A Molecular Analysis of the Aminopeptidase P-Related Domain of PID-5 from Caenorhabditis elegans.

A novel protein, PID-5, has been shown to be a requirement for germline immortality and has recently been implicated in RNA-induced epigenetic silencing in the Caenorhabditis elegans embryo. Importantly, it has been shown to contain both an eTudor and aminopeptidase P-related domain. However, the silencing mechanism has not yet been fully characterised. In this study, bioinformatic tools were used to compare pre-existing aminopeptidase P molecular structures to the AlphaFold2-predicted aminopeptidase P-related domain of PID-5 (PID-5 APP-RD). Structural homology, metal composition, inhibitor-bonding interactions, and the potential for dimerisation were critically assessed through computational techniques, including structural superimposition and protein-ligand docking. Results from this research suggest that the metallopeptidase-like domain shares high structural homology with known aminopeptidase P enzymes and possesses the canonical 'pita-bread fold'. However, the absence of conserved metal-coordinating residues indicates that only a single Zn2+ may be bound at the active site. The PID-5 APP-RD may form transient interactions with a known aminopeptidase P inhibitor and may therefore recognise substrates in a comparable way to the known structures. However, loss of key catalytic residues suggests the domain will be inactive. Further evidence suggests that heterodimerisation with C. elegans aminopeptidase P is feasible and therefore PID-5 is predicted to regulate proteolytic cleavage in the silencing pathway. PID-5 may interact with PID-2 to bring aminopeptidase P activity to the Z-granule, where it could influence WAGO-4 activity to ensure the balanced production of 22G-RNA signals for transgenerational silencing. Targeted experiments into APPs implicated in malaria and cancer are required in order to build upon the biological and therapeutic significance of this research.

Conserved Characteristics of NMPylation Activities of Alpha- and Betacoronavirus NiRAN Domains.

Coronavirus genome replication and expression are mediated by the viral replication-transcription complex (RTC) which is assembled from multiple nonstructural proteins (nsp). Among these, nsp12 represents the central functional subunit. It harbors the RNA-directed RNA polymerase (RdRp) domain and contains, at its N terminus, an additional domain called NiRAN which is widely conserved in coronaviruses and other nidoviruses. In this study, we produced bacterially expressed coronavirus nsp12s to investigate and compare NiRAN-mediated NMPylation activities from representative alpha- and betacoronaviruses. We found that the four coronavirus NiRAN domains characterized to date have a number of conserved properties, including (i) robust nsp9-specific NMPylation activities that appear to operate largely independently of the C-terminal RdRp domain, (ii) nucleotide substrate preference for UTP followed by ATP and other nucleotides, (iii) dependence on divalent metal ions, with Mn2+ being preferred over Mg2+, and (iv) a key role of N-terminal residues (particularly Asn2) of nsp9 for efficient formation of a covalent phosphoramidate bond between NMP and the N-terminal amino group of nsp9. In this context, a mutational analysis confirmed the conservation and critical role of Asn2 across different subfamilies of the family Coronaviridae, as shown by studies using chimeric coronavirus nsp9 variants in which six N-terminal residues were replaced with those from other corona-, pito- and letovirus nsp9 homologs. The combined data of this and previous studies reveal a remarkable degree of conservation among coronavirus NiRAN-mediated NMPylation activities, supporting a key role of this enzymatic activity in viral RNA synthesis and processing. IMPORTANCE There is strong evidence that coronaviruses and other large nidoviruses evolved a number of unique enzymatic activities, including an additional RdRp-associated NiRAN domain, that are conserved in nidoviruses but not in most other RNA viruses. Previous studies of the NiRAN domain mainly focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggested different functions for this domain, such as NMPylation/RNAylation of nsp9, RNA guanylyltransferase activities involved in canonical and/or unconventional RNA capping pathways, and other functions. To help resolve partly conflicting information on substrate specificities and metal ion requirements reported previously for the SARS-CoV-2 NiRAN NMPylation activity, we extended these earlier studies by characterizing representative alpha- and betacoronavirus NiRAN domains. The study revealed that key features of NiRAN-mediated NMPylation activities, such as protein and nucleotide specificity and metal ion requirements, are very well conserved among genetically divergent coronaviruses, suggesting potential avenues for future antiviral drug development targeting this essential viral enzyme.

