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MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of the MET intracellular kinase domain in two fusion protein backgrounds: wild-type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase ⍺C-helix, pointing to potential differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a β5 motif that acts as a structural pivot for the kinase domain in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.

The protein phosphatase 5 (PP5) is normally recruited to its substrates by the molecular chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). This interaction requires the tetratricopeptide repeat (TPR) domain of PP5, which binds to an EEVD motif at the extreme C termini of cytosolic Hsp70 and Hsp90 isoforms. In addition to bringing PP5 into proximity with chaperone-bound substrates, this interaction also relieves autoinhibition in PP5s catalytic domain, promoting its phosphatase activity. To better understand the molecular determinants of this process, we screened a large, pentapeptide library for binding to PP5. This screen identified the amino acid preferences at each position, which we validated by showing that the optimal sequences bind 4- to 7-fold tighter than the natural EEVD motifs and stimulate PP5s enzymatic activity. The enhanced affinity for PP5s TPR domain was confirmed using a protein-adaptive differential scanning fluorimetry assay. Using this increased knowledge of structure-activity relationships, we re-examined affinity proteomics results to look for potential EEVD-like motifs in the C termini of known PP5-binding partners. This search identified elongator acetyltransferase complex subunit 1 (IKBKAP) as a putative partner, and indeed, we found that its C-terminal sequence, LSLLD, binds directly to PP5s TPR domain in vitro. Consistent with this idea, mutation of elongator acetyltransferase complex subunit 1s terminal aspartate was sufficient to interrupt the interaction with PP5 in vitro and in cells. Together, these findings reveal the sequence preferences of PP5s TPR domain and expand the scope of PP5s functions to include chaperone-independent complexes.

The phytochrome superfamily comprises three groups of photoreceptors sharing a conserved GAF (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA) domain that uses a covalently attached linear tetrapyrrole (bilin) chromophore to sense light. Knotted red/far-red phytochromes are widespread in both bacteria and eukaryotes, but cyanobacteria also contain knotless red/far-red phytochromes and cyanobacteriochromes (CBCRs). Unlike typical phytochromes, CBCRs require only the GAF domain for bilin binding, chromophore ligation, and full, reversible photoconversion. CBCRs can sense a wide range of wavelengths (ca. 330-750 nm) and can regulate phototaxis, second messenger metabolism, and optimization of the cyanobacterial light-harvesting apparatus. However, the origins of CBCRs are not well understood: we do not know when or why CBCRs evolved, or what selective advantages led to retention of early CBCRs in cyanobacterial genomes. In the current work, we use the increasing availability of genomes and metagenome-assembled-genomes from early-branching cyanobacteria to explore the origins of CBCRs. We reaffirm the earliest branches in CBCR evolution. We also show that early-branching cyanobacteria contain late-branching CBCRs, implicating early appearance of CBCRs during cyanobacterial evolution. Moreover, we show that early-branching CBCRs behave as integrators of light and pH, providing a potential unique function for early CBCRs that led to their retention and subsequent diversification. Our results thus provide new insight into the origins of these diverse cyanobacterial photoreceptors.

N-capping (N-cap) and C-capping (C-cap) in biologically active peptides, including specific amino acids or unconventional group motifs, have been shown to modulate activity against pharmacological targets by interfering with the peptide's secondary structure, thus generating unusual scaffolds. The insertion of capping motifs in linear peptides has been shown to prevent peptide degradation by reducing its susceptibility to proteolytic cleavage, and the replacement of some functional groups by unusual groups in N- or C-capping regions in linear peptides has led to optimized peptide variants with improved secondary structure and enhanced activity. Furthermore, some essential amino acid residues that, when placed in antimicrobial peptide (AMP) capping regions, are capable of complexing metals such as Cu2+, Ni2+, and Zn2+, give rise to the family known as metallo-AMPs, which are capable of boosting antimicrobial efficacy, as well as other activities. Therefore, this review presents and discusses the different strategies for creating N- and C-cap motifs in AMPs, aiming at fine-tuning this class of antimicrobials. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) functions downstream of cyclic GMP-AMP synthase in DNA sensing or as a direct receptor for bacterial cyclic dinucleotides and small molecules to activate immunity during infection, cancer and immunotherapy1-10. Precise regulation of STING is essential to ensure balanced immune responses and prevent detrimental autoinflammation11-16. After activation, STING, a transmembrane protein, traffics from the endoplasmic reticulum to the Golgi, where its phosphorylation by the protein kinase TBK1 enables signal transduction17-20. The mechanism that ends STING signalling at the Golgi remains unknown. Here we show that adaptor protein complex 1 (AP-1) controls the termination of STING-dependent immune activation. We find that AP-1 sorts phosphorylated STING into clathrin-coated transport vesicles for delivery to the endolysosomal system, where STING is degraded21. We identify a highly conserved dileucine motif in the cytosolic C-terminal tail (CTT) of STING that, together with TBK1-dependent CTT phosphorylation, dictates the AP-1 engagement of STING. A cryo-electron microscopy structure of AP-1 in complex with phosphorylated STING explains the enhanced recognition of TBK1-activated STING. We show that suppression of AP-1 exacerbates STING-induced immune responses. Our results reveal a structural mechanism of negative regulation of STING and establish that the initiation of signalling is inextricably associated with its termination to enable transient activation of immunity.

Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling1. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes2-5. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .

N-capping (N-cap) and C-capping (C-cap) in biologically active peptides, including specific amino acids or unconventional group motifs, have been shown to modulate activity against pharmacological targets by interfering with the peptide’s secondary structure, thus generating unusual scaffolds. The insertion of capping motifs in linear peptides has been shown to prevent peptide degradation by reducing its susceptibility to proteolytic cleavage, and the replacement of some functional groups by unusual groups in N- or C-capping regions in linear peptides has led to optimized peptide variants with improved secondary structure and enhanced activity. Furthermore, some essential amino acid residues that, when placed in antimicrobial peptide (AMP) capping regions, are capable of complexing metals such as Cu2+, Ni2+, and Zn2+, give rise to the family known as metallo-AMPs, which are capable of boosting antimicrobial efficacy, as well as other activities. Therefore, this review presents and discusses the different strategies for creating N- and C-cap motifs in AMPs, aiming at fine-tuning this class of antimicrobials.

