impacts remodeling of that site.

Protein translation is a highly regulated process influenced by multiple factors at the initiation, elongation, and termination stages. One notable regulatory element of the ribosome is the CAR interaction surface, a three-residue motif in the structure of the ribosome composed of C1274 and A1427 of S. cerevisiae 18S rRNA (corresponding to C1054 and A1196 in E. coli 16S rRNA) and R146 of ribosomal protein Rps3. CAR is highly conserved and positioned adjacent to the amino-acyl (A site) decoding center. It establishes hydrogen bonds with the +1 codon next in line to enter the ribosome A site, acting as an extension of the tRNA anticodon and forming base-stacking interactions with nucleotide 34 of the tRNA. However, despite CARs enzymatically strategic positioning within the ribosome, its functional relationship with the A site remains poorly characterized. Using molecular dynamics (MD) simulations, we examined the interplay between the A site and CAR site, revealing sequence-dependent modulation of H-bonding and {pi}-stacking interactions within and between the two sites. These findings highlight the interplay between the A site and CAR site, suggesting a structural and functional connection between these two regions of the ribosome that may contribute to mRNA sequence-specific tuning of translation elongation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/714784v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1d783d3org.highwire.dtl.DTLVardef@f9cd8org.highwire.dtl.DTLVardef@102667corg.highwire.dtl.DTLVardef@967c56_HPS_FORMAT_FIGEXP M_FIG C_FIG

Eukaryotes have distinct nuclear genes for tryptophanyl-tRNA synthetase (TrpRS). Human mitochondrial (Hmt) TrpRS (also WARS2) shares only 14% sequence identity with human cytoplasmic (Hc)TrpRS, but 41% with Bacillus stearothermophilus (Bs)TrpRS. Tryptophan binding to BsTrpRS is largely promoted by hydrophobic interactions and recognition of the indole nitrogen by side chains of Met129 and Asp132. The non-reactive analog indolmycin can recruit unique polar interactions to form an active-site metal coordination that lies off the normal mechanistic path, enhancing affinity to BsTrpRS and other prokaryotic TrpRS enzymes by 1500-fold over its tryptophan substrate. By contrast, human WARS2, complements nonpolar interactions for tryptophan binding with additional electrostatic and hydrogen bonding interactions that are inconsistent with indolmycin binding. We report here a 1.82 [A] crystal structure of an HmtTrpRS* indolmycin*Mn2+*ATP complex, showing that mitochondrial and bacterial enzymes use similar determinants to bind both ATP and indolmycin. ATP forms tight electrostatic interactions between the catalytic metal ion and a non-bridging oxygen atom from each phosphate group. Hydrogen bonds between the oxazolinone group and active-site residues create an off-path ground-state configuration. This arrangement closely mimics that in the corresponding BsTrpRS complex but varies greatly from ATP binding to HcTrpRS, Moreover, isothermal titration calorimetry demonstrates that, as for BsTrpRS, Mg2+*ATP, but not ATP alone, enhances indolmycin binding affinity [~]100-fold with a supplemental {Delta}({Delta}G) of [~] -3 kcal/mol. Structural, thermodynamic, and kinetic similarities confirm our previous conclusion that a reinforced ground-state Mg2+ ion configuration contributes to the high indolmycin affinity in the bacterial system.

Co-translational folding is a critical, yet poorly understood, aspect of protein biogenesis due to its transient, heterogeneous, and experimentally inaccessible nature. Using a myoglobin variant engineered towards increased domain swapping, we show that stable dimers formed during heterologous E. Coli expression revert to the monomeric state following denaturation - renaturation and that domain swapping propensity is significantly affected by synonymous coding. Wider implications for the role of synonymous coding in aggregation and disease are discussed.

