The LexA-RecA* structure reveals a cryptic lock-and-key mechanism for SOS activation.

The bacterial SOS response plays a key role in adaptation to DNA damage, including genomic stress caused by antibiotics. SOS induction begins when activated RecA*, an oligomeric nucleoprotein filament that forms on single-stranded DNA, binds to and stimulates autoproteolysis of the repressor LexA. Here, we present the structure of the complete Escherichia coli SOS signal complex, constituting full-length LexA bound to RecA*. We uncover an extensive interface unexpectedly including the LexA DNA-binding domain, providing a new molecular rationale for ordered SOS gene induction. We further find that the interface involves three RecA subunits, with a single residue in the central engaged subunit acting as a molecular key, inserting into an allosteric binding pocket to induce LexA cleavage. Given the pro-mutagenic nature of SOS activation, our structural and mechanistic insights provide a foundation for developing new therapeutics to slow the evolution of antibiotic resistance.

Structure of a HIV-1 IN-Allosteric inhibitor complex at 2.93 Å resolution: Routes to inhibitor optimization.

HIV integrase (IN) inserts viral DNA into the host genome and is the target of the strand transfer inhibitors (STIs), a class of small molecules currently in clinical use. Another potent class of antivirals is the allosteric inhibitors of integrase, or ALLINIs. ALLINIs promote IN aggregation by stabilizing an interaction between the catalytic core domain (CCD) and carboxy-terminal domain (CTD) that undermines viral particle formation in late replication. Ongoing challenges with inhibitor potency, toxicity, and viral resistance motivate research to understand their mechanism. Here, we report a 2.93 Å X-ray crystal structure of the minimal ternary complex between CCD, CTD, and the ALLINI BI-224436. This structure reveals an asymmetric ternary complex with a prominent network of π-mediated interactions that suggest specific avenues for future ALLINI development and optimization.

Transient Prenyltransferase-Cyclase Association in Fusicoccadiene Synthase, an Assembly-Line Terpene Synthase.

Fusicoccadiene synthase from the fungus Phomopsis amygdali (PaFS) is an assembly-line terpene synthase that catalyzes the first two steps in the biosynthesis of Fusiccocin A, a diterpene glycoside. The C-terminal prenyltransferase domain of PaFS catalyzes the condensation of one molecule of C5 dimethylallyl diphosphate and three molecules of C5 isopentenyl diphosphate to form C20 geranylgeranyl diphosphate, which then transits to the cyclase domain for cyclization to form fusicoccadiene. Previous structural studies of PaFS using electron microscopy (EM) revealed a central octameric prenyltransferase core with eight cyclase domains tethered in random distal positions through flexible 70-residue linkers. However, proximal prenyltransferase-cyclase configurations could be captured by covalent cross-linking and observed by cryo-EM and mass spectrometry. Here, we use cryo-EM to show that proximally configured prenyltransferase-cyclase complexes are observable even in the absence of covalent cross-linking; moreover, such complexes can involve multiple cyclase domains. A conserved basic patch on the prenyltransferase domain comprises the primary touchpoint with the cyclase domain. These results support a model for transient prenyltransferase-cyclase association in which the cyclase domains of PaFS are in facile equilibrium between proximal associated and random distal positions relative to the central prenyltransferase octamer. The results of biophysical measurements using small-angle X-ray scattering, analytical ultracentrifugation, dynamic light scattering, and size-exclusion chromatography in-line with multi-angle light scattering are consistent with this model. This model accordingly provides a framework for understanding substrate transit between the prenyltransferase and cyclase domains as well as the cooperativity observed for geranylgeranyl diphosphate cyclization.

Unique structural features govern the activity of a human mitochondrial AAA+ disaggregase, Skd3.

The AAA+ protein, Skd3 (human CLPB), solubilizes proteins in the mitochondrial intermembrane space, which is critical for human health. Skd3 variants with defective protein-disaggregase activity cause severe congenital neutropenia (SCN) and 3-methylglutaconic aciduria type 7 (MGCA7). How Skd3 disaggregates proteins remains poorly understood. Here, we report a high-resolution structure of a Skd3-substrate complex. Skd3 adopts a spiral hexameric arrangement that engages substrate via pore-loop interactions in the nucleotide-binding domain (NBD). Substrate-bound Skd3 hexamers stack head-to-head via unique, adaptable ankyrin-repeat domain (ANK)-mediated interactions to form dodecamers. Deleting the ANK linker region reduces dodecamerization and disaggregase activity. We elucidate apomorphic features of the Skd3 NBD and C-terminal domain that regulate disaggregase activity. We also define how Skd3 subunits collaborate to disaggregate proteins. Importantly, SCN-linked subunits sharply inhibit disaggregase activity, whereas MGCA7-linked subunits do not. These advances illuminate Skd3 structure and mechanism, explain SCN and MGCA7 inheritance patterns, and suggest therapeutic strategies.

