Search

Phosphatidylglycerol is a crucial phospholipid found ubiquitously in biological membranes of prokaryotic and eukaryotic cells. The phosphatidylglycerol phosphate (PGP) synthase (PgsA), a membrane-embedded enzyme, catalyzes the primary reaction of phosphatidylglycerol biosynthesis. Mutations in pgsA frequently correlate with daptomycin resistance in Staphylococcus aureus and other prevalent infectious pathogens. Here we report the crystal structures of S. aureus PgsA (SaPgsA) captured at two distinct states of the catalytic process, with lipid substrate (cytidine diphosphate-diacylglycerol, CDP-DAG) or product (PGP) bound to the active site within a trifurcated amphipathic cavity. The hydrophilic head groups of CDP-DAG and PGP occupy two different pockets in the cavity, inducing local conformational changes. An elongated membrane-exposed surface groove accommodates the fatty acyl chains of CDP-DAG/PGP and opens a lateral portal for lipid entry/release. Remarkably, the daptomycin resistance-related mutations mostly cluster around the active site, causing reduction of enzymatic activity. Our results provide detailed mechanistic insights into the dynamic catalytic process of PgsA and structural frameworks beneficial for development of antimicrobial agents targeting PgsA from pathogenic bacteria., Graphical abstract Image 1, Highlights • PgsA uses a trifurcated amphipathic cavity for binding of substrates or products. • Conversion of CDP-DAG to PGP induces local conformational changes in PgsA. • Daptomycin-resistant mutations of PgsA mostly lead to reduced catalytic activity. • A structure-based five-state model is proposed for the synthesis of PGP by PgsA.

An essential step for SARS‐CoV‐2 infection is the attachment to the host cell receptor by its Spike receptor‐binding domain (RBD). Most of the existing RBD‐targeting neutralizing antibodies block the receptor‐binding motif (RBM), a mutable region with the potential to generate neutralization escape mutants. Here, we isolated and structurally characterized a non‐RBM‐targeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a K D of 5.6 nM, neutralizes SARS‐CoV‐2 including the current Variants of Concern such as B.1.1.7, B.1.351, P.1, and B.1.617.2 (Delta), displays modest cross‐reactivity against SARS‐CoV, and reduces viral replication in hamsters. The epitope coincides with a predicted “ideal” vulnerability site with high functional and structural constraints. Mutation of the residues of the conserved epitope variably affects FD20‐binding but confers little or no resistance to neutralization. Finally, in vitro mode‐of‐action characterization and negative‐stain electron microscopy suggest a neutralization mechanism by which FD20 destructs the Spike. Our results reveal a conserved vulnerability site in the SARS‐CoV‐2 Spike for the development of potential antiviral drugs., A monoclonal antibody (FD20) from convalescent COVID‐19 patients has been isolated and structurally and biologically characterized. Various SARS‐CoV‐2 strains, including the Alpha, Beta, Gamma, and Delta variants, and naturally occurring epitope mutants, can be neutralized by FD20 with similar potency.

The evolutionarily conserved Hedgehog (Hh) signaling pathway is crucial for programmed cell differentiation and proliferation. Dispatched (Disp) is a 12-transmembrane protein that plays a critical role in the Hedgehog (Hh) signaling pathway by releasing the dually lipidated ligand HhN from the membrane, a prerequisite step to the downstream signaling cascade. In this study, we focus on the Disp from water bear, a primitive animal known as the most indestructible on Earth. Using a zebrafish model, we show that the water bear homolog possesses the function of Disp. We have solved its structure to a 6.5-Å resolution using single-particle cryogenic electron microscopy. Consistent with the evolutional conservation of the pathway, the water bear Disp structure is overall similar to the previously reported structures of the fruit fly and human homologs. Although not revealing much detail at this resolution, the water bear Disp shows a different conformation compared to published structures, suggesting that they represent different functional snapshots.

