Structure-based mechanism of riboregulation of the metabolic enzyme SHMT1.

RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme's reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.

Simple engineering of hybrid cellulose nanocrystal-gold nanoparticles results in a functional glyconanomaterial with biomolecular recognition properties.

Cellulose nanocrystal and gold nanoparticles are assembled, in a unique way, to yield a novel modular glyconanomaterial whose surface is then easily engineered with one or two different headgroups, by exploiting a robust click chemistry route. We demonstrate the potential of this approach by conjugating monosaccharide headgroups to the glyconanomaterial and show that the sugars retain their binding capability to C-type lectin receptors, as also directly visualized by cryo-TEM.

Beyond the standard model of solubilization: Non-ionic surfactants induce collapse of lipid vesicles into rippled bilamellar nanodiscs.

HYPOTHESIS: Although solubilization of lipid membranes has been studied extensively, questions remain regarding the structural pathways and metastable structures involved. This study investigated whether the non-ionic detergent Triton X-100 follows the classical solubilization pathway or if intermediate nanostructures are formed. EXPERIMENTS: Small angle X-ray and neutron scattering (SAXS/SANS) was used in combination with transmission electron cryo-microscopy and cryo-tomography to deduce the structure of mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles and Triton X-100. Time-resolved SAXS and dynamic light scattering were used to investigate the kinetics of the process. FINDINGS: Upon addition of moderate detergent amounts at low temperatures, the lipid vesicles implode into ordered rippled bilamellar disc structures. The bilayers arrange in a ripple phase to accommodate packing constraints caused by inserted TX-100 molecules. The collapse is suggested to occur through a combination of water structure destabilization by detergents flipping across the membrane and osmotic pressure causing interbilayer attraction internally. The subsequently induced ripples then stabilize the aggregates and prevent solubilization, supported by the observation that negatively charged vesicles undergo a different pathway upon TX-100 addition, forming large bicelles. The findings demonstrate the richness in assembly pathways of simple lipids and detergents and stimulate considerations for the use of certain detergents in membrane solubilization.

Priming mycobacterial ESX-secreted protein B to form a channel-like structure.

ESX-1 is a major virulence factor of Mycobacterium tuberculosis, a secretion machinery directly involved in the survival of the microorganism from the immune system defence. It disrupts the phagosome membrane of the host cell through a contact-dependent mechanism. Recently, the structure of the inner-membrane core complex of the homologous ESX-3 and ESX-5 was resolved; however, the elements involved in the secretion through the outer membrane or those acting on the host cell membrane are unknown. Protein substrates might form this missing element. Here, we describe the oligomerisation process of the ESX-1 substrate EspB, which occurs upon cleavage of its C-terminal region and is favoured by an acidic environment. Cryo-electron microscopy data shows that quaternary structure of EspB is conserved across slow growing species, but not in the fast growing M. smegmatis. EspB assembles into a channel with dimensions and characteristics suitable for the transit of ESX-1 substrates, as shown by the presence of another EspB trapped within. Our results provide insight into the structure and assembly of EspB, and suggests a possible function as a structural element of ESX-1.

Ensemble description of the intrinsically disordered N-terminal domain of the Nipah virus P/V protein from combined NMR and SAXS.

Using SAXS and NMR spectroscopy, we herein provide a high-resolution description of the intrinsically disordered N-terminal domain (PNT, aa 1-406) shared by the Nipah virus (NiV) phosphoprotein (P) and V protein, two key players in viral genome replication and in evasion of the host innate immune response, respectively. The use of multidimensional NMR spectroscopy allowed us to assign as much as 91% of the residues of this intrinsically disordered domain whose size constitutes a technical challenge for NMR studies. Chemical shifts and nuclear relaxation measurements provide the picture of a highly flexible protein. The combination of SAXS and NMR information enabled the description of the conformational ensemble of the protein in solution. The present results, beyond providing an overall description of the conformational behavior of this intrinsically disordered region, also constitute an asset for obtaining atomistic information in future interaction studies with viral and/or cellular partners. The present study can thus be regarded as the starting point towards the design of inhibitors that by targeting crucial protein-protein interactions involving PNT might be instrumental to combat this deadly virus.

Structural dynamics of the GluK3-kainate receptor neurotransmitter binding domains revealed by cryo-EM.

