Illuminating the mechanism and allosteric behavior of NanoLuc luciferase.

NanoLuc, a superior ß-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques are combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Structurally, binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O2 via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters.

Atypical homodimerization revealed by the structure of the (S)-enantioselective haloalkane dehalogenase DmmarA from Mycobacterium marinum.

Haloalkane dehalogenases (HLDs) are a family of α/ß-hydrolase fold enzymes that employ SN2 nucleophilic substitution to cleave the carbon-halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Atomic-level knowledge of both the inner organization and supramolecular complexation of HLDs is thus crucial to understand their catalytic and noncatalytic functions. Here, crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85 Šresolution. The structures show a canonical αßα-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate-histidine-aspartate) and uncommon active-site pocket reveal the molecular specificities of a catalytic apparatus that exhibits a rare (S)-enantiopreference. Additionally, the structures reveal a previously unobserved mode of symmetric homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering. Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its uncommon enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution.

Advancing Enzyme's Stability and Catalytic Efficiency through Synergy of Force-Field Calculations, Evolutionary Analysis, and Machine Learning.

Thermostability is an essential requirement for the use of enzymes in the bioindustry. Here, we compare different protein stabilization strategies using a challenging target, a stable haloalkane dehalogenase DhaA115. We observe better performance of automated stabilization platforms FireProt and PROSS in designing multiple-point mutations over the introduction of disulfide bonds and strengthening the intra- and the inter-domain contacts by in silico saturation mutagenesis. We reveal that the performance of automated stabilization platforms was still compromised due to the introduction of some destabilizing mutations. Notably, we show that their prediction accuracy can be improved by applying manual curation or machine learning for the removal of potentially destabilizing mutations, yielding highly stable haloalkane dehalogenases with enhanced catalytic properties. A comparison of crystallographic structures revealed that current stabilization rounds were not accompanied by large backbone re-arrangements previously observed during the engineering stability of DhaA115. Stabilization was achieved by improving local contacts including protein-water interactions. Our study provides guidance for further improvement of automated structure-based computational tools for protein stabilization.

Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and x-ray crystallography.

Haloalkane dehalogenase (HLD) enzymes employ an SN 2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD-like enzyme from the archaeon Haloferax mediterranei, by combining cryo-electron microscopy (cryo-EM) and x-ray crystallography. We show that full-length wild-type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring-like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three-dimensional reconstructions of an oligomeric species by single-particle cryo-EM. Moreover, we engineered a crystallizable mutant (DhmeAΔGG ) that provided diffraction-quality crystals. The 3.3 Å crystal structure reveals that DhmeAΔGG forms a ring-like 20-mer structure with outer and inner diameter of ~200 and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization, and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates.

Influence of Structure Development on Performance of Copper Composites Processed via Intensive Plastic Deformation.

Designing a composite, possibly strengthened by a dispersion of (fine) oxides, is a favorable way to improve the mechanical characteristics of Cu while maintaining its advantageous electric conductivity. The aim of this study was to perform mechanical alloying of a Cu powder with a powder of Al2O3 oxide, seal the powder mixture into evacuated Cu tubular containers, i.e., cans, and apply gradual direct consolidation via rotary swaging at elevated temperatures, as well as at room temperature (final passes) to find the most convenient way to produce the designed Al2O3 particle-strengthened Cu composite. The composites swaged with the total swaging degree of 1.83 to consolidated rods with a diameter of 10 mm were subjected to measurements of electroconductivity, investigations of mechanical behavior via compression testing, and detailed microstructure observations. The results revealed that the applied swaging degree was sufficient to fully consolidate the canned powders, even at moderate and ambient temperatures. In other words, the final structures, featuring ultra-fine grains, did not exhibit voids or remnants of unconsolidated powder particles. The swaged composites featured favorable plasticity regardless of the selected processing route. The flow stress curves exhibited the establishment of steady states with increasing strain, regardless of the applied strain rate. The electroconductivity of the composite swaged at elevated temperatures, featuring homogeneous distribution of strengthening oxide particles and the average grain size of 1.8 µm2, reaching 80% IACS (International Annealed Copper Standard).

Domino-like effect of C112R mutation on ApoE4 aggregation and its reduction by Alzheimer's Disease drug candidate.

