Two distinct archaeal type IV pili structures formed by proteins with identical sequence.

Type IV pili (T4P) represent one of the most common varieties of surface appendages in archaea. These filaments, assembled from small pilin proteins, can be many microns long and serve diverse functions, including adhesion, biofilm formation, motility, and intercellular communication. Here, we determine atomic structures of two distinct adhesive T4P from Saccharolobus islandicus via cryo-electron microscopy (cryo-EM). Unexpectedly, both pili were assembled from the same pilin polypeptide but under different growth conditions. One filament, denoted mono-pilus, conforms to canonical archaeal T4P structures where all subunits are equivalent, whereas in the other filament, the tri-pilus, the same polypeptide exists in three different conformations. The three conformations in the tri-pilus are very different from the single conformation found in the mono-pilus, and involve different orientations of the outer immunoglobulin-like domains, mediated by a very flexible linker. Remarkably, the outer domains rotate nearly 180° between the mono- and tri-pilus conformations. Both forms of pili require the same ATPase and TadC-like membrane pore for assembly, indicating that the same secretion system can produce structurally very different filaments. Our results show that the structures of archaeal T4P appear to be less constrained and rigid than those of the homologous archaeal flagellar filaments that serve as helical propellers.

Beyond the Triple Helix: Exploration of the Hierarchical Assembly Space of Collagen-like Peptides.

The de novo design of self-assembling peptides has garnered significant attention in scientific research. While alpha-helical assemblies have been extensively studied, exploration of polyproline type II (PPII) helices, such as those found in collagen, remains relatively limited. In this study, we focused on understanding the sequence-structure relationship in hierarchical assemblies of collagen-like peptides, using defense collagen SP-A as a model. By dissecting the sequence derived from SP-A and synthesizing short collagen-like peptides, we successfully constructed a discrete bundle of hollow triple helices. Mutation studies pinpointed amino acid sequences, including hydrophobic and charged residues that are critical for oligomer formation. These insights guided the de novo design of collagen-like peptides, resulting in the formation of diverse quaternary structures, including discrete and heterogenous bundled oligomers, 2D nanosheets, and pH-responsive nanoribbons. Our study represents a significant advancement in the understanding and harnessing of collagen higher-order assemblies beyond the triple helix.

Tight-packing of large pilin subunits provides distinct structural and mechanical properties for the Myxococcus xanthus type IVa pilus.

Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While major pilins of structurally characterized T4aP have lengths of <165 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a conserved N-terminal domain and a variable C-terminal domain, and the additional residues of PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4aP (T4aPMx) at a resolution of 3.0 Å using cryo-EM. The T4aPMx follows the structural blueprint of other T4aP with the pilus core comprised of the interacting N-terminal α1-helices, while the globular domains decorate the T4aP surface. The atomic model of PilA built into this map shows that the large C-terminal domain has more extensive intersubunit contacts than major pilins in other T4aP. As expected from these greater contacts, the bending and axial stiffness of the T4aPMx is significantly higher than that of other T4aP and supports T4aP-dependent motility on surfaces of different stiffnesses. Notably, T4aPMx variants with interrupted intersubunit interfaces had decreased bending stiffness, pilus length, and strongly reduced motility. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4aP that expands the environmental conditions in which the T4aP system functions.

Cryo-EM structure of flagellotropic bacteriophage Chi.

The flagellotropic bacteriophage χ (Chi) infects bacteria via the flagellar filament. Despite years of study, its structural architecture remains partly characterized. Through cryo-EM, we unveil χ's nearly complete structure, encompassing capsid, neck, tail, and tail tip. While the capsid and tail resemble phage YSD1, the neck and tail tip reveal new proteins and their arrangement. The neck shows a unique conformation of the tail tube protein, forming a socket-like structure for attachment to the neck. The tail tip comprises four proteins, including distal tail protein (DTP), two baseplate hub proteins (BH1P and BH2P), and tail tip assembly protein (TAP) exhibiting minimal organization compared to other siphophages. Deviating from the consensus in other siphophages, DTP in χ forms a trimeric assembly, reducing tail symmetry from 6-fold to 3-fold at the tip. These findings illuminate the previously unexplored structural organization of χ's neck and tail tip.

