Protein Architecture Research Articles

Molecular resolution imaging reveals step-wise reduction of axonemal diameter and ultrastructural architecture of intraflagellar transport proteins.

Primary cilia are microtubule-based organelles essential for mediating mechano-/photo-/chemo-sensations in mammalian cells. Intraflagellar transport (IFT) proteins are crucial components to support ciliary assembly, maintenance, and cellular signaling. The ciliary microtubule doublets of the ‘9 + 0’ arrangement serve as tracks for molecular motor-based transport along cilia. Although the tapering structure of primary cilia has been examined by electron microscopy, the spatial correlation between the IFT pattern and axonemal structure remains elusive due to insufficient light resolution. Here, we integrated single-molecule localization microscopy and sample expansion techniques achieving two-color imaging with effective localization precision of sub-5 nm to explore the axonemal ultrastructure and molecular organization of IFT and IFT-associated proteins in retinal pigment epithelial cilia. Our results revealed the step-wise reduction of ciliary diameter, different from the well-known paradigm showing a gradual change in the width of axonemal bundles. Surprisingly, our data showed that the IFT proteins congested near the end of the microtubule doubles within the ciliary compartment. In addition, we resolved the molecular architecture of IFT complexes, cargo, and motor proteins to characterize their relative localizations. We found that IFT proteins presented distinct distributions, including isolated units, paired configurations, or alternating arrangements in IFT trains. Together, our ultrastructural light nanoscopic imaging enables us to perform morphological analysis of primary cilia, which guides the localization of IFT complexes at segmented sites of cilia, suggesting the asynchronous axonemal growth of microtubule doubles.

Simulated microgravity leads to altered intracellular calcium homeostasis and protein expression in C2C12 cultures.

Skeletal muscle has the role to maintain body posture against a constant gravitational load. During aging, in low physical activity or even in spaceflight, the muscle mass is decreasing, resulting in impaired myogenesis and regeneration. The cytoskeleton, mechanosensitive ion channels, and calcium homeostasis play crucial role in maintaining normal myogenic processes. Simulated microgravity (SM) with 3D clinorotation is one of the accepted techniques to model gravitational unloading in vitro. C2C12 mouse myoblast cell line is a verified in vitro model system of myogenesis. Using RPM 2.0, a Random Positioning Machine as a partial g simulator, by randomly rotating the accommodated experiment package around the Earth's gravity vector, we are able to investigate alteration of physiological processes caused by SM. In our experiments, territorial gravity is compared to 0 g, respectively. However, fusion of myoblasts was preserved with SM, the formation of myotubes was different as compared to the control environment. Expression of Myosin Heavy Chain 2 (MYH2) protein, which is an important structural protein and a marker molecule of myotube differentiation program, was significantly reduced in samples kept in RPM. Immunocytochemistry of myogenic cultures revealed decreased size and average nuclei content in differentiated myotubes. The expression level of Piezo1 channels, which are mechanically activated cation channels are also decreased under SM. Altered calcium transients evoked by KCl have been observed in myotubes formed under simulated microgravity. Our results are in coherence with the recently published data. Our future goal is to analyze the architecture of cytoskeletal Septin7 protein and investigate how intracellular calcium concentrations and mechanotransduction is regulated by Piezo1 channels under microgravity condition.

Ancestral acetylcholine receptor beta-subunit forms homopentamers that prime before opening spontaneously.

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from two α-subunits, and one each of the β-, δ-, and ε-subunits. To form functional channels, the subunits must assemble with one another in a precise stoichiometry and arrangement. Despite being different, the four subunits share a common ancestor that is presumed to have formed homopentamers. The extent to which the properties of the modern-day receptor result from its subunit complexity is unknown. Here we discover that a reconstructed ancestral muscle-type β-subunit can unexpectedly form homopentameric ion channels. These homopentamers open spontaneously and display single-channel hallmarks of muscle-type acetylcholine receptor activity. Our findings attest to the homopentameric origin of the muscle-type acetylcholine receptor, and demonstrate that signature features of its function are both independent of agonist and do not necessitate the complex heteropentameric architecture of the modern-day protein.

Unraveling the encrypted lipophilicity of disulfide bridges in the molecular architecture of proteins and peptides: A machine learning approach in structural bioinformatics.

