Structural basis of human NOX5 activation.

NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which - in particular, how calcium triggers electron transfer from NADPH to FAD - is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.

Unveiling the structure-emulsifying function relationship of truncated recombinant forms of the SA01-OmpA protein opens up a new vista in bioemulsifiers.

The emulsifying ability of SA01-OmpA (outer membrane protein A from Acinetobacter sp. SA01) was found to be constrained by challenges like low production efficiency and high costs associated with protein recovery from E. coli inclusion bodies, as described in our previous study. The present study sought to benefit from the advantages of the targeted truncating of SA01-OmpA protein, taking into account the reduced propensity of protein expression as inclusion bodies and cytotoxicity. Here, the structure and activity relationship of two truncated recombinant forms of SA01-OmpA protein was unraveled through a hybrid approach based on experimental data and computational methodologies, representing an innovative bioemulsifier with advantageous emulsifying activity. The recombinant truncated SA01-OmpA variants were cloned and heterologously expressed in E. coli host cells and subsequently purified. The results showed increased emulsifying activity of N-terminally truncated SA01-OmpA (NT-OmpA) compared to full-length SA01-OmpA. Molecular dynamics (MD) simulations analysis demonstrated a direct correlation between the C-terminally truncated SA01-OmpA (CT-OmpA) and its expression as inclusion bodies. Analysis of the structure-activity relationship of truncated variants of SA01-OmpA revealed that, compared to the full-length protein, deletion of the ß-barrel portion from the N-terminal of SA01-OmpA increased the emulsifying activity of NT-OmpA while lowering its expression as inclusion bodies. Contrary to the full-length protein, the N-terminally truncated SA01-OmpA was not as cytotoxic, according to the MTT assay, FCM analysis, and AO/EB staining. The findings of this extensive study advance our knowledge of SA01-OmpA at the molecular level as well as the design and development of efficient bioemulsifiers.IMPORTANCEPrevious research (Shahryari et al. 2021, mSystems 6: e01175-20) introduced and characterized the SA01-OmpA protein as a multifaceted protein with a variety of functions, including maintaining cellular homeostasis under oxidative stress conditions, biofilm formation, outer membrane vesicles (OMV) biogenesis, and beneficial emulsifying capacity. By truncating the SA01-OmpA protein, the current study presents a unique method for developing protein-type bioemulsifiers. The findings indicate that the N-terminally truncated SA01-OmpA (NT-OmpA) has the potential to fully replace full-length SA01-OmpA as a novel bioemulsifier with significant emulsifying activity. This study opens up a new frontier in bioemulsifiers, shedding light on a possible relationship between the structure and activity of SA01-OmpA truncated forms.

GproteinDb in 2024: new G protein-GPCR couplings, AlphaFold2-multimer models and interface interactions.

G proteins are the major signal proteins of ∼800 receptors for medicines, hormones, neurotransmitters, tastants and odorants. GproteinDb offers integrated genomic, structural, and pharmacological data and tools for analysis, visualization and experiment design. Here, we present the first major update of GproteinDb greatly expanding its coupling data and structural templates, adding AlphaFold2 structure models of GPCR-G protein complexes and advancing the interactive analysis tools for their interfaces underlying coupling selectivity. We present insights on coupling agreement across datasets and parameters, including constitutive activity, agonist-induced activity and kinetics. GproteinDb is accessible at https://gproteindb.org.

Applications of fluorescent protein tagging in structural studies of membrane proteins.

Generating active, pure, and monodisperse protein remains a major bottleneck for structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM). The current methodology heavily relies on overexpressing the recombinant protein fused with a histidine tag in conventional expression systems and evaluating the quality and stability of purified protein using size exclusion chromatography (SEC). This requires a large amount of protein and can be highly laborious and time consuming. Therefore, this approach is not suitable for high-throughput screening and low-expressing macromolecules, particularly eukaryotic membrane proteins. Using fluorescent proteins fused to the target protein (applicable to both soluble and membrane proteins) enables rapid and efficient screening of expression level and monodispersity of tens of unpurified constructs using fluorescence-based size exclusion chromatography (FSEC). Moreover, FSEC proves valuable for screening multiple detergents to identify the most stabilizing agent in the case of membrane proteins. Additionally, FSEC can facilitate nanodisc reconstitution by determining the optimal ratio of membrane scaffold protein (MSP), lipids, and target protein. The distinct advantages offered by FSEC indicate that fluorescent proteins can serve as a viable alternative to commonly used affinity tags for both characterization and purification purposes. In this review, I will summarize the advantages of this technique using examples from my own work. It should be noted that this article is not intended to provide an exhaustive review of all available literature, but rather to offer representative examples of FSEC applications.

GPCRdb in 2023: state-specific structure models using AlphaFold2 and new ligand resources.

G protein-coupled receptors (GPCRs) are physiologically abundant signaling hubs routing hundreds of extracellular signal substances and drugs into intracellular pathways. The GPCR database, GPCRdb supports >5000 interdisciplinary researchers every month with reference data, analysis, visualization, experiment design and dissemination. Here, we present our fifth major GPCRdb release setting out with an overview of the many resources for receptor sequences, structures, and ligands. This includes recently published additions of class D generic residue numbers, a comparative structure analysis tool to identify functional determinants, trees clustering GPCR structures by 3D conformation, and mutations stabilizing inactive/active states. We provide new state-specific structure models of all human non-olfactory GPCRs built using AlphaFold2-MultiState. We also provide a new resource of endogenous ligands along with a larger number of surrogate ligands with bioactivity, vendor, and physiochemical descriptor data. The one-stop-shop ligand resources integrate ligands/data from the ChEMBL, Guide to Pharmacology, PDSP Ki and PubChem database. The GPCRdb is available at https://gpcrdb.org.

