Helix Bundle Research Articles

A Chemo-Mechanically Coupled DNA Origami Clamp Capable of Generating Robust Compression Forces.

DNA nanostructures have been utilized to study biological mechanical processes and construct artificial nanosystems. Many application scenarios necessitate nanodevices able to robustly generate large single molecular forces. However, most existing dynamic DNA nanostructures are triggered by probabilistic hybridization reactions between spatially separated DNA strands, which only non-deterministically generate relatively small compression forces (≈0.4 piconewtons (pN)). Here, an intercalator-triggered dynamic DNA origami nanostructure is developed, where large amounts of local binding reactions between intercalators and the nanostructure collectively lead to the robust generation of relatively large compression forces (≈11.2 pN). Biomolecular loads with different stiffnesses, 3, 4, and 6-helix DNA bundles are efficiently bent by the compression forces. This work provides a robust and powerful force-generation tool for building highly chemo-mechanically coupled molecular machines in synthetic nanosystems.

P-080 Unveiling the molecular determinants of sperm endpiece stabilization

Abstract Study question How are microtubules at the endpiece of the mammalian sperm tail stabilized? Summary answer SAX01 and SPACA9 are microtubule inner proteins that binds at the endpieces. In addition, singlet microtubules are capped by a complex of proteins. What is known already At the core of the sperm flagellum is the axoneme, a supramolecular assembly of nine doublet microtubules around a central pair of singlet microtubules. At the endpiece, axoneme structure is lost and only singlet microtubules remain. Using cryo-electron microscopy (cryo-EM) our group showed that the singlet microtubules contain species-specific microtubule inner proteins in a helical arrangement. Furthermore, we observed a protein density capping the microtubules plus-ends in pig, horse, and mouse sperm. Study design, size, duration To elucidate the function of the sperm endpiece plug and its associated proteins, we apply single-particle analysis cryo-EM. Participants/materials, setting, methods - Main results and the role of chance We revealed that the microtubule inner proteins in the singlets are SAX01 and SPACA9. Our high-resolution structure shows that SPACA9 forms a discontinuously intraluminal spiral. It consists of a bundle of alpha helices that binds across the intradimer α/β-tubulin interface as well as between adjacent protofilaments. Specifically, SPACA9 interacts with both α- and β-tubulin within a dimer while also interacting with the M-loop of α-tubulin from the neighbouring protofilament. Our structure further shows that SAX01 binds longitudinally along individual protofilaments with its Mn motif, a 5- to 6-residue helix flanked by a Tyr/Phe residue on one end and by a Ser/Thr on the other that binds the shoulder of the α -tubulin S9/S10 loop. We further observed that microtubule termini are capped with yet to be identified protein complexes. Taken together, we show that microtubule inner proteins longitudinally and laterally reinforce the microtubule lattice, while capped in their termini. Limitations, reasons for caution Limitations include species-specific variations as we previously observed between mouse sperm endpieces and porcine or horse. Cryo-EM structures represent averages across singlet microtubules, so individual variation within particles may be lost. Wider implications of the findings Investigating the mechanism behind stabilization of the endpiece singlet microtubules using cryo-EM provides better understanding of sperm tail stabilization that holds potential for progress in understanding male infertility and can be utilized for a development of male contraception. Trial registration number not applicable

An Azospirillum brasilense chemoreceptor that mediates nitrate chemotaxis has conditional roles in the colonization of plant roots.

