Structure

Conformational changes in the central helix at the inter-domain cleft of bacterial treadmilling motor protein FtsZ are coupled to polymerization. Central helix of mycobacterial FtsZ interconverts between a bent and a straight form, with an unknown mechanism. We probed the mechanism of this conformational switching in the central helix of mycobacterial FtsZ using multi-temperature synchrotron crystallography at 20 {o}C, 30 {o}C, 37 {o}C and -173 {o}C temperatures. A comparison of the resultant crystal structures of FtsZ revealed altered conformations at the bending pivot of the bent central helix inside the inter-domain cleft. Further, ensemble modeling of FtsZ structure shows that this bending pivot is labile at near-physiological temperatures. Conformational fluctuations in this pivot region resulted in breakage of regular alpha helical hydrogen bonds that likely made the central helix easily bendable. These fluctuations are largely arrested in the straightened form of the central helix in comparison to the bent form. To summarize, multi-temperature crystallography combined with ensemble modeling suggest that conformational heterogeneity and associated perturbations of helix-forming interactions in the bending pivot can trigger bent-to-straight conformational transition in the central helix of mycobacterial FtsZ. This work demonstrates the effectiveness of multi-temperature crystallography in delineating the mechanisms of conformational changes in protein machines.

Glutamate-like receptors (GLRs) in plants play an important role in a number of physiological processes, including wound response, stomatal aperture control, seed germination, root development, innate immune responses, pollen tube growth and morphogenesis. GLRs share amino acid sequence similarity with ionotropic glutamate receptors (iGluRs) that mediate neurotransmission in the nervous system of vertebrates. In contrast to iGluRs, however, for which numerous full-length structures are available, the structural information about the plant GLRs has been missing. Here we determine crystal structures of Arabidopsis thaliana GLR3.2 ligand-binding domain (LBD) in complex with glycine and methionine to 1.57 and 1.86 [A] resolution, respectively. Our structures show a fold similar to iGluRs, with several secondary structure elements either missing or different. The closed clamshell conformation of GLR3.2 LBD suggests that both glycine and methionine act as agonists. The structures reveal molecular determinants of ligand binding and explain the promiscuity of GLRs ligand activation compared to iGluRs. Structural similarities of LBDs confirm an evolutionary relationship between GLRs and iGluRs and predict common molecular principles of their gating mechanisms that are driven by the bilobed clamshell-like LBDs.

Cilia are ubiquitous eukaryotic organelles important to cellular motility, signalling and sensory reception. Cilium formation requires intraflagellar transport for trafficking of structural and signalling components. The large MDa IFT-B complex constitutes the backbone of polymeric IFT trains that carry ciliary cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B sub-complexes leaving >50% of the IFT-B complex structurally uncharacterized. We have used recent advances in protein structure prediction as implemented in Alphafold to assemble a structural model for the 15-subunit IFT-B complex. The model was validated using crosslinking/MS data on reconstituted IFT-B complexes, X-ray scattering in solution and diffraction from crystals as well as site-directed mutagenesis and protein binding assays. The IFT-B structural model reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The >400[A] long IFT-B complex can roughly be divided into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our structural modelling and crosslinking/MS results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of two different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.

The aminoacylation of lipid head group in many bacteria is carried out by bi-functional enzymes called MprF, which encode for a soluble synthase domain that typically transfers lysine or alanine from a tRNA to lipid head groups, and the modified lipid is translocated across the leaflets by a transmembrane domain. This modification of the lipids probably evolved to adapt to the environment where the microbes reside. Here, we describe the cryoEM structures of MprF enzyme from Pseudomonas aeruginosa revealing a dimeric enzyme with a distinct architecture when compared with the homologous Rhizobium enzymes and validate this arrangement with biochemical analysis. The cryoEM maps and the models in detergent micelle and nanodisc reveal a conformational change of the terminal helix of the synthase domain, highlighting the dynamic elements in the enzyme that might facilitate catalysis. Several lipid-like densities are observed in the cryoEM maps, which might indicate the path taken by the lipids and the coupling function of the two functional domains. Thus, the structure of a well-characterised PaMprF lays a platform for understanding the mechanism of amino acid transfer to a lipid head group and subsequent flipping across the leaflet that changes the property of the membrane.