SARS-CoV-2 and its variants continue to threaten public health. The virus recognizes the host cell by attaching its Spike receptor-binding domain (RBD) to the host receptor ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here we report the isolation, and biological and structural characterization of two single-chain antibodies (nanobodies, DL4 and DL28) from RBD-immunized alpaca. Both nanobodies bind Spike with affinities that exceeded the detection limit (picomolar) of the biolayer interferometry assay and neutralize the original SARS-CoV- 2 strain with IC50of 0.086 μg mL-1(DL4) and 0.385 μg mL-1(DL28). DL4 and a more potent, rationally designed mutant, neutralizes the Alpha variant as potently as the original strain but only displays marginal activity against the Beta variant. By contrast, the neutralizing activity of DL28, when in the Fc-fused divalent form, was less affected by the mutations in the Beta variant (IC50of 0.414 μg mL-1for Alpha, 1.060 μg mL-1for Beta). Crystal structure studies reveal that DL4 blocks ACE2-binding by direct competition, while DL28 neutralizes SARS-CoV-2 by an uncommon mechanism through which DL28 distorts the receptor-binding motif in RBD and hence prevents ACE2-binding. Our work provides two neutralizing nanobodies for potential therapeutic development and reveals an uncommon mechanism to design and screen novel neutralizing antibodies against SARS-CoV-2.

SARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1–6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7–17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 μg mL−1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak., Here, the authors report the engineering, structural and biological characterization of synthetic nanobodies (sybodies) that display potent therapeutic activity against SARS-CoV-2 infection in animal models via targeting the virus receptor-binding domain.

Breast cancer is a heterogeneous disease. In particular, triple-negative breast cancer (TNBC) comprises various molecular subgroups with unclear identities and currently has few targeted treatment options. Our previous study identified protein C receptor (Procr) as a surface marker on mammary stem cells (MaSCs) located in the basal layer of the normal mammary gland. Given the possible connection of TNBC with basal layer stem cells, we conducted comparative analyses of Procr in breast cancers of mouse and human origin. In mouse mammary tumors, we showed that Procr(+) cells are enriched for cancer stem cells (CSCs) in Wnt1 basal-like tumors, but not in Brca1 basal-like tumors or PyVT luminal tumors. In human cancers, PROCR was robustly expressed in half of TNBC cases. Experiments with patient-derived xenografts (PDXs) revealed that PROCR marks CSCs in this discrete subgroup (referred to as PROCR(+) TNBC). Interfering with the function of PROCR using an inhibitory nanobody reduced the CSC numbers, arrested tumor growth and prevented rapid tumor recurrence. Our data suggest a key role of MaSC in breast tumorigenesis. Moreover, our work indicates that PROCR can be used as a biomarker to stratify TNBC into clinically relevant subgroups and may provide a novel targeted treatment strategy for this clinically important tumor subtype.

Phospholipid biosynthesis begins with the acylation of glycerol 3-phosphate (G3P). In most Gram-positive bacteria including many pathogens, a membrane protein called PlsY is the only acylt...

SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22-7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8-18. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 μg mL-1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL-1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22–7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8–18. Llama-derived single-domain antibodies (nanobodies, ∼15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 μg mL−1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL−1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

The membrane-integral glycerol 3-phosphate (G3P) acyltransferase PlsY catalyses the committed and essential step in bacterial phospholipid biosynthesis by acylation of G3P, forming lysophosphatidic acid. It contains no known acyltransferase motifs, lacks eukaryotic homologs, and uses the unusual acyl-phosphate as acyl donor, as opposed to acyl-CoA or acyl-carrier protein for other acyltransferases. Previous studies have identified several PlsY inhibitors as potential antimicrobials. Here we determine the crystal structure of PlsY at 1.48 Å resolution, revealing a seven-transmembrane helix fold. Four additional substrate- and product-bound structures uncover the atomic details of its relatively inflexible active site. Structure and mutagenesis suggest a different acylation mechanism of ‘substrate-assisted catalysis’ that, unlike other acyltransferases, does not require a proteinaceous catalytic base to complete. The structure data and a high-throughput enzymatic assay developed in this work should prove useful for virtual and experimental screening of inhibitors against this vital bacterial enzyme., The first step in bacterial phospholipid biosynthesis is the acylation of glycerol 3-phosphate to form lysophosphatidic acid. Here, the authors present the high resolution crystal structure of the glycerol 3-phosphate acyltransferase PlsY, a membrane protein and give insights into its catalytical mechanism.

