Engineered RecA Constructs Reveal the Minimal SOS Activation Complex.

The SOS response is a bacterial DNA damage response pathway that has been heavily implicated in bacteria's ability to evolve resistance to antibiotics. Activation of the SOS response is dependent on the interaction between two bacterial proteins, RecA and LexA. RecA acts as a DNA damage sensor by forming lengthy oligomeric filaments (RecA*) along single-stranded DNA (ssDNA) in an ATP-dependent manner. RecA* can then bind to LexA, the repressor of SOS response genes, triggering LexA degradation and leading to induction of the SOS response. Formation of the RecA*-LexA complex therefore serves as the key "SOS activation signal." Given the challenges associated with studying a complex involving multiple macromolecular interactions, the essential constituents of RecA* that allow LexA cleavage are not well defined. Here, we leverage head-to-tail linked and end-capped RecA constructs as tools to define the minimal RecA* filament that can engage LexA. In contrast to previously postulated models, we found that as few as three linked RecA units are capable of ssDNA binding, LexA binding, and LexA cleavage. We further demonstrate that RecA oligomerization alone is insufficient for LexA cleavage, with an obligate requirement for ATP and ssDNA binding to form a competent SOS activation signal with the linked constructs. Our minimal system for RecA* highlights the limitations of prior models for the SOS activation signal and offers a novel tool that can inform efforts to slow acquired antibiotic resistance by targeting the SOS response.

Exploration of inhibitors of the bacterial LexA repressor-protease.

Resistant and tolerant bacterial infections lead to billions in healthcare costs and cause hundreds of thousands of deaths each year. The bulk of current antibiotic research efforts focus on molecules which, although novel, are not immune from acquired resistance and seldomly affect tolerant populations. The bacterial SOS response has been implicated in several resistance and tolerance mechanisms, making it an attractive antibiotic target. Using small molecule inhibitors targeting a key step in the deployment of the SOS response, our approach focused on preventing the deployment of mechanisms such as biofilm formation, horizontal gene transfer, and error-prone DNA repair. Herein we report the synthesis and testing of analogs of a triazole-containing tricyclic inhibitor of LexA proteolysis, the key event in the SOS response. Our results hint that our inhibitor's may function by adopting a ß-hairpin conformation, reminiscent of the native cleavage loop of LexA.

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.

Flux Crystal Growth and Optical Properties of Two Uranium-Containing Silicates: A2USiO6 (A = Cs, Rb).

Single crystals of two uranium silicates, Cs2USiO6 and Rb2USiO6, have been grown from molten fluoride fluxes and structurally characterized by single-crystal X-ray diffraction. Cs2USiO6 crystallizes in the body-centered orthorhombic space group, Immm, with a = 8.5812(4) Å, b = 13.0011(6) Å, and c = 13.8811(7) Å. The size of Rb is slightly too small to fit into this structural framework without effecting slight structural changes that result in a 6-fold superstructure. Sharp satellite peaks were observed in the single-crystal X-ray diffraction data, indicating the existing of a superstructure. The crystals were examined by electron diffraction, the results of which suggest that the structure can be thought of as the Immm isotype (a = 8.4916(6) Å, b = 12.6678(9) Å, and c = 13.5077(9) Å) on average, with an approximately 6-fold superstructure along the c axis. The materials were further characterized by UV-vis reflectance spectroscopy.

A2MnU3O11 (A = K, Rb) and Li3.2Mn1.8U6O22: Three New Alkali-Metal Manganese Uranium(VI) Oxides Related to Natrotantite.