The LL-37 domain: A clue to cathelicidin immunomodulatory response?

Host defense peptides (HDPs) are naturally occurring polypeptide sequences that, in addition to being active against bacteria, fungi, viruses, and other parasites, may stimulate immunomodulatory responses. Cathelicidins, a family of HDPs, are produced by diverse animal species, such as mammals, fish, birds, amphibians, and reptiles, to protect them against pathogen infections. These peptides have variable C-terminal domains responsible for their antimicrobial and immunomodulatory activities and a highly conserved N-terminal pre-pro region homologous to cathelin. Although cathelicidins are the major components of innate immunity, the molecular basis by which they induce an immune response is still unclear. In this review, we will address the role of the LL-37 domain and its SK-24, IV-20, FK-13 and LL-37 fragments in the immunity response. Other cathelicidins also share structural and functional characteristics with the LL-37 domain, suggesting that these fragments may be responsible for interaction between these peptides and receptors in humans. Fragments of the LL-37 domain can give us clues about how homologous cathelicidins, in general, induce an immune response.

Unraveling the mechanism of [4Fe-4S] cluster assembly on the N-terminal cluster binding site of NUBP1.

[4Fe-4S]2+ cluster assembly in human cytosol requires both a [2Fe-2S] cluster chaperone being able to donate two [2Fe-2S]2+ clusters and an electron donor providing two electrons to reductively couple the two [2Fe-2S]2+ clusters into a [4Fe-4S]2+ cluster. The mechanism through which the cytosolic [4Fe-4S]2+ cluster assembly works is still not defined. Here, we show that a hetero-tetrameric complex formed by two molecules of cluster-reduced [2Fe-2S]+ 2 -anamorsin and one molecule of dimeric cluster-oxidized [2Fe-2S]2+ 2 -GLRX32 orchestrates the assembly of a [4Fe-4S]2+ cluster on the N-terminal cluster binding site of the cytosolic protein NUBP1. We demonstrate that the hetero-tetrameric complex is able to synergically provide two [2Fe-2S]2+ clusters from GLRX3 and two electrons from anamorsin for the assembly of the [4Fe-4S]2+ cluster on the N-terminal cluster binding site of NUBP1. We also showed that only one of the two [2Fe-2S] clusters bound to anamorsin, that is, that bound to the CX8 CX2 CXC motif, provides the electrons required to form the [4Fe-4S]2+ cluster. Our study contributes to the molecular understanding of the mechanism of [4Fe-4S] protein biogenesis in the cytosol.

The SPOC domain is a phosphoserine binding module that bridges transcription machinery with co- and post-transcriptional regulators.

The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulators as well as RNA capping, splicing and 3'end processing factors. The SPOC domain of PHF3 was recently identified as a CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP (also known as SPEN) and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP SPOC in complex with CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with writers and readers of m6A, the most abundant RNA modification. RBM15 positively regulates m6A levels and mRNA stability in a SPOC-dependent manner, while SHARP SPOC is essential for its localization to inactive X-chromosomes. Our findings suggest that the SPOC domain is a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes.

Staphylococcal Periscope proteins Aap, SasG, and Pls project noncanonical legume-like lectin adhesin domains from the bacterial surface.

Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG, and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonization; they comprise a repetitive region ("B region") and an N-terminal host colonization domain within the "A region," predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG, and Pls) form elongated "stalks" that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap, and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain had been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, while the Aap, SasG, and Pls lectin domains bind a metal ion, they lack the nonproline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of noncanonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.

α-synuclein and phosphoinositide-binding proteins: α-synuclein inhibits the association of PX- but not FYVE-containing proteins with vesicles in vivo.