Motile plant-associated bacteria use chemotaxis and dedicated chemoreceptors to navigate gradients in their surroundings and to colonize host plant surfaces. Here, we characterize a chemoreceptor that we named Tlp2 in the soil alphaproteobacterium Azospirillum brasilense. We show that the Tlp2 ligand-binding domain is related to the 4-helix bundle family and is conserved in chemoreceptors found in the genomes of many soil- and sediment-dwelling alphaproteobacteria. The promoter of tlp2 is regulated in an NtrC- and RpoN-dependent manner and is most upregulated under conditions of nitrogen fixation or in the presence of nitrate. Using fluorescently tagged Tlp2 (Tlp2-YFP), we show that this chemoreceptor is present in low abundance in chemotaxis-signaling clusters and is prone to degradation. We also obtained evidence that the presence of ammonium rapidly disrupts Tlp2-YFP localization. Behavioral experiments using a strain lacking Tlp2 and variants of Tlp2 lacking conserved arginine residues suggest that Tlp2 mediates chemotaxis in gradients of nitrate and nitrite, with the R159 residue being essential for Tlp2 function. We also provide evidence that Tlp2 is essential for root surface colonization of some plants (teff, red clover, and cowpea) but not others (wheat, sorghum, alfalfa, and pea). These results highlight the selective role of nitrate sensing and chemotaxis in plant root surface colonization and illustrate the relative contribution of chemoreceptors to chemotaxis and root surface colonization.IMPORTANCEBacterial chemotaxis mediates host-microbe associations, including the association of beneficial bacteria with the roots of host plants. Dedicated chemoreceptors specify sensory preferences during chemotaxis. Here, we show that a chemoreceptor mediating chemotaxis to nitrate is important in the beneficial soil bacterium colonization of some but not all plant hosts tested. Nitrate is the preferred nitrogen source for plant nutrition, and plants sense and tightly control nitrate transport, resulting in varying nitrate uptake rates depending on the plant and its physiological state. Nitrate is thus a limiting nutrient in the rhizosphere. Chemotaxis and dedicated chemoreceptors for nitrate likely provide motile bacteria with a competitive advantage to access this nutrient in the rhizosphere.

De novo design of alpha-beta repeat proteins.

Proteins composed of a single structural unit tandemly repeated multiple times carry out a wide range of functions in biology. There has hence been considerable interest in designing such repeat proteins; previous approaches have employed strict constraints on secondary structure types and relative geometries, and most characterized designs either mimic a known natural topology, adhere closely to a parametric helical bundle architecture, or exploit very short repetitive sequences. Here, we describe Rosetta-based and deep learning hallucination methods for generating novel repeat protein architectures featuring mixed alpha-helix and beta-strand topologies, and 25 new highly stable alpha-beta proteins designed using these methods. We find that incorporation of terminal caps which prevent beta strand mediated intermolecular interactions increases the solubility and monomericity of individual designs as well as overall design success rate.

Selection of a promiscuous minimalist cAMP phosphodiesterase from a library of de novo designed proteins

The ability of unevolved amino acid sequences to become biological catalysts was key to the emergence of life on Earth. However, billions of years of evolution separate complex modern enzymes from their simpler early ancestors. To probe how unevolved sequences can develop new functions, we use ultrahigh-throughput droplet microfluidics to screen for phosphoesterase activity amidst a library of more than one million sequences based on a de novo designed 4-helix bundle. Characterization of hits revealed that acquisition of function involved a large jump in sequence space enriching for truncations that removed >40% of the protein chain. Biophysical characterization of a catalytically active truncated protein revealed that it dimerizes into an α-helical structure, with the gain of function accompanied by increased structural dynamics. The identified phosphodiesterase is a manganese-dependent metalloenzyme that hydrolyses a range of phosphodiesters. It is most active towards cyclic AMP, with a rate acceleration of ~109 and a catalytic proficiency of >1014 M−1, comparable to larger enzymes shaped by billions of years of evolution.

Staphylococcal peroxidase inhibitor (SPIN): Residue-level investigation of the helical bundle domain

Myeloperoxidase is a critical component of the antibacterial arsenal of neutrophils, whereby it consumes H2O2 as an oxidant to convert halogen and pseudohalogen anions into cytotoxic hypohalous acids. Following phagocytosis by neutrophils, the human pathogen Staphylococcus aureus secretes a potent myeloperoxidase inhibitory protein, called SPIN, as part of its immune evasion repertoire. The matured S. aureus SPIN polypeptide consists of only 73 residues yet contains two functional domains: whereas the 60 residue C-terminal helical bundle domain is responsible for MPO binding, the 13 residue N-terminal domain is required to inhibit MPO. Previous studies have informed understanding of the SPIN N-terminal domain, but comparatively little is known about the helical domain insofar as the contribution of individual residues is concerned. To address this limitation, we carried out a residue-level structure/function investigation on the helical bundle domain of S. aureus SPIN. Using sequence conservation and existing structures of SPIN bound to human MPO as a guide, we selected residues L49, E50, H51, E52, Y55, and Y75 for interrogation by site-directed mutagenesis. We found that loss of L49 or E52 reduced SPIN activity by roughly an order of magnitude, but that loss of Y55 or H51 caused progressively greater loss of inhibitory potency. Direct binding studies by SPR showed that loss of inhibitory potency in these SPIN mutants resulted from a diminished initial interaction between the inhibitor and MPO. Together, our studies provide new insights into the structure/function relationships of SPIN and identify positions Y55 and H51 as critical determinants of SPIN function.