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin-nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.

The interactions of proteins with surfaces are important in both biological processes and biotechnologies. In contrast to decades of study regarding the biophysics of proteins in bulk solution, however, our mechanistic understanding of the biophysics of proteins interacting with surfaces remains largely qualitative. In response, we have set to explore quantitatively the thermodynamics of protein-surface interactions. In this work, we explore systematically the role of electrostatics in modulating the interaction between proteins and charged surfaces. In particular, we use electrochemistry to explore the extent to which a macroscopic, hydroxyl-coated surface held at a slightly negative potential affects the folding thermodynamics of surface-attached protein variants with different composition of charged amino acids. Doing so, we find that attachment to the surface generally leads to a net stabilization, presumably due to excluded volume effects that reduce the entropy of the unfolded state. The magnitude of this stabilization, however, is strongly correlated with the charged-residue content of the protein. In particular, we find statistically significant correlations with both the net charge of the protein, with greater negative charge leading to less stabilization by the surface, and with the number of arginines, with more arginines leading to greater stabilization. Such findings refine our understanding of protein-surface interactions, providing in turn a guiding rationale to achieve the functional deposition of proteins on artificial surfaces for implementation in, for example, protein-based biotechnologies.

Organisms orchestrate cellular functions through transcription factor (TF) interactions with their target genes, although these regulatory relationships are largely unknown in most species. Here we report a high-throughput approach for characterizing TF-target gene interactions across species and its application to 354 TFs across 48 bacteria, generating 17,000 genome-wide binding maps. This dataset revealed themes of ancient conservation and rapid evolution of regulatory modules. We observed rewiring, where the TF sensing and regulatory role is maintained while the arrangement and identity of target genes diverges, in some cases encoding entirely new functions. We further integrated phenotypic information to define new functional regulatory modules and pathways. Finally, we identified 242 new TF DNA binding motifs, including a 70% increase of known Escherichia coli motifs and the first annotation in Pseudomonas simiae, revealing deep conservation in bacterial promoter architecture. Our method provides a versatile tool for functional characterization of genetic pathways in prokaryotes and eukaryotes.

BackgroundSonic Hedgehog (Shh) has a catalytic cleft characteristic for zinc metallopeptidases and has significant sequence similarities with some bacterial peptidoglycan metallopeptidases defining a subgroup within the M15A family that, besides having the characteristic zinc coordination motif, can bind two calcium ions. Extracellular matrix (ECM) components in animals include heparan-sulfate proteoglycans, which are analogs of bacterial peptidoglycan and are involved in the extracellular distribution of Shh.ResultsWe found that the zinc-coordination center of Shh is required for its association to the ECM as well as for non-cell autonomous signaling. Association with the ECM requires the presence of at least 0.1 μM zinc and is prevented by mutations affecting critical conserved catalytical residues. Consistent with the presence of a conserved calcium binding domain, we find that extracellular calcium inhibits ECM association of Shh.ConclusionsOur results indicate that the putative intrinsic peptidase activity of Shh is required for non-cell autonomous signaling, possibly by enzymatically altering ECM characteristics.

Chemical synaptic transmission represents the most sophisticated dynamic process and is highly regulated with optimized neurotransmitter balance. Imbalanced transmitters can lead to transmission impairments, for example, intracellular zinc accumulation is a hallmark of degenerating neurons. However, the underlying mechanisms remain elusive. Postsynaptic density protein-95 (PSD-95) is a primary postsynaptic membrane-associated protein and the major scaffolding component in the excitatory postsynaptic densities, which performs substantial functions in synaptic development and maturation. Its membrane association induced by palmitoylation contributes largely to its regulatory functions at postsynaptic sites. Unlike other structural domains in PSD-95, the N-terminal region (PSD-95NT) is flexible and interacts with various targets, which modulates its palmitoylation of two cysteines (C3/C5) and glutamate receptor distributions in postsynaptic densities. PSD-95NT contains a putative zinc-binding motif (C2H2) with undiscovered functions. This study is the first effort to investigate the interaction between Zn2+ and PSD-95NT. The NMR titration of 15 N-labeled PSD-95NT by ZnCl2 was performed and demonstrated Zn2+ binds to PSD-95NT with a binding affinity (Kd ) in the micromolar range. The zinc binding was confirmed by fluorescence and mutagenesis assays, indicating two cysteines and two histidines (H24, H28) are critical residues for the binding. These results suggested the concentration-dependent zinc binding is likely to influence PSD-95 palmitoylation since the binding site overlaps the palmitoylation sites, which was verified by the mimic PSD-95 palmitoyl modification and intact cell palmitoylation assays. This study reveals zinc as a novel modulator for PSD-95 postsynaptic membrane association by chelating its N-terminal region, indicative of its importance in postsynaptic signaling.

Membraneless organelles (MLOs) are vital and dynamic reaction centers in cells that compartmentalize the cytoplasm in the absence of a membrane. Multivalent interactions between protein low-complexity domains contribute to MLO organization. Previously, we used computational methods to identify structural motifs termed low-complexity amyloid-like reversible kinked segments (LARKS) that promote phase transition to form hydrogels and that are common in human proteins that participate in MLOs. Here, we searched for LARKS in the proteomes of six model organisms: Homo sapiens, Drosophila melanogaster, Plasmodium falciparum, Saccharomyces cerevisiae, Mycobacterium tuberculosis, and Escherichia coli to gain an understanding of the distribution of LARKS in the proteomes of various species. We found that LARKS are abundant in M. tuberculosis, D. melanogaster, and H. sapiens but not in S. cerevisiae or P. falciparum. LARKS have high glycine content, which enables kinks to form as exemplified by the known LARKS-rich amyloidogenic structures of TDP43, FUS, and hnRNPA2, three proteins that are known to participate in MLOs. These results support the idea of LARKS as an evolved structural motif. Based on these results, we also established the LARKSdb Web server, which permits users to search for LARKS in their protein sequences of interest.