Disulfide bridges can be considered the concertmaster in a molecular orchestra that directs folding or inhibits unfolding in the molecular architecture of proteins and peptides, so standing for key structural element in protein stability. Its formation requires that two side chain Sγ atoms of cysteine residues are spatially proximal forming the new cystine residues (CRs). Although cystine-containing biomolecules such as antimicrobial peptides (defensines and lantibiotics), toxins (snake venoms and conopeptides), hyperstable peptides (cyclotides and lasso peptides) are well represented in databases and a wide diversity of residue-based lipophilicity scales are reported in the literature, there are no experimental or theoretical studies that describe this fundamental property in CRs. Here, with the motivation to complement previous studies focused on the lipophilicity of amino acids, we have studied the conformational richness of CRs in the biomolecules mentioned above characterizing structural details such as chiralities of the disulfide bridge but also, computing the QM-based lipophilicity (logP), unraveling for the first time the encrypted lipophilicities of different conformations of CRs. Interestingly, we have found a very diverse lipophilicity profiles of CRs in the peptides and proteins studied, thus indicating a hydrophobic fingerprint that could explain molecular recognition events and the unique properties of these biomolecules. In addition, we have developed a machine learning model capable of recognizing whether a cystine residue is hydrophilic or hydrophobic using chiral descriptors of the disulfide bridge. Our results have the potential to contribute to a better understanding of the implications of CRs on the properties and functions of cystine-containing biomolecules.

Experimental reconstruction of membrane transporter evolution.

Cellular homeostasis relies on solutes crossing cell membranes against their concentration gradients. Membrane transporters catalyze this energetically unfavorable movement using energy from the downhill movement of ions. Often, related transporters couple to either Na+ or H+ gradients; it can be difficult to glean the mechanistic reasons between such differences because the ability to use ions with such different physicochemical properties might require many changes in the transporter. Since evolutionary adaptations to environmental constraints such as salt concentrations and pH likely gave rise to the switch between sodium- and proton-motive forces, we hypothesized that phylogenetic reconstruction of ion-coupling may be a method to study such complex changes. We used phylogenetic analysis to reconstruct the evolution of prokaryotic glutamate transporters and deduce stepwise amino acid sequence changes resulting in the emergence of H+-coupled transporters from Na+-coupled ancestors. The analysis suggests that in addition to changing protonatable residues, introducing H+-dependence increases the hydrophobicity of previously polar regions proximal to the substrate and Na+ binding sites. To contextualize these changes, we used ancestral sequence reconstruction (ASR) to infer transporter sequences before and after the emergence of H+-coupling. We expressed and purified the inferred ancestral transporters, enabling their biochemical characterization and high-resolution cryo-EM structures. Surprisingly, the ancestral transporter before the emergence of H+-coupling shows Na+-independent substrate binding even though its Na+-binding pockets are structurally near-identical to Na+-coupled homologs. The emergence of H+-coupling preserves the protein architecture while subtly restructuring these critical regions. Our results suggest that changes in local structure and complex global allosteric properties underlie the diversification of the ion-motive forces used within the glutamate transporter family. The approach we developed might be broadly applicable to dissecting complex allosteric properties of membrane proteins.

An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F.hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F.hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

Semi-automated 3D fluorescence speckle analyzer (3D-Speckler) for microscope calibration and nanoscale measurement.

The widespread use of fluorescence microscopy has prompted the ongoing development of tools aiming to improve resolution and quantification accuracy for study of biological questions. Current calibration and quantification tools for fluorescence images face issues with usability/user experience, lack of automation, and comprehensive multidimensional measurement/correction capabilities. Here, we developed 3D-Speckler, a versatile, and high-throughput image analysis software that can provide fluorescent puncta quantification measurements such as 2D/3D particle size, spatial location/orientation, and intensities through semi-automation in a single, user-friendly interface. Integrated analysis options such as 2D/3D local background correction, chromatic aberration correction, and particle matching/filtering are also encompassed for improved precision and accuracy. We demonstrate 3D-Speckler microscope calibration capabilities by determining the chromatic aberrations, field illumination uniformity, and response to nanometer-scale emitters above and below the diffraction limit of our imaging system using multispectral beads. Furthermore, we demonstrated 3D-Speckler quantitative capabilities for offering insight into protein architectures and composition in cells.