Kainate receptors belong to the ionotropic glutamate receptor family and play critical roles in the regulation of synaptic networks. The kainate receptor subunit GluK3 has unique functional properties and contributes to presynaptic facilitation at the hippocampal mossy fiber synapses along with roles at the post-synapses. To gain structural insights into the unique functional properties and dynamics of GluK3 receptor, we imaged them via electron microscopy in the apo-state and in complex with either agonist kainate or antagonist UBP301. Our analysis of all the GluK3 full-length structures not only provides insights into the receptor transitions between desensitized and closed states but also reveals a "non-classical" conformation of neurotransmitter binding domain in the closed-state distinct from that observed in AMPA and other kainate receptor structures. We show by molecular dynamics simulations that Asp759 influences the stability of the LBD dimers and hence could be responsible for the observed conformational variability and dynamics of the GluK3 via electron microscopy. Lower dimer stability could explain faster desensitization and low agonist sensitivity of GluK3. In overview, our work helps to associate biochemistry and physiology of GluK3 receptors with their structural biology and offers structural insights into the unique functional properties of these atypical receptors.

Structure-specific recognition protein-1 (SSRP1) is an elongated homodimer that binds histones.

The histone chaperone complex facilitates chromatin transcription (FACT) plays important roles in DNA repair, replication, and transcription. In the formation of this complex, structure-specific recognition protein-1 (SSRP1) heterodimerizes with suppressor of Ty 16 (SPT16). SSRP1 also has SPT16-independent functions, but how SSRP1 functions alone remains elusive. Here, using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS) techniques, we characterized human SSRP1 and that from the amoeba Dictyostelium discoideum and show that both orthologs form an elongated homodimer in solution. We found that substitutions in the SSRP1 pleckstrin homology domain known to bind SPT16 also disrupt SSRP1 homodimerization. Moreover, AUC and SAXS analyses revealed that SSRP1 homodimerization and heterodimerization with SPT16 (resulting in FACT) involve the same SSRP1 surface, namely the PH2 region, and that the FACT complex contains only one molecule of SSRP1. These observations suggest that SSRP1 homo- and heterodimerization might be mutually exclusive. Moreover, isothermal titration calorimetry analyses disclosed that SSRP1 binds both histones H2A-H2B and H3-H4 and that disruption of SSRP1 homodimerization decreases its histone-binding affinity. Together, our results provide evidence for regulation of SSRP1 by homodimerization and suggest a potential role for homodimerization in facilitating SPT16-independent functions of SSRP1.

Casitas B-lineage lymphoma linker helix mutations found in myeloproliferative neoplasms affect conformation.

BACKGROUND: Casitas B-lineage lymphoma (Cbl or c-Cbl) is a RING ubiquitin ligase that negatively regulates protein tyrosine kinase (PTK) signalling. Phosphorylation of a conserved residue (Tyr371) on the linker helix region (LHR) between the substrate-binding and RING domains is required to ubiquitinate PTKs, thereby flagging them for degradation. This conserved Tyr is a mutational hotspot in myeloproliferative neoplasms. Previous studies have revealed that select point mutations in Tyr371 can potentiate transformation in cells and mice but not all possible mutations do so. To trigger oncogenic potential, Cbl Tyr371 mutants must perturb the LHR-substrate-binding domain interaction and eliminate PTK ubiquitination. Although structures of native and pTyr371-Cbl are available, they do not reveal how Tyr371 mutations affect Cbl's conformation. Here, we investigate how Tyr371 mutations affect Cbl's conformation in solution and how this relates to Cbl's ability to potentiate transformation in cells. RESULTS: To explore how Tyr371 mutations affect Cbl's properties, we used surface plasmon resonance to measure Cbl mutant binding affinities for E2 conjugated with ubiquitin (E2-Ub), small angle X-ray scattering studies to investigate Cbl mutant conformation in solution and focus formation assays to assay Cbl mutant transformation potential in cells. Cbl Tyr371 mutants enhance E2-Ub binding and cause Cbl to adopt extended conformations in solution. LHR flexibility, RING domain accessibility and transformation potential are associated with the extent of LHR-substrate-binding domain perturbation affected by the chemical nature of the mutation. More disruptive mutants like Cbl Y371D or Y371S are more extended and the RING domain is more accessible, whereas Cbl Y371F mimics native Cbl in solution. Correspondingly, the only Tyr371 mutants that potentiate transformation in cells are those that perturb the LHR-substrate-binding domain interaction. CONCLUSIONS: c-Cbl's LHR mutations are only oncogenic when they disrupt the native state and fail to ubiquitinate PTKs. These findings provide new insights into how LHR mutations deregulate c-Cbl.

A New Method for Determining Structure Ensemble: Application to a RNA Binding Di-Domain Protein.