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA-binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn directs the relative trajectories of the CC motifs on each subunit of the att-bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal-length CC motifs and 14 residues surrounding the tip where the pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggest that molecular interactions between CC motif tips may differ in integrative (attP × attB) and excisive (attL × attR) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminately, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after the initial assembly of the integrase synaptic tetramer. IMPORTANCE The robust and exquisitely regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage and, in the case of the A118 prophage, are an important virulence factor of Listeria monocytogenes. The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

Multicomponent immune receptors are essential complexes in which distinct ligand-recognition and signaling subunits are held together by interactions between acidic and basic residues of their transmembrane helices. A 2:1 acidic-to-basic motif in the transmembrane domains of the subunits is necessary and sufficient to assemble these receptor complexes. Here, we study a prototype for these receptors, a DAP12-NKG2C 2:1 heterotrimeric complex, in which the two DAP12 subunits each contribute a single transmembrane Asp residue, and the NKG2C subunit contributes a Lys to form the complex. DAP12 can also associate with 20 other subunits using a similar motif. Here, we use molecular-dynamics simulations to understand the basis for the high affinity and diversity of interactions in this group of receptors. Simulations of the transmembrane helices with differing protonation states of the Asp-Asp-Lys triad identified a structurally stable interaction in which a singly-protonated Asp-Asp pair forms a hydrogen-bonded carboxyl-carboxylate clamp that clasps onto a charged Lys side chain. This polar motif was also supported by density functional theory and a Protein Data Bank-wide search. In contrast, the helices are dynamic at sites distal to the stable carboxyl-carboxylate clamp motif. Such a locally stable but globally dynamic structure is well suited to accommodate the sequence and structural variations in the transmembrane helices of multicomponent receptors, which mix and match subunits to create combinatorial functional diversity from a limited number of subunits. It also supports a signaling mechanism based on multisubunit clustering rather than propagation of rigid conformational changes through the membrane.

Microbial sulfur metabolism contributes to biogeochemical cycling on global scales. Sulfur metabolizing microbes are infected by phages that can encode auxiliary metabolic genes (AMGs) to alter sulfur metabolism within host cells but remain poorly characterized. Here we identified 191 phages derived from twelve environments that encoded 227 AMGs for oxidation of sulfur and thiosulfate (dsrA, dsrC/tusE, soxC, soxD and soxYZ). Evidence for retention of AMGs during niche-differentiation of diverse phage populations provided evidence that auxiliary metabolism imparts measurable fitness benefits to phages with ramifications for ecosystem biogeochemistry. Gene abundance and expression profiles of AMGs suggested significant contributions by phages to sulfur and thiosulfate oxidation in freshwater lakes and oceans, and a sensitive response to changing sulfur concentrations in hydrothermal environments. Overall, our study provides fundamental insights on the distribution, diversity, and ecology of phage auxiliary metabolism associated with sulfur and reinforces the necessity of incorporating viral contributions into biogeochemical configurations.

To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.

Telomerase is unique among the reverse transcriptases in containing a noncoding RNA (known as telomerase RNA (TER)) that includes a short template that is used for the processive synthesis of G-rich telomeric DNA repeats at the 3' ends of most eukaryotic chromosomes1. Telomerase maintains genomic integrity, and its activity or dysregulation are critical determinants of human longevity, stem cell renewal and cancer progression2,3. Previous cryo-electron microscopy structures have established the general architecture, protein components and stoichiometries of Tetrahymena and human telomerase, but our understandings of the details of DNA-protein and RNA-protein interactions and of the mechanisms and recruitment involved remain limited4-6. Here we report cryo-electron microscopy structures of active Tetrahymena telomerase with telomeric DNA at different steps of nucleotide addition. Interactions between telomerase reverse transcriptase (TERT), TER and DNA reveal the structural basis of the determination of the 5' and 3' template boundaries, handling of the template-DNA duplex and separation of the product strand during nucleotide addition. The structure and binding interface between TERT and telomerase protein p50 (a homologue of human TPP17,8) define conserved interactions that are required for telomerase activation and recruitment to telomeres. Telomerase La-related protein p65 remodels several regions of TER, bridging the 5' and 3' ends and the conserved pseudoknot to facilitate assembly of the TERT-TER catalytic core.

The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x3-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCβII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.

Gene regulatory elements are central drivers of phenotypic variation and thus of critical importance towards understanding the genetics of complex traits. The Functional Annotation of Animal Genomes consortium was formed to collaboratively annotate the functional elements in animal genomes, starting with domesticated animals. Here we present an expansive collection of datasets from eight diverse tissues in three important agricultural species: chicken (Gallus gallus), pig (Sus scrofa), and cattle (Bos taurus). Comparative analysis of these datasets and those from the human and mouse Encyclopedia of DNA Elements projects reveal that a core set of regulatory elements are functionally conserved independent of divergence between species, and that tissue-specific transcription factor occupancy at regulatory elements and their predicted target genes are also conserved. These datasets represent a unique opportunity for the emerging field of comparative epigenomics, as well as the agricultural research community, including species that are globally important food resources.

Both nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations are routinely used in understanding the conformational space sampled by peptides in the solution state. To investigate the role of single-residue change in the ensemble of conformations sampled by a set of heptapeptides, AEVXEVG with X = L, F, A, or G, comprehensive NMR, and MD simulations were performed. The rationale for selecting the particular model peptides is based on the high variability in the occurrence of tri-peptide E*L between the transmembrane β-barrel (TMB) than in globular proteins. The ensemble of conformations sampled by E*L was compared between the three sets of ensembles derived from NMR spectroscopy, MD simulations with explicit solvent, and the random coil conformations. In addition to the estimation of global determinants such as the radius of gyration of a large sample of structures, the ensembles were analyzed using principal component analysis (PCA). In general, the results suggest that the -EVL- peptide indeed adopts a conformational preference that is distinctly different not only from a random distribution but also from other peptides studied here. The relatively straightforward approach presented herein could help understand the conformational preferences of small peptides in the solution state.

SARS-CoV-2 antibodies develop within two weeks of infection, but wane relatively rapidly post-infection, raising concerns about whether antibody responses will provide protection upon re-exposure. Here we revisit T-B cooperation as a prerequisite for effective and durable neutralizing antibody responses centered on a mutationally constrained RBM B cell epitope. T-B cooperation requires co-processing of B and T cell epitopes by the same B cell and is subject to MHC-II restriction. We evaluated MHC-II constraints relevant to the neutralizing antibody response to a mutationally-constrained B cell epitope in the receptor binding motif (RBM) of the spike protein. Examining common MHC-II alleles, we found that peptides surrounding this key B cell epitope are predicted to bind poorly, suggesting a lack MHC-II support in T-B cooperation, impacting generation of high-potency neutralizing antibodies in the general population. Additionally, we found that multiple microbial peptides had potential for RBM cross-reactivity, supporting previous exposures as a possible source of T cell memory.