Modular architecture and functional annotation of human RNA-binding proteins containing RNA recognition motif

RNA-binding proteins (RBPs) are structurally and functionally diverse macromolecules with significant involvement in several post-transcriptional gene regulatory processes and human diseases. RNA recognition motif (RRM) is one of the most abundant RNA-binding domains in human RBPs. The unique modular architecture of each RBP containing RRM is crucial for its diverse target recognition and function. Genome-wide study of these structurally conserved and functionally diverse domains can enhance our understanding of their functional implications. In this study, modular architecture of RRM containing RBPs in human proteome is identified and systematically analysed. We observe that 30% of human RBPs with RNA-binding function contain RRM in single or multiple repeats or with other domains with maximum of six repeats. Zinc-fingers are the most frequently co-occurring domain partner of RRMs. Human RRM containing RBPs mostly belong to RNA metabolism class of proteins and are significantly enriched in two functional pathways including spliceosome and mRNA surveillance. Various human diseases are associated with 18% of the RRM containing RBPs. Single RRM containing RBPs are highly enriched in disorder regions. Gene ontology (GO) molecular functions including poly(A), poly(U) and miRNA binding are highly depleted in RBPs with single RRM, indicating the significance of modular nature of RRMs in specific function. The current study reports all the possible domain architectures of RRM containing human RBPs and their functional enrichment. The idea of domain architecture, and how they confer specificity and new functionalities to RBPs, can help in re-designing of modular RRM containing RBPs with re-engineered function.

A spatial statistical framework for the parametric study of fiber networks: Application to fibronectin deposition by normal and activated fibroblasts.

Due to the complex architectural diversity of biological networks, there is an increasing need to complement statistical analyses with a qualitative and local description of their spatial properties. One such network is the extracellular matrix (ECM), a biological scaffold for which changes in its spatial organization significantly impact tissue functions in health and disease. Quantifying variations in the fibrillar architecture of major ECM proteins should considerably advance our understanding of the link between tissue structure and function. Inspired by the analysis of functional magnetic resonance imaging (fMRI) images, we propose a novel statistical analysis approach embedded into a machine learning paradigm, to measure and detect local variations of meaningful ECM parameters. We show that parametric maps representing fiber length and pore directionality can be analyzed within the proposed framework to differentiate among various tissue states. The parametric maps are derived from graph-based representations that reflect the network architecture of fibronectin (FN) fibers in a normal, or disease-mimicking in vitro setting. Such tools can potentially lead to a better characterization of dynamic matrix networks within fibrotic tumor microenvironments and contribute to the development of better imaging modalities for monitoring their remodeling and normalization following therapeutic intervention.

Phylogenetic Analysis Provides Insight Into the Molecular Evolution of Nociception and Pain-Related Proteins.

Nociception and pain sensation are important neural processes in humans to avoid injury. Many proteins are involved in nociception and pain sensation in humans; however, the evolution of these proteins in animals is unknown. Here, we chose nociception- and pain-related proteins, including G protein-coupled receptors (GPCRs), ion channels (ICs), and neuropeptides (NPs), which are reportedly associated with nociception and pain in humans, and identified their homologs in various animals by BLAST, phylogenetic analysis and protein architecture comparison to reveal their evolution from protozoans to humans. We found that the homologs of transient receptor potential channel A 1 (TRPA1), TRAPM, acid-sensing IC (ASIC), and voltage-dependent calcium channel (VDCC) first appear in Porifera. Substance-P receptor 1 (TACR1) emerged from Coelenterata. Somatostatin receptor type 2 (SSTR2), TRPV1 and voltage-dependent sodium channels (VDSC) appear in Platyhelminthes. Calcitonin gene-related peptide receptor (CGRPR) was first identified in Nematoda. However, opioid receptors (OPRs) and most NPs were discovered only in vertebrates and exist from agnatha to humans. The results demonstrated that homologs of nociception and pain-related ICs exist from lower animal phyla to high animal phyla, and that most of the GPCRs originate from low to high phyla sequentially, whereas OPRs and NPs are newly evolved in vertebrates, which provides hints of the evolution of nociception and pain-related proteins in animals and humans.

How good are AlphaFold models for docking-based virtual screening?