Atomic structures of naphthalene dipeptide micelles unravel mechanisms of assembly and gelation.

Peptide-based biopolymers have gained increasing attention due to their versatile applications. A naphthalene dipeptide (2NapFF) can form chirality-dependent tubular micelles, leading to supramolecular gels. The precise molecular arrangement within these micelles and the mechanism governing gelation have remained enigmatic. We determined, at near-atomic resolution, cryoelectron microscopy structures of the 2NapFF micelles LL-tube and LD-tube, generated by the stereoisomers (l,l)-2NapFF and (l,d)-2NapFF, respectively. The structures reveal that the fundamental packing of dipeptides is driven by the systematic π-π stacking of aromatic rings and that same-charge repulsion between the carbonyl groups is responsible for the stiffness of both tubes. The structural analysis elucidates how a single residue's altered chirality gives rise to markedly distinct tubular structures and sheds light on the mechanisms underlying the pH-dependent gelation of LL- and LD-tubes. The understanding of dipeptide packing and gelation mechanisms provides insights for the rational design of 2NapFF derivatives, enabling the modulation of micellar dimensions.

Stabilization of F-actin by Salmonella effector SipA resembles the structural effects of inorganic phosphate and phalloidin.

Entry of Salmonella into host enterocytes relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a 1:2 stoichiometry with sub-nanomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the groove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved by a combination of fast association via the core and very slow dissociation dictated by the arm. Similar to Pi, BeF3, and phalloidin, SipA potently inhibited actin depolymerization by actin depolymerizing factor (ADF)/cofilin, which correlated with increased filament stiffness, supporting the hypothesis that F-actin's mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxin's effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.

Helical reconstruction, again.

Many protein and nucleoprotein complexes exist as helical polymers. As a result, much effort has been invested in developing methods for using electron microscopy to determine the structure of these assemblies. With the revolution in cryo-electron microscopy (cryo-EM), it has now become routine to reach a near-atomic level of resolution for these structures, and it is the exception when this is not possible. However, the greatest challenge is frequently determining the correct symmetry. This review focuses on why this can be so difficult and the current absence of a better approach than trial-and-error.

Near-atomic-resolution structure of J-aggregated helical light-harvesting nanotubes.

Cryo-electron microscopy has delivered a resolution revolution for biological self-assemblies, yet only a handful of structures have been solved for synthetic supramolecular materials. Particularly for chromophore supramolecular aggregates, high-resolution structures are necessary for understanding and modulating the long-range excitonic coupling. Here, we present a 3.3 Å structure of prototypical biomimetic light-harvesting nanotubes derived from an amphiphilic cyanine dye (C8S3-Cl). Helical 3D reconstruction directly visualizes the chromophore packing that controls the excitonic properties. Our structure clearly shows a brick layer arrangement, revising the previously hypothesized herringbone arrangement. Furthermore, we identify a new non-biological supramolecular motif-interlocking sulfonates-that may be responsible for the slip-stacked packing and J-aggregate nature of the light-harvesting nanotubes. This work shows how independently obtained native-state structures complement photophysical measurements and will enable accurate understanding of (excitonic) structure-function properties, informing materials design for light-harvesting chromophore aggregates.

An extensive disulfide bond network prevents tail contraction in Agrobacterium tumefaciens phage Milano.

A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness. The contractile tail of Agrobacterium tumefaciens bacteriophage Milano is flexible and bent to varying degrees, which sets it apart from other contractile tail-like systems. Here, we report structures of the Milano tail including the sheath-tube complex, baseplate, and putative receptor-binding proteins. The flexible-to-rigid transformation of the Milano tail upon contraction can be explained by unique electrostatic properties of the tail tube and sheath. All components of the Milano tail, including sheath subunits, are crosslinked by disulfides, some of which must be reduced for contraction to occur. The putative receptor-binding complex of Milano contains a tailspike, a tail fiber, and at least two small proteins that form a garland around the distal ends of the tailspikes and tail fibers. Despite being flagellotropic, Milano lacks thread-like tail filaments that can wrap around the flagellum, and is thus likely to employ a different binding mechanism.