Starch branching enzymes (SBEs) are key determinants of the structure and amount of the starch in plant organs, and as such, they have the capacity to influence plant growth, developmental, and fitness processes, and in addition, the industrial end-use of starch. However, little is known about the role of SBEs in determining starch structure-function relations in economically important horticultural crops such as fruit and leafy greens, many of which accumulate starch transiently. Further, a full understanding of the biological function of these types of starches is lacking. Because of this gap in knowledge, this minireview aims to provide an overview of SBEs in horticultural crops, to investigate the potential role of starch in determining postharvest quality. A systematic examination of SBE sequences in 43 diverse horticultural species, identified SBE1, 2 and 3 isoforms in all species examined except apple, olive, and Brassicaceae, which lacked SBE1, but had a duplicated SBE2. Among our findings after a comprehensive and critical review of published data, was that as apple, banana, and tomato fruits ripens, the ratio of the highly digestible amylopectin component of starch increases relative to the more digestion-resistant amylose fraction, with parallel increases in SBE2 transcription, fruit sugar content, and decreases in starch. It is tempting to speculate that during the ripening of these fruit when starch degradation occurs, there are rearrangements made to the structure of starch possibly via branching enzymes to increase starch digestibility to sugars. We propose that based on the known action of SBEs, and these observations, SBEs may affect produce quality, and shelf-life directly through starch accumulation, and indirectly, by altering sugar availability. Further studies where SBE activity is fine-tuned in these crops, can enrich our understanding of the role of starch across species and may improve horticulture postharvest quality.

Binding of the intracellular adapter proteins talin and its cofactor, kindlin, to the integrin receptors induces integrin activation and clustering. These processes are essential for cell adhesion, migration, and organ development. Although the talin head, the integrin-binding segment in talin, possesses a typical FERM-domain sequence, a truncated form has been crystallized in an unexpected, elongated form. This form, however, lacks a C-terminal fragment and possesses reduced β3-integrin binding. Here, we present a crystal structure of a full-length talin head in complex with the β3-integrin tail. The structure reveals a compact FERM-like conformation and a tightly associated N-P-L-Y motif of β3-integrin. A critical C-terminal poly-lysine motif mediates FERM interdomain contacts and assures the tight association with the β3-integrin cytoplasmic segment. Removal of the poly-lysine motif or disrupting the FERM-folded configuration of the talin head significantly impairs integrin activation and clustering. Therefore, structural characterization of the FERM-folded active talin head provides fundamental understanding of the regulatory mechanism of integrin function.

BackgroundThe circadian clock drives endogenous 24-h rhythms that allow organisms to adapt and prepare for predictable and repeated changes in their environment throughout the day-night (diurnal) cycle. Many components of the circadian clock in Arabidopsis thaliana have been functionally characterized, but comparatively little is known about circadian clocks in grass species including major crops like maize and sorghum.ResultsComparative research based on protein homology and diurnal gene expression patterns suggests the function of some predicted clock components in grasses is conserved with their Arabidopsis counterparts, while others have diverged in function. Our analysis of diurnal gene expression in three panicoid grasses sorghum, maize, and foxtail millet revealed conserved and divergent evolution of expression for core circadian clock genes and for the overall transcriptome. We find that several classes of core circadian clock genes in these grasses differ in copy number compared to Arabidopsis, but mostly exhibit conservation of both protein sequence and diurnal expression pattern with the notable exception of maize paralogous genes. We predict conserved cis-regulatory motifs shared between maize, sorghum, and foxtail millet through identification of diurnal co-expression clusters for a subset of 27,196 orthologous syntenic genes. In this analysis, a Cochran-Mantel-Haenszel based method to control for background variation identified significant enrichment for both expected and novel 6-8 nucleotide motifs in the promoter regions of genes with shared diurnal regulation predicted to function in common physiological activities.ConclusionsThis study illustrates the divergence and conservation of circadian clocks and diurnal regulatory networks across syntenic orthologous genes in panacoid grass species. Further, conserved local regulatory sequences contribute to the architecture of these diurnal regulatory networks that produce conserved patterns of diurnal gene expression.

Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin. Using an inducible complementation system, we find that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remains at CTCF sites in this mutant, albeit with reduced enrichment. Given the orientation of CTCF motifs presents the N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites translates into genome folding patterns.

RNA helicases catalyze the ATP-dependent destabilization of RNA duplexes. DEAD-box helicases share a helicase core that mediates ATP binding and hydrolysis, RNA binding and unwinding. Most members of this family contain domains flanking the core that can confer RNA substrate specificity and guide the helicase to a specific RNA. However, the in vivo RNA substrates of most helicases are currently not defined. The DEAD-box helicase Hera from Thermus thermophilus contains a helicase core, followed by a dimerization domain and an RNA binding domain that folds into an RNA recognition motif (RRM). The RRM mediates high affinity binding to an RNA hairpin, and an adjacent duplex is then unwound by the helicase core. Hera is a cold-shock protein, and has been suggested to act as an RNA chaperone under cold-shock conditions. Using crosslinking immunoprecipitation of Hera/RNA complexes and sequencing, we show that Hera binds to a large fraction of T. thermophilus RNAs under normal-growth and cold-shock conditions without a strong sequence preference, in agreement with a structure-specific recognition of RNAs and a general function in RNA metabolism. Under cold-shock conditions, Hera is recruited to RNAs with high propensities to form stable secondary structures. We show that selected RNAs identified, including a set of tRNAs, bind to Hera in vitro, and activate the Hera helicase core. Gene ontology analysis reveals an enrichment of genes related to translation, including mRNAs of ribosomal proteins, tRNAs, tRNA ligases, and tRNA-modifying enzymes, consistent with a key role of Hera in ribosome and tRNA metabolism.

Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19 is a newly recognized condition in children with recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. These children and adult patients with severe hyperinflammation present with a constellation of symptoms that strongly resemble toxic shock syndrome, an escalation of the cytotoxic adaptive immune response triggered upon the binding of pathogenic superantigens to T cell receptors (TCRs) and/or major histocompatibility complex class II (MHCII) molecules. Here, using structure-based computational models, we demonstrate that the SARS-CoV-2 spike (S) glycoprotein exhibits a high-affinity motif for binding TCRs, and may form a ternary complex with MHCII. The binding epitope on S harbors a sequence motif unique to SARS-CoV-2 (not present in other SARS-related coronaviruses), which is highly similar in both sequence and structure to the bacterial superantigen staphylococcal enterotoxin B. This interaction between the virus and human T cells could be strengthened by a rare mutation (D839Y/N/E) from a European strain of SARS-CoV-2. Furthermore, the interfacial region includes selected residues from an intercellular adhesion molecule (ICAM)-like motif shared between the SARS viruses from the 2003 and 2019 pandemics. A neurotoxin-like sequence motif on the receptor-binding domain also exhibits a high tendency to bind TCRs. Analysis of the TCR repertoire in adult COVID-19 patients demonstrates that those with severe hyperinflammatory disease exhibit TCR skewing consistent with superantigen activation. These data suggest that SARS-CoV-2 S may act as a superantigen to trigger the development of MIS-C as well as cytokine storm in adult COVID-19 patients, with important implications for the development of therapeutic approaches.

Sialic acid-binding immunoglobulin-like lectins (Siglec)-like domains of streptococcal serine-rich repeat (SRR) adhesins recognize sialylated glycans on human salivary, platelet, and plasma glycoproteins via a YTRY sequence motif. The SRR adhesin from Streptococcus sanguinis strain SK1 has tandem sialoglycan-binding domains and has previously been shown to bind sialoglycans with high affinity. However, both domains contain substitutions within the canonical YTRY motif, making it unclear how they interact with host receptors. To identify how the S. sanguinis strain SK1 SRR adhesin affects interactions with sialylated glycans and glycoproteins, we determined high-resolution crystal structures of the binding domains alone and with purified trisaccharides. These structural studies determined that the ligands still bind at the noncanonical binding motif, but with fewer hydrogen-bonding interactions to the protein than is observed in structures of other Siglec-like adhesins. Complementary biochemical studies identified that each of the two binding domains has a different selectivity profile. Interestingly, the binding of SK1 to platelets and plasma glycoproteins identified that the interaction to some host targets is dominated by the contribution of one binding domain, whereas the binding to other host receptors is mediated by both binding domains. These results provide insight into outstanding questions concerning the roles of tandem domains in targeting host receptors and suggest mechanisms for how pathogens can adapt to the availability of a range of related but nonidentical host receptors. They further suggest that the definition of the YTRY motif should be changed to ϕTRX, a more rigorous description of this sialic acid-recognition motif given recent findings.

Tumor-infiltrating leukocytes, in particular macrophages, play an important role in tumor behavior and clinical outcome. The spectrum of macrophage subtypes ranges from antitumor 'M1'-type to protumor 'M2'-type macrophages. Tumor-associated macrophages (TAMs) typically display phenotypic features of both M1 and M2, and the population distribution is thought to be dynamic and evolves as the tumor progresses. However, our understanding of how TAMs impact the tumor microenvironment remains limited by the lack of appropriate 3D in vitro models that can capture cell-cell dynamics at high spatial and temporal resolution. Using our recently developed microphysiological 'tumor-on-a-chip' (TOC) device, we present here our findings on the impact of defined macrophage subsets on tumor behavior. The TOC device design contains three adjacent and connected chambers in which both the upper and lower chambers are loaded with tumor cells, whereas the central chamber contains a dynamic, perfused, living microvascular network. Introduction of human pancreatic or colorectal cancer cells together with M1-polarized macrophages significantly inhibited tumor growth and tumor-induced angiogenesis. Protein analysis and antibody-based neutralization studies confirmed that these effects were mediated through production of C-X-C motif chemokines (CXCL9), CXCL10 and CXCL11. By contrast, M2-macrophages mediated increased tumor cell migration into the vascularized chamber and did not inhibit tumor growth or angiogenesis. In fact, single-cell RNA sequencing showed that M2 macrophages further segregated endothelial cells into two distinct subsets, corresponding to static cells in vessels versus active cells involved in angiogenesis. The impact of M2 macrophages was mediated mostly by production of matrix metalloproteinase 7 and angiopoietin 2. In summary, our data demonstrate the utility of the TOC device to mechanistically probe biological questions in a 3D in vitro microenvironment.

Proline-rich domains (PRDs) are among the most prevalent signaling modules of eukaryotes but often unexplored by biophysical techniques as their heterologous recombinant expression poses significant difficulties. Using a "divide-and-conquer" approach, we present a detailed investigation of a PRD (166 residues; ∼30% prolines) belonging to a human protein ALIX, a versatile adaptor protein involved in essential cellular processes including ESCRT-mediated membrane remodeling, cell adhesion, and apoptosis. In solution, the N-terminal fragment of ALIX-PRD is dynamically disordered. It contains three tandem sequentially similar proline-rich motifs that compete for a single binding site on its signaling partner, TSG101-UEV, as evidenced by heteronuclear NMR spectroscopy. Global fitting of relaxation dispersion data, measured as a function of TSG101-UEV concentration, allowed precise quantitation of these interactions. In contrast to the soluble N-terminal portion, the C-terminal tyrosine-rich fragment of ALIX-PRD forms amyloid fibrils and viscous gels validated using dye-binding assays with amyloid-specific probes, congo red and thioflavin T (ThT), and visualized by transmission electron microscopy. Remarkably, fibrils dissolve at low temperatures (2 to 6 °C) or upon hyperphosphorylation with Src kinase. Aggregation kinetics monitored by ThT fluorescence shows that charge repulsion dictates phosphorylation-mediated fibril dissolution and that the hydrophobic effect drives fibril formation. These data illuminate the mechanistic interplay between interactions of ALIX-PRD with TSG101-UEV and polymerization of ALIX-PRD and its central role in regulating ALIX function. This study also demonstrates the broad functional repertoires of PRDs and uncovers the impact of posttranslational modifications in the modulation of reversible amyloids.