A crucial component in structure-based drug discovery is the availability of high-quality three-dimensional structures of the protein target. Whenever experimental structures were not available, homology modeling has been, so far, the method of choice. Recently, AlphaFold (AF), an artificial-intelligence-based protein structure prediction method, has shown impressive results in terms of model accuracy. This outstanding success prompted us to evaluate how accurate AF models are from the perspective of docking-based drug discovery. We compared the high-throughput docking (HTD) performance of AF models with their corresponding experimental PDB structures using a benchmark set of 22 targets. The AF models showed consistently worse performance using four docking programs and two consensus techniques. Although AlphaFold shows a remarkable ability to predict protein architecture, this might not be enough to guarantee that AF models can be reliably used for HTD, and post-modeling refinement strategies might be key to increase the chances of success.

Proteomic Profiling of Major Peanut Allergens and Their Post-Translational Modifications Affected by Roasting.

Post-translational modifications (PTMs) are covalent changes occurring on amino acid side chains of proteins and yet are neglected structural and functional aspects of protein architecture. The objective was to detect differences in PTM profiles that take place after roasting using open PTM search. We conducted a bottom-up proteomic study to investigate the impact of peanut roasting on readily soluble allergens and their PTM profiles. Proteomic PTM profiling of certain modifications was confirmed by Western blotting with a series of PTM-specific antibodies. In addition to inducing protein aggregation and denaturation, roasting may facilitate change in their PTM pattern and relative profiling. We have shown that Ara h 1 is the most modified major allergen in both samples in terms of modification versatility and extent. The most frequent PTM was methionine oxidation, especially in roasted samples. PTMs uniquely found in roasted samples were hydroxylation (Trp), formylation (Arg/Lys), and oxidation or hydroxylation (Asn). Raw and roasted peanut extracts did not differ in the binding of IgE from the serum of peanut-sensitised individuals done by ELISA. This study provides a better understanding of how roasting impacts the PTM profile of major peanut allergens and provides a good foundation for further exploration of PTMs.

Increased Ca2+-dependent intrinsic tone and arterial stiffness in mesenteric microvessels of hypertensive pregnant rats

Preeclampsia is a pregnancy-related hypertensive disorder (HTN-Preg) with unclear mechanisms. We have shown increased vascular reactivity to extrinsic vasoconstrictors in HTN-Preg rats. Here, we test whether microvascular intrinsic tone and arterial stiffness could contribute to HTN-Preg, and examined the underlying cellular mechanisms. On gestational day 19, BP was recorded in normal pregnant (Preg) rats and Preg rats with reduced uterine perfusion pressure (RUPP), and mesenteric microvessels were mounted on a pressure myograph for measurement of intrinsic tone, simultaneous changes in [Ca2+]i (fura-2 340/380 ratio), and arterial stiffness. Arteries were incubated in Ca2+-containing and 0 Ca2+ (2 mM EGTA) Krebs, pressurized at 10 to 110 mmHg in 10 mmHg increments, and the % change in vessel diameter from initial diameter at 10 mmHg was analyzed for measurement of total (active + passive) intrinsic tone and passive intrinsic response, respectively. The passive response was then subtracted from the total intrinsic tone to determine the active myogenic tone. The strain–stress relationship was also constructed as a measure of arterial stiffness. BP was higher in RUPP vs Preg rats. In Ca2+-containing Krebs, increases in intraluminal pressure caused smaller increases in diameter and greater increases in [Ca2+]i in microvessels of RUPP vs Preg rats, suggesting increased Ca2+-dependent myogenic tone. In 0 Ca2+ Krebs, increases in pressure also caused less increases in diameter in microvessels of RUPP vs Preg rats, but with no changes in [Ca2+]i, suggesting changes in the structure and mechanics of the arterial wall. The total and passive strain–stress relationship was shifted to the left in microvessels of RUPP vs Preg rats, suggesting increased arterial wall stiffness. Histology and immunohistochemistry showed greater vascular wall thickness and collagen-I staining in RUPP vs Preg rats, supporting changes in the wall architecture and structural proteins. The increased active myogenic tone and underlying increases in Ca2+ signaling as well as the increased passive intrinsic response, arterial stiffness and collagen-I in the mesenteric microvessels could play a role in the regulation of blood flow to the splanchnic region and the increased vascular resistance and BP in HTN-Preg.

