Nuclear Magnetic Resonance Structure Research Articles

EFG-CS: Predicting chemical shifts from amino acid sequences with protein structure prediction using machine learning and deep learning models.

Nuclear magnetic resonance (NMR) crystallography is one of the main methods in structural biology for analyzing protein stereochemistry and structure. The chemical shift of the resonance frequency reflects the effect of the protons in a molecule producing distinct NMR signals in different chemical environments. Apprehending chemical shifts from NMR signals can be challenging since having an NMR structure does not necessarily provide all the required chemical shift information, making predictive models essential for accurately deducing chemical shifts, either from protein structures or, more ideally, directly from amino acid sequences. Here, we present EFG-CS, a web server that specializes in chemical shift prediction. EFG-CS employs a machine learning-based transfer prediction model for backbone atom chemical shift prediction, using ESMFold-predicted protein structures. Additionally, ESG-CS incorporates a graph neural network-based model to provide comprehensive side-chain atom chemical shift predictions. Our method demonstrated reliable performance in backbone atom prediction, achieving comparable accuracy levels with root mean square errors (RMSE) of 0.30 ppm for H, 0.22 ppm for Hα, 0.89 ppm for C, 0.89 ppm for Cα, 0.84 ppm for Cβ, and 1.69 ppm for N. Moreover, our approach also showed predictive capabilities in side-chain atom chemical shift prediction achieving RMSE values of 0.71 ppm for Hβ, 0.74-1.15 ppm for Hδ, and 0.58-0.94 ppm for Hγ, solely utilizing amino acid sequences without homology or feature curation. This work shows for the first time that generative AI protein models can predict NMR shifts nearly comparable to experimental models. This web server is freely available at https://biosig.lab.uq.edu.au/efg_cs, and the chemical shift prediction results can be downloaded in tabular format and visualized in 3D format.

Unraveling Copper Exchange in the Atox1-Cu(I)-Mnk1 Heterodimer: A Simulation Approach.

Copper, an essential metal for various cellular processes, requires tight regulation to prevent cytotoxicity. Intracellular pathways crucial for maintaining optimal copper levels involve soluble and membrane transporters, namely, metallochaperones and P-type ATPases, respectively. In this study, we used a simulation workflow based on free-energy perturbation (FEP) theory and parallel bias metadynamics (PBMetaD) to predict the Cu(I) exchange mechanism between the human Cu(I) chaperone, Atox1, and one of its two physiological partners, ATP7A. ATP7A, also known as the Menkes disease protein, is a transmembrane protein and one of the main copper-transporting ATPases. It pumps copper into the trans-Golgi network for the maturation of cuproenzymes and is also essential for the efflux of excess copper across the plasma membrane. In this analysis, we utilized the nuclear magnetic resonance (NMR) structure of the Cu(I)-mediated complex between Atox1 and the first soluble domain of the Menkes protein (Mnk1) as a starting point. Independent free-energy simulations were conducted to investigate the dissociation of both Atox1 and Mnk1. The calculations revealed that the two dissociations require free energy values of 6.3 and 6.2 kcal/mol, respectively, following a stepwise dissociation mechanism.

Decoding the mechanism governing the structural stability of wheat germ agglutinin and its isolated domains: A combined calorimetric, NMR, and MD simulation study.

Wheat germ agglutinin (WGA) demonstrates potential as an oral delivery agent owing to its selective binding to carbohydrates and its capacity to traverse biological membranes. In this study, we employed differential scanning calorimetry and molecular dynamics simulations to comprehensively characterize the thermal unfolding process of both the complete lectin and its four isolated domains. Furthermore, we present the nuclear magnetic resonance structures of three domains that were previously lacking experimental structures in their isolated forms. Our results provide a collective understanding of the energetic and structural factors governing the intricate unfolding mechanism of the complete agglutinin, shedding light on the specific role played by each domain in this process. The analysis revealed negligible interdomain cooperativity, highlighting instead significant coupling between dimer dissociation and the unfolding of the more labile domains. By comparing the dominant interactions, we rationalized the stability differences among the domains. Understanding the structural stability of WGA opens avenues for enhanced drug delivery strategies, underscoring its potential as a promising carrier throughout the gastrointestinal environment.