By an unknown mechanism, alpha-synuclein (α-syn) inhibits autophagy in yeast and human cells. Herein, using the yeast Saccharomyces cerevisiae, we tested the hypothesis that α-syn disrupts autophagy by inhibiting the required association of sorting nexin 4 (Snx4) with phagophores. Snx4 contains a phox (PX) homology domain that selectively binds membranes enriched in phosphatidylinositol 3-phosphate (PI3P). Using fluorescence microscopy, we show that upon nitrogen starvation, 70% of the cells exhibited green puncta (phagophores); whereas identically treated cells expressing α-syn exhibited a significantly lower percentage of cells (30%) with such puncta. Our interpretation is that α-syn outcompetes Snx4 for binding to membranes enriched in PI3P, resulting in fewer phagophores and consequently inefficient induction of autophagy. As a control, we tested whether α-syn disrupts the binding of Vps27-GFP to late endosomes/multivesicular bodies (MVBs). Vps27 contains a PI3P-binding domain called FYVE. α-Syn did not disrupt the binding of Vps27-GFP to late endosomes. α-Syn likely inhibits the binding of PX- but not FYVE-containing proteins to PI3P because FYVE domains bind more than two-orders of magnitude tighter than PX domains. We propose that in all cells, whether yeast or human, α-syn has the potential to inhibit protein trafficking pathways that are dependent on PX-domain proteins such as sorting nexins.

Biochemical and Structural Insights into the Winged Helix Domain of P150, the Largest Subunit of the Chromatin Assembly Factor 1.

The Chromatin Assembly Factor 1 is a heterotrimeric complex responsible for the nucleosome assembly during DNA replication and DNA repair. In humans, the largest subunit P150 is the major actor of this process. It has been recently considered as a tumor-associated protein due to its overexpression in many malignancies. Structural and functional studies targeting P150 are still limited and only scarce information about this subunit is currently available. Literature data and bioinformatics analysis assisted the identification of a stable DNA binding domain, encompassing residues from 721 to 860 of P150 within the full-length protein. This domain was recombinantly produced and in vitro investigated. An acidic region modulating its DNA binding ability was also identified and characterized. Results showed similarities and differences between the P150 and its yeast homologue, namely Cac-1, suggesting that, although sharing a common biological function, the two proteins may also possess different features.

A novel effect of PDLIM5 in α7 nicotinic acetylcholine receptor upregulation and surface expression.

Nicotinic acetylcholine receptors (nAChRs) are widespread throughout the central nervous system. Signaling through nAChRs contributes to numerous higher-order functions, including memory and cognition, as well as abnormalities such as nicotine addiction and neurodegenerative disorders. Although recent studies indicate that the PDZ-containing proteins comprising PSD-95 family co-localize with nicotinic acetylcholine receptors and mediate downstream signaling in the neurons, the mechanisms by which α7nAChRs are regulated remain unclear. Here, we show that the PDZ-LIM domain family protein PDLIM5 binds to α7nAChRs and plays a role in nicotine-induced α7nAChRs upregulation and surface expression. We find that chronic exposure to 1 µM nicotine upregulated α7, ß2-contained nAChRs and PDLIM5 in cultured hippocampal neurons, and the upregulation of α7nAChRs and PDLIM5 is increased more on the cell membrane than the cytoplasm. Interestingly, in primary hippocampal neurons, α7nAChRs and ß2nAChRs display distinct patterns of expression, with α7nAChRs colocalized more with PDLIM5. Furthermore, PDLIM5 interacts with α7nAChRs, but not ß2nAChRs in native brain neurons. Knocking down of PDLIM5 in SH-SY5Y abolishes nicotine-induced upregulation of α7nAChRs. In primary hippocampal neurons, using shRNA against PDLIM5 decreased both surface clustering of α7nAChRs and α7nAChRs-mediated currents. Proteomics analysis and isothermal titration calorimetry (ITC) results show that PDLIM5 interacts with α7nAChRs through the PDZ domain, and the interaction between PDLIM5 and α7nAChRs can be promoted by nicotine. Collectively, our data suggest a novel cellular role of PDLIM5 in the regulation of α7nAChRs, which may be relevant to plastic changes in the nervous system.