Recoverin is a key calcium sensor that controls the desensitization of the visual rhodopsin by GRK1. Previous studies have traditionally been conducted on bovine protein (bRec), while data on human ortholog (hRec) remain scarce. Here, we combine X-ray crystallography, X-ray absorption spectroscopy (XANES), quantum mechanical calculations, molecular dynamics, and functional assays to provide an integrated characterization of hRec. The 2Ca2+-bound hRec structure was solved at 1.60 [A], showing that, unlike bRec, hRec interacts with ROS membranes at physiologically relevant submicromolar Ca2+ levels, due to a species-specific charge distribution that might influence membrane interactions. Both recoverins form a set of Ca2+/Zn2+-bound conformers with improved functional performance. X-ray crystallography (1.85 [A]) and XANES revealed a specific tetrahedral Zn2+ site in 1Ca2+-bound hRec, the first such site reported in the NCS family. In 1Ca2+-bound hRec, zinc promotes the formation of active state, whereas in 2Ca2+-state of bRec, it significantly enhances GRK1 binding, as the latter can complement the Zn2+ coordination. These data refine our understanding of recoverin function in humans and highlight its role as a key link between calcium and zinc signaling in mammalian photoreceptors under normal and pathological conditions.

Purified proteins are routinely flash frozen for use in functional and structural studies, providing a convenient way to reproduce results across complex experiments. Rho guanine-nucleotide exchange factors (RhoGEFs) are no exception to this practice, yet the effects of freezing on their activity and stability remain largely uncharacterized. This gap potentially affects the characterization of these important enzymes and how results are interpreted with respect to their prospective use as therapeutic targets. Here, we tested the isolated DH/PH tandems of P-Rex1, P-Rex2, and PRG under different cryoprotectant conditions and monitored activity and thermostability over time after flash freezing. Our results show a clear divergence between the activity of fresh and frozen purified RhoGEF protein samples in as little as one week for some conditions. Specifically, the variability in data collected on frozen samples was greatly increased. Despite these differences, thermostability seems to be preserved for much longer timepoints across RhoGEFs. Moreover, despite eventual changes in both activity and thermostability with respect to freezing, there are no obvious changes in global conformation between fresh and frozen samples of the isolated P-Rex2 DH/PH tandem. From our data, there are few generalizable trends between the different RhoGEFs and no single cryoprotective agent tested was a silver bullet to preserve both activity and thermostability across RhoGEFs. Overall, our findings emphasize the unpredictable effects of freezing RhoGEFs. As such, RhoGEF freezing should be carefully characterized for each protein and critically viewed when comparing analyses between different studies.

Escherichia coli YicC enzyme is the founding member of a family of endoribonucleases that is encoded in virtually all bacterial species. Previous structural studies revealed that this ribonuclease binds RNA by a novel mechanism in which the hexameric apoprotein presents an open channel that undergoes a large rotation upon RNA binding and clamps down on the RNA. The current study follows up on these findings by examining the cleavage of various oligonucleotide substrates designed to probe recognition elements required for YicC binding and cleavage. A 26-nucleotide RNA oligomer (oligo), with a KD in the low micromolar range, was the standard to which numerous oligos with altered sequence were compared. In vitro RNase assays and fluorescence anisotropy binding measurements indicated that the preferred substrates for YicC were relatively small RNAs that contain some secondary structure. Larger RNAs or highly structured RNAs were less-than-optimal substrates. Similarly, RyhB RNA, a [~]90-nucleotide, iron-responsive RNA of E. coli, which has been described as a target of YicC binding and/or cleavage, was a poor YicC substrate in our assays. These results suggest that the native substrates for YicC-family members are very small RNAs or RNA fragments derived from larger RNAs.

Proteins with intrinsically disordered regions (IDRs) migrate at a higher apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) complicating their analysis and identification. Here, we investigate the sequence determinants of the hypomobility of IDRs using a series of synthetic low complexity domains. We find that negative charge increases the apparent molecular weight, but neutral polar tracts also have abnormally slow migration. Positive charge and hydrophobic residues decrease the apparent molecular weight, although lysine residues show a biphasic effect with decreased migration at high fractional contents. Combinations of residues show that different sequence contributions to the apparent molecular weight are not additive. The results can be rationalized by the protein-decorated micelle model by considering both SDS binding and the compaction of protein SDS-complexes.

Intrinsically disordered proteins (IDPs) mediate many cellular functions through interactions with structured protein partners, but predicting the corresponding binding sites on the structured partner remains challenging. Here, we present IDBSpred, a sequence-based method for residue-level prediction of IDP-binding sites on structured proteins. Training and test data were collected from the DIBS database, which contains more than 700 non-redundant IDP-protein complexes. Residue-level embeddings of structured partner sequences were generated using the ESM-2 protein language model and used as input to a multilayer perceptron classifier for binary prediction of binding versus non-binding residues. Analysis of amino acid composition showed that IDP-binding sites are enriched in aromatic residues, especially Trp, Tyr, and Phe, as well as several charged and polar residues, whereas Ala and several small or conformationally restrictive residues are depleted. The classifier achieved an ROC AUC of 0.87 and an average precision of 0.61. Structural case studies further showed that the predicted sites largely recapitulate the major experimentally defined binding interfaces. These results demonstrate that protein language model embeddings plus machine learning algorithms can effectively capture sequence features associated with IDP recognition on structured proteins. IDBSpred provides a practical framework for studying IDP-mediated interfaces and identifying potential therapeutic hotspots.