Recognition of the TDP-43 nuclear localization signal by importin α1/ß.

Cytoplasmic mislocalization of the TAR-DNA binding protein of 43 kDa (TDP-43) leads to large, insoluble aggregates that are a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. Here, we study how importin α1/ß recognizes TDP-43 bipartite nuclear localization signal (NLS). We find that the NLS makes extensive contacts with importin α1, especially at the minor NLS-binding site. NLS binding results in steric clashes with the C terminus of importin α1 that disrupts the TDP-43 N-terminal domain (NTD) dimerization interface. A putative phosphorylation site in the proximity of TDP-43 R83 at the minor NLS site destabilizes binding to importins by reducing the NLS backbone dynamics. Based on these data, we explain the pathogenic role of several post-translational modifications and mutations in the proximity of TDP-43 minor NLS site that are linked to disease and shed light on the chaperone activity of importin α1/ß.

Structural basis of cyclic oligoadenylate binding to the transcription factor Csa3 outlines cross talk between type III and type I CRISPR systems.

RNA interference by type III CRISPR systems results in the synthesis of cyclic oligoadenylate (cOA) second messengers, which are known to bind and regulate various CARF domain-containing nuclease receptors. The CARF domain-containing Csa3 family of transcriptional factors associated with the DNA-targeting type I CRISPR systems regulate expression of various CRISPR and DNA repair genes in many prokaryotes. In this study, we extend the known receptor repertoire of cOA messengers to include transcriptional factors by demonstrating specific binding of cyclic tetra-adenylate (cA4) to Saccharolobus solfataricus Csa3 (Csa3Sso). Our 2.0-Å resolution X-ray crystal structure of cA4-bound full-length Csa3Sso reveals the binding of its CARF domain to an elongated conformation of cA4. Using cA4 binding affinity analyses of Csa3Sso mutants targeting the observed Csa3Sso•cA4 structural interface, we identified a Csa3-specific cA4 binding motif distinct from a more widely conserved cOA-binding CARF motif. Using a rational surface engineering approach, we increased the cA4 binding affinity of Csa3Sso up to ∼145-fold over the wildtype, which has potential applications for future second messenger-driven CRISPR gene expression and editing systems. Our in-solution Csa3Sso structural analysis identified cA4-induced allosteric and asymmetric conformational rearrangement of its C-terminal winged helix-turn-helix effector domains, which could potentially be incompatible to DNA binding. However, specific in vitro binding of the purified Csa3Sso to its putative promoter (PCas4a) was found to be cA4 independent, suggesting a complex mode of Csa3Sso regulation. Overall, our results support cA4-and Csa3-mediated cross talk between type III and type I CRISPR systems.

Structures of full-length VanR from Streptomyces coelicolor in both the inactive and activated states.

Vancomycin has historically been used as a last-resort treatment for serious bacterial infections. However, vancomycin resistance has become widespread in certain pathogens, presenting a serious threat to public health. Resistance to vancomycin is conferred by a suite of resistance genes, the expression of which is controlled by the VanR-VanS two-component system. VanR is the response regulator in this system; in the presence of vancomycin, VanR accepts a phosphoryl group from VanS, thereby activating VanR as a transcription factor and inducing expression of the resistance genes. This paper presents the X-ray crystal structures of full-length VanR from Streptomyces coelicolor in both the inactive and activated states at resolutions of 2.3 and 2.0 Å, respectively. Comparison of the two structures illustrates that phosphorylation of VanR is accompanied by a disorder-to-order transition of helix 4, which lies within the receiver domain of the protein. This transition generates an interface that promotes dimerization of the receiver domain; dimerization in solution was verified using analytical ultracentrifugation. The inactive conformation of the protein does not appear intrinsically unable to bind DNA; rather, it is proposed that in the activated form DNA binding is enhanced by an avidity effect contributed by the receiver-domain dimerization.

Assembly of higher-order SMN oligomers is essential for metazoan viability and requires an exposed structural motif present in the YG zipper dimer.