Glucose-Triggered Gelation of Supramolecular Peptide Nanocoils with Glucose-Binding Motifs.

Peptide self-assembly is a powerful tool to prepare functional materials at the nanoscale. Often, the resulting materials have high aspect-ratio, with intermolecular ß-sheet formation underlying 1D fibrillar structures. Inspired by dynamic structures in nature, peptide self-assembly is increasingly moving toward stimuli-responsive designs wherein assembled structures are formed, altered, or dissipated in response to a specific cue. Here, a peptide bearing a prosthetic glucose-binding phenylboronic acid (PBA) is demonstrated to self-assemble into an uncommon nanocoil morphology. These nanocoils arise from antiparallel ß-sheets, with molecules aligned parallel to the long axis of the coil. The binding of glucose to the PBA motif stabilizes and elongates the nanocoil, driving entanglement and gelation at physiological glucose levels. The glucose-dependent gelation of these materials is then explored for the encapsulation and release of a therapeutic agent, glucagon, that corrects low blood glucose levels. Accordingly, the release of glucagon from the nanocoil hydrogels is inversely related to glucose level. When evaluated in a mouse model of severe acute hypoglycemia, glucagon delivered from glucose-stabilized nanocoil hydrogels demonstrates increased protection compared to delivery of the agent alone or within a control nanocoil hydrogel that is not stabilized by glucose.

The mating pilus of E. coli pED208 acts as a conduit for ssDNA during horizontal gene transfer.

IMPORTANCE: Bacteria are constantly exchanging DNA, which constitutes horizontal gene transfer. While some of these occurs by a non-specific process called natural transformation, some occurs by a specific mating between a donor and a recipient cell. In specific conjugation, the mating pilus is extended from the donor cell to make contact with the recipient cell, but whether DNA is actually transferred through this pilus or by another mechanism involving the type IV secretion system complex without the pilus has been an open question. Using Escherichia coli, we show that DNA can be transferred through this pilus between a donor and a recipient cell that has not established a tight mating junction, providing a new picture for the role of this pilus.

Hierarchical Assembly of Intrinsically Disordered Short Peptides.

The understanding on how short peptide assemblies transit from disorder to order remains limited due to the lack of atomistic structures. Here we report cryo-EM structure of the nanofibers short intrinsically disordered peptides (IDPs). Upon lowering pH or adding calcium ions, the IDP transitions from individual nanoparticles to nanofibers containing an aromatic core and a disordered periphery comprised of 2 to 5 amino acids. Protonating the phosphate or adding more metal ions further assembles the nanofibers into filament bundles. The assemblies of the IDP analogs with controlled chemistry, such as phosphorylation site, hydrophobic interactions, and sequences indicate that metal ions interact with the flexible periphery of the nanoparticles of the IDPs to form fibrils and enhance the interfibrillar interactions to form filament bundles. Illustrating that an IDP self-assembles from disorder to order, this work offers atomistic molecular insights to understand assemblies of short peptides driven by noncovalent interactions.

Tad and toxin-coregulated pilus structures reveal unexpected diversity in bacterial type IV pili.