BACKGROUND: Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer's Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. METHODS: Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. RESULTS: We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. CONCLUSIONS: Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.

Study of Protein Conformational Dynamics Using Hydrogen/Deuterium Exchange Mass Spectrometry.

Intrinsic protein dynamics contribute to their biological functions. Rational engineering of protein dynamics is extremely challenging with only a handful of successful examples. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) represents a powerful technique for quantitative analysis of protein dynamics. Here we provide a detailed description of the preparation of protein samples, collection of high-quality data, and their in-depth analysis using various computational tools. We illustrate the application of HDX-MS for the study of protein dynamics in the rational engineering of flexible loops in the reconstructed ancestor of haloalkane dehalogenase and Renilla luciferase. These experiments provided unique and valuable data rigorously describing the modification of protein dynamics upon grafting of the loop-helix element. Tips and tricks are provided to stimulate the wider use of HDX-MS to study and engineer protein dynamics.

In cellulo and in vivo assays for compound testing against Trypanosoma cruzi.

Here, we describe a combined in cellulo and in vivo approach to identify compounds with higher potential for efficient inhibition of Trypanosoma cruzi. Phase I of in cellulo assays is designed to exclude inactive or toxic compounds, while phase II is designed for accurate IC50, CC50, and selective index (SI) determination. Compounds showing high SI are tested using in vivo infection models in parallel with benznidazole to assess their efficacy relative to a reference drug used for Chagas disease treatment. For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).1.

Hidden Potential of Highly Efficient and Widely Accessible Thrombolytic Staphylokinase.

Stroke burden is substantially increasing but current therapeutic drugs are still far from ideal. Here we highlight the vast potential of staphylokinase as an efficient, fibrin-selective, inexpensive, and evolvable thrombolytic agent. The emphasis is escalated by new recent findings. Staphylokinase nonimmunogenic variant was proven noninferior to alteplase in a clinical trial, with decreased risk of intracranial hemorrhage and the advantage of single bolus administration. Furthermore, our detailed kinetic analysis revealed a new staphylokinase limiting bottleneck whose elimination might provide up to 1000-fold higher activity than the clinically approved alteplase. This knowledge of limitations unlocks new possibilities for improvements that are now achievable by the community of protein engineers who have the required expertise and are ready to transform staphylokinase into a powerful molecule. Collectively, the noninferiority and safety of nonimmunogenic staphylokinase together with the newly identified effectivity limitation make staphylokinase a perfect candidate for further exploration, modification, and advancement to make it the next-generation widely accessible thrombolytic drug effectively treating stroke all around the world, including middle- and low-income countries.

Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling.

HaloTag labeling technology has introduced unrivaled potential in protein chemistry and molecular and cellular biology. A wide variety of ligands have been developed to meet the specific needs of diverse applications, but only a single protein tag, DhaAHT, is routinely used for their incorporation. Following a systematic kinetic and computational analysis of different reporters, a tetramethylrhodamine- and three 4-stilbazolium-based fluorescent ligands, we showed that the mechanism of incorporating different ligands depends both on the binding step and the efficiency of the chemical reaction. By studying the different haloalkane dehalogenases DhaA, LinB, and DmmA, we found that the architecture of the access tunnels is critical for the kinetics of both steps and the ligand specificity. We showed that highly efficient labeling with specific ligands is achievable with natural dehalogenases. We propose a simple protocol for selecting the optimal protein tag for a specific ligand from the wide pool of available enzymes with diverse access tunnel architectures. The application of this protocol eliminates the need for expensive and laborious protein engineering.

Increased occurrence of Treponema spp. and double-species infections in patients with Alzheimer's disease.