Assembly of short amphiphilic peptoids into nanohelices with controllable supramolecular chirality.

A long-standing challenge in bioinspired materials is to design and synthesize synthetic materials that mimic the sophisticated structures and functions of natural biomaterials, such as helical protein assemblies that are important in biological systems. Herein, we report the formation of a series of nanohelices from a type of well-developed protein-mimetics called peptoids. We demonstrate that nanohelix structures and supramolecular chirality can be well-controlled through the side-chain chemistry. Specifically, the ionic effects on peptoids from varying the polar side-chain groups result in the formation of either single helical fiber or hierarchically stacked helical bundles. We also demonstrate that the supramolecular chirality of assembled peptoid helices can be controlled by modifying assembling peptoids with a single chiral amino acid side chain. Computational simulations and theoretical modeling predict that minimizing exposure of hydrophobic domains within a twisted helical form presents the most thermodynamically favorable packing of these amphiphilic peptoids and suggests a key role for both polar and hydrophobic domains on nanohelix formation. Our findings establish a platform to design and synthesize chiral functional materials using sequence-defined synthetic polymers.

Lanmodulin's EF 2-3 Domain: Insights from Infrared Spectroscopy and Simulations.

Lanmodulins are small, ∼110-residue proteins with four EF-hand motifs that demonstrate a picomolar affinity for lanthanide ions, making them efficient in the recovery and separation of these technologically important metals. In this study, we examine the thermodynamic and structural complexities of lanthanide ion binding to a 41-residue domain, EF 2-3, that constitutes the two highest-affinity metal-binding sites in the lanmodulin protein from Methylorubrum extorquens. Using a combination of circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we characterize the metal binding capabilities of EF 2-3. ITC demonstrates that binding occurs between peptide and lanthanides with conditional dissociation constants (Kd) in the range 20-30 μM, with no significant differences in the Kd values for La3+, Eu3+, and Tb3+ at pH 7.4. In addition, CD spectroscopy suggests that only one binding site of EF 2-3 undergoes a significant conformational change in the presence of lanthanides. 2D IR spectroscopy demonstrates the presence of both mono- and bidentate binding configurations in EF 2-3 with all three lanthanides. MD simulations, supported by Eu3+ luminescence measurements, explore these results, suggesting a competition between water-lanthanide and carboxylate-lanthanide interactions in the EF 2-3 domain. These results underscore the role of the core helical bundle of the protein architecture in influencing binding affinities and communication between the metal-binding sites in the full-length protein.

Utility of protein–protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display

Over the past three decades a diverse collection of small protein domains have been used as scaffolds to generate general-purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK), and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and Next Generation Sequencing (NGS), we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low uM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were insertion into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general-purpose protein-binding reagents.

Ykt6 functionally overlaps with vacuolar and exocytic R-SNAREs in the yeast Saccharomyces cerevisiae