Type IV pili (T4P) are ubiquitous in both bacteria and archaea. They are polymers of the major pilin protein, which has an extended and protruding N-terminal helix, α1, and a globular C-terminal domain. Cryo-EM structures have revealed key differences between the bacterial and archaeal T4P in their C-terminal domain structure and in the packing and continuity of α1. This segment forms a continuous α-helix in archaeal T4P but is partially melted in all published bacterial T4P structures due to a conserved helix breaking proline at position 22. The tad (tight adhesion) T4P are found in both bacteria and archaea and are thought to have been acquired by bacteria through horizontal transfer from archaea. Tad pilins are unique among the T4 pilins, being only 40 to 60 residues in length and entirely lacking a C-terminal domain. They also lack the Pro22 found in all high-resolution bacterial T4P structures. We show using cryo-EM that the bacterial tad pilus from Caulobacter crescentus is composed of continuous helical subunits that, like the archaeal pilins, lack the melted portion seen in other bacterial T4P and share the packing arrangement of the archaeal T4P. We further show that a bacterial T4P, the Vibrio cholerae toxin coregulated pilus, which lacks Pro22 but is not in the tad family, has a continuous N-terminal α-helix, yet its α1 s are arranged similar to those in other bacterial T4P. Our results highlight the role of Pro22 in helix melting and support an evolutionary relationship between tad and archaeal T4P.

Neck and capsid architecture of the robust Agrobacterium phage Milano.

Large gaps exist in our understanding of how bacteriophages, the most abundant biological entities on Earth, assemble and function. The structure of the "neck" region, where the DNA-filled capsid is connected to the host-recognizing tail remains poorly understood. We describe cryo-EM structures of the neck, the neck-capsid and neck-tail junctions, and capsid of the Agrobacterium phage Milano. The Milano neck 1 protein connects the 12-fold symmetrical neck to a 5-fold vertex of the icosahedral capsid. Comparison of Milano neck 1 homologs leads to four proposed classes, likely evolved from the simplest one in siphophages to more complex ones in myo- and podophages. Milano neck is surrounded by the atypical collar, which covalently crosslinks the tail sheath to neck 1. The Milano capsid is decorated with three types of proteins, a minor capsid protein (mCP) and two linking proteins crosslinking the mCP to the major capsid protein. The extensive network of disulfide bonds within and between neck, collar, capsid and tail provides an exceptional structural stability to Milano.

Two dramatically distinct archaeal type IV pili structures formed by the same pilin.

Type IV pili (T4P) represent one of the most common varieties of surface appendages in archaea. These filaments, assembled from relatively small pilin proteins, can be many microns long and serve diverse functions, including adhesion, biofilm formation, motility, and intercellular communication. Using cryo-electron microscopy (cryo-EM), we determined atomic structures of two dramatically different T4P from Saccharolobus islandicus REY15A. Unexpectedly, both pili were assembled from the same pilin protein but under different growth conditions. One filament, denoted mono-pilus, conforms to canonical archaeal T4P structures where all subunits are equivalent, whereas in the other filament, the tri-pilus, the same protein exists in three different conformations. The three conformations involve different orientations of the outer immunoglobulin (Ig)-like domains, mediated by a very flexible linker, and all three of these conformations are very different from the single conformation found in the mono-pilus. Remarkably, the outer domains rotate nearly 180° between the mono- and tri-pilus conformations, formally similar to what has been shown for outer domains in bacterial flagellar filaments, despite lack of homology between bacterial flagella and archaeal T4P. Interestingly, both forms of pili require the same ATPase and TadC-like membrane pore for assembly, indicating that the same secretion system can produce structurally very different filaments. However, the expression of the ATPase and TadC genes was significantly different under the conditions yielding mono- and tri-pili. While archaeal T4P are homologs of archaeal flagellar filaments, our results show that in contrast to the rigid supercoil that the flagellar filaments must adopt to serve as helical propellers, archaeal T4P are likely to have fewer constraints on their structure and enjoy more internal degrees of freedom.

Large pilin subunits provide distinct structural and mechanical properties for the Myxococcus xanthus type IV pilus.