The glycan shield of the beta-coronavirus (β-CoV) Spike (S) glycoprotein provides protection from host immune responses, acting as a steric block to potentially neutralizing antibody responses. The conformationally dynamic S-protein is the primary immunogenic target of vaccine design owing to its role in host-cell fusion, displaying multiple receptor binding domain (RBD) ‘up’ and ‘down’ state configurations. Here, we investigated the potential for RBD adjacent, N-terminal domain (NTD) glycans to influence the conformational equilibrium of these RBD states. Using a combination of antigenic screens and high-resolution cryo-EM structure determination, we show that an N-glycan deletion at position 234 results in a dramatically reduced population of the ‘up’ state RBD position. Conversely, glycan deletion at position N165 results in a discernable increase in ‘up’ state RBDs. This indicates the glycan shield acts not only as a passive hinderance to antibody meditated immunity but also as a conformational control element. Together, our results demonstrate this highly dynamic conformational machine is responsive to glycan modification with implications in viral escape and vaccine design.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Type IV pili (T4P) mediate surface motility, host interactions, and DNA uptake through cycles of extension and retraction. While the primary retraction ATPase PilT has been extensively characterized, its homolog PilU remains less well understood despite being demonstrated as a PilT-dependent retraction ATPase. Here, we determined six PilU structures by cryo-electron microscopy and X-ray crystallography. The structures reveal a homohexameric assembly stabilized by interactions between the C-terminal catalytic domain of one subunit and the N-terminal PAS-like domain of a neighboring subunit. PilU adopts multiple conformational states, exhibiting digerent combinations of open and closed interfaces even in the absence of nucleotide. Comparison with PilT highlights structural features that likely underlie PilUs weak ATPase activity and its dependence on PilT for function. Together, these findings provide a structural framework for understanding PilUs role within the T4P retraction machinery.

Wag31, or DivIVA, is an essential protein and a drug target in human pathogen Mycobacterium tuberculosis that self-assembles at the negatively curved membrane surface to form a higher-order structural scaffold, maintains rod-shaped cellular morphology, and localizes key cell-wall synthesizing proteins at the pole for exclusive polar growth. We determined the crystal structure of N-terminal membrane anchoring domain of mycobacterial Wag31 at 2.3 [A] resolution using molecular replacement method. Crystal packing analysis revealed a previously unseen dimer-of-dimer assembly state of N-terminal Wag31 with C2 point group symmetry, which is formed by antiparallel stacking of two coiled coil dimers. Size-exclusion column chromatography-coupled small angle solution X-ray scattering data showed a tetrameric form as a major assembly state of N-terminal Wag31 in solution, further supporting the crystal structure. Plausible models of linear self-assembling, and branching, of Wag31 filaments consistent with available data are suggested.

Fusing a variant of the sterile alpha motif domain of the human translocation ETS leukaemia protein (TELSAM) to a protein of interest has been shown to significantly enhance crystallization propensity. TELSAM is a pH-dependent, polymer-forming protein crystallization chaperone which, when covalently fused to a protein of interest, forms a stable, well-ordered crystal lattice. However, despite its success, a challenge persists in that crystal quality and diffraction limits appear to be heavily dependent on the choice of linker between TELSAM and the protein of interest, with identification of a functional linker relying on trial-and-error methods. Likewise, previous studies revealed that the 10xHis tag at the TELSAM N-terminus can either facilitate or hinder the ordered crystallization of target proteins attached via flexible or semi-flexible linkers. To address these challenges, we designed multiple constructs with several types of linkers--rigid (helical fusion), semi-flexible (Pro-Alan), and flexible (poly-Gly)--of varying lengths to fuse a designed ankyrin repeat protein (DARPin) to the TELSAM C-terminus. Semi-flexible and flexible linker constructs were made with and without the 10xHis tag. Our findings indicate that short semi-flexible and rigid linkers consistently yield large crystals within 24 hours with a DARPin target protein, but that flexible linkers perform best with a TNK1 UBA domain target protein. Removing the 10xHis tag enhanced crystallization rates, improved crystal morphology, and increased the crystallization propensity of semi-flexible and flexible linker constructs. While removing the His tag did not have a significant effect on crystal size, it improved the diffraction limits and crystal quality of the 1TEL-PA-DARPin construct. These results suggest that the ideal linker selection primarily depends on the properties of the target protein. Our data support the recommendation to use a short yet flexible or semi-flexible linker between TELSAM and the target protein to facilitate protein crystallization and high-resolution structure determination. SynopsisIn this study, we examine the effect of short to medium-length flexible, semi-flexible, and rigid linkers on the crystallization of a DARPin fused to the 1TEL protein crystallization chaperone, demonstrating that while rigid linkers impair crystallization and reduce diffraction quality, the ideal linker character remain target-protein dependent. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/672704v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@b90d1org.highwire.dtl.DTLVardef@1508e7dorg.highwire.dtl.DTLVardef@1baf0d1org.highwire.dtl.DTLVardef@1843df_HPS_FORMAT_FIGEXP M_FIG C_FIG