Ubiquitin-like protein 3 (UBL3) is a post-translational modifier that sorts proteins into small extracellular vesicles and regulates the trafficking of disease-associated proteins such as -synuclein. The structural and dynamic features of the UBL domain that underlie these functions, however, remain poorly understood. Here we performed in silico structural dynamics analysis of the UBL3 UBL domain using an NMR structure ensemble combined with anisotropic network modeling (ANM) and perturbation response scanning (PRS). Principal component analysis and residue-wise fluctuation analysis consistently revealed high flexibility in the C-terminal region of UBL3. Comparative ANM analysis across 20 ubiquitin-like proteins (UBLs) further showed that C-terminal flexibility is a conserved yet variable property within the UBL family. PRS analysis demonstrated that residues forming the central -helix of the {beta}-grasp fold exert greater dynamic control over collective motions than {beta}-sheet residues. Notably, UBL3 displayed the highest helix/sheet PRS effectiveness ratio among all UBLs analyzed, highlighting the prominent dynamic contribution of helix residues in this domain. Together, these results provide a structural basis for understanding UBL3-dependent protein interactions and disease-related mechanisms, and suggest that helix-centered dynamic control in the UBL domain may represent a potential target for modulating UBL3 function.

RNA thermometers (RNATs) are temperature-responsive structures in 5' untranslated regions (UTRs) of bacterial messenger RNA (mRNA) that control translation by modulating ribosome access. The Listeria monocytogenes prfA RNAT represses translation of PrfA (positive regulatory factor A), the master virulence regulator, at ambient temperature and activates it near the human host temperature ([~]37 {degrees}C) by modulating ribosome binding site (RBS) accessibility. However, the prfA RNAT shares no homology with known RNAT classes, and its unfolding mechanism remains unclear. Here, we used analytical ultracentrifugation and single-molecule kinetic analysis of RNA transient structure (SiM-KARTS) to map prfA RNAT unfolding. SiM-KARTS analysis demonstrates that thermal opening occurs predominantly at the RBS, while the upper helix of the RNAT hairpin remains largely folded at 37 {degrees}C. The RBS binding kinetics increases with temperature in parallel with translation output, establishing a quantitative link between structural unfolding and function. Mutations in the upper helix impair thermal regulation, indicating that this region tunes switching even as it stays structured at host temperature. Together, these data reveal a hierarchical unfolding pathway in which initial RBS opening triggers activation, whereas the upper helix remotely tunes temperature sensitivity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/717274v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@a95daborg.highwire.dtl.DTLVardef@144bc42org.highwire.dtl.DTLVardef@1a39bf6org.highwire.dtl.DTLVardef@545e60_HPS_FORMAT_FIGEXP M_FIG C_FIG

c-di-AMP is a bacterial second messenger with the crucial role of regulating turgor and osmotic adaptation. Due to the importance of intracellular K+ for osmotic balance, c-di-AMP controls the import and export of K+ by regulating the activity and transcription level of K+ transporters and channels. It has been postulated that c-di-AMP inactivates K+ import and activates K+ export. To gain a full understanding of the properties the K+ machinery in the Gram-positive model organism Bacillus subtilis and in particular, of how the machinery is regulated by c-di-AMP, we characterized the molecular properties of CpaA, a cation/H+ antiporter that has been shown to bind the dinucleotide. We determined the crystal structure of the cytosolic RCK domain with and without c-di-AMP and performed a functional characterization of full-length CpaA using a fluorescence-based flux assay. We found that c-di-AMP binds on the interface of the RCK-C subdomain but only small structural differences are detected between the apo- and holo-structure. We determined that CpaA is more active at high pH and that it slightly favors K+ over Na+ for exchange with H+. Unexpectedly, CpaA is inactivated by c-di-AMP with a K1/2 of inactivation around 1 {micro}M. Our results reinforce the emerging view that regulation of the bacterial K+ machinery by c-di-AMP is more complex than previously thought and that a detailed characterization of the molecular properties of the individual protein components and of how their activity is integrated is necessary for a complete view of the machinery physiological function.