Type IV pili (T4P) are ubiquitous bacterial cell surface filaments important for surface motility, adhesion to biotic and abiotic surfaces, DNA uptake, biofilm formation, and virulence. T4P are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While the major pilins of structurally characterized T4P have lengths of up to 161 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a highly conserved N-terminal domain and a highly variable C-terminal domain, and the additional residues in the M. xanthus PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4P (T4P Mx ) at a resolution of 3.0 Å using cryo-electron microscopy (cryo-EM). The T4P Mx follows the structural blueprint observed in other T4P with the pilus core comprised of the extensively interacting N-terminal α1-helices while the globular domains decorate the T4P surface. The atomic model of PilA built into this map shows that the large C-terminal domain has much more extensive intersubunit contacts than major pilins in other T4P. As expected from these greater contacts, the bending and axial stiffness of the T4P Mx is significantly higher than that of other T4P and supports T4P-dependent motility on surfaces of different stiffnesses. Notably, T4P Mx variants with interrupted intersubunit interfaces had decreased bending stiffness and strongly reduced motility on all surfaces. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4P that expands the environmental conditions in which the T4P system functions.

The evolution of archaeal flagellar filaments.

Flagellar motility has independently arisen three times during evolution: in bacteria, archaea, and eukaryotes. In prokaryotes, the supercoiled flagellar filaments are composed largely of a single protein, bacterial or archaeal flagellin, although these two proteins are not homologous, while in eukaryotes, the flagellum contains hundreds of proteins. Archaeal flagellin and archaeal type IV pilin are homologous, but how archaeal flagellar filaments (AFFs) and archaeal type IV pili (AT4Ps) diverged is not understood, in part, due to the paucity of structures for AFFs and AT4Ps. Despite having similar structures, AFFs supercoil, while AT4Ps do not, and supercoiling is essential for the function of AFFs. We used cryo-electron microscopy to determine the atomic structure of two additional AT4Ps and reanalyzed previous structures. We find that all AFFs have a prominent 10-strand packing, while AT4Ps show a striking structural diversity in their subunit packing. A clear distinction between all AFF and all AT4P structures involves the extension of the N-terminal α-helix with polar residues in the AFFs. Additionally, we characterize a flagellar-like AT4P from Pyrobaculum calidifontis with filament and subunit structure similar to that of AFFs which can be viewed as an evolutionary link, showing how the structural diversity of AT4Ps likely allowed for an AT4P to evolve into a supercoiling AFF.

Extracellular cytochrome nanowires appear to be ubiquitous in prokaryotes.

Electrically conductive appendages from the anaerobic bacterium Geobacter sulfurreducens, recently identified as extracellular cytochrome nanowires (ECNs), have received wide attention due to numerous potential applications. However, whether other organisms employ similar ECNs for electron transfer remains unknown. Here, using cryoelectron microscopy, we describe the atomic structures of two ECNs from two major orders of hyperthermophilic archaea present in deep-sea hydrothermal vents and terrestrial hot springs. Homologs of Archaeoglobus veneficus ECN are widespread among mesophilic methane-oxidizing Methanoperedenaceae, alkane-degrading Syntrophoarchaeales archaea, and in the recently described megaplasmids called Borgs. The ECN protein subunits lack similarities in their folds; however, they share a common heme arrangement, suggesting an evolutionarily optimized heme packing for efficient electron transfer. The detection of ECNs in archaea suggests that filaments containing closely stacked hemes may be a common and widespread mechanism for long-range electron transfer in both prokaryotic domains of life.

Cell spheroid creation by transcytotic intercellular gelation.

Cell spheroids bridge the discontinuity between in vitro systems and in vivo animal models. However, inducing cell spheroids by nanomaterials remains an inefficient and poorly understood process. Here we use cryogenic electron microscopy to determine the atomic structure of helical nanofibres self-assembled from enzyme-responsive D-peptides and fluorescent imaging to show that the transcytosis of D-peptides induces intercellular nanofibres/gels that potentially interact with fibronectin to enable cell spheroid formation. Specifically, D-phosphopeptides, being protease resistant, undergo endocytosis and endosomal dephosphorylation to generate helical nanofibres. On secretion to the cell surface, these nanofibres form intercellular gels that act as artificial matrices and facilitate the fibrillogenesis of fibronectins to induce cell spheroids. No spheroid formation occurs without endo- or exocytosis, phosphate triggers or shape switching of the peptide assemblies. This study-coupling transcytosis and morphological transformation of peptide assemblies-demonstrates a potential approach for regenerative medicine and tissue engineering.