Protein oligomerization is one mechanism by which homogenous solutions can separate into distinct liquid phases, enabling assembly of membraneless organelles. Survival Motor Neuron (SMN) is the eponymous component of a large macromolecular complex that chaperones biogenesis of eukaryotic ribonucleoproteins and localizes to distinct membraneless organelles in both the nucleus and cytoplasm. SMN forms the oligomeric core of this complex, and missense mutations within its YG box domain are known to cause Spinal Muscular Atrophy (SMA). The SMN YG box utilizes a unique variant of the glycine zipper motif to form dimers, but the mechanism of higher-order oligomerization remains unknown. Here, we use a combination of molecular genetic, phylogenetic, biophysical, biochemical and computational approaches to show that formation of higher-order SMN oligomers depends on a set of YG box residues that are not involved in dimerization. Mutation of key residues within this new structural motif restricts assembly of SMN to dimers and causes locomotor dysfunction and viability defects in animal models.

Allosteric HIV Integrase Inhibitors Promote Formation of Inactive Branched Polymers via Homomeric Carboxy-Terminal Domain Interactions.

The major effect of allosteric HIV integrase (IN) inhibitors (ALLINIs) is observed during virion maturation, where ALLINI treatment interrupts IN-RNA interactions via drug-induced IN aggregation, leading to the formation of aberrant virions. To understand the structural changes that accompany drug-induced aggregation, we determined the soft matter properties of ALLINI-induced IN aggregates. Using small-angle neutron scattering, SEM, and rheology, we have discovered that the higher-order aggregates induced by ALLINIs have the characteristics of weak three-dimensional gels with a fractal-like character. Their formation is inhibited by the host factor LEDGF/p75, as well as ex vivo resistance substitutions. Mutagenesis and biophysical analyses reveal that homomeric carboxy-terminal domain interactions are required to achieve the branched-polymer nature of the ALLINI-induced aggregates. These studies provide key insight into the mechanisms of ALLINI action and resistance in the context of the crowded virion environment where ALLINIs exert their effect.

Manipulation of a cation-π sandwich reveals conformational flexibility in phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) is an allosteric enzyme that maintains phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria, an inborn error of amino acid metabolism. Wild type (WT) PAH equilibrates among resting-state (RS-PAH) and activated (A-PAH) conformations, whose equilibrium position depends upon allosteric Phe binding. The RS-PAH conformation of WT rat PAH (rPAH) contains a cation-π sandwich involving Phe80 that cannot exist in the A-PAH conformation. Phe80 variants F80A, F80D, F80L, and F80R were prepared and evaluated using native PAGE, size exclusion chromatography, ion exchange behavior, intrinsic protein fluorescence, enzyme kinetics, and limited proteolysis, each as a function of [Phe]. Like WT rPAH, F80A and F80D show allosteric activation by Phe while F80L and F80R are constitutively active. Maximal activity of all variants suggests relief of a rate-determining conformational change. Limited proteolysis of WT rPAH (minus Phe) reveals facile cleavage within a 4-helix bundle that is buried in the RS-PAH tetramer interface, reflecting dynamic dissociation of that tetramer. This cleavage is not seen for the Phe80 variants, which all show proteolytic hypersensitivity in a linker that repositions during the RS-PAH to A-PAH interchange. Hypersensitivity is corrected by addition of Phe such that all variants become like WT rPAH and achieve the A-PAH conformation. Thus, manipulation of Phe80 perturbs the conformational space sampled by PAH, increasing sampling of on-pathway intermediates in the RS-PAH and A-PAH interchange. The behavior of the Phe80 variants mimics that of disease-associated R68S and suggests a molecular basis for proteolytic susceptibility in PKU-associated human PAH variants.

Influence of the amino-terminal sequence on the structure and function of HIV integrase.

BACKGROUND: Antiretroviral therapy (ART) can mitigate the morbidity and mortality caused by the human immunodeficiency virus (HIV). Successful development of ART can be accelerated by accurate structural and biochemical data on targets and their responses to inhibitors. One important ART target, HIV integrase (IN), has historically been studied in vitro in a modified form adapted to bacterial overexpression, with a methionine or a longer fusion protein sequence at the N-terminus. In contrast, IN present in viral particles is produced by proteolytic cleavage of the Pol polyprotein, which leaves a phenylalanine at the N-terminus (IN 1F). Inspection of available structures suggested that added residues on the N-terminus might disrupt proper protein folding and formation of multimeric complexes. RESULTS: We purified HIV-1 IN 1F1-212 and solved its structure at 2.4 Å resolution, which showed extension of an N-terminal helix compared to the published structure of IN1-212. Full-length IN 1F showed increased in vitro catalytic activity in assays of coupled joining of the two viral DNA ends compared to two IN variants containing additional N-terminal residues. IN 1F was also altered in its sensitivity to inhibitors, showing decreased sensitivity to the strand-transfer inhibitor raltegravir and increased sensitivity to allosteric integrase inhibitors. In solution, IN 1F exists as monomers and dimers, in contrast to other IN preparations which exist as higher-order oligomers. CONCLUSIONS: The structural, biochemical, and biophysical characterization of IN 1F reveals the conformation of the native HIV-1 IN N-terminus and accompanying unique biochemical and biophysical properties. IN 1F thus represents an improved reagent for use in integration reactions in vitro and the development of antiretroviral agents.