Outer membrane-anchoring enables LpoB to regulate peptidoglycan synthesis rate.

Peptidoglycan (PG) is an essential component of the cell envelope in most bacteria, responsible for maintaining the shape of the cell and protecting the cell from environmental stresses. The growth of the PG layer during cell elongation and division is facilitated by the coordinated activities of PG synthases and hydrolases. PG synthases are regulated from inside the cell by components of the elongasome and divisome complexes driven by the cytoskeletal proteins MreB and FtsZ. In Escherichia coli the PG synthases PBP1A and PBP1B require the activation by outer membrane (OM)-anchored lipoproteins LpoA and LpoB, respectively. These have an elongated structure and are capable to span the periplasm to reach their cognate, cytoplasmic membrane (CM)-anchored PG synthase through the PG layer. Presumably, the Lpo proteins activate the PBPs at sites where the PG mesh is stretched or defective, resulting in coupling of PG synthase activation with cell growth or PG repair. Here we investigated the importance of OM-anchoring on the function of Lpo proteins in regulating PG synthesis in response to environmental stresses. We investigated the effects of an artificially CM-tethered LpoB on cell morphology and PG synthesis. Our results indicate that mis-localization of LpoB affects the growth and morphology of cells in high osmolarity growth medium, and PG synthesis rate upon an osmotic upshift.

Crystal structures of bacterial small multidrug resistance transporter EmrE in complex with structurally diverse substrates.

Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here, we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.

A guide to membrane protein X-ray crystallography.

Membrane proteins play critical physiological roles in all organisms, from ion transport and signal transduction to multidrug resistance. Elucidating their 3D structures is essential for understanding their functions, and this information can also be exploited for structure-aided drug discovery efforts. In this regard, X-ray crystallography has been the most widely used technique for determining the high-resolution 3D structures of membrane proteins. However, the success of this technique is dependent on efficient protein extraction, solubilization, stabilization, and generating diffracting crystals. Each of these steps can impose great challenges for membrane protein crystallographers. In this review, the process of generating membrane protein crystals from protein extraction and solubilization to structure determination is discussed. In addition, the current methods for precrystallization screening and a few strategies to increase the chance of crystallizing challenging membrane proteins are introduced.

The structural basis of promiscuity in small multidrug resistance transporters.

By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported membrane electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family's extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the membrane for the hydrophobic substituents of transported drug-like cations.

Membrane Protein Production in Escherichia coli.

Escherichia coli is the workhorse of the structural biology lab. In addition to routine cloning and molecular biology, E. coli can be used as a factory for the production of recombinant membrane proteins. Purification of homogeneous samples of membrane protein expressed in E. coli is a significant bottleneck for researchers, and the protocol we present here for the overexpression and purification of membrane proteins in E. coli will provide a solid basis to develop lab- and protein-specific protocols for your membrane protein of interest. We additionally provide extensive notes on the purification process, as well as the theory surrounding principles of purification.

Crystal structure of the TreS:Pep2 complex, initiating α-glucan synthesis in the GlgE pathway of mycobacteria.

A growing body of evidence implicates the mycobacterial capsule, the outermost layer of the mycobacterial cell envelope, in modulation of the host immune response and virulence of mycobacteria. Mycobacteria synthesize the dominant capsule component, α(1→4)-linked glucan, via three interconnected and potentially redundant metabolic pathways. Here, we report the crystal structure of the Mycobacterium smegmatis TreS:Pep2 complex, containing trehalose synthase (TreS) and maltokinase (Pep2), which converts trehalose to maltose 1-phosphate as part of the TreS:Pep2-GlgE pathway. The structure, at 3.6 Å resolution, revealed that a diamond-shaped TreS tetramer forms the core of the complex and that pairs of Pep2 monomers bind to opposite apices of the tetramer in a 4 + 4 configuration. However, for the M. smegmatis orthologues, results from isothermal titration calorimetry and analytical ultracentrifugation experiments indicated that the prevalent stoichiometry in solution is 4 TreS + 2 Pep2 protomers. The observed discrepancy between the crystallized complex and the behavior in the solution state may be explained by the relatively weak affinity of Pep2 for TreS (Kd 3.5 µm at mildly acidic pH) and crystal packing favoring the 4 + 4 complex. Proximity of the ATP-binding site in Pep2 to the complex interface provides a rational basis for rate enhancement of Pep2 upon binding to TreS, but the complex structure appears to rule out substrate channeling between the active sites of TreS and Pep2. Our findings provide a structural model for the trehalose synthase:maltokinase complex in M. smegmatis that offers critical insights into capsule assembly.

Guanidinium export is the primal function of SMR family transporters.

The small multidrug resistance (SMR) family of membrane proteins is prominent because of its rare dual topology architecture, simplicity, and small size. Its best studied member, EmrE, is an important model system in several fields related to membrane protein biology, from evolution to mechanism. But despite decades of work on these multidrug transporters, the native function of the SMR family has remained a mystery, and many highly similar SMR homologs do not transport drugs at all. Here we establish that representative SMR proteins, selected from each of the major clades in the phylogeny, function as guanidinium ion exporters. Drug-exporting SMRs are all clustered in a single minority clade. Using membrane transport experiments, we show that these guanidinium exporters, which we term Gdx, are very selective for guanidinium and strictly and stoichiometrically couple its export with the import of two protons. These findings draw important mechanistic distinctions with the notably promiscuous and weakly coupled drug exporters like EmrE.