Insertions and deletions mediated functional divergence of Rossmann fold enzymes

Nucleobase-containing coenzymes are hypothesized to be relics of an early RNA-based world that preceded the emergence of proteins. Despite the importance of coenzyme-protein synergisms, their emergence and evolution remain understudied. An excellent target to address this issue is the Rossmann fold, the most catalytically diverse and abundant protein architecture in nature. We investigated two main Rossmann lineages: the nicotinamide adenine dinucleotide phosphate (NAD(P)) and the S-adenosyl methionine (SAM)- binding superfamilies. To identify the evolutionary changes that lead to a coenzyme specificity switch on these superfamilies, we performed structural and sequence-based Hidden Markov model analysis to systematically search for key motifs in their coenzyme-binding pockets. Our analyses revealed that through insertions and deletions (InDels) and a residue substitution, the ancient β1-loop-α1 coenzyme-binding structure of NAD(P) could be reshaped into the SAM-binding β1-loop-α1 structure. To experimentally prove this obsevation, we removed three amino acids from the NAD(P)-binding pocket and solved the structure of the resulting mutant, revealing the characteristic loop features of the SAM-binding pocket. To confirm the binding to SAM, we performed isothermal titration calorimetry measurements. Molecular dynamics simulations also corroborated the role of InDels in abolishing NAD binding and acquiring SAM binding. Our results uncovered how nature may have utilized insertions and deletions to optimize the different coenzyme-binding pockets and the distinct functionalities observed for Rossmann superfamilies. This work also proposes a general mechanism by which protein templates could have been recycled through the course of evolution to adopt different coenzymes and confer distinct chemistries.

Chromatin Architecture Protein CTCF Plays a Role in Determining 5-Azacytidine Response of Leukemic Cells By Potentially Regulating Hoxb Genes

Chromatin Architecture Protein CTCF Plays a Role in Determining 5-Azacytidine Response of Leukemic Cells By Potentially Regulating Hoxb Genes

Atomic Force Microscopy-Based Measurements of Retinal Microvessel Stiffness in Mice with Endothelial-Specific Deletion of CCN1.

Vascular stiffness is an independent predictor of human vascular diseases and is linked to ischemia, diabetes, high blood pressure, hyperlipidemia, and/or aging. Blood vessel stiffening increases owing to changes in the microscale architecture and/or content of extracellular, cytoskeletal, and nuclear matrix proteins. These alterations, while best appreciated in large blood vessels, also gradually occur in the microvasculature and play an important role in the initiation and progression of numerous microangiopathies including diabetic retinopathy. Although macroscopic measurements of arterial stiffness by pulse wave velocity are often used for clinical diagnosis, stiffness changes of intact microvessels and their causative factors have not been characterized. Herein, we describe the use of atomic force microscopy (AFM) to determine stiffness of mouse retinal capillaries and assess its regulation by the cellular communication network (CCN) 1, a stiffness-sensitive gene-encoded matricellular protein. AFM yields reproducible measurements of retinal capillary stiffness in lightly fixed freshly isolated retinal flat mounts. AFM measurements also show significant changes in compliance properties of the retinal microvasculature of mice with endothelial-specific deletion of CCN1, indicating that CCN1 expression, or lack thereof, affects the mechanical properties of microvascular cells in vivo. Thus, AFM has the force sensitivity and the spatial resolution necessary to measure the local modulus of retinal capillaries in situ and eventually to investigate microvascular compliance heterogeneities as key components of disease pathogenesis.

Membrane Fusion Mediated by Non-covalent Binding of Re-engineered Cholera Toxin Assemblies to Glycolipids.

Membrane fusion is essential for the transport of macromolecules and viruses across membranes. While glycan-binding proteins (lectins) often initiate cellular adhesion, subsequent fusion events require additional protein machinery. No mechanism for membrane fusion arising from simply a protein binding to membrane glycolipids has been described thus far. Herein, we report that a biotinylated protein derived from cholera toxin becomes a fusogenic lectin upon cross-linking with streptavidin. This novel reengineered protein brings about hemifusion and fusion of vesicles as demonstrated by mixing of fluorescently labeled lipids between vesicles as well as content mixing of liposomes filled with fluorescently labeled dextran. Exclusion of the complex at vesicle-vesicle interfaces could also be observed, indicating the formation of hemifusion diaphragms. Discovery of this fusogenic lectin complex demonstrates that new emergent properties can arise from simple changes in protein architecture and provides insights into new mechanisms of lipid-driven fusion.