Single crystals of three new alkali-metal manganese uranium oxides, K2MnU3O11, Rb2MnU3O11, and Li3.2Mn1.8U6O22, have been grown from molten chloride fluxes and structurally characterized by single-crystal X-ray diffraction. The first two compounds crystallize in the trigonal space group, R3̅c, in the three-dimensional (3D), natrotantite structure composed of α-U3O8-topological layers connected via MnO6 octahedra. The Li-containing compound crystallizes in the monoclinic space group, Cc, with a related 3D structure, composed of ß-U3O8-topological sheets connected via irregular MnO7 polyhedra. All three compounds exhibit typical uranyl, UO2(2+), coordination environments consisting of either UO7 pentagonal bipyramids or UO6 flattened octahedra. The lattice parameters of the new oxides are K2MnU3O11, a = 6.8280(2) Å, c = 36.8354(17) Å; Rb2MnU3O11, a = 6.8407(2) Å, c = 37.5520(17) Å; and Li3.2Mn1.8U6O22, a = 11.8958(8) Å, b = 10.9639(7) Å, c = 13.3269(8) Å, and ß = 91.442(4)°. The magnetic susceptibilities of the K and Rb phases are discussed.

Modification of Humeral Component Results in Increased Impingement Free Range of Motion in a Reverse Shoulder Arthroplasty Model.

Introduction: Shoulder arthroplasties have been demonstrated to provide reliable pain relief as well as functional benefits. The advent of the reverse shoulder arthroplasty allowed for expanded indications for shoulder replacement. Several studies comparing the outcomes of anatomic and reverse total shoulder arthroplasties have demonstrated decreased range of motion in the reverse arthroplasty cohort, especially in internal rotation. The authors hypothesized that slight modifications to the humeral component of a reverse shoulder arthroplasty could result in increased impingement free range of motion without significant sacrifices to stability. Methods: A reverse shoulder arthroplasty model was fashioned to mimic a setting of anterior mechanical impingement after replacement. Sequential resections were taken from the anterior aspect of the polyethylene up to a resection of 10 mm. A solid modeling software was utilized to compare the experimental group to the control group with regard to impingement free motion. Finite element analysis was subsequently utilized to assess stability of the construct in comparison to the nonmodified polyethylene. Results: Impingement free internal rotation increased minimally at 3 mm of resection but considerably at each further increase in resection. A resection of 10 mm resulted roughly 30% improvement in impingement free internal rotation. Instability in this model increased with modifications beyond 7 mm. Conclusion: Slight alterations to the geometry of the humeral tray and polyethene components can result in improvements in impingement-free internal rotation without substantial increased instability in this model. Further work is needed to determine in vivo implications of modifications to the humeral tray and polyethylene.

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.

FRETing about the details: Case studies in the use of a genetically encoded fluorescent amino acid for distance-dependent energy transfer.

Förster resonance energy transfer (FRET) is a valuable method for monitoring protein conformation and biomolecular interactions. Intrinsically fluorescent amino acids that can be genetically encoded, such as acridonylalanine (Acd), are particularly useful for FRET studies. However, quantitative interpretation of FRET data to derive distance information requires careful use of controls and consideration of photophysical effects. Here we present two case studies illustrating how Acd can be used in FRET experiments to study small molecule induced conformational changes and multicomponent biomolecular complexes.

Kinetic dissection of macromolecular complex formation with minimally perturbing fluorescent probes.

The formation of macromolecular complexes containing multiple protein binding partners is at the core of many biochemical pathways. Studying the kinetics of complex formation can offer significant biological insights and complement static structural snapshots or approaches that reveal thermodynamic affinities. However, determining the kinetics of macromolecular complex formation can be difficult without significant manipulations to the system. Fluorescence anisotropy using a fluorophore-labeled constituent of the biologic complex offers potential advantages in obtaining time-resolved signals tracking complex assembly. However, an inherent challenge of traditional post-translational protein labeling is the orthogonality of labeling chemistry with regards to protein target and the potential disruption of complex formation. In this chapter, we will discuss the application of unnatural amino acid labeling as a means for generating a minimally perturbing reporter. We then describe the use of fluorescence anisotropy to define the kinetics of complex formation, using the key protein-protein-nucleic acid complex governing the bacterial DNA damage response-RecA nucleoprotein filaments binding to LexA-as a model system. We will also show how this assay can be expanded to ask questions about the kinetics of complex formation for unlabeled variants, thus assessing assembly kinetics in more native contexts and broadening its utility. We discuss the optimization process for our model system and offer guidelines for applying the same principles to other macromolecular systems.