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex forms a 4-helix coiled-coil bundle consisting of 16 layers of interacting side chains upon membrane fusion. The central layer (layer 0) is highly conserved and comprises three glutamines (Q) and one arginine (R), and thus SNAREs are classified into Qa-, Qb-, Qc-, and R-SNAREs. Homotypic vacuolar fusion in Saccharomyces cerevisiae requires the SNAREs Vam3 (Qa), Vti1 (Qb), Vam7 (Qc), and Nyv1 (R). However, the yeast strain lacking NYV1 (nyv1Δ) shows no vacuole fragmentation, whereas the vam3Δ and vam7Δ strains display fragmented vacuoles. Here, we provide genetic evidence that the R-SNAREs Ykt6 and Nyv1 are functionally redundant in vacuole homotypic fusion in vivo using a newly isolated ykt6 mutant. We observed the ykt6-104 mutant showed no defect in vacuole morphology, but the ykt6-104 nyv1Δ double mutant had highly fragmented vacuoles. Furthermore, we show the defect in homotypic vacuole fusion caused by the vam7-Q284R mutation was compensated by the nyv1-R192Q or ykt6-R165Q mutations, which maintained the 3Q:1R ratio in the layer 0 of the SNARE complex, indicating that Nyv1 is exchangeable with Ykt6 in the vacuole SNARE complex. Unexpectedly, we found Ykt6 assembled with exocytic Q-SNAREs when the intrinsic exocytic R-SNAREs Snc1 and its paralog Snc2 lose their ability to assemble into the exocytic SNARE complex. These results suggest that Ykt6 may serve as a backup when other R-SNAREs become dysfunctional and that this flexible assembly of SNARE complexes may help cells maintain the robustness of the vesicular transport network.

De novo GRIN variants in M3 helix associated with neurological disorders control channel gating of NMDA receptor

N-methyl-d-aspartate receptors (NMDARs) are members of the glutamate receptor family and participate in excitatory postsynaptic transmission throughout the central nervous system. Genetic variants in GRIN genes encoding NMDAR subunits are associated with a spectrum of neurological disorders. The M3 transmembrane helices of the NMDAR couple directly to the agonist-binding domains and form a helical bundle crossing in the closed receptors that occludes the pore. The M3 functions as a transduction element whose conformational change couples ligand binding to opening of an ion conducting pore. In this study, we report the functional consequences of 48 de novo missense variants in GRIN1, GRIN2A, and GRIN2B that alter residues in the M3 transmembrane helix. These de novo variants were identified in children with neurological and neuropsychiatric disorders including epilepsy, developmental delay, intellectual disability, hypotonia and attention deficit hyperactivity disorder. All 48 variants in M3 for which comprehensive testing was completed produce a gain-of-function (28/48) compared to loss-of-function (9/48); 11 variants had an indeterminant phenotype. This supports the idea that a key structural feature of the M3 gate exists to stabilize the closed state so that agonist binding can drive channel opening. Given that most M3 variants enhance channel gating, we assessed the potency of FDA-approved NMDAR channel blockers on these variant receptors. These data provide new insight into the structure–function relationship of the NMDAR gate, and suggest that variants within the M3 transmembrane helix produce a gain-of-function.

Structure of the intact tail machine of Anabaena myophage A-1(L)

The Myoviridae cyanophage A-1(L) specifically infects the model cyanobacteria Anabaena sp. PCC 7120. Following our recent report on the capsid structure of A-1(L), here we present the high-resolution cryo-EM structure of its intact tail machine including the neck, tail and attached fibers. Besides the dodecameric portal, the neck contains a canonical hexamer connected to a unique pentadecamer that anchors five extended bead-chain-like neck fibers. The 1045-Å-long contractile tail is composed of a helical bundle of tape measure proteins surrounded by a layer of tube proteins and a layer of sheath proteins, ended with a five-component baseplate. The six long and six short tail fibers are folded back pairwise, each with one end anchoring to the baseplate and the distal end pointing to the capsid. Structural analysis combined with biochemical assays further enable us to identify the dual hydrolytic activities of the baseplate hub, in addition to two host receptor binding domains in the tail fibers. Moreover, the structure of the intact A-1(L) also helps us to reannotate its genome. These findings will facilitate the application of A-1(L) as a chassis cyanophage in synthetic biology.

Proline Isomerization and Molten Globular Property of TgPDCD5 Secreted from Toxoplasma gondii Confers Its Regulation of Heparin Sulfate Binding.

Toxoplasmosis, caused by Toxoplasma gondii, poses risks to vulnerable populations. TgPDCD5, a secreted protein of T. gondii, induces apoptosis through heparan sulfate-mediated endocytosis. The entry mechanism of TgPDCD5 has remained elusive. Here, we present the solution structure of TgPDCD5 as a helical bundle with an extended N-terminal helix, exhibiting molten globule characteristics. NMR perturbation studies reveal heparin/heparan sulfate binding involving the heparan sulfate/heparin proteoglycans-binding motif and the core region, influenced by proline isomerization of P107 residue. The heterogeneous proline recruits a cyclophilin TgCyp18, accelerating interconversion between conformers and regulating heparan/heparin binding. These atomic-level insights elucidate the binary switch's functionality, expose novel heparan sulfate-binding surfaces, and illuminate the unconventional cellular entry of pathogenic TgPDCD5.