Expansive discovery of chemically diverse structured macrocyclic oligoamides.

Small macrocycles with four or fewer amino acids are among the most potent natural products known, but there is currently no way to systematically generate such compounds. We describe a computational method for identifying ordered macrocycles composed of alpha, beta, gamma, and 17 other amino acid backbone chemistries, which we used to predict 14.9 million closed cycles composed of >42,000 monomer combinations. We chemically synthesized 18 macrocycles predicted to adopt single low-energy states and determined their x-ray or nuclear magnetic resonance structures; 15 of these were very close to the design models. We illustrate the therapeutic potential of these macrocycle designs by developing selective inhibitors of three protein targets of current interest. By opening up a vast space of readily synthesizable drug-like macrocycles, our results should considerably enhance structure-based drug design.

Experimental Investigation of the Impact of Blended Fibers on the Mechanical Properties and Microstructure of Aeolian Sand Concrete.

To investigate the effect of hybrid fibers on the compressive strength of aeolian sand concrete, compressive strength tests were conducted on aeolian sand concrete with single polypropylene fibers and aeolian sand concrete with mixed polypropylene fibers and calcium carbonate whisker, and their variation rules were studied. Using scanning electron microscopy and nuclear magnetic resonance, the microstructure and pore structure of specimens were analyzed, and a mathematical model of the relationship between compressive strength and pore structure was established with gray entropy analysis. The results show that the compressive strength of hybrid fiber aeolian sand concrete first increases and then decreases with an increase in whisker content. When the replacement rate of wind-accumulated sand is 80% and the fiber content is 0.1%, the optimal volume content of whisker is 0.4%, and the 28 d compressive strength of whisker is 24.8% higher than that of aeolian sand concrete. The average relative errors of compressive strength at 7 d and 28 d are 8.16% and 7.48%, respectively, using the GM (1,3) model. This study can provide effective theoretical support for the application of calcium carbonate whisker and polypropylene fibers in aeolian sand concrete.

Structural insight into the ZFAND1–p97 interaction involved in stress granule clearance

Arsenite-induced stress granule (SG) formation can be cleared by the ubiquitin-proteasome system aided by the ATP-dependent unfoldase p97. ZFAND1 participates in this pathway by recruiting p97 to trigger SG clearance. ZFAND1 contains two An1-type zinc finger domains (ZF1 and ZF2), followed by a ubiquitin-like domain (UBL); but their structures are not experimentally determined. To shed light on the structural basis of the ZFAND1-p97 interaction, we determined the atomic structures of the individual domains of ZFAND1 by solution-state nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. We further characterized the interaction between ZFAND1 and p97 by methyl NMR spectroscopy and cryo-electron microscopy (cryo-EM). 15N spin relaxation dynamics analysis indicated independent domain motions for ZF1, ZF2, and UBL. The crystal structure and NMR structure of UBL showed a conserved β-grasp fold homologus to ubiquitin and other UBLs. Nevertheless, the UBL of ZFAND1 contains an additional N-terminal helix that adopts different conformations in the crystalline and solution states. ZFAND1 uses the C-terminal UBL to bind to p97, evidenced by the pronounced line-broadening of the UBL domain during the p97 titration monitored by methyl NMR spectroscopy. ZFAND1 binding induces pronounced conformational heterogeneity in the N-terminal domain (NTD) of p97, leading to a partial loss of the cryo-EM density of the NTD of p97. In conclusion, this work paved the way for a better understanding of the interplay between p97 and ZFAND1 in the context of SG clearance.

A rationally designed antimicrobial peptide from structural and functional insights of Clostridioides difficile translation initiation factor 1.