Structure and Peptidomes of Swine MHC Class I with Long Peptides Reveal the Cross-Species Characteristics of the Novel N-Terminal Extension Presentation Mode.

Antigenic peptide presentation by the MHC is essential for activating T cells. The current view is that the peptide termini are tethered within the closed Ag-binding groove of MHC class I (MHC-I). Recently, the N-terminal extension mode of peptide presentation has been observed in human MHC-I (HLA-I). In this study, we found that the N terminus of the long peptide can extend beyond the groove of swine MHC-I (SLA-1*0401), confirming that this phenomenon can occur across species. Removal of the N-terminal extra (P-1) residue of the RW12 peptide significantly reduced the folding efficiency of the complex, but truncation of the second half of the peptide did not. Consistent with previous reports, the second (P1) residue of the peptide is twisted, and its side chain is inserted into the A pocket to form two hydrogen bonds with polymorphic E63 and conserved Y159. Mutations of E63 disrupt the binding of the peptide, indicating that E63 is necessary for this peptide-binding mode. Compared with W167, which exists in most MHC-Is, SLA-I-specific S167 ensures an open N-terminal groove of SLA-1*0401, enabling the P-1 residue to extend from the groove. In this MHC class II-like peptide-binding mode, the A pocket is restrictive to the P1 residue and is affected by the polymorphic residues. The peptidomes and refolding data indicated that the open N-terminal groove of SLA-1*0401 allows one to three residues to extend out of the Ag-binding groove. These cross-species comparisons can help us better understand the characteristics of this N-terminal extension presentation mode.

Is amyloid fibrillation related to 3D domain swapping for the C-terminal domain of SARS-CoV main protease?

The C-terminal domain of SARS-CoV main protease (Mpro-C) can form 3D domain-swapped dimer by exchanging the α1-helices fully buried inside the protein hydrophobic core, under non-denaturing conditions. Here, we report that Mpro-C can also form amyloid fibrils under the 3D domain-swappable conditions in vitro, and the fibrils are not formed through runaway/propagated domain swapping. It is found that there are positive correlations between the rates of domain swapping dimerization and amyloid fibrillation at different temperatures, and for different mutants. However, some Mpro-C mutants incapable of 3D domain swapping can still form amyloid fibrils, indicating that 3D domain swapping is not essential for amyloid fibrillation. Furthermore, NMR H/D exchange data and molecular dynamics simulation results suggest that the protofibril core region tends to unpack at the early stage of 3D domain swapping, so that the amyloid fibrillation can proceed during the 3D domain swapping process. We propose that 3D domain swapping makes it possible for the unpacking of the amyloidogenic fragment of the protein and thus accelerates the amyloid fibrillation process kinetically, which explains the well-documented correlations between amyloid fibrillation and 3D domain swapping observed in many proteins.

The CBM48 domain-containing protein FLO6 regulates starch synthesis by interacting with SSIVb and GBSS in rice.

KEY MESSAGE: FLO6 is involved in starch synthesis by interacting with SSIVb and GBSS in rice. Starch synthesized and stored in plastids including chloroplasts and amyloplasts plays a vital role in plant growth and provides the major energy for human diet. However, the molecular mechanisms by which regulate starch synthesis remain largely unknown. In this study, we identified and characterized a rice floury endosperm mutant M39, which exhibited defective starch granule formation in pericarp and endosperm, accompanied by the decreased starch content and amylose content. The abnormal starch accumulation in M39 pollen grains caused a significant decrease in plant fertility. Chloroplasts in M39 leaves contained no or only one large starch granule. Positional cloning combined with complementary experiment demonstrated that the mutant phenotypes were restored by the FLOURY ENDOSPERM6 (FLO6). FLO6 was generally expressed in various tissues, including leaf, anther and developing endosperm. FLO6 is a chloroplast and amyloplast-localized protein that is able to bind to starch by its carbohydrate-binding module 48 (CBM48) domain. Interestingly, we found that FLO6 interacted with starch synthase IVb (SSIVb) and granule-bound starch synthase (GBSSI and GBSSII). Together, our results suggested that FLO6 plays a critical role in starch synthesis through cooperating with several starch synthesis enzymes throughout plant growth and development.