β- and γ-herpesviruses include the oncogenic human viruses Kaposi's sarcoma-associated virus (KSHV) and Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV), which is a significant cause of congenital disease. Near the end of their replication cycle, these viruses transcribe their late genes in a manner distinct from host transcription. Late gene transcription requires six virally encoded proteins, one of which is a functional mimic of host TATA-box-binding protein (TBP) that is also involved in recruitment of RNA polymerase II (Pol II) via unknown mechanisms. Here, we applied biochemical protein interaction studies together with electron microscopy-based imaging of a reconstituted human preinitiation complex to define the mechanism underlying Pol II recruitment. These data revealed that the herpesviral TBP, encoded by ORF24 in KSHV, makes a direct protein-protein contact with the C-terminal domain of host RNA polymerase II (Pol II), which is a unique feature that functionally distinguishes viral from cellular TBP. The interaction is mediated by the N-terminal domain (NTD) of ORF24 through a conserved motif that is shared in its β- and γ-herpesvirus homologs. Thus, these herpesviruses employ an unprecedented strategy in eukaryotic transcription, wherein promoter recognition and polymerase recruitment are facilitated by a single transcriptional activator with functionally distinct domains.

The ESCRT complexes drive membrane scission in HIV-1 release, autophagosome closure, multivesicular body biogenesis, cytokinesis, and other cell processes. ESCRT-I is the most upstream complex and bridges the system to HIV-1 Gag in virus release. The crystal structure of the headpiece of human ESCRT-I comprising TSG101-VPS28-VPS37B-MVB12A was determined, revealing an ESCRT-I helical assembly with a 12-molecule repeat. Electron microscopy confirmed that ESCRT-I subcomplexes form helical filaments in solution. Mutation of VPS28 helical interface residues blocks filament formation in vitro and autophagosome closure and HIV-1 release in human cells. Coarse-grained (CG) simulations of ESCRT assembly at HIV-1 budding sites suggest that formation of a 12-membered ring of ESCRT-I molecules is a geometry-dependent checkpoint during late stages of Gag assembly and HIV-1 budding and templates ESCRT-III assembly for membrane scission. These data show that ESCRT-I is not merely a bridging adaptor; it has an essential scaffolding and mechanical role in its own right.

Human catechol O-methyltransferase (COMT) has emerged as a model for understanding enzyme-catalyzed methyl transfer from S-adenosylmethionine (AdoMet) to small-molecule catecholate acceptors. Mutation of a single residue (tyrosine 68) behind the methyl-bearing sulfonium of AdoMet was previously shown to impair COMT activity by interfering with methyl donor-acceptor compaction within the activated ground state of the wild type enzyme [J. Zhang, H. J. Kulik, T. J. Martinez, J. P. Klinman, Proc. Natl. Acad. Sci. U.S.A. 112, 7954-7959 (2015)]. This predicts the involvement of spatially defined protein dynamical effects that further tune the donor/acceptor distance and geometry as well as the electrostatics of the reactants. Here, we present a hydrogen/deuterium exchange (HDX)-mass spectrometric study of wild type and mutant COMT, comparing temperature dependences of HDX against corresponding kinetic and cofactor binding parameters. The data show that the impaired Tyr68Ala mutant displays similar breaks in Arrhenius plots of both kinetic and HDX properties that are absent in the wild type enzyme. The spatial resolution of HDX below a break point of 15-20 °C indicates changes in flexibility across ∼40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20 °C, Tyr68Ala behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Ala can be understood in the context of a mutationally induced alteration in protein motions that becomes manifest along and perpendicular to the primary group transfer coordinate.

In a radical departure from the classical E1-E2-E3 three-enzyme mediated ubiquitination of eukaryotes, the recently described bacterial enzymes of the SidE family of Legionella pneumophila effectors utilize NAD+ to ligate ubiquitin onto target substrate proteins. This outcome is achieved via a two-step mechanism involving (1) ADP ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Here, using fluorescent NAD+ analogues as well as synthetic substrate mimics, we have developed continuous assays enabling real-time monitoring of both steps of this mechanism. These assays are amenable to biochemical studies and high-throughput screening of inhibitors of these effectors, and the discovery and characterization of putative enzymes similar to members of the SidE family in other organisms. We also show their utility in studying enzymes that can reverse and inhibit this post-translational modification.

Key messageThe non-intrinsic ABC proteins ABCI20 and ABCI21 are induced by light under HY5 regulation, localize to the ER, and ameliorate cytokinin-driven growth inhibition in young Arabidopsis thaliana seedlings. The plant ATP-binding cassette (ABC) I subfamily (ABCIs) comprises heterogeneous proteins containing any of the domains found in other ABC proteins. Some ABCIs are known to function in basic metabolism and stress responses, but many remain functionally uncharacterized. ABCI19, ABCI20, and ABCI21 of Arabidopsis thaliana cluster together in a phylogenetic tree, and are suggested to be targets of the transcription factor ELONGATED HYPOCOTYL 5 (HY5). Here, we reveal that these three ABCIs are involved in modulating cytokinin responses during early seedling development. The ABCI19, ABCI20 and ABCI21 promoters harbor HY5-binding motifs, and ABCI20 and ABCI21 expression was induced by light in a HY5-dependent manner. abci19 abci20 abci21 triple and abci20 abci21 double knockout mutants were hypersensitive to cytokinin in seedling growth retardation assays, but did not show phenotypic differences from the wild type in either control medium or auxin-, ABA-, GA-, ACC- or BR-containing media. ABCI19, ABCI20, and ABCI21 were expressed in young seedlings and the three proteins interacted with each other, forming a large protein complex at the endoplasmic reticulum (ER) membrane. These results suggest that ABCI19, ABCI20, and ABCI21 fine-tune the cytokinin response at the ER under the control of HY5 at the young seedling stage.