A significant increase of hospital-acquired bacterial infections during the COVID-19 pandemic has become an urgent medical problem. Clostridioides difficile is an urgent antibiotic-resistant bacterial pathogen and a leading causative agent of nosocomial infections. The increasing recurrence of C. difficile infection and antibiotic resistance in C. difficile has led to an unmet need for the discovery of new compounds distinctly different from present antimicrobials, while antimicrobial peptides as promising alternatives to conventional antibiotics have attracted growing interest recently. Protein synthesis is an essential metabolic process in all bacteria and a validated antibiotic target. Initiation factor 1 from C. difficile (Cd-IF1) is the smallest of the three initiation factors that acts to establish the 30S initiation complex to initiate translation during protein biosynthesis. Here, we report the solution nuclear magnetic resonance (NMR) structure of Cd-IF1 which adopts a typical β-barrel fold and consists of a five-stranded β-sheet and one short α-helix arranged in the sequential order β1-β2-β3-α1-β4-β5. The interaction of Cd-IF1 with the 30S ribosomal subunit was studied by NMR titration for the construction of a structural model of Cd-IF1 binding with the 30S subunit. The short α-helix in IF1 was found to be critical for IF1 ribosomal binding. A peptide derived from this α-helix was tested and displayed a high ability to inhibit the growth of C. difficile and other bacterial strains. These results provide a clue for the rational design of new antimicrobials.IMPORTANCEBacterial infections continue to represent a major worldwide health hazard due to the emergence of drug-resistant strains. Clostridioides difficile is a common nosocomial pathogen and the causative agent in many infections resulting in an increase in morbidity and mortality. Bacterial protein synthesis is an essential metabolic process and an important target for antibiotic development; however, the precise structural mechanism underlying the process in C. difficile remains unknown. This study reports the solution structure of C. difficile translation initiation factor 1 (IF1) and its interaction with the 30S ribosomal subunit. A short α-helix in IF1 structure was identified as critically important for ribosomal binding and function in regulating the translation initiation, which allowed a rational design of a new peptide. The peptide demonstrated a high ability to inhibit bacterial growth with broad-spectrum antibacterial activity. This study provides a new clue for the rational design of new antimicrobials against bacterial infections.

High-Resolution NMR Structures of Intrastrand Hairpins Formed by CTG Trinucleotide Repeats.

The CAG and CTG trinucleotide repeat expansions cause more than 10 human neurodegenerative diseases. Intrastrand hairpins formed by trinucleotide repeats contribute to repeat expansions, establishing them as potential drug targets. High-resolution structural determination of CAG and CTG hairpins poses as a long-standing goal to aid drug development, yet it has not been realized due to the intrinsic conformational flexibility of repetitive sequences. We herein investigate the solution structures of CTG hairpins using nuclear magnetic resonance (NMR) spectroscopy and found that four CTG repeats with a clamping G-C base pair was able to form a stable hairpin structure. We determine the first solution NMR structure of dG(CTG)4C hairpin and decipher a type I folding geometry of the TGCT tetraloop, wherein the two thymine residues form a T·T loop-closing base pair and the first three loop residues continuously stack. We further reveal that the CTG hairpin can be bound and stabilized by a small-molecule ligand, and the binding interferes with replication of a DNA template containing CTG repeats. Our determined high-resolution structures lay an important foundation for studying molecular interactions between native CTG hairpins and ligands, and benefit drug development for trinucleotide repeat expansion diseases.

Structural investigation of pathogenic RFC1 AAGGG pentanucleotide repeats reveals a role of G-quadruplex in dysregulated gene expression in CANVAS.

An expansion of AAGGG pentanucleotide repeats in the replication factor C subunit 1 (RFC1) gene is the genetic cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), and it also links to several other neurodegenerative diseases including the Parkinson's disease. However, the pathogenic mechanism of RFC1 AAGGG repeat expansion remains enigmatic. Here, we report that the pathogenic RFC1 AAGGG repeats form DNA and RNA parallel G-quadruplex (G4) structures that play a role in impairing biological processes. We determine the first high-resolution nuclear magnetic resonance (NMR) structure of a bimolecular parallel G4 formed by d(AAGGG)2AA and reveal how AAGGG repeats fold into a higher-order structure composed of three G-tetrad layers, and further demonstrate the formation of intramolecular G4s in longer DNA and RNA repeats. The pathogenic AAGGG repeats, but not the nonpathogenic AAAAG repeats, form G4 structures to stall DNA replication and reduce gene expression via impairing the translation process in a repeat-length-dependent manner. Our results provide an unprecedented structural basis for understanding the pathogenic mechanism of AAGGG repeat expansion associated with CANVAS. In addition, the high-resolution structures resolved in this study will facilitate rational design of small-molecule ligands and helicases targeting G4s formed by AAGGG repeats for therapeutic interventions.