Subtle changes in protein sequence can equate to large changes in function, such as enabling pathogens to evade the immune system, hindering antibody recognition of antigens, or conferring antibiotic resistance. Even single amino acid substitutions may alter ligand binding affinity, enzymatic activity, and protein stability. Yet, due to limitations in time and resources, proteins closely related in sequence to those already characterized often remain unexamined. AlphaFold has emerged as a promising tool for protein structure prediction, though its utility in modeling single amino acid substitutions remains uncertain. In this study, we assessed AlphaFold 3s accuracy in modeling natural variants of the Helicobacter pylori catalase KatA by comparing its predictions to a novel high-resolution crystal structure of KatA from strain SS1. This variant contains key substitutions at residues 234, 237, 255, and 421 relative to the well-characterized strain 26695. AlphaFold 3 models accurately reproduced the global structure and local conformations of most variant residues, with high fidelity in conservative substitutions but variable accuracy in more flexible or interface-exposed sites. We further explored how user inputs, such as incorrect oligomeric states or sequence modifications, influence prediction quality. While AlphaFold 3 consistently produced high-quality models, deviations at variant sites occurred when incorrect oligomeric states were specified. Our findings highlight both the strengths and limitations of AlphaFold 3 in modeling natural protein variants and underscore the importance of accurate user input for reliable structural predictions. ImportanceExperimental structure determination is rarely performed for natural protein variants possessing only minor amino acid differences from published structures, even though small substitutions can significantly impact structure and function. Here, we present a case study showing that AlphaFold 3 can accurately model the structures of natural protein variants. However, providing an incorrect oligomeric state can reduce model accuracy--an error that non-expert users may easily make.

The core component of the actin cytoskeleton is the globular protein G-actin, which reversibly polymerises into filaments (F-actin). Budding yeast possesses a single actin which shares 87-89% sequence identity with vertebrate actin isoforms. Previous structural studies indicate very close overlap of main-chain backbones. Intriguingly however, substitution of yeast ACT1 with vertebrate {beta}-cytoplasmic actin severely disrupts cell function and substitution with a skeletal muscle isoform is lethal. Here we report a 2.5 [A] structure of budding yeast F-actin. Previously unresolved side-chain information now highlights four main differences in the comparison of yeast and vertebrate ADP F-actins: a more open nucleotide binding pocket; a more solvent exposed C-terminus; a rearrangement of intersubunit binding interactions in the vicinity of the D-loop and changes in the hydrogen bonding network in the vicinity of histidine 73 (yeast actin) and methyl-histidine 73 (vertebrate actin).

Nanobodies or VHHs (Variable Heavy domains of Heavy chain) are single domain antibodies that comprise three antigenic complementary determining regions (CDR). Nanobodies are used in numerous scientific applications including, bio-imaging, diagnosis, therapeutics, and macromolecular crystallography. We obtained crystals of a [~]14 kDa nanobody specific for the NEIL1 DNA glycosylase (hereafter called A5) in 0.5 M ammonium sulfate, 0.1 M sodium citrate tribasic dihydrate pH 5.6, and 1.0 M lithium sulfate monohydrate from the Crystal HT Hampton Research screen that were further optimized. Here, we describe the structure determination and refinement of the A5 crystals to a resolution of 2.1 [A]. The data collected were complicated by the presence of anisotropy and twinning, and while initial space group determination pointed to a higher apparent tetragonal crystal system, the data statistics suggested twinning, placing the crystal in an orthorhombic system. Twinning was confirmed by the Padilla and Yeates test, H-test, and Britton test based on local intensity differences with a twin fraction of 0.4. Molecular replacement produced the best solution in the orthorhombic space group P21212 with four molecules in the asymmetric unit and we were able to model over 96% of the residues in the electron density with a final Rwork and Rfree of 0.1988 and 0.2289 upon refinement. SynopsisThe crystal structure of a specific nanobody against NEIL1 was determined to 2.1 [A]. The structure was ultimately solved in an orthorhombic space group after diffraction data analysis revealed mild anisotropy as well as pseudo-merohedral twinning

Methodological improvements in cryo-electron microscopy (cryoEM) have made it a useful tool in ligand-bound structure determination for biology and drug design. However, determining the conformation and identity of bound ligands is still challenging at the resolutions typical for cry-oEM. Automated methods can aid in ligand conformational modeling, but current ligand identification tools -- developed for X-ray crystallography data -- perform poorly at resolutions common for cryoEM. Here, we present EMERALD-ID, a method capable of docking and evaluating small molecule conformations for ligand identification. EMERALD-ID identifies 43% of common ligands exactly and identifies closely related ligands in 66% of cases. We then use this tool to discover possible ligand identification errors, as well as previously unidentified ligands. Furthermore, we show EMERALD-ID is capable of identifying ligands from custom ligand libraries of various small molecule types, including human metabolites and drug fragments. Our method provides a valuable addition to cryoEM modeling tools to improve small molecule model accuracy and quality.