Structures of NPAS4-ARNT and NPAS4-ARNT2 heterodimers reveal new dimerization modalities in the bHLH-PAS transcription factor family

Neuronal PER-ARNT-SIM (PAS) domain protein 4 (NPAS4) is a protective transcriptional regulator whose dysfunction has been linked to a variety of neuropsychiatric and metabolic diseases. As a member of the basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) transcription factor family, NPAS4 is distinguished by an ability to form functional heterodimers with aryl hydrocarbon receptor nuclear translocator (ARNT) and ARNT2, both of which are also bHLH-PAS family members. Here, we describe the quaternary architectures of NPAS4-ARNT and NPAS4-ARNT2 heterodimers in complexes involving DNA response elements. Our crystallographic studies reveal a uniquely interconnected domain conformation for the NPAS4 protein itself, as well as its differentially configured heterodimeric arrangements with both ARNT and ARNT2. Notably, the PAS-A domains of ARNT and ARNT2 exhibit variable conformations within these two heterodimers. The ARNT PAS-A domain also forms a set of interfaces with the PAS-A and PAS-B domains of NPAS4, different from those previously noted in ARNT heterodimers formed with other class I bHLH-PAS family proteins. Our structural observations together with biochemical and cell-based interrogations of these NPAS4 heterodimers provide molecular glimpses of the NPAS4 protein architecture and extend the known repertoire of heterodimerization patterns within the bHLH-PAS family. The PAS-B domains of NPAS4, ARNT, and ARNT2 all contain ligand-accessible pockets with appropriate volumes required for small-molecule binding. Given NPAS4's linkage to human diseases, the direct visualization of these PAS domains and the further understanding of their relative positioning and interconnections within the NPAS4-ARNT and NPAS4-ARNT2 heterodimers may provide a road map for therapeutic discovery targeting these complexes.

Native Planar Asymmetric Suspended Membrane for Single-Molecule Investigations: Plasma Membrane on a Chip.

Cellular plasma membranes, in their role as gatekeepers to the external environment, host numerous protein assemblies and lipid domains that manage the movement of molecules into and out of cells, regulate electric potential, and direct cell signaling. The ability to investigate these roles on the bilayer at a single-molecule level in a controlled, in vitro environment while preserving lipid and protein architectures will provide deeper insights into how the plasma membrane works. A tunable silicon microarray platform that supports stable, planar, and asymmetric suspended lipid membranes (SLIM) using synthetic and native plasma membrane vesicles for single-molecule fluorescence investigations is developed. Essentially, a "plasma membrane-on-a-chip" system that preserves lipid asymmetry and protein orientation is created. By harnessing the combined potential of this platform with total internal reflection fluorescence (TIRF) microscopy, the authors are able to visualize protein complexes with single-molecule precision. This technology has widespread applications in biological processes that happen at the cellular membranes and will further the knowledge of lipid and protein assemblies.

A chromosome-encoded T4SS independently contributes to horizontal gene transfer in Enterococcus faecalis.

Bacterial type IV secretion systems (T4SSs) are the specific devices that mediate the dissemination of antibiotic resistant genes via horizontal gene transfer (HGT). Multi-drug-resistant Enterococcus faecalis (E.faecalis) represents a clinical public health threat because of its transferable plasmid with a functional plasmid-encoded (PE)-T4SS. Here, we report a chromosome-encoded (CE)-T4SS that exists in 40% of E.faecalis isolates. Compared with the PE-T4SS, CE-T4SS displays distinct characteristics in protein architecture and is capable of mediating large and genome-wide gene transfer in an imprecise manner. Reciprocal exchange of CE-T4SS- or PE-T4SS-associated origin of transfer (oriT) could disrupt HGT function, indicating that CE-T4SS is an independent system compared with PE-T4SS. Taken together, the CE-T4SS sheds light on the knowledge of HGT in gram-positive bacteria and triggers us to explore more evolutionary mechanisms in E.faecalis.