Antiviral peptides inhibiting the main protease of SARS-CoV-2 investigated by computational screening and in vitro protease assay.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in viral replication and transcription and received great attention as a vital target for drug/peptide development. Therapeutic agents such as small-molecule drugs or peptides that interact with the Cys-His present in the catalytic site of Mpro are an efficient way to inhibit the protease. Although several emergency-approved vaccines showed good efficacy and drastically dropped the infection rate, evolving variants are still infecting and killing millions of people globally. While a small-molecule drug (Paxlovid) received emergency approval, small-molecule drugs have low target specificity and higher toxicity. Besides small-molecule drugs, peptide therapeutics are thus gaining increasing popularity as they are easy to synthesize and highly selective and have limited side effects. In this study, we investigated the therapeutic value of 67 peptides targeting Mpro using molecular docking. Subsequently, molecular dynamics (MD) simulations were implemented on eight protein-peptide complexes to obtain molecular-level information on the interaction between these peptides and the Mpro active site, which revealed that temporin L, indolicidin, and lymphocytic choriomeningitis virus (LCMV) GP1 are the best candidates in terms of stability, interaction, and structural compactness. These peptides were synthesized using the solid-phase peptide synthesis protocol, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and authenticated by mass spectrometry (MS). The in vitro fluorometric Mpro activity assay was used to validate the computational results, where temporin L and indolicidin were observed to be very active against SARS-CoV-2 Mpro with IC50 values of 38.80 and 87.23 μM, respectively. A liquid chromatography-MS (LC-MS) assay was developed, and the IC50 value of temporin L was measured at 23.8 μM. The solution-state nuclear magnetic resonance (NMR) structure of temporin L was determined in the absence of sodium dodecyl sulfate (SDS) micelles and was compared to previous temporin structures. This combined investigation provides critical insights and assists us to further develop peptide inhibitors of SARS-CoV-2 Mpro through structural guided investigation.

Structural basis of metabolite transport by the chloroplast outer envelope channel OEP21

Triose phosphates (TPs) are the primary products of photosynthetic CO2 fixation in chloroplasts, which need to be exported into the cytosol across the chloroplast inner envelope (IE) and outer envelope (OE) membranes to sustain plant growth. While transport across the IE is well understood, the mode of action of the transporters in the OE remains unclear. Here we present the high-resolution nuclear magnetic resonance (NMR) structure of the outer envelope protein 21 (OEP21) from garden pea, the main exit pore for TPs in C3 plants. OEP21 is a cone-shaped β-barrel pore with a highly positively charged interior that enables binding and translocation of negatively charged metabolites in a competitive manner, up to a size of ~1 kDa. ATP stabilizes the channel and keeps it in an open state. Despite the broad substrate selectivity of OEP21, these results suggest that control of metabolite transport across the OE might be possible.

Periodicity of the Affinity of Lanmodulin for Trivalent Lanthanides and Actinides: Structural and Electronic Insights from Quantum Chemical Calculations.