Lower complication rates associated with transradial versus transfemoral flow diverting stent placement.

BACKGROUND: Currently, there are no large-scale studies in the neurointerventional literature comparing safety between transradial (TRA) and transfemoral (TFA) approaches for flow diversion procedures. This study aims to assess complication rates in a large multicenter registry for TRA versus TFA flow diversion. METHODS: We retrospectively analyzed flow diversion cases for cerebral aneurysms from 14 institutions from 2010 to 2019. Pooled analysis of proportions was calculated using weighted analysis with 95% CI to account for results from multiple centers. Access site complication rate and overall complication rate were compared between the two approaches. RESULTS: A total of 2,285 patients who underwent flow diversion were analyzed, with 134 (5.86%) treated with TRA and 2151 (94.14%) via TFA. The two groups shared similar patient and aneurysm characteristics. Crossover from TRA to TFA was documented in 12 (8.63%) patients. There were no access site complications in the TRA group. There was a significantly higher access site complication rate in the TFA cohort as compared with TRA (2.48%, 95% CI 2.40% to 2.57%, vs 0%; p=0.039). One death resulted from a femoral access site complication. The overall complications rate was also higher in the TFA group (9.02%, 95% CI 8.15% to 9.89%) compared with the TRA group (3.73%, 95% CI 3.13% to 4.28%; p=0.035). CONCLUSION: TRA may be a safer approach for flow diversion to treat cerebral aneurysms at a wide range of locations. Both access site complication rate and overall complication rate were lower for TRA flow diversion compared with TFA in this large series.

The Kinetic and Molecular Basis for the Interaction of LexA and Activated RecA Revealed by a Fluorescent Amino Acid Probe.

The bacterial DNA damage response (the SOS response) is a key pathway involved in antibiotic evasion and a promising target for combating acquired antibiotic resistance. Activation of the SOS response is controlled by two proteins: the repressor LexA and the DNA damage sensor RecA. Following DNA damage, direct interaction between RecA and LexA leads to derepression of the SOS response. However, the exact molecular details of this interaction remain unknown. Here, we employ the fluorescent unnatural amino acid acridonylalanine (Acd) as a minimally perturbing probe of the E. coli RecA:LexA complex. Using LexA labeled with Acd, we report the first kinetic model for the reversible binding of LexA to activated RecA. We also characterize the effects that specific amino acid truncations or substitutions in LexA have on RecA:LexA binding strength and demonstrate that a mobile loop encoding LexA residues 75-84 comprises a key recognition interface for RecA. Beyond insights into SOS activation, our approach also further establishes Acd as a sensitive fluorescent probe for investigating the dynamics of protein-protein interactions in other complex systems.

Clinical outcomes and cost-effectiveness analysis for the treatment of basilar tip aneurysms.

BACKGROUND: Endovascular treatment of basilar tip aneurysms is less invasive than microsurgical clipping, but requires closer follow-up. OBJECTIVE: To characterize the additional costs associated with endovascular treatment of basilar tip aneurysms rather than microsurgical clipping. MATERIALS AND METHODS: We obtained clinical records and billing information for 141 basilar tip aneurysms treated with clip ligation (n=48) or endovascular embolization (n=93). Costs included direct and indirect costs associated with index hospitalization, as well as re-treatments, follow-up visits, imaging studies, rehabilitation, and disability. Effectiveness of treatment was quantified by converting functional outcomes (modified Rankin Scale (mRS) score) into quality-adjusted life-years (QALYs). Cost-effectiveness was performed using cost/QALY ratios. RESULTS: Average index hospitalization costs were significantly higher for patients with unruptured aneurysms treated with clip ligation ($71 400 ± $47 100) compared with coil embolization ($33 500 ± $22 600), balloon-assisted coiling ($26 200 ± $11 600), and stent-assisted coiling ($38 500 ± $20 900). Multivariate predictors for higher index hospitalization cost included vasospasm requiring endovascular intervention, placement of a ventriculoperitoneal shunt, longer length of stay, larger aneurysm neck and width, higher Hunt-Hess grade, and treatment-associated complications. At 1 year, endovascular treatment was associated with lower cost/QALY than clip ligation in unruptured aneurysms ($52 000/QALY vs $137 000/QALY, respectively, p=0.006), but comparable rates in ruptured aneurysms ($193 000/QALY vs $233 000/QALY, p=0.277). Multivariate predictors for higher cost/QALY included worse mRS score at discharge, procedural complications, and larger aneurysm width. CONCLUSIONS: Coil embolization of basilar tip aneurysms is associated with a lower cost/QALY. This effect is sustained during follow-up. Clinical condition at discharge is the most significant predictor of overall cost/QALY at 1 year.