The protein data bank (PDB) is one of the richest databases in biology. The structural models deposited have provided insights into protein folds, relationships to evolution, energy functions of structures, and most recently, protein structure prediction, connecting sequence to structure. However, the X-ray crystallography (and cryo-EM) models deposited in the PDB are determined by a combination of refinement algorithms and manual modeling. The intervention of human modeling leads to the possibility that within a single structure, there can be differences in how well parts of a structure are modeled and/or fit the underlying experimental data. We identified that small molecule binding sites are more carefully modeled and better match the underlying experimental data than the rest of the protein structural model. This trend persisted irrespective of the structures resolution or its overall agreement with the experimental data. The variation of modeling has implications for how we interpret protein structural models and use structural models in explaining mechanisms, structural bioinformatics, simulations, docking, and structure prediction, especially when drawing conclusions about binding sites compared to the rest of the protein.

Diverse aminoacyl-tRNA synthetase (AARS) gene fusions are now recognized as a common mechanism for enhancing genetic diversity across all domains of life. The metS gene from Mycoplasma penetrans is a striking example of such an evolutionary mechanism because although M. penetrans has a condensed genome, the metS gene is nearly twice the size of a typical bacterial gene encoding methionyl tRNA synthetase (MetRS). We used cryo-EM to analyze the structure of the MpMetRS gene product to show that it is the product of three distinct enzyme domains: an N-terminal nucleotidyl transferase, a dimeric alanine-glyoxylate aminotransferase, and a MetRS. Only the N-terminal domains show two-fold symmetry, and the MetRS domain is only partially resolved. Modelling the full structure shows that a conformational change must occur to accommodate a tRNA-bound MetRS domain. A further rearrangement of the catalytic domains would also be necessary to bring the active sites adjacent to one another if this unique assembly of catalytic domains functions to channel substrates to MetRS.

The small size and flexibility of G protein-coupled receptors (GPCRs) have long posed a significant challenge to determining their structures for research and therapeutic applications. Single particle cryogenic electron microscopy (cryoEM) is often out of reach due to the small size of the receptor without a signaling partner. Crystallization of GPCRs in lipidic cubic phase (LCP) often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers. Here, we determine the previously unknown structure of the human vasopressin 1B receptor (V1BR) using microcrystal electron diffraction (MicroED). To achieve this, we grew V1BR microcrystals in LCP and transferred the material directly onto electron microscopy grids. The protein was labeled with a fluorescent dye prior to crystallization to locate the microcrystals using cryogenic fluorescence microscopy, and then the surrounding material was removed using a plasma-focused ion beam to thin the sample to a thickness amenable to MicroED. MicroED data from 14 crystalline lamellae were used to determine the 3.2 [A] structure of the receptor in the crystallographic space group P 1. These results demonstrate the use of MicroED to determine previously unknown GPCR structures that, despite significant effort, were not tractable by other methods.

-Conotoxins (-CTX) are disulfide-rich peptide antagonists with exceptional potency and subtype selectivity across nicotinic acetylcholine receptors (nAChRs), making them promising leads for therapeutic development. The molecular basis of their specificity has remained unresolved for decades, limiting rational drug design. Here, we present the first high-resolution cryo-EM structures of full-length muscle-type nAChR bound to three distinct -CTXs, revealing their precise binding modes at both acetylcholine binding sites. Our integrated approach, combining structural biology, pharmacological profiling, and computational hydration mapping, uncovers conserved pharmacophore features, water-mediated interactions, and subunit-specific determinants that govern potency and selectivity. These findings provide a long-sought molecular framework for -conotoxin recognition and inhibition, addressing a critical gap required to realise the full potential of conotoxins as lead compounds in drug discovery.