Lanmodulin (LanM) is the first identified macrochelator that has naturally evolved to sequester ions of rare earth elements (REEs) such as Y and all lanthanides, reversibly. This natural protein showed a 106 times better affinity for lanthanide cations than for Ca, which is a naturally abundant and biologically relevant element. Recent experiments have shown that its metal ion binding activity can be further extended to some actinides, like Np, Pu, and Am. For this reason, it was thought that LanM could be adopted for the separation of REE ions and actinides, thus increasing the interest in its potential use for industry-oriented applications. In this work, a systematic study of the affinity of LanM for lanthanides and actinides has been carried out, taking into account all trivalent ions belonging to the 4f (from La to Lu) and 5f (from Ac to Lr) series, starting from their chemistry in solution. On the basis of a recently published nuclear magnetic resonance structure, a model of the LanM-binding site was built and a detailed structural and electronic description of initial aquo- and LanM-metal ion complexes was provided. The obtained binding energies are in agreement with the available experimental data. A possible reason that could explain the origin of the affinity of LanM for these metal ions is also discussed.

Solution NMR structure of cementum protein 1 derived peptide (CEMP1-p1) and its role in the mineralization process.

We report the characterization of the three-dimensional structure of the CEMP1-p1 peptide [MGTSSTDSQQAQHRRCSTSN: corresponding to residues 1-20 of the N-terminus of cementum protein 1 (CEMP1)]. This peptide imitates the capacity of CEMP1 to stimulate hydroxyapatite (HA) crystal nucleation and growth, and promotes the differentiation of periodontal ligament cells into a cementoblastic phenotype. Additionally, in experimental models of critical-sized calvarial defects in Wistar rats, CEMP1-p1 has shown osteogenic properties that enhanced the physiological deposition and maturation of newly formed bone. In this work, studies of CEMP1-p1 by circular dichroism (CD) and nuclear magnetic resonance (NMR) were performed in trifluoroethanol D2 (TFED2) and aqueous solution to determine the 3D structure of the peptide. Using the 3D model, experimental data from HA crystals formation and calcium fluorescence emission, we explain the biological mechanisms involved in CEMP1-p1 activity to promote calcium recruitment and its affinity to HA crystals. This information is valuable because it proposes, for the first time, a plausible molecular mechanism during the mineralization process, from a specific cementum protein-derived peptide.

Recent Advances in NMR Protein Structure Prediction with ROSETTA.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful method for studying the structure and dynamics of proteins in their native state. For high-resolution NMR structure determination, the collection of a rich restraint dataset is necessary. This can be difficult to achieve for proteins with high molecular weight or a complex architecture. Computational modeling techniques can complement sparse NMR datasets (<1 restraint per residue) with additional structural information to elucidate protein structures in these difficult cases. The Rosetta software for protein structure modeling and design is used by structural biologists for structure determination tasks in which limited experimental data is available. This review gives an overview of the computational protocols available in the Rosetta framework for modeling protein structures from NMR data. We explain the computational algorithms used for the integration of different NMR data types in Rosetta. We also highlight new developments, including modeling tools for data from paramagnetic NMR and hydrogen-deuterium exchange, as well as chemical shifts in CS-Rosetta. Furthermore, strategies are discussed to complement and improve structure predictions made by the current state-of-the-art AlphaFold2 program using NMR-guided Rosetta modeling.

Transmembrane dimers of type 1 receptors sample alternate configurations: MD simulations using coarse grain Martini 3 versus AlphaFold2 Multimer

Structures and dynamics of transmembrane (TM) receptor regions are key to understanding their signaling mechanism across membranes. Here we examine configurations of TM region dimers, assembled using the recent Martini 3 force field for coarse-grain (CG) molecular dynamics simulations. At first glance, our results show only a reasonable agreement with ab initio predictions using PREDDIMER and AlphaFold2 Multimer and with nuclear magnetic resonance (NMR)-derived structures. 5 of 11 CG TM structures are similar to the NMR structures (within <3.5Å root-mean-square deviation [RMSD]) compared with 10 and 9 using PREDDIMER and AlphaFold2, respectively (with 8 structures of the later within 1.5Å). Surprisingly, AlphaFold2 predictions are closer to NMR structures when the 2001 instead of 2020 database is used for training. The CG simulations reveal that alternative configurations of TM dimers readily interconvert with a predominant population. The implications for transmembrane signaling are discussed, including for the development of peptide-based pharmaceuticals.

Conformation and Orientation of Antimicrobial Peptides MSI-594 and MSI-594A in a Lipid Membrane.