Full and Partial Agonism of a Designed Enzyme Switch.

Chemical biology has long sought to build protein switches for use in molecular diagnostics, imaging, and synthetic biology. The overarching challenge for any type of engineered protein switch is the ability to respond in a selective and predictable manner that caters to the specific environments and time scales needed for the application at hand. We previously described a general method to design switchable proteins, called "chemical rescue of structure", that builds de novo allosteric control sites directly into a protein's functional domain. This approach entails first carving out a buried cavity in a protein via mutation, such that the protein's structure is disrupted and activity is lost. An exogenous ligand is subsequently added to substitute for the atoms that were removed by mutation, restoring the protein's structure and thus its activity. Here, we begin to ask what principles dictate such switches' response to different activating ligands. Using a redesigned ß-glycosidase enzyme as our model system, we find that the designed effector site is quite malleable and can accommodate both larger and smaller ligands, but that optimal rescue comes only from a ligand that perfectly replaces the deleted atoms. Guided by these principles, we then altered the shape of this cavity by using different cavity-forming mutations, and predicted different ligands that would better complement these new cavities. These findings demonstrate how the protein switch's response can be tuned via small changes to the ligand with respect to the binding cavity, and ultimately enabled us to design an improved switch. We anticipate that these insights will help enable the design of future systems that tune other aspects of protein activity, whereby, like evolved protein receptors, remolding the effector site can also adjust additional outputs such as substrate selectivity and activation of downstream signaling pathways.

Expression, preparation, and high-throughput screening of caspase-8: discovery of redox-based and steroid diacid inhibition.

Because of the intimate role of caspase-8 in apoptosis signaling pathways from FAS, TNFR1, and other death receptors, the enzyme is a potentially important therapeutic target. We have generated an Escherichia coli expression construct for caspase-8 in which a His-tag sequence is inserted ahead of codon 217 of caspase-8. The strain produced a significant amount of soluble His-tagged 31-kDa inactive single-chain enzyme precursor. This 31-kDa protein could be purified to 98% purity. Hydroxyapatite resolved the enzyme into two species, one with the appropriate 31,090 relative mass and the other with 178 units additional mass. The latter proved to result from E. coli-based modification of the His-tag with one equivalent of glucono-1,5-lactone. The purified proteins could be activated by autoproteolysis to the appropriate 19- plus 11-kDa enzyme by the addition of dithiothreitol in appropriate buffer conditions. This yielded an enzyme with specific activity of 4-5 units/mg against 200 microM Ac-IETD-pNA at 25 degrees C. The fully active protein was used in a high-throughput screen for inhibitors of caspase-8. A preliminary robustness screen demonstrated that caspase-8 is susceptible to reactive oxygen-based inactivation in the presence of dithiothreitol (DTT) but not in the presence of cysteine. Investigation into the mechanism of this inactivation showed that quinone-like compounds were reduced by DTT establishing a reactive oxygen generating redox cycle the products of which (likely H(2)O(2)) inactivated the enzyme. A new class of caspase-8 inhibitors, steroid-derived diacids, with affinity in the low micromolar range were discovered in the refined screen. Structure--activity investigation of the inhibitors showed that both the steroid template and the acid moieties were required for activity.