Phylogenetic Analysis with Prediction of Cofactor or Ligand Binding for Pseudomonas aeruginosa PAS and Cache Domains.

PAS domains are omnipresent building blocks of multidomain proteins in all domains of life. Bacteria possess a variety of PAS domains in intracellular proteins and the related Cache domains in periplasmic or extracellular proteins. PAS and Cache domains are predominant in sensory systems, often carry cofactors or bind ligands, and serve as dimerization domains in protein association. To aid our understanding of the wide distribution of these domains, we analyzed the proteome of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 in silico. The ability of this bacterium to survive under different environmental conditions, to switch between planktonic and sessile/biofilm lifestyle, or to evade stresses, notably involves c-di-GMP regulatory proteins or depends on sensory pathways involving multidomain proteins that possess PAS or Cache domains. Maximum likelihood phylogeny was used to group PAS and Cache domains on the basis of amino acid sequence. Conservation of cofactor- or ligand-coordinating amino acids aided by structure-based comparison was used to inform function. The resulting classification presented here includes PAS domains that are candidate binders of carboxylic acids, amino acids, fatty acids, flavin adenine dinucleotide (FAD), 4-hydroxycinnamic acid, and heme. These predictions are put in context to previously described phenotypic data, often generated from deletion mutants. The analysis predicts novel functions for sensory proteins and sheds light on functional diversification in a large set of proteins with similar architecture. IMPORTANCE To adjust to a variety of life conditions, bacteria typically use multidomain proteins, where the modular structure allows functional differentiation. Proteins responding to environmental cues and regulating physiological responses are found in chemotaxis pathways that respond to a wide range of stimuli to affect movement. Environmental cues also regulate intracellular levels of cyclic-di-GMP, a universal bacterial secondary messenger that is a key determinant of bacterial lifestyle and virulence. We study Pseudomonas aeruginosa, an organism known to colonize a broad range of environments that can switch lifestyle between the sessile biofilm and the planktonic swimming form. We have investigated the PAS and Cache domains, of which we identified 101 in 70 Pseudomonas aeruginosa PAO1 proteins, and have grouped these by phylogeny with domains of known structure. The resulting data set integrates sequence analysis and structure prediction to infer ligand or cofactor binding. With this data set, functional predictions for PAS and Cache domain-containing proteins are made.

The sixth transmembrane region of a pheromone G-protein coupled receptor, Map3, is implicated in discrimination of closely related pheromones in Schizosaccharomyces pombe.

Most sexually reproducing organisms have the ability to recognize individuals of the same species. In ascomycete fungi including yeasts, mating between cells of opposite mating type depends on the molecular recognition of two peptidyl mating pheromones by their corresponding G-protein coupled receptors (GPCRs). Although such pheromone/receptor systems are likely to function in both mate choice and prezygotic isolation, very few studies have focused on the stringency of pheromone receptors. The fission yeast Schizosaccharomyces pombe has two mating types, Plus (P) and Minus (M). Here, we investigated the stringency of the two GPCRs, Mam2 and Map3, for their respective pheromones, P-factor and M-factor, in fission yeast. First, we switched GPCRs between S. pombe and the closely related species Schizosaccharomyces octosporus, which showed that SoMam2 (Mam2 of S. octosporus) is partially functional in S. pombe, whereas SoMap3 (Map3 of S. octosporus) is not interchangeable. Next, we swapped individual domains of Mam2 and Map3 with the respective domains in SoMam2 and SoMap3, which revealed differences between the receptors both in the intracellular regions that regulate the downstream signaling of pheromones and in the activation by the pheromone. In particular, we demonstrated that two amino acid residues of Map3, F214 and F215, are key residues important for discrimination of closely related M-factors. Thus, the differences in these two GPCRs might reflect the significantly distinct stringency/flexibility of their respective pheromone/receptor systems; nevertheless, species-specific pheromone recognition remains incomplete.