Although the domains of cis-acyltransferase (cis-AT) modular polyketide synthases (PKSs) have been understood at atomic resolution for over a decade, the domain-domain interactions responsible for the architectures and activities of these giant molecular assembly lines remain largely uncharacterized. The multimeric structure of the 6{beta}6 fungal fatty acid synthase (FAS) provides 6 equivalent reaction chambers for its acyl carrier protein (ACP) domains to shuttle carbon building blocks and the growing acyl chain between surrounding, oriented enzymatic domains. The presumed homodimeric oligomerization of cis-AT assembly lines is insufficient to provide similar reaction chambers; however, the crystal structure of a ketosynthase (KS)+AT didomain presented here and three already reported show an interaction between the AT domains appropriate for lateral multimerization. This interaction was used to construct a framework for the pikromycin PKS from its KS, AT, and docking domains that contains highly-ordered reaction chambers. Its AT domains also mediate vertical interactions, both with upstream KS domains and downstream docking domains.

In Gram-negative bacteria, several trans-envelope complexes (TECs) have been identified that span the periplasmic space in order to facilitate lipid transport between the inner- and outer- membranes. While partial or near-complete structures of some of these TECs have been solved by conventional experimental techniques, most remain incomplete. Here we describe how a combination of computational approaches, constrained by experimental data, can be used to build complete atomic models for four TECs implicated in lipid transport in Escherichia coli. We use DeepMinds protein structure prediction algorithm, AlphaFold2, and a variant of it designed to predict protein complexes, AF2Complex, to predict the oligomeric states of key components of TECs and their likely interfaces with other components. After obtaining initial models of the complete TECs by superimposing predicted structures of subcomplexes, we use the membrane orientation prediction algorithm OPM to predict the likely orientations of the inner- and outer- membrane components in each TEC. Since, in all cases, the predicted membrane orientations in these initial models are tilted relative to each other, we devise a novel molecular mechanics-based strategy that we call "membrane morphing" that adjusts each TEC model until the two membranes are properly aligned with each other and separated by a distance consistent with estimates of the periplasmic width in E. coli. The study highlights the potential power of combining computational methods, operating within limits set by both experimental data and by cell physiology, for producing useable atomic structures of very large protein complexes.

The eukaryotic COMMD family of proteins are core subunits of the Commander protein complex, with a central role in endosomal membrane trafficking and signalling. Previous crystal and cryoEM structures show that COMMD and COMMD-like proteins form homo-oligomeric and hetero-decameric assemblies with a central ring structure formed by their small C-terminal COMM domains. Their -helical N-terminal (HN) domains decorate each side of these rings, and in the eukaryotic Commander complex engage the coiled-coil proteins CCDC22 and CCDC93. Here, we have determined new crystal structures of the isolated HN domains of human Commd4, Commd9 and Commd10, and find that all three proteins form domain swapped structures with a remarkably consistent topology. This occurs via a conformational change in a hinge-loop between helices 2 and 3, leading to exchange of helices 3 to 6 between protomers. The hinge-loops of Commd9 and Commd10 possess several serine and threonine residues that can be phosphorylated, and we find that specific phospho-mimicking mutations in Commd10 can promote or inhibit domain swapping. Whether the unique COMMD HN domains play any roles beyond assembly with CCDC proteins is unclear, but this work suggests a common conformational switch exists with a potential to regulate their function.

Myelin protein P2 is a peripheral membrane protein of the fatty acid binding protein family. It functions in the formation and maintenance of the peripheral nerve myelin sheath, and several P2 mutations causing human Charot-Marie-Tooth neuropathy have been reported. Here, electron cryomicroscopy of myelin-like proteolipid multilayers revealed a three-dimensionally ordered lattice of P2 molecules between stacked lipid bilayers, visualizing its possible assembly at the myelin major dense line. A single layer of P2 is inserted between two bilayers in a tight intermembrane space of [~]3 nm, implying direct interactions between P2 and two membrane surfaces. Further details on lateral protein organization were revealed through X-ray diffraction from bicelles stacked by P2. Surface mutagenesis of P2 coupled to structural and functional experiments revealed a role for both the portal region and the opposite face of P2 in membrane interactions. Atomistic molecular dynamics simulations of P2 on myelin-like and model membrane surfaces suggested that Arg88 is an important residue for P2-membrane interactions, in addition to the helical lid domain on the opposite face of the molecule. Negatively charged myelin lipid headgroups anchor P2 stably on the bilayer surface. Membrane binding may be accompanied by opening of the P2 {beta} barrel structure and ligand exchange with the apposing lipid bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane protein from human myelin. This is an important step towards deciphering the 3-dimensional assembly of a mature myelin sheath at the molecular level.