Structure ensemble determination is the basis of understanding the structure-function relationship of a multidomain protein with weak domain-domain interactions. Paramagnetic relaxation enhancement has been proven a powerful tool in the study of structure ensembles, but there exist a number of challenges such as spin-label flexibility, domain dynamics, and overfitting. Here we propose a new (to our knowledge) method to describe structure ensembles using a minimal number of conformers. In this method, individual domains are considered rigid; the position of each spin-label conformer and the structure of each protein conformer are defined by three and six orthogonal parameters, respectively. First, the spin-label ensemble is determined by optimizing the positions and populations of spin-label conformers against intradomain paramagnetic relaxation enhancements with a genetic algorithm. Subsequently, the protein structure ensemble is optimized using a more efficient genetic algorithm-based approach and an overfitting indicator, both of which were established in this work. The method was validated using a reference ensemble with a set of conformers whose populations and structures are known. This method was also applied to study the structure ensemble of the tandem di-domain of a poly (U) binding protein. The determined ensemble was supported by small-angle x-ray scattering and nuclear magnetic resonance relaxation data. The ensemble obtained suggests an induced fit mechanism for recognition of target RNA by the protein.

Structural insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution.

Infection by the four serotypes of Dengue virus (DENV-1 to DENV-4) causes an important arthropod-borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all four Dengue virus serotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full-length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV-1 to DENV-4 NS5 forms are elongated and flexible in solution, with DENV-4 NS5 being more compact relative to NS5 from DENV-1, DENV-2 and DENV-3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten-residue linker region to the flexibility of the entire NS5. Swapping the ten-residue linker between DENV-4 NS5FL and DENV-3 NS5FL demonstrated its importance in MTase-RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented.

Maturation of 6S regulatory RNA to a highly elongated structure.

As bacterial populations leave the exponential growth phase and enter the stationary phase, their patterns of gene expression undergo marked changes. A key effector of this change is 6S RNA, which is a highly conserved regulatory RNA that impedes the transcription of genes associated with exponential growth by forming an inactivating ternary complex with RNA polymerase and sigma factor σ(70) (σ(70)-RNAP). In Escherichia coli, the endoribonuclease RNase E generates 6S RNA by specific cleavage of a precursor that is nearly twice the size of the 58 kDa mature form. We have explored recognition of the precursor by RNase E, and observed that processing is inhibited under conditions of excess substrate. This finding supports a largely distributive mechanism, meaning that each round of catalysis results in enzyme dissociation and re-binding to the substrate. We show that the precursor molecule and the mature 6S share a structural core dominated by an A-type helix, indicating that processing is not accompanied by extensive refolding. Both precursor and mature forms of 6S have a highly stable secondary structure, adopt an elongated shape, and show the potential to form dimers under specific conditions; nonetheless, 6S has a high structural plasticity that probably enables it to be structurally adapted upon binding to its cognate protein partners. Analysis of the 6S-σ(70)-RNAP complex by native mass spectrometry reveals a stable association with a stoichiometry of 1:1:1. A theoretical 3D model of mature 6S is presented, which is consistent with the experimental data and supports a previously proposed structure with a small stem-loop inside the central bubble.

Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering.

Dynamic ensembles of macromolecules mediate essential processes in biology. Understanding the mechanisms driving the function and molecular interactions of 'unstructured' and flexible molecules requires alternative approaches to those traditionally employed in structural biology. Small-angle X-ray scattering (SAXS) is an established method for structural characterization of biological macromolecules in solution, and is directly applicable to the study of flexible systems such as intrinsically disordered proteins and multi-domain proteins with unstructured regions. The Ensemble Optimization Method (EOM) [Bernadó et al. (2007 ▶). J. Am. Chem. Soc. 129, 5656-5664] was the first approach introducing the concept of ensemble fitting of the SAXS data from flexible systems. In this approach, a large pool of macromolecules covering the available conformational space is generated and a sub-ensemble of conformers coexisting in solution is selected guided by the fit to the experimental SAXS data. This paper presents a series of new developments and advancements to the method, including significantly enhanced functionality and also quantitative metrics for the characterization of the results. Building on the original concept of ensemble optimization, the algorithms for pool generation have been redesigned to allow for the construction of partially or completely symmetric oligomeric models, and the selection procedure was improved to refine the size of the ensemble. Quantitative measures of the flexibility of the system studied, based on the characteristic integral parameters of the selected ensemble, are introduced. These improvements are implemented in the new EOM version 2.0, and the capabilities as well as inherent limitations of the ensemble approach in SAXS, and of EOM 2.0 in particular, are discussed.

Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular c-terminal domain in complex with calmodulin.

Kv7 channels tune neuronal and cardiomyocyte excitability. In addition to the channel membrane domain, they also have a unique intracellular C-terminal (CT) domain, bound constitutively to calmodulin (CaM). This CT domain regulates gating and tetramerization. We investigated the structure of the membrane proximal CT module in complex with CaM by X-ray crystallography. The results show how the CaM intimately hugs a two-helical bundle, explaining many channelopathic mutations. Structure-based mutagenesis of this module in the context of concatemeric tetramer channels and functional analysis along with in vitro data lead us to propose that one CaM binds to one individual protomer, without crosslinking subunits and that this configuration is required for proper channel expression and function. Molecular modeling of the CT/CaM complex in conjunction with small-angle X-ray scattering suggests that the membrane proximal region, having a rigid lever arm, is a critical gating regulator.