There is significant interest in the development of antimicrobial compounds to overcome the increasing bacterial resistance to conventional antibiotics. Studies have shown that naturally occurring and de novo-designed antimicrobial peptides could be promising candidates. MSI-594 is a synthetic linear, cationic peptide that has been reported to exhibit a broad spectrum of antimicrobial activities. Investigation into how MSI-594 disrupts the cell membrane is important for better understanding the details of this antimicrobial peptide (AMP)'s action against bacterial cells. In this study, we used two different synthetic lipid bilayers: zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 7:3 POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (POPG). Sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) were used to determine the orientations of MSI-594 and its analogue MSI-594A associated with zwitterionic POPC and anionic 7:3 POPC/POPG lipid bilayers. The simulated ATR-FTIR and SFG spectra using nuclear magnetic resonance (NMR)-determined structures were compared with experimental spectra to optimize the bent angle between the N- (1-11) and C- (12-24) termini helices and the membrane orientations of the helices; since the NMR structure of the peptide was determined from lipopolysaccharide (LPS) micelles, the optimization was needed to find the most suitable conformation and orientation in lipid bilayers. The reported experimental results indicate that the optimized MSI-594 helical hairpin structure adopts a complete lipid bilayer surface-bound orientation (denoted "face-on") in both POPC and 7:3 POPC/POPG lipid bilayers. The analogue peptide, MSI-584A, on the other hand, exhibited a larger bent angle between the N- (1-11) and C- (12-24) termini helices with the hydrophobic C-terminal helix inserted into the hydrophobic region of the bilayer (denoted "membrane-inserted") when interacting with both POPC and 7:3 POPC/POPG lipid bilayers. These experimental findings on the membrane orientations suggest that both peptides are likely to disrupt the cell membrane through the carpet mechanism.

Abstract 3842: Berberine recognition of the dGMP-bound PDGFR-β promoter G-quadruplex: Structural insight into specific drug targeting

Abstract PDGFR-β (Platelet-Derived Growth Factor Receptor beta) is a receptor tyrosine kinase whose overexpression drives the progression and metastasis of many cancers. It is an established anticancer target and PDGFR-β inhibition via kinase inhibitors has been actively sought-after. However, PDGFR-β kinase inhibitors often lack specificity. The PDGFR-β gene promoter forms DNA G-quadruplexes that are transcription silencer whose stabilization by small molecules represses PDGFR-β expression. Therefore, targeting PDGFR-β promoter G-quadruplexes for transcriptional silencing is a promising alternative strategy for cancer therapy. G-quadruplex is an exciting family of non-canonical DNA secondary structures. DNA G-quadruplex within the PDGFR-β gene promoter forms novel vacancy pockets (vG4) that can be filled-in by guanine metabolites such as dGMP and cGMP. This presents an attractive target for small molecule targeting. Herein, we present the binding of the natural product berberine with the unique PDGFR-β dGMP-vG4 binary complex to form a novel ternary complex, the first case observed in DNA G-quadruplexes. Using circular dichroism, fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, we demonstrated that berberine binds with high affinity and strongly stabilizes the dGMP-fill-in PDGFR-β vG4. Our determined high-resolution NMR structure of this ternary complex in K+-containing solution reveals a 2:1 binding stoichiometry of berberine to the dGMP-fill-in PDGFR-β vG4. Significantly, one berberine directly stacks upon and extensively covers the dGMP. This direct binding mode provides structural basis for targeting the unique PDGFR-β promoter G-quadruplex by small molecules and suggests an intriguing strategy of designing drug-like, guanine-conjugated molecules to selectively silence the PDGFR-β gene. Because vG4 formation and dGMP fill-in is a unique property of the PDGFR-β promoter G-quadruplex, our study provides a structural basis for rationally designing small molecule drugs to specifically target the PDGFR-β promoter G-quadruplex to inhibit oncogenic PDGFR-β for cancer therapeutics. Citation Format: Kai-Bo Wang, Jonathan Dickerhoff, Yichen Han, Danzhou Yang. Berberine recognition of the dGMP-bound PDGFR-β promoter G-quadruplex: Structural insight into specific drug targeting. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3842.