The cellular protein SERINC5 inhibits the infectivity of diverse retroviruses, and its activity is counteracted by the glycosylated Gag (glycoGag) protein of murine leukemia virus (MLV), the S2 protein of equine infectious anemia virus (EIAV), and the Nef protein of human immunodeficiency virus type 1 (HIV-1). Determining the regions within SERINC5 that provide restrictive activity or Nef sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. Here, we report that deletion of the conserved sequence EDTEE, which is located within a cytoplasmic loop of SERINC5 and which is reminiscent of an acidic-cluster membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef, while it has no effect on the intrinsic activity of the protein as an inhibitor of infectivity. These effects correlated with enhanced removal of the ΔEDTEE mutant relative to that of wild-type SERINC5 from the cell surface and with enhanced exclusion of the mutant protein from virions by Nef. Mutational analysis indicated that the acidic residues, but not the threonine, within the EDTEE motif are important for the relative resistance to Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef specific. These results are consistent with the reported mapping of the cytoplasmic loop that contains the EDTEE sequence as a general determinant of Nef responsiveness, but they further indicate that sequences inhibitory to as well as supportive of Nef activity reside in this region. We speculate that the EDTEE motif might have evolved to mediate resistance against retroviruses that use Nef-like proteins to antagonize SERINC5.IMPORTANCE Cellular membrane proteins in the SERINC family, especially SERINC5, inhibit the infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, specifically, HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-pathogen "arms race" is a compensatory change in the host antiviral protein as it evolves to escape the effects of viral antagonists. This is often reflected in a genetic signature, positive selection, which is conspicuously missing in SERINC5 Here we show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless provides relative resistance to antagonism by HIV-1 Nef.

The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.

Aggregation of α-synuclein is a defining molecular feature of Parkinson's disease, Lewy body dementia, and multiple systems atrophy. Hereditary mutations in α-synuclein are linked to both Parkinson's disease and Lewy body dementia; in particular, patients bearing the E46K disease mutation manifest a clinical picture of parkinsonism and Lewy body dementia, and E46K creates more pathogenic fibrils in vitro. Understanding the effect of these hereditary mutations on α-synuclein fibril structure is fundamental to α-synuclein biology. We therefore determined the cryo-electron microscopy (cryo-EM) structure of α-synuclein fibrils containing the hereditary E46K mutation. The 2.5-Å structure reveals a symmetric double protofilament in which the molecules adopt a vastly rearranged, lower energy fold compared to wild-type fibrils. We propose that the E46K misfolding pathway avoids electrostatic repulsion between K46 and K80, a residue pair which form the E46-K80 salt bridge in the wild-type fibril structure. We hypothesize that, under our conditions, the wild-type fold does not reach this deeper energy well of the E46K fold because the E46-K80 salt bridge diverts α-synuclein into a kinetic trap-a shallower, more accessible energy minimum. The E46K mutation apparently unlocks a more stable and pathogenic fibril structure.

The human ether-á-go-go-related gene (hERG1) channel conducts small outward K+ currents that are critical for cardiomyocyte membrane repolarization. The gain-of-function mutation N629D at the outer mouth of the selectivity filter (SF) disrupts inactivation and K+-selective transport in hERG1, leading to arrhythmogenic phenotypes associated with long-QT syndrome. Here, we combined computational electrophysiology with Markov state model analysis to investigate how SF-level gating modalities control selective cation transport in wild-type (WT) and mutant (N629D) hERG1 variants. Starting from the recently reported cryogenic electron microscopy (cryo-EM) open-state channel structure, multiple microseconds-long molecular-dynamics (MD) trajectories were generated using different cation configurations at the filter, voltages, electrolyte concentrations, and force-field parameters. Most of the K+ permeation events observed in hERG1-WT simulations occurred at microsecond timescales, influenced by the spontaneous dehydration/rehydration dynamics at the filter. The SF region displayed conductive, constricted, occluded, and dilated states, in qualitative agreement with the well-documented flickering conductance of hERG1. In line with mutagenesis studies, these gating modalities resulted from dynamic interaction networks involving residues from the SF, outer-mouth vestibule, P-helices, and S5-P segments. We found that N629D mutation significantly stabilizes the SF in a state that is permeable to both K+ and Na+, which is reminiscent of the SF in the nonselective bacterial NaK channel. Increasing the external K+ concentration induced "WT-like" SF dynamics in N629D, in qualitative agreement with the recovery of flickering currents in experiments. Overall, our findings provide an understanding of the molecular mechanisms controlling selective transport in K+ channels with a nonconventional SF sequence.

Epistasis emerges when the effects of an amino acid depend on the identities of interacting residues. This phenomenon shapes fitness landscapes, which have the power to reveal evolutionary paths and inform evolution of desired functions. However, there is a need for easily implemented, high-throughput methods to capture epistasis particularly at distal sites. Here, we combine deep mutational scanning (DMS) with a straightforward data processing step to bridge reads in distal sites within genes (BRIDGE). We use BRIDGE, which matches non-overlapping reads to their cognate templates, to uncover prevalent epistasis within the binding pocket of a human G protein-coupled receptor (GPCR) yielding variants with 4-fold greater affinity to a target ligand. The greatest functional improvements in our screen result from distal substitutions and substitutions that are deleterious alone. Our results corroborate findings of mutational tolerance in GPCRs, even in conserved motifs, but reveal inherent constraints restricting tolerated substitutions due to epistasis.

Late gene transcription in the beta- and gammaherpesviruses depends on a set of virally encoded transcriptional activators (vTAs) that hijack the host transcriptional machinery and direct it to a subset of viral genes that are required for completion of the viral replication cycle and capsid assembly. In Kaposi's sarcoma-associated herpesvirus (KSHV), these vTAs are encoded by ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. Assembly of the vTAs into a complex is critical for late gene transcription, and thus, deciphering the architecture of the complex is central to understanding its transcriptional regulatory activity. Here, we generated an ORF66-null virus and confirmed that it fails to produce late genes and infectious virions. We show that ORF66 is incorporated into the vTA complex primarily through its interaction with ORF34, which is dependent upon a set of four conserved cysteine-rich motifs in the C-terminal domain of ORF66. While both ORF24 and ORF66 occupy the canonical K8.1 late gene promoter, their promoter occupancy requires the presence of the other vTAs, suggesting that sequence-specific, stable binding requires assembly of the entire complex on the promoter. Additionally, we found that ORF24 expression is impaired in the absence of a stable vTA complex. This work extends our knowledge about the architecture of the KSHV viral preinitiation complex and suggests that it functions as a complex to recognize late gene promoters.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that is the causative agent of multiple human cancers. The release of infectious virions requires the production of capsid proteins and other late genes, whose production is transcriptionally controlled by a complex of six virally encoded proteins that hijack the host transcription machinery. It is poorly understood how this complex assembles or what function five of its six components play in transcription. Here, we demonstrate that ORF66 is an essential component of this complex in KSHV and that its inclusion in the complex depends upon its C-terminal domain, which contains highly conserved cysteine-rich motifs reminiscent of zinc finger motifs. Additionally, we examined the assembly of the viral preinitiation complex at late gene promoters and found that while sequence-specific binding of late gene promoters requires ORF24, it additionally requires a fully assembled viral preinitiation complex.