TRPC3 is a calcium-permeable, non-selective cation channel that is activated by DAG. It is expressed in several tissues, especially in the cerebellum, and has been implicated in various human diseases. Despite recent progress in understanding the structural mechanism of TRPC3, how the channel opens remains elusive. Here, we present structures of hTRPC3 in an agonist-free resting state, determined using a DAG-binding site mutant. We also present the structure of hTRPC3 in a DAG-bound open state, determined using a constitutively active "moonwalker" (T561A) mutant. These structures, together with electrophysiological results, reveal that the T561A mutation activates hTRPC3 by disrupting a polar interaction with N652. A newly formed {pi}-bulge in S6 leads to rotation and outward tilting of the lower half of S6, resulting in dilation of the pore and thus channel opening. Agonist DAG stabilizes hTRPC3 in the open conformation. BTDM exerts its inhibitory effect by pushing S5 and S6 back to the center to close the pore, while preserving the {pi}-bulge. These results shed light on the opening mechanism of hTRPC3.

Although machine learning has transformed protein structure prediction of folded protein ground states with remarkable accuracy, intrinsically disordered proteins and regions (IDPs/IDRs) are defined by diverse and dynamical structural ensembles that are predicted with low confidence by algorithms such as AlphaFold and RoseTTAFold. We present a new machine learning method, IDPForge (Intrinsically Disordered Protein, FOlded and disordered Region GEnerator), that exploits a transformer protein language diffusion model to create all-atom IDP ensembles and IDR disordered ensembles that maintains the folded domains. IDPForge does not require sequence-specific training, back transformations from coarse-grained representations, nor ensemble reweighting, as in general the created IDP/IDR conformational ensembles show good agreement with solution experimental data, and options for biasing with experimental restraints are provided if desired. We envision that IDPForge with these diverse capabilities will facilitate integrative and structural studies for proteins that contain intrinsic disorder, and is available as an open source resource for general use.

Coat protein (CP) tertiary structures and their capsid organization of spherical viruses are highly conserved within each virus family. While AlphaFold successfully predicts the tertiary structures of individual CPs, their association to form proper quaternary assemblies cannot be easily accomplished. Here, we report a generalized methodology and associated web-based utility (https://foldavirus.org) that combines AlphaFold predictions of CPs with the knowledge on corresponding icosahedral architectures (e.g., T=1, 3, 4...) based on the known structures from the same virus family to generate associated capsids. The resulting assemblies are subjected to Amber energy minimization to relieve any steric clashes at the inter-subunit interfaces. Significantly, the capsid models are validated by calculating robust Mahalanobis distance using the residue annotations categorized as interface, core and surface amino acids with respect to those observed in the experimentally determined analogous structures. Given the amino acid sequence of CP(s), we successfully generated capsids up to T=9 icosahedral symmetry, including those of Picornaviruses that display pseudo-T=3 symmetry comprising different CPs. As the number of currently available CP sequences are 3-4 orders of magnitude larger than the experimentally determined 3D-structures, this approach bridges the huge gap that exists between the corresponding sequence and structure space.

Nicotinic acetylcholine receptors (nAChRs) belong to the pentameric ligand-gated ion channel superfamily (pLGICs). Among them, the neuronal homomeric 7 nAChR is highly permeable to calcium and plays critical roles in synaptic transmission, cell signaling, and inflammation modulation. The biogenesis of 7 nAChRs is enhanced by the chaperone proteins RIC-3 and NACHO. Previously, we reported a motif in the 5-HT3A receptor, another pLGIC, involved in RIC-3 modulation. Residues in this motif are conserved and also found within the L1-MX segment of the 7 nACh subunit. We therefore explored the regulatory roles of these conserved residues in the biogenesis of 7 nAChRs using multiple approaches, including heterologous expression in Xenopus laevis oocytes, mutagenesis, pull-down assays, cell-surface labeling, and two-electrode voltage-clamp (TEVC) recordings. We find that synthetic 7 L1-MX peptide interacts with both RIC-3 and NACHO. In particular, conserved residues W330, R332, and L336 in the L1-MX positively regulates the assembly of 7 oligomers and the biogenesis of 7nAChR. In presence of residues W330, R332, and L336, NACHO promotes an assembly of an 7 pentamer which is resistant to strong denaturing conditions. NACHO-promoted 7 pentamer is also resistant to Endo H enzyme. Sensitivity of the pentamer to moderate temperatures (37 {degrees}C, 45 {degrees}C, and 50 {degrees}C) suggests that NACHO stabilizes the pentamer via non-covalent interactions. In contrast, Ala replacements at these residues disrupt the biogenesis and abolish 7 current. NACHO and RIC-3 co-expression yields partial rescue of functional expression for some Ala replacement constructs. SUMMARYThis work identifies regulatory roles of conserved residues W330, R332, and L336 in the biogenesis of 7 nAChR. This discovery positions MX subdomain as a promising target for future drug development that can minimize adverse effects.