Structural and Functional Analysis of SsaV Cytoplasmic Domain and Variable Linker States in the Context of the InvA-SsaV Chimeric Protein.

The type III secretion (T3S) injectisome is a syringe-like protein-delivery nanomachine widely utilized by Gram-negative bacteria. It can deliver effector proteins directly from bacteria into eukaryotic host cells, which is crucial for the bacterial-host interaction. Intracellular pathogen Salmonella enterica serovar Typhimurium encodes two sets of T3S injectisomes from Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), which are critical for its host invasion and intracellular survival, respectively. The inner membrane export gate protein, SctV (InvA in SPI-1 and SsaV in SPI-2), is the largest component of the injectisome and is essential for assembly and function of T3SS. Here, we report the 2.11 Å cryo-EM structure of the SsaV cytoplasmic domain (SsaVC) in the context of a full-length SctV chimera consisting of the transmembrane region of InvA, the linker of SsaV (SsaVL) and SsaVC. The structural analysis shows that SsaVC exists in a semi-open state and SsaVL exhibits two major orientations, implying a highly dynamic process of SsaV for the substrate selection and secretion in a full-length context. A biochemical assay indicates that SsaVL plays an essential role in maintaining the nonameric state of SsaV. This study offers near atomic-level insights into how SsaVC and SsaVL facilitate the assembly and function of SsaV and may lead to the development of potential anti-virulence therapeutics against T3SS-mediated bacterial infection. IMPORTANCE Type III secretion system (T3SS) is a multicomponent nanomachine and a critical virulence factor for a wide range of Gram-negative bacterial pathogens. It can deliver numbers of effectors into the host cell to facilitate the bacterial host infection. Export gate protein SctV, as one of the engines of T3SS, is at the center of T3SS assembly and function. In this study, we show the high-resolution atomic structure of the cytosolic domain of SctV in the nonameric state with variable linker conformations. Our first observation of conformational changes of the linker region of SctV and the semi-open state of the cytosolic domain of SctV in the full-length context further support that the substrate selection and secretion process of SctV is highly dynamic. These findings have important implications for the development of therapeutic strategies targeting SctV to combat T3SS-mediated bacterial infection.

Class-specific interactions between Sis1 J-domain protein and Hsp70 chaperone potentiate disaggregation of misfolded proteins.

Protein homeostasis is constantly being challenged with protein misfolding that leads to aggregation. Hsp70 is one of the versatile chaperones that interact with misfolded proteins and actively support their folding. Multifunctional Hsp70s are harnessed to specific roles by J-domain proteins (JDPs, also known as Hsp40s). Interaction with the J-domain of these cochaperones stimulates ATP hydrolysis in Hsp70, which stabilizes substrate binding. In eukaryotes, two classes of JDPs, Class A and Class B, engage Hsp70 in the reactivation of aggregated proteins. In most species, excluding metazoans, protein recovery also relies on an Hsp100 disaggregase. Although intensely studied, many mechanistic details of how the two JDP classes regulate protein disaggregation are still unknown. Here, we explore functional differences between the yeast Class A (Ydj1) and Class B (Sis1) JDPs at the individual stages of protein disaggregation. With real-time biochemical tools, we show that Ydj1 alone is superior to Sis1 in aggregate binding, yet it is Sis1 that recruits more Ssa1 molecules to the substrate. This advantage of Sis1 depends on its ability to bind to the EEVD motif of Hsp70, a quality specific to most of Class B JDPs. This second interaction also conditions the Hsp70-induced aggregate modification that boosts its subsequent dissolution by the Hsp104 disaggregase. Our results suggest that the Sis1-mediated chaperone assembly at the aggregate surface potentiates the entropic pulling, driven polypeptide disentanglement, while Ydj1 binding favors the refolding of the solubilized proteins. Such subspecialization of the JDPs across protein reactivation improves the robustness and efficiency of the disaggregation machinery.