After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ATP-binding cassette (ABC) transporter LptB2FG extracts LPS molecules from the IM and transports them to the outer membrane. Here we report the crystal structure of nucleotide-free LptB2FG from Pseudomonas aeruginosa. The structure reveals that lipopolysaccharide transport proteins LptF and LptG each contain a transmembrane domain (TMD), a periplasmic β-jellyroll-like domain and a coupling helix that interacts with LptB on the cytoplasmic side. The LptF and LptG TMDs form a large outward-facing V-shaped cavity in the IM. Mutational analyses suggest that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the β-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptB2FG that is distinct from those of classical ABC transporters that transport substrates across the IM.

Energy-coupling factor (ECF) transporters are a large family of ATP-binding cassette transporters recently identified in microorganisms. Responsible for micronutrient uptake from the environment, ECF transporters are modular transporters composed of a membrane substrate-binding component EcfS and an ECF module consisting of an integral membrane scaffold component EcfT and two cytoplasmic ATP binding/hydrolysis components EcfA/A'. ECF transporters are classified into groups I and II. Currently, the molecular understanding of group-I ECF transporters is very limited, partly due to a lack of transporter complex structural information. Here, we present structures and structure-based analyses of the group-I cobalt ECF transporter CbiMNQO, whose constituting subunits CbiM/CbiN, CbiQ, and CbiO correspond to the EcfS, EcfT, and EcfA components of group-II ECF transporters, respectively. Through reconstitution of different CbiMNQO subunits and determination of related ATPase and transporter activities, the substrate-binding subunit CbiM was found to stimulate CbiQO's basal ATPase activity. The structure of CbiMQO complex was determined in its inward-open conformation and that of CbiO in β, γ-methyleneadenosine 5'-triphosphate-bound closed conformation. Structure-based analyses revealed interactions between different components, substrate-gating function of the L1 loop of CbiM, and conformational changes of CbiO induced by ATP binding and product release within the CbiMNQO transporter complex. These findings enabled us to propose a working model of the CbiMNQO transporter, in which the transport process requires the rotation or toppling of both CbiQ and CbiM, and CbiN might function in coupling conformational changes between CbiQ and CbiM.

Membrane proteins are generally challenging to work with because of their notorious instability. Protein engineering has been used increasingly to thermostabilize labile membrane proteins such as G-protein coupled receptors for structural and functional studies in recent years. Two major strategies exist. Scanning mutagenesis systematically eliminates destabilizing residues, whereas the consensus approach assembles mutants with the most frequent residues among selected homologs, bridging sequence conservation with stability. Here we applied the consensus concept to stabilize a fungal homolog of the human sterol Δ8-7 isomerase, a 26.4-kDa protein with five transmembrane helices. The isomerase is also called emopamil binding protein (EBP) as it binds this anti-ischemic drug with high affinity. The wild-type had an apparent melting temperature (Tm) of 35.9 °C as measured by the fluorescence-detection size-exclusion chromatography (FSEC)-based thermostability assay. A total of 87 consensus mutations sourced from 22 homologs gained expression level and thermostability, increasing the apparent Tm to 69.9 °C at the cost of partial function loss. Assessing the stability and activity of several systematic chimeric constructs identified a construct with an apparent Tm of 79.8 °C and two regions for function rescue. Further back-mutations of the chimeric construct in the two target regions yielded the final construct with similar apparent activity to the wild-type and an elevated Tm of 88.8 °C, totaling an increase of 52.9 °C. The consensus approach was effective and efficient because it involves fewer constructs compared to scanning mutagenesis. Our results should encourage more use of the consensus strategy for membrane protein thermostabilization.