Human mitochondrial genome (mtDNA) encodes multiple proteins in the oxidative phosphorylation complexes as well as the ribosomal and transfer RNAs (tRNAs) needed for in situ translation. These genes are transcribed from only three promoters, producing polycistronic transcripts that are co-transcriptionally cleaved by mitochondrial RNase enzymes to release majority of individual gene products. tRNAs separate many of these genes and are thought to serve as "punctuation" marks that enable RNase recognition, binding, and hydrolysis of the 5' "leader" and 3' "trailer" sequences flanking the tRNA. Mutations in the tRNA genes dominate the mtDNA-linked mitochondrial pathologies; yet a systematic study of the impact of tRNA sequence variation on the RNase-catalyzed processing is lacking. Here, we employed human mitochondrial tRNATyr as a model system to dissect the effect of tRNA variants on the in vitro 5' leader and 3' trailer hydrolysis. We found that nucleotide variations located near the catalytic interfaces - particularly within or near the tRNA acceptor stem - showed the strongest defects in 5' processing and prevented release of the downstream tRNA in a tRNA cluster where multiple tRNAs are transcribed in tandem. This work provides mechanistic insight into how mutations disrupt coordinated mitochondrial tRNA processing and establish a framework for predicting variant effects based on their structural position relative to the processing enzymes.

GPI-anchored proteins are crucial cell surface proteins with diverse, organism-specific functions, in eukaryotes. They are produced when the GPI transamidase (GPIT), a five-subunit membrane-bound enzyme complex, attaches a pre-formed GPI anchor to the C-terminal end of nascent proteins on the lumenal face of the endoplasmic reticulum. This process requires the removal of a C-terminal signal sequence (SS) on the substrate protein by the action of an endopeptidase subunit of the GPIT, Gpi8/ PIG-K. Using an AMC-tagged peptide in a cell free (post-mitochondrial fraction) assay, this manuscript studies the steady state kinetics of enzymatic cleavage of the substrate by GPIT of the human pathogenic fungus, C. albicans. We show that Mn+2 enhances activity by improving substrate binding but plays no direct role in substrate cleavage per se. Molecular dynamics simulations suggest that the divalent cation binds at a site away from the active site but provides compactness and stability to Gpi8. It also enables a conformation in which a flexible loop (219-244 residues) in the vicinity of the catalytic pocket is able to interact with and position the scissile bond for cleavage by Cys202. Steady state kinetics also indicate that peptides of lengths 7-mer to 9-mer are better bound than 4-mer or 15-mer peptide substrates. A bulky residue at the site of cleavage reduces the catalytic activity of the GPIT. This is the first detailed steady state kinetics study on the endopeptidase activity of a GPIT from any organism.

The coronavirus envelope (E) protein is a viroporin that plays a key role in viral assembly, release, budding and pathogenesis. E protein forms oligomeric ion channels that can activate immune responses. However, high-resolution structural data for its extramembrane domains is limited. The C-terminal domain of SARS-CoV has been shown previously to form amyloid fibers, and here we show that these fibers can modulate the shape of liposomes. The propensity to form fibrils, and their effect on liposomes, was examined for sequences belonging to the four clades of coronaviruses. Electron microscopy data shows that the C-terminal domain in E protein adopts a filamentous structure. These findings demonstrate the potential of these peptides to modulate membranes, providing a possible mechanism by which E protein interacts with membranes in the host cell.