Ycs4 Subunit of Saccharomyces cerevisiae Condensin Binds DNA and Modulates the Enzyme Turnover.

Condensins play a key role in higher order chromosome organization. In budding yeast Saccharomyces cerevisiae, a condensin complex consists of five subunits: two conserved structural maintenance of chromosome subunits, Smc2 and Smc4, a kleisin Brn1, and two HEAT repeat subunits, Ycg1, which possesses a DNA binding activity, and Ycs4, which can transiently associate with Smc4 and thereby disrupt its association with the Smc2 head. We characterized here DNA binding activity of the non-SMC subunits using an agnostic, model-independent approach. To this end, we mapped the DNA interface of the complex using sulfo-NHS biotin labeling. Besides the known site on Ycg1, we found a patch of lysines at the C-terminal domain of Ycs4 that were protected from biotinylation in the presence of DNA. Point mutations at the predicted protein-DNA interface reduced both Ycs4 binding to DNA and the DNA stimulated ATPase activity of the reconstituted condensin, whereas overproduction of the mutant Ycs4 was detrimental for yeast viability. Notably, the DNA binding site on Ycs4 partially overlapped with its interface with SMC4, revealing an intricate interplay between DNA binding, engagement of the Smc2-Smc4 heads, and ATP hydrolysis and suggesting a mechanism for ATP-modulated loading and translocation of condensins on DNA.

Molecular interactions contributing to FUS SYGQ LC-RGG phase separation and co-partitioning with RNA polymerase II heptads.

The RNA-binding protein FUS (Fused in Sarcoma) mediates phase separation in biomolecular condensates and functions in transcription by clustering with RNA polymerase II. Specific contact residues and interaction modes formed by FUS and the C-terminal heptad repeats of RNA polymerase II (CTD) have been suggested but not probed directly. Here we show how RGG domains contribute to phase separation with the FUS N-terminal low-complexity domain (SYGQ LC) and RNA polymerase II CTD. Using NMR spectroscopy and molecular simulations, we demonstrate that many residue types, not solely arginine-tyrosine pairs, form condensed-phase contacts via several interaction modes including, but not only sp2-π and cation-π interactions. In phases also containing RNA polymerase II CTD, many residue types form contacts, including both cation-π and hydrogen-bonding interactions formed by the conserved human CTD lysines. Hence, our data suggest a surprisingly broad array of residue types and modes explain co-phase separation of FUS and RNA polymerase II.

The A39G FF domain folds on a volcano-shaped free energy surface via separate pathways.

Conformational dynamics play critical roles in protein folding, misfolding, function, misfunction, and aggregation. While detecting and studying the different conformational states populated by protein molecules on their free energy surfaces (FESs) remain a challenge, NMR spectroscopy has emerged as an invaluable experimental tool to explore the FES of a protein, as conformational dynamics can be probed at atomic resolution over a wide range of timescales. Here, we use chemical exchange saturation transfer (CEST) to detect "invisible" minor states on the energy landscape of the A39G mutant FF domain that exhibited "two-state" folding kinetics in traditional experiments. Although CEST has mostly been limited to studies of processes with rates between ∼5 to 300 s-1 involving sparse states with populations as low as ∼1%, we show that the line broadening that is often associated with minor state dips in CEST profiles can be exploited to inform on additional conformers, with lifetimes an order of magnitude shorter and populations close to 10-fold smaller than what typically is characterized. Our analysis of CEST profiles that exploits the minor state linewidths of the 71-residue A39G FF domain establishes a folding mechanism that can be described in terms of a four-state exchange process between interconverting states spanning over two orders of magnitude in timescale from ∼100 to ∼15,000 µs. A similar folding scheme is established for the wild-type domain as well. The study shows that the folding of this small domain proceeds through a pair of sparse, partially structured intermediates via two discrete pathways on a volcano-shaped FES.