Although the link between microbial infections and Alzheimer's disease (AD) has been demonstrated in multiple studies, the involvement of pathogens in the development of AD remains unclear. Here, we investigated the frequency of the 10 most commonly cited viral (HSV-1, EBV, HHV-6, HHV-7, and CMV) and bacterial (Chlamydia pneumoniae, Helicobacter pylori, Borrelia burgdorferi, Porphyromonas gingivalis, and Treponema spp.) pathogens in serum, cerebrospinal fluid (CSF) and brain tissues of AD patients. We have used an in-house multiplex PCR kit for simultaneous detection of five bacterial and five viral pathogens in serum and CSF samples from 50 AD patients and 53 healthy controls (CTRL). We observed a significantly higher frequency rate of AD patients who tested positive for Treponema spp. compared to controls (AD: 62.2 %; CTRL: 30.3 %; p-value = 0.007). Furthermore, we confirmed a significantly higher occurrence of cases with two or more simultaneous infections in AD patients compared to controls (AD: 24 %; CTRL 7.5 %; p-value = 0.029). The studied pathogens were detected with comparable frequency in serum and CSF. In contrast, Borrelia burgdorferi, human herpesvirus 7, and human cytomegalovirus were not detected in any of the studied samples. This study provides further evidence of the association between microbial infections and AD and shows that paralleled analysis of multiple sample specimens provides complementary information and is advisable for future studies.

Efficient CRISPR-Cas9-mediated genome editing for characterization of essential genes in Trypanosoma cruzi.

This protocol outlines a new genetic complementation strategy to investigate gene function in Trypanosoma cruzi, the parasite causing Chagas disease. We combine CRISPR-Cas9 technology with recombination of variants of the target gene containing the desired mutations that are resistant to Cas9-cleavage, which enables detailed investigation of protein function. This experimental strategy overcomes some of the limitations associated with gene knockouts in T. cruzi. For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).

Computer-aided engineering of staphylokinase toward enhanced affinity and selectivity for plasmin.

Cardio- and cerebrovascular diseases are leading causes of death and disability, resulting in one of the highest socio-economic burdens of any disease type. The discovery of bacterial and human plasminogen activators and their use as thrombolytic drugs have revolutionized treatment of these pathologies. Fibrin-specific agents have an advantage over non-specific factors because of lower rates of deleterious side effects. Specifically, staphylokinase (SAK) is a pharmacologically attractive indirect plasminogen activator protein of bacterial origin that forms stoichiometric noncovalent complexes with plasmin, promoting the conversion of plasminogen into plasmin. Here we report a computer-assisted re-design of the molecular surface of SAK to increase its affinity for plasmin. A set of computationally designed SAK mutants was produced recombinantly and biochemically characterized. Screening revealed a pharmacologically interesting SAK mutant with ∼7-fold enhanced affinity toward plasmin, ∼10-fold improved plasmin selectivity and moderately higher plasmin-generating efficiency in vitro. Collectively, the results obtained provide a framework for SAK engineering using computational affinity-design that could pave the way to next-generation of effective, highly selective, and less toxic thrombolytics.

Characterising Correlations between Electric Conductivity and Structural Features in Rotary Swaged Al/Cu Laminated Conductors.

This study aims to characterize the correlations between electric characteristics and selected structural features of newly designed Al/Cu laminated conductors manufactured via room temperature rotary swaging. After swaging, the laminates with diameters of 15 mm were subjected to two different post-process annealing treatments. Structure analyses performed to evaluate the effects of thermomechanical processing were performed via scanning and transmission electron microscopies. Electric conductivity and resistivity of the laminates were experimentally measured and numerically simulated using models designed according to the real conditions. The results showed that the electric resistivity was affected by the grain size, bimodal grains' distribution (where observed), the presence of twins, and, last but not least, dislocation density. Among the influencing factors were the area fractions of Al and Cu at the cross-sections of the of the laminated conductors, too. The results revealed that fabrication of the laminate via the technology of rotary swaging introduced more advantageous combinations of electric and mechanical properties than fabrication by conventional manufacturing techniques. The lowest specific electric resistivity of 20.6 Ωm × 10-9 was measured for the laminated conductor subjected to the post-process annealing treatment at 350 °C, which imparted significant structure restoration (confirmed by the presence of fine, equiaxed, randomly oriented grains).

Species-selective targeting of pathogens revealed by the atypical structure and active site of Trypanosoma cruzi histone deacetylase DAC2.