The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility.

The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic and glycinergic synapses is controlled by the scaffold protein gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin-based protein interaction network that controls the gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin-2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans-synaptic neuroligin-dependent adhesion and phosphoinositide signaling with gephyrin recruitment, the collybistin-based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses.

The Salmonella enterica ZinT structure, zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc.

BACKGROUND: In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT-ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim. METHODS: ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy). RESULTS: The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a KD value of 22±2nM for ZinT, while those with ZnuA point to one high affinity (KD<20nM) and one low affinity Zn(II) binding site (KD in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT-ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other. CONCLUSIONS: ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA. GENERAL SIGNIFICANCE: Knowledge of the ZinT-ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.

Structural characterization of gephyrin by AFM and SAXS reveals a mixture of compact and extended states.

Gephyrin is a trimeric protein involved in the final steps of molybdenum-cofactor (Moco) biosynthesis and in the clustering of inhibitory glycine and GABAA receptors at postsynaptic specializations. Each protomer consists of stably folded domains (referred to as the G and E domains) located at either terminus and connected by a proteolytically sensitive linker of ∼150 residues. Both terminal domains can oligomerize in their isolated forms; however, in the context of the full-length protein only the G-domain trimer is permanently present, whereas E-domain dimerization is prevented. Atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) reveal a high degree of flexibility in the structure of gephyrin. The results imply an equilibrium between compact and extended conformational states in solution, with a preference for compact states. CD spectroscopy suggests that a partial compaction is achieved by interactions of the linker with the G and E domains. Taken together, the data provide a rationale for the role of the linker in the overall structure and the conformational dynamics of gephyrin.

Small angle X-ray scattering studies of mitochondrial glutaminase C reveal extended flexible regions, and link oligomeric state with enzyme activity.

Glutaminase C is a key metabolic enzyme, which is unregulated in many cancer systems and believed to play a central role in the Warburg effect, whereby cancer cells undergo changes to an altered metabolic profile. A long-standing hypothesis links enzymatic activity to the protein oligomeric state, hence the study of the solution behavior in general and the oligomer state in particular of glutaminase C is important for the understanding of the mechanism of protein activation and inhibition. In this report, this is extensively investigated in correlation to enzyme concentration or phosphate level, using a high-throughput microfluidic-mixing chip for the SAXS data collection, and we confirm that the oligomeric state correlates with activity. The in-depth solution behavior analysis further reveals the structural behavior of flexible regions of the protein in the dimeric, tetrameric and octameric state and investigates the C-terminal influence on the enzyme solution behavior. Our data enable SAXS-based rigid body modeling of the full-length tetramer states, thereby presenting the first ever experimentally derived structural model of mitochondrial glutaminase C including the N- and C-termini of the enzyme.

A structural model of PpoA derived from SAXS-analysis-implications for substrate conversion.

In plants and mammals, oxylipins may be synthesized via multi step processes that consist of dioxygenation and isomerization of the intermediately formed hydroperoxy fatty acid. These processes are typically catalyzed by two distinct enzyme classes: dioxygenases and cytochrome P450 enzymes. In ascomycetes biosynthesis of oxylipins may proceed by a similar two-step pathway. An important difference, however, is that both enzymatic activities may be combined in a single bifunctional enzyme. These types of enzymes are named Psi-factor producing oxygenases (Ppo). Here, the spatial organization of the two domains of PpoA from Aspergillus nidulans was analyzed by small-angle X-ray scattering and the obtained data show that the enzyme exhibits a relatively flat trimeric shape. Atomic structures of the single domains were obtained by template-based structure prediction and docked into the enzyme envelope of the low resolution structure obtained by SAXS. EPR-based distance measurements between the tyrosyl radicals formed in the activated dioxygenase domain of the enzyme supported the trimeric structure obtained from SAXS and the previous assignment of Tyr374 as radical-site in PpoA. Furthermore, two phenylalanine residues in the cytochrome P450 domain were shown to modulate the specificity of hydroperoxy fatty acid rearrangement.

New developments in the ATSAS program package for small-angle scattering data analysis.

New developments in the program package ATSAS (version 2.4) for the processing and analysis of isotropic small-angle X-ray and neutron scattering data are described. They include (i) multiplatform data manipulation and display tools, (ii) programs for automated data processing and calculation of overall parameters, (iii) improved usage of high- and low-resolution models from other structural methods, (iv) new algorithms to build three-dimensional models from weakly interacting oligomeric systems and complexes, and (v) enhanced tools to analyse data from mixtures and flexible systems. The new ATSAS release includes installers for current major platforms (Windows, Linux and Mac OSX) and provides improved indexed user documentation. The web-related developments, including a user discussion forum and a widened online access to run ATSAS programs, are also presented.