The mitochondrial calcium uniporter (MCU) plays a critical role in the mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multi-component system including the pore-forming subunit MCU and several regulators (MICU1, MICU2, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE.Dictyostelium discoideum(Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologs. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as oligomers, most probably as a pentamer or hexamer. Mutagenesis showed that the acidic residues Asp60, Glu72 and Glu74, which appeared to mediate the parallel interface as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex readily dissociated to lower-order oligomers in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency., Author summary SARS-CoV-2 relies on the receptor-binding domain (RBD) of its envelope Spike protein to recognize and infect host cells. RBD is therefore an immunological hotspot to generate antibodies for therapeutic and detection purposes. Binding affinity is one of the key characteristics of such antibodies. Here, we report a single-chain antibody (nanobody, ~14 kDa) that binds RBD with nanomolar affinity. The nanobody, dubbed SR31, binds RBD at an epitope distal to the receptor-binding motif (RBM) which is the target of most neutralizing antibodies. SR31 can therefore bind RBD in addition to RBM-binders, and increase their affinity and potency by avidity effects when used as a fusion partner. Compared to other in vitro affinity maturation techniques such as library screening and structure-based design, the fusion strategy offers advantages in speed and simplicity. In addition, SR31, together with RBD-targeting nanobodies recognizing a wide spectrum of epitopes, provides a useful toolkit to probe epitopes of uncharacterized antibodies by competitive binding.

The successful structure solution of the integral membrane diacylglycerol kinase and the CRISPR-associated endonuclease RNA–DNA complex by native SAD phasing is demonstrated. The structures were solved with a combined low-dose multi-orientation, multi-crystal data-collection strategy., Recent improvements in data-collection strategies have pushed the limits of native SAD (single-wavelength anomalous diffraction) phasing, a method that uses the weak anomalous signal of light elements naturally present in macromolecules. These involve the merging of multiple data sets from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose. Both approaches yield data of high multiplicity while minimizing radiation damage and systematic error, thus ensuring accurate measurements of the anomalous differences. Here, the combined use of these two strategies is described to solve cases of native SAD phasing that were particular challenges: the integral membrane diacylglycerol kinase (DgkA) with a low Bijvoet ratio of 1% and the large 200 kDa complex of the CRISPR-associated endonuclease (Cas9) bound to guide RNA and target DNA crystallized in the low-symmetry space group C2. The optimal native SAD data-collection strategy based on systematic measurements performed on the 266 kDa multiprotein/multiligand tubulin complex is discussed.

Phospholipids constitute a major and essential part of biomembranes and provide mechanical support for membrane proteins. As well, they participate in important cellular activities by interacting with membrane proteins and regulating their functions. In the past decades, enzymes responsible for phospholipid synthesis have been largely identified, purified and characterized, mostly using Escherichia coli and Bacillus subtilis as the model systems. Ever-increasing genome sequencing projects revealed similar enzymes in other microorganisms, as well as new enzymes and pathways for lipids that are traditionally regarded as eukaryotic lipids such as phosphatidylcholine and phosphatidylinositol. Enzymes involved in the phospholipid biosynthesis pathway are mostly associated with the membrane, either peripherally or as an integral part. Structural biology for the pathway had been lacking for these hydrophobic enzymes but are catching up owing to recent technological advances in the membrane protein structural biology field. Here, the biosynthesis pathway of bacterial phospholipids, starting from glycerol 3-phosphate, is summarized. Architecture of the enzymes and mechanisms for substrate binding and catalysis are also illustrated for those whose structural information are available.