Membrane Proteins Have Distinct Fast Internal Motion and Residual Conformational Entropy.

The internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side-chain dynamics of the α-helical sensory rhodopsin II and the ß-barrel outer membrane protein W have been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Despite their differing topologies, both proteins have a similar distribution of methyl-bearing side-chain motion that is largely independent of membrane mimetic. The methyl-bearing side chains of both proteins are, on average, more dynamic in the ps-ns timescale than any soluble protein characterized to date. Accordingly, both proteins retain an extraordinary residual conformational entropy in the folded state, which provides a counterbalance to the absence of the hydrophobic effect. Furthermore, the high conformational entropy could greatly influence the thermodynamics underlying membrane-protein functions, including ligand binding, allostery, and signaling.

Higher-order oligomerization of a chimeric αßγ bifunctional diterpene synthase with prenyltransferase and class II cyclase activities is concentration-dependent.

The unusual diterpene (C20) synthase copalyl diphosphate synthase from Penicillium verruculosum (PvCPS) is the first bifunctional terpene synthase identified with both prenyltransferase and class II cyclase activities in a single polypeptide chain with αßγ domain architecture. The C-terminal prenyltransferase α domain generates geranylgeranyl diphosphate which is then cyclized to form copalyl diphosphate at the N-terminal ßγ domain interface. We now demonstrate that PvCPS exists as a hexamer at high concentrations - a unique quaternary structure for known αßγ terpene synthases. Hexamer assembly is corroborated by a 2.41 Å-resolution crystal structure of the α domain prenyltransferase obtained from limited proteolysis of full-length PvCPS, as well as the ab initio model of full-length PvCPS derived from small-angle X-ray scattering data. Hexamerization of the prenyltransferase α domain appears to drive the hexamerization of full-length PvCPS. The PvCPS hexamer dissociates into lower-order species at lower concentrations, as evidenced by size-exclusion chromatography in-line with multiangle light scattering, sedimentation velocity analytical ultracentrifugation, and native polyacrylamide gel electrophoresis experiments, suggesting that oligomerization is concentration dependent. Even so, PvCPS oligomer assembly does not affect prenyltransferase activity in vitro.

Structural Characterization of Tau in Fuzzy Tau:Tubulin Complexes.

Tau is a neuronal microtubule (MT)-associated protein of significant interest due to its association with several neurodegenerative disorders. Tau's intrinsic disorder and the dynamic nature of its interactions with tubulin and MTs make its structural characterization challenging. Here, we use an environmentally sensitive fluorophore as a site-specific probe of tau bound to soluble tubulin. Comparison of our results with a recently published tau:MT cryoelectron microscopy model reveals structural similarities between tubulin- and MT-bound tau. Analysis of residues across the repeat regions reveals a hierarchy in tubulin occupancy, which may be relevant to tau's ability to differentiate between tubulin and MTs. As binding to soluble tubulin is a critical first step in MT polymerization, our characterization of the structural features of tau in dynamic, fuzzy tau:tubulin assemblies advances our understanding of how tau functions in the cell and how function may be disrupted in disease.

Biophysical characterization of full-length human phenylalanine hydroxylase provides a deeper understanding of its quaternary structure equilibrium.