Proteins are molecular machines whose function depends on their ability to achieve complex folds with precisely defined structural and dynamic properties. The rational design of proteins from first-principles, or de novo, was once considered to be impossible, but today proteins with a variety of folds and functions have been realized. We review the evolution of the field from its earliest days, placing particular emphasis on how this endeavor has illuminated our understanding of the principles underlying the folding and function of natural proteins, and is informing the design of macromolecules with unprecedented structures and properties. An initial set of milestones in de novo protein design focused on the construction of sequences that folded in water and membranes to adopt folded conformations. The first proteins were designed from first-principles using very simple physical models. As computers became more powerful, the use of the rotamer approximation allowed one to discover amino acid sequences that stabilize the desired fold. As the crystallographic database of protein structures expanded in subsequent years, it became possible to construct proteins by assembling short backbone fragments that frequently recur in Nature. The second set of milestones in de novo design involves the discovery of complex functions. Proteins have been designed to bind a variety of metals, porphyrins, and other cofactors. The design of proteins that catalyze hydrolysis and oxygen-dependent reactions has progressed significantly. However, de novo design of catalysts for energetically demanding reactions, or even proteins that bind with high affinity and specificity to highly functionalized complex polar molecules remains an importnant challenge that is now being achieved. Finally, the protein design contributed significantly to our understanding of membrane protein folding and transport of ions across membranes. The area of membrane protein design, or more generally of biomimetic polymers that function in mixed or non-aqueous environments, is now becoming increasingly possible.

Biological systems consist of multiple components of different physical and chemical properties that require complex and dynamic regulatory loops to function efficiently. The discovery of ever more novel interacting sites in complex proteins suggests that we are only beginning to understand how cellular and biological functions are integrated and tuned at the molecular and systems levels. Here we review recently discovered interacting sites which have been identified through rationally designed amino acid motifs diagnostic for specific molecular functions, including enzymatic activities and ligand-binding properties. We specifically discuss the nature of the latter using as examples, novel hormone recognition and gas sensing sites that occur in moonlighting protein complexes. Drawing evidence from the current literature, we discuss the potential implications at the cellular, tissue, and/or organismal levels of such non-catalytic interacting sites and provide several promising avenues for the expansion of amino acid motif searches to discover hitherto unknown protein interactants and interaction networks. We believe this knowledge will unearth unexpected functions in both new and well-characterized proteins, thus filling existing conceptual gaps or opening new avenues for applications either as drug targets or tools in pharmacology, cell biology and bio-catalysis. Beyond this, motif searches may also support the design of novel, effective and sustainable approaches to crop improvements and the development of new therapeutics.

High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR β-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1+ TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1-encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag.

Francisella bacteria rely on a phylogenetically distinct type VI secretion system (T6SS) to escape host phagosomes and cause the fatal disease tularemia, but the structural and molecular mechanisms involved are unknown. Here we report the atomic structure of the Francisella T6SS central spike complex, obtained by cryo-electron microscopy. Our structural and functional studies demonstrate that, unlike the single-protein spike composition of other T6SS subtypes, Francisella T6SS's central spike is formed by two proteins, PdpA and VgrG, akin to T4-bacteriophage gp27 and gp5, respectively, and that PdpA has unique characteristics, including a putative cargo within its cavity and an N-terminal helical lid. Structure-guided mutagenesis demonstrates that the PdpA N-terminal lid and C-terminal spike are essential to Francisella T6SS function. PdpA is thus both an adaptor, connecting VgrG to the tube, and a likely carrier of secreted cargo. These findings are important to understanding Francisella pathogenicity and designing therapeutics to combat tularemia.

Previous models suggested that regulation of telomere length in Saccharomyces cerevisiae by Tel1(ATM) and Mec1(ATR) would parallel the established pathways regulating the DNA damage response. Here, we provide evidence that telomere length regulation differs from the DNA damage response in both the Tel1 and Mec1 pathways. We found that Rad53 mediates a Mec1 telomere length regulation pathway but is dispensable for Tel1 telomere length regulation, whereas in the DNA damage response, Rad53 is regulated by both Mec1 and Tel1 Using epistasis analysis with a Tel1 hypermorphic allele, Tel1-hy909, we found that the MRX complex is not required downstream of Tel1 for telomere elongation but is required downstream of Tel1 for the DNA damage response. Our data suggest that nucleolytic telomere end processing is not a required step for telomerase to elongate telomeres.

To date, 12 protein lysine methyltransferases that modify translational elongation factors and ribosomal proteins (Efm1-7 and Rkm 1-5) have been identified in the yeast Saccharomyces cerevisiae. Of these 12, five (Efm1 and Efm4-7) appear to be specific to elongation factor 1A (EF1A), the protein responsible for bringing aminoacyl-tRNAs to the ribosome. In S. cerevisiae, the functional implications of lysine methylation in translation are mostly unknown. In this work, we assessed the physiological impact of disrupting EF1A methylation in a strain where four of the most conserved methylated lysine sites are mutated to arginine residues and in strains lacking either four or five of the Efm lysine methyltransferases specific to EF1A. We found that loss of EF1A methylation was not lethal but resulted in reduced growth rates, particularly under caffeine and rapamycin stress conditions, suggesting EF1A interacts with the TORC1 pathway, as well as altered sensitivities to ribosomal inhibitors. We also detected reduced cellular levels of the EF1A protein, which surprisingly was not reflected in its stability in vivo. We present evidence that these Efm methyltransferases appear to be largely devoted to the modification of EF1A, finding no evidence of the methylation of other substrates in the yeast cell. This work starts to illuminate why one protein can need five different methyltransferases for its functions and highlights the resilience of yeast to alterations in their posttranslational modifications.