Structural analysis of phosphoribosyltransferase-mediated cell wall precursor synthesis in Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl pyrophosphate to decaprenyl phosphate, to generate 5-phospho-β-ribosyl-1-phosphoryldecaprenol. Despite Rv3806c being an attractive drug target, structural and molecular mechanistic insight into this PRTase is lacking. Here we report cryogenic electron microscopy structures for Rv3806c in the donor- and acceptor-bound states. In a lipidic environment, Rv3806c is trimeric, creating a UbiA-like fold. Each protomer forms two helical bundles, which, alongside the bound lipids, are required for PRTase activity in vitro. Mutational and functional analyses reveal that decaprenyl phosphate and phosphoribosyl pyrophosphate bind the intramembrane and extramembrane cavities of Rv3806c, respectively, in a distinct manner to that of UbiA superfamily enzymes. Our data suggest a model for Rv3806c-catalysed phosphoribose transfer through an inverting mechanism. These findings provide a structural basis for cell wall precursor biosynthesis that could have potential for anti-tuberculosis drug development.

Investigating vulnerability of the conserved SARS-CoV-2 spike's heptad repeat 2 as target for fusion inhibitors using chimeric miniproteins

Inhibition of SARS-CoV-2 membrane fusion is a highly desired target to combat COVID-19. The interaction between the spike's heptad repeat (HR) regions 1 (HR1) and 2 (HR2) is a crucial step during the fusion process and these highly conserved HR regions constitute attractive targets for fusion inhibitors. However, the relative importance of each subregion of the long HR1-HR2 interface for viral inhibition remains unclear. Here, we designed, produced, and characterized a series of chimeric miniproteins that mimic two different half subdomains of HR1. The proteins were designed as single polypeptide chains that spontaneously fold into antiparallel trimeric helical bundles aimed at structurally imitate the molecular surface of each HR1 half subregion. All the miniproteins folded stably as helical structures and could bind complementary HR2 peptides with moderate affinity. However, only the miniproteins mimicking the N-terminal HR1 half subdomain, but not those imitating C-terminal one, could inhibit cell infection by SARS-COV-2 real viruses in cell cultures. Most interestingly, the inhibitory activity of the miniproteins correlated with their structural stability, but not with their relative binding affinity for HR2 peptides. These results are highly relevant for designing more focused and active fusion inhibitors targeting the highly conserved HR2 region of the Spike.

Crystal Structures of Fusion Cores from CCoV-HuPn-2018 and SADS-CoV.

Cross-species spillover to humans of coronaviruses (CoVs) from wildlife animal reservoirs poses marked and global threats to human and animal health. Recently, sporadic infection of canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) in hospitalized patients with pneumonia genetically related to canine and feline coronavirus were identified. In addition, swine acute diarrhea syndrome coronavirus (SADS-CoV) had the capability of broad tropism to cultured cells including from humans. Together, the transmission of Alphacoronaviruses that originated in wildlife to humans via intermediate hosts was responsible for the high-impact emerging zoonosis. Entry of CoV is mainly mediated by Spike and formation of a typical six helix bundle (6-HB) structure in the postfusion state of Spike is pivotal. Here, we present the complete fusion core structures of CCoV-HuPn-2018 and SADS-CoV from Alphacoronavirus at 2.10 and 2.59 Å, respectively. The overall structure of the CCoV-HuPn-2018 fusion core is similar to Alphacoronavirus like HCoV-229E, while SADS-CoV is analogous to Betacoronavirus like SARS-CoV-2. Collectively, we provide a structural basis for the development of pan-CoV small molecules and polypeptides based on the HR1-HR2 complex, concerning CCoV-HuPn-2018 and SADS-CoV.