Discovery and Rational Mutagenesis of Methionine Sulfoxide Reductase Biocatalysts To Expand the Substrate Scope of the Kinetic Resolution of Chiral Sulfoxides.

Methionine sulfoxide reductase A (MsrA) enzymes have recently found applications as nonoxidative biocatalysts in the enantioselective kinetic resolution of racemic sulfoxides. This work describes the identification of selective and robust MsrA biocatalysts able to catalyze the enantioselective reduction of a variety of aromatic and aliphatic chiral sulfoxides at 8-64 mM concentration with high yields and excellent ees (up to 99%). Moreover, with the aim to expand the substrate scope of MsrA biocatalysts, a library of mutant enzymes has been designed via rational mutagenesis utilizing in silico docking, molecular dynamics, and structural nuclear magnetic resonance (NMR) studies. The mutant enzyme MsrA33 was found to catalyze the kinetic resolution of bulky sulfoxide substrates bearing non-methyl substituents on the sulfur atom with ees up to 99%, overcoming a significant limitation of the currently available MsrA biocatalysts.

Pmu1a, a novel spider toxin with dual inhibitory activity at pain targets hNaV 1.7 and hCaV 3 voltage-gated channels.

Venom-derived peptides targeting ion channels involved in pain are regarded as a promising alternative to current, and often ineffective, chronic pain treatments. Many peptide toxins are known to specifically and potently block established therapeutic targets, among which the voltage-gated sodium and calcium channels are major contributors. Here, we report on the discovery and characterization of a novel spider toxin isolated from the crude venom of Pterinochilus murinus that shows inhibitory activity at both hNaV 1.7 and hCaV 3.2 channels, two therapeutic targets implicated in pain pathways. Bioassay-guided HPLC fractionation revealed a 36-amino acid peptide with three disulfide bridges named μ/ω-theraphotoxin-Pmu1a (Pmu1a). Following isolation and characterization, the toxin was chemically synthesized, and its biological activity was further assessed using electrophysiology, revealing Pmu1a to be a toxin that potently blocks both hNaV 1.7 and hCaV 3. NMR structure determination of Pmu1a shows an inhibitor cystine knot (ICK) fold that is characteristic of many spider peptides. Combined, these data show the potential of Pmu1a as a basis for the design of compounds with dual activity at the therapeutically relevant hCaV 3.2 and hNaV 1.7 voltage-gated channels.

New TSPO Crystal Structures of Mutant and Heme-Bound Forms with Altered Flexibility, Ligand Binding, and Porphyrin Degradation Activity.

The ancient protein TSPO (translocator protein 18kD) is found in all kingdoms and was originally identified as a binding site of benzodiazepine drugs. Its physiological function remains unclear, although porphyrins are conserved ligands. Several crystal structures of bacterial TSPO and nuclear magnetic resonance structures of a mouse form have revealed monomer and dimer configurations, but there have been no reports of structures with a physiological ligand. Here, we present the first X-ray structures of Rhodobacter sphaeroides TSPO with a physiological ligand bound. Two different variants (substituting threonine for alanine at position 139 (A139T) and phenylalanine for alanine at position 138 (A138F)) yielded well-diffracting crystals giving structures of both apo- and heme-containing forms. Both variants have wild-type micromolar affinity for heme and protoporphyrin IX, but A139T has very low ability to accelerate the breakdown of porphyrin in the presence of light and oxygen. The binding of heme to one protomer of the dimer of either mutant induces a more rigid structure, both in the heme-binding protomer and the protomer without heme bound, demonstrating an allosteric response. Ensemble refinement of the X-ray data reveals distinct regions of altered flexibility in response to single heme binding to the dimer. The A139T variant shows a more rigid structure overall, which may relate to extra hydrogen bonding of waters captured in the heme crevice. As TSPO has been suggested to have a role in heme delivery from mitochondria to the cytoplasm, the new structures provide potential clues regarding the structural basis of such activity.