Dysfunction of human phenylalanine hydroxylase (hPAH, EC 1.14.16.1) is the primary cause of phenylketonuria, the most common inborn error of amino acid metabolism. The dynamic domain rearrangements of this multimeric protein have thwarted structural study of the full-length form for decades, until now. In this study, a tractable C29S variant of hPAH (C29S) yielded a 3.06 Å resolution crystal structure of the tetrameric resting-state conformation. We used size-exclusion chromatography in line with small-angle X-ray scattering (SEC-SAXS) to analyze the full-length hPAH solution structure both in the presence and absence of Phe, which serves as both substrate and allosteric activators. Allosteric Phe binding favors accumulation of an activated PAH tetramer conformation, which is biophysically distinct in solution. Protein characterization with enzyme kinetics and intrinsic fluorescence revealed that the C29S variant and hPAH are otherwise equivalent in their response to Phe, further supported by their behavior on various chromatography resins and by analytical ultracentrifugation. Modeling of resting-state and activated forms of C29S against SAXS data with available structural data created and evaluated several new models for the transition between the architecturally distinct conformations of PAH and highlighted unique intra- and inter-subunit interactions. Three best-fitting alternative models all placed the allosteric Phe-binding module 8-10 Å farther from the tetramer center than do all previous models. The structural insights into allosteric activation of hPAH reported here may help inform ongoing efforts to treat phenylketonuria with novel therapeutic approaches.

Water loading driven size, shape, and composition of cetyltrimethylammonium/hexanol/pentane reverse micelles.

Cetyltrimethylammonium bromide (CTAB)/hexanol reverse micelles have found a variety of applications that demand control over physical parameters. Water content or loading is among the most basic tunable components and is the major driver of the physical properties of these systems. This study uses small-angle scattering with contrast variation to characterize these systems as a function of water loading. The scattering data were analyzed with a variety of approaches, resulting in converging physical specifications. Equations that describe basic physical parameters were determined that allow for characterization and manipulation of the CTAB/hexanol reverse micelle surfactant system. The shape of the reverse micelles was revealed to be slightly ellipsoidal and varies slightly through the water loading range. The surfactant shell is shown to contain a higher fraction of hexanol upon addition of water. Analysis reveals that the size, shape, and surfactant/cosurfactant composition are directly tunable by variation of the water content and that these properties are consequences of the balance of forces present in the reverse micelles.

Serine Integrase attP Binding and Specificity.

Serine integrases catalyze the site-specific insertion of viral DNA into a host's genome. The minimal requirements and irreversible nature of this integration reaction have led to the use of serine integrases in applications ranging from bacterial memory storage devices to gene therapy. Our understanding of how the integrase proteins recognize the viral (attP) and host (attB) attachment sites is limited, with structural data available for only a Listeria integrase C-terminal domain (CTD) bound to an attP half-site. Here we report quantitative binding and saturation mutagenesis analyses for the Listeria innocua prophage attP site and a new 2.8-Šcrystal structure of the CTD•attP half site. We find that Int binds with high affinity to attP (6.9 nM), but the Int CTD binds to attP half-sites with only 7- to 10-fold lower affinity, supporting the idea that free energy is expended to open an Int dimer for attP binding. Despite the 50-bp Int-attP interaction surface, only 20 residues are sensitive to mutagenesis, and of these, only 6 require a specific residue for efficient Int binding and integration activity. One of the integrase DNA-binding domains, the recombinase domain, appears to be primarily non-specific. Several substitutions result in an improved attP site, indicating that higher-efficiency attachment sites can be obtained through site engineering. These findings advance our understanding of serine integrase function and provide important data for efforts towards engineering this family of enzymes for a variety of biotechnology applications.

Functional activity of the H3.3 histone chaperone complex HIRA requires trimerization of the HIRA subunit.

The HIRA histone chaperone complex deposits the histone variant H3.3 onto chromatin in a DNA synthesis-independent manner. It comprises three identified subunits, HIRA, UBN1 and CABIN1, however the functional oligomerization state of the complex has not been investigated. Here we use biochemical and crystallographic analysis to show that the HIRA subunit forms a stable homotrimer that binds two subunits of CABIN1 in vitro. A HIRA mutant that is defective in homotrimer formation interacts less efficiently with CABIN1, is not enriched at DNA damage sites upon UV irradiation and cannot rescue new H3.3 deposition in HIRA knockout cells. The structural homology with the homotrimeric replisome component Ctf4/AND-1 enables the drawing of parallels and discussion of the functional importance of the homotrimerization state of the HIRA subunit.

Neutron scattering in the biological sciences: progress and prospects.

The scattering of neutrons can be used to provide information on the structure and dynamics of biological systems on multiple length and time scales. Pursuant to a National Science Foundation-funded workshop in February 2018, recent developments in this field are reviewed here, as well as future prospects that can be expected given recent advances in sources, instrumentation and computational power and methods. Crystallography, solution scattering, dynamics, membranes, labeling and imaging are examined. For the extraction of maximum information, the incorporation of judicious specific deuterium labeling, the integration of several types of experiment, and interpretation using high-performance computer simulation models are often found to be particularly powerful.