Comparing effects of attractive interactions in crowded systems: nonspecific, hydrophobic, and hydrogen bond interactions

The equilibrium stability of a protein is determined by its amino acid sequence and the solution conditions, such as temperature, pH and presence of chemical denaturant. The stability of a single protein in two identical solutions can nonetheless differ if other macromolecules, termed cosolutes or crowders, are present in one of the solutions at concentrations high enough to occupy a substantial fraction of the solution volume. This effect, due to the presence of the crowders, decreases or increases the stability depending on the interactions between the protein and crowders. Hard-core steric repulsions, which are responsible for the reduction in free volume, are expected to entropically stabilize the protein while attractive interactions can be destabilizing. Here we use a coarse-grained protein model to assess the impact of different types of crowder-protein interactions on the stability of a 35-amino acid model sequence folding into a helical bundle. We find that, for the same interaction strength and concentration, spherical crowders with a hydrophobic character are more destabilizing than crowders interacting nonspecifically with the protein. However, the two types of interactions differ in the degree of association between crowders and protein. At an interaction strength for which the attractive interactions roughly counteracts the stabilizing hard-core repulsions, the nonspecific interactions lead to much stronger crowder-protein association than the hydrophobic interactions. Additionally, we study crowders in the form of polypeptide chains, which are capable of hydrogen bonding with the protein. These peptide crowders have a destabilizing effect even at relatively low crowder concentrations, especially if the sequence of the peptide crowders includes hydrophobic amino acids. Our findings emphasize the importance of the interplay between different types of attractive crowder-protein interactions and entropic effects in determining the net effect on protein stability.

Doa10/MARCH6 architecture interconnects E3 ligase activity with lipid-binding transmembrane channel to regulate SQLE

Transmembrane E3 ligases play crucial roles in homeostasis. Much protein and organelle quality control, and metabolic regulation, are determined by ER-resident MARCH6 E3 ligases, including Doa10 in yeast. Here, we present Doa10/MARCH6 structural analysis by cryo-EM and AlphaFold predictions, and a structure-based mutagenesis campaign. The majority of Doa10/MARCH6 adopts a unique circular structure within the membrane. This channel is established by a lipid-binding scaffold, and gated by a flexible helical bundle. The ubiquitylation active site is positioned over the channel by connections between the cytosolic E3 ligase RING domain and the membrane-spanning scaffold and gate. Here, by assaying 95 MARCH6 variants for effects on stability of the well-characterized substrate SQLE, which regulates cholesterol levels, we reveal crucial roles of the gated channel and RING domain consistent with AlphaFold-models of substrate-engaged and ubiquitylation complexes. SQLE degradation further depends on connections between the channel and RING domain, and lipid binding sites, revealing how interconnected Doa10/MARCH6 elements could orchestrate metabolic signals, substrate binding, and E3 ligase activity.

Oligourea helix bundle binds detergents with diverse polar head groups.

Here we report a series of crystal structures (and accompanying biophysical data) of an array of diverse detergent guests bound to an oligourea foldamer helix bundle. These results significantly increase our structural and chemical understanding of aqueous guest recognition by oligourea foldamers and will aid the design of further functionalised oligourea-based self-assemblies.

AlphaFold2 captures the conformational landscape of the HAMP signaling domain.

In this study, we present a conformational landscape of 5000 AlphaFold2 models of the Histidine kinases, Adenyl cyclases, Methyl-accepting proteins and Phosphatases (HAMP) domain, a short helical bundle that transduces signals from sensors to effectors in two-component signaling proteins such as sensory histidine kinases and chemoreceptors. The landscape reveals the conformational variability of the HAMP domain, including rotations, shifts, displacements, and tilts of helices, many combinations of which have not been observed in experimental structures. HAMP domains belonging to a single family tend to occupy a defined region of the landscape, even when their sequence similarity is low, suggesting that individual HAMP families have evolved to operate in a specific conformational range. The functional importance of this structural conservation is illustrated by poly-HAMP arrays, in which HAMP domains from families with opposite conformational preferences alternate, consistent with the rotational model of signal transduction. The only poly-HAMP arrays that violate this rule are predicted to be of recent evolutionary origin and structurally unstable. Finally, we identify a family of HAMP domains that are likely to be dynamic due to the presence of a conserved pi-helical bulge. All code associated with this work, including a tool for rapid sequence-based prediction of the rotational state in HAMP domains, is deposited at https://github.com/labstructbioinf/HAMPpred.