The prokaryotic lysine-specific permease (LysP) belongs to the amino acid–polyamine–organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP fromPseudomonas aeruginosawas recombinantly expressed inEscherichia coliand purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space groupP21, with unit-cell parametersa= 169.53,b= 169.53,c= 290.13 Å, γ = 120°.

Recombinant expression of human membrane proteins in large quantities remains a major challenge. Expression host is an important variable to screen for high-level production of membrane proteins. Using the green fluorescent protein (GFP) as a reporter, we screened the expression of a human multi-pass membrane protein called sterol Δ8-Δ7 isomerase in three different hosts: Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. The expression of the His-tagged isomerase was exceptionally high in P. pastoris, reaching ~200 mg L−1 in standard flasks, and ~1,000 mg L−1 in condensed culture that mimics fermentation. The heterogeneously expressed isomerase could be extracted fully with dodecyl maltoside, and the solubilized protein in the form of GFP fusion showed a sharp and symmetric peak on fluorescence-detection size exclusion chromatography. Our work provides a useful source for the purification of the recombinant isomerase.

The conversion of 7-dehydrocholesterol to cholesterol, the final step of cholesterol synthesis in the Kandutsch-Russell pathway, is catalyzed by the enzyme 7-dehydrocholesterol reductase (DHCR7). Homozygous or compound heterozygous mutations in DHCR7 lead to the developmental disease Smith-Lemli-Opitz syndrome, which can also result in fetal mortality, highlighting the importance of this enzyme in human development and survival. Besides serving as a substrate for DHCR7, 7-dehydrocholesterol is also a precursor of vitamin D via the action of ultraviolet light on the skin. Thus, DHCR7 exerts complex biological effects, involved in both cholesterol and vitamin D production. Indeed, we argue that DHCR7 can act as a switch between cholesterol and vitamin D synthesis. This review summarizes current knowledge about the critical enzyme DHCR7, highlighting recent findings regarding its structure, transcriptional and post-transcriptional regulation, and its links to vitamin D synthesis. Greater understanding about DHCR7 function, regulation and its place within cellular metabolism will provide important insights into its biological roles.

Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution., Diacylglycerol kinase is a small bacterial membrane-bound trimer that catalyses diacylglycerol conversion to phosphatidic acid. Here, the authors solve the crystal structure of the kinase bound to a lipid substrate and an ATP analogue, and show that the active site arose through convergent evolution.

Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-A resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a similar to 20 degrees rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

This study presents the crystal structures of three functional forms of diacylglycerol kinase, an integral membrane protein that catalyses a crucial step in oligosaccharide and lipopolysaccharide synthesis and assembly; these X-ray structures are markedly different from the only other structure available for this unique kinase that was solved using solution NMR. The integral membrane enzyme diacylglycerol kinase is involved in the synthesis of periplasmic-membrane-derived oligosaccharide and outer-membrane lipopolysaccharide in Gram-negative bacteria. Containing only 121 amino acid residues, this is the smallest known kinase and it has become a model for the study membrane protein behaviour and enzymology. Martin Caffrey and colleagues report here the X-ray crystal structure of native diacylglycerol kinase and of two thermostable, functional mutants at high resolution. Of interest is the fact that the crystal structures are at variance with the only other published structure of diacylglycerol kinase, that solved using solution NMR. In an accompanying News & Views, Jimin Zheng and Zongchao Jia discuss the factors that may have contributed to the discrepancy between the NMR and crystal structures. Diacylglycerol kinase catalyses the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane-derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria1. For half a century, this 121-residue kinase has served as a model for investigating membrane protein enzymology1,2,3,4,5,6, folding7,8, assembly9,10,11,12 and stability1,13. Here we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type. These reveal a homo-trimeric enzyme with three transmembrane helices and an amino-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site that is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are, however, at variance with a published solution NMR model14 in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.