Writing and erasing of posttranslational modifications are crucial to phenotypic plasticity and antigenic variation of eukaryotic pathogens. Targeting pathogens' modification machineries, thus, represents a valid approach to fighting parasitic diseases. However, identification of parasitic targets and the development of selective anti-parasitic drugs still represent major bottlenecks. Here, we show that the zinc-dependent histone deacetylases (HDACs) of the protozoan parasite Trypanosoma cruzi are key regulators that have significantly diverged from their human counterparts. Depletion of T. cruzi class I HDACs tcDAC1 and tcDAC2 compromises cell-cycle progression and division, leading to cell death. Notably, tcDAC2 displays a deacetylase activity essential to the parasite and shows major structural differences with human HDACs. Specifically, tcDAC2 harbors a modular active site with a unique subpocket targeted by inhibitors showing substantial anti-parasitic effects in cellulo and in vivo. Thus, the targeting of the many atypical HDACs in pathogens can enable anti-parasitic selective chemical impairment.

Analyses of Influence on Chromium Coating after Grinding from the View of Final Microstructure and Microhardness in the Surface Layer.

The article deals with the analysis of chromium layer grinding on a steel substrate, where this issue was addressed with regard to the requirements of the manufacturing sector, specifically in the aerospace industry. The experimental samples were chromium-plated and ground under different cutting conditions by the grooving method of grinding. Two types of grinding wheels for grinding were used, grinding wheel based on SG (solgel) a grinding wheel based on SiC. The resulting microstructure and microhardness in the machined layer were evaluated with using of confocal laser microscopy, inverted materials microscopy, and hardness testing. Based on the results, recommendations were made regarding a suitable approach to grinding the chromium coating. We used a confocal laser microscope and hardness tester for the evaluation of presented values. It was found that, on the base of analyses values, with both grinding wheel and using cutting conditions used, good results have been achieved. This could be stated, because the analysis of the samples microstructure after grinding for the given cutting conditions showed that it is possible that a small influence is completely acceptable from the point of the final product view and there are no major negative phenomena. Measurements of surface microhardness after grinding showed similar results for all samples. The SiC-based grinding wheel showed slightly better results, but both grinding wheels can be used without problems for the presented cutting conditions, and the presented cutting conditions with both grinding wheels can be recommended for the grinding of the given material.

Engineering the protein dynamics of an ancestral luciferase.

Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.

The structure of the mouse ADAT2/ADAT3 complex reveals the molecular basis for mammalian tRNA wobble adenosine-to-inosine deamination.

Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2.

Structures of hyperstable ancestral haloalkane dehalogenases show restricted conformational dynamics.

Ancestral sequence reconstruction is a powerful method for inferring ancestors of modern enzymes and for studying structure-function relationships of enzymes. We have previously applied this approach to haloalkane dehalogenases (HLDs) from the subfamily HLD-II and obtained thermodynamically highly stabilized enzymes (ΔT m up to 24 °C), showing improved catalytic properties. Here we combined crystallographic structural analysis and computational molecular dynamics simulations to gain insight into the mechanisms by which ancestral HLDs became more robust enzymes with novel catalytic properties. Reconstructed ancestors exhibited similar structure topology as their descendants with the exception of a few loop deviations. Strikingly, molecular dynamics simulations revealed restricted conformational dynamics of ancestral enzymes, which prefer a single state, in contrast to modern enzymes adopting two different conformational states. The restricted dynamics can potentially be linked to their exceptional stabilization. The study provides molecular insights into protein stabilization due to ancestral sequence reconstruction, which is becoming a widely used approach for obtaining robust protein catalysts.

Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family.

Engineering enzyme catalytic properties is important for basic research as well as for biotechnological applications. We have previously shown that the reshaping of enzyme access tunnels via the deletion of a short surface loop element may yield a haloalkane dehalogenase variant with markedly modified substrate specificity and enantioselectivity. Here, we conversely probed the effects of surface loop-helix transplantation from one enzyme to another within the enzyme family of haloalkane dehalogenases. Precisely, we transplanted a nine-residue long extension of L9 loop and α4 helix from DbjA into the corresponding site of DbeA. Biophysical characterization showed that this fragment transplantation did not affect the overall protein fold or oligomeric state, but lowered protein stability (ΔT m = -5 to 6 °C). Interestingly, the crystal structure of DbeA mutant revealed the unique structural features of enzyme access tunnels, which are known determinants of catalytic properties for this enzyme family. Biochemical data confirmed that insertion increased activity of DbeA with various halogenated substrates and altered its enantioselectivity with several linear ß-bromoalkanes. Our findings support a protein engineering strategy employing surface loop-helix transplantation for construction of novel protein catalysts with modified catalytic properties.