Synthesis of lipid-linked precursors of the bacterial cell wall is governed by a feedback control mechanism in Pseudomonas aeruginosa.

Many bacterial surface glycans such as the peptidoglycan (PG) cell wall are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting carrier is distributed among competing pathways has remained unclear. Here we describe the isolation of hyperactive variants of Pseudomonas aeruginosa MraY, the enzyme that forms the first lipid-linked PG precursor. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in vitro. The activated MraY variants have substitutions that map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural and molecular dynamics results suggest that this cavity is a binding site for externalized lipid II. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism that prevents the sequestration of lipid carrier in the PG biogenesis pathway.

A feedback control mechanism governs the synthesis of lipid-linked precursors of the bacterial cell wall.

Many bacterial surface glycans such as the peptidoglycan (PG) cell wall, O-antigens, and capsules are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting lipid carrier is effectively distributed among competing pathways has remained unclear for some time. Here, we describe the isolation and characterization of hyperactive variants of Pseudomonas aeruginosa MraY, the essential and conserved enzyme catalyzing the formation of the first lipid-linked PG precursor called lipid I. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in a purified system. Amino acid substitutions within the activated MraY variants unexpectedly map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural evidence and molecular dynamics simulations suggest that the cavity is a binding site for lipid II molecules that have been transported to the outer leaflet of the membrane. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism to prevent the sequestration of lipid carrier in the PG biogenesis pathway. MraY belongs to the broadly distributed polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase (PNPT) superfamily of enzymes. We therefore propose that similar feedback mechanisms may be widely employed to coordinate precursor supply with demand by polymerases, thereby optimizing the partitioning of lipid carriers between competing glycan biogenesis pathways.

The mechanism of the phage-encoded protein antibiotic from ΦX174.

The historically important phage ΦX174 kills its host bacteria by encoding a 91-residue protein antibiotic called protein E. Using single-particle electron cryo-microscopy, we demonstrate that protein E bridges two bacterial proteins to form the transmembrane YES complex [MraY, protein E, sensitivity to lysis D (SlyD)]. Protein E inhibits peptidoglycan biosynthesis by obstructing the MraY active site leading to loss of lipid I production. We experimentally validate this result for two different viral species, providing a clear model for bacterial lysis and unifying previous experimental data. Additionally, we characterize the Escherichia coli MraY structure-revealing features of this essential enzyme-and the structure of the chaperone SlyD bound to a protein. Our structures provide insights into the mechanism of phage-mediated lysis and for structure-based design of phage therapeutics.

Intradimeric Walker A ATPases: Conserved Features of A Functionally Diverse Family.

Nucleoside-triphosphate hydrolases (NTPases) are a diverse, but essential group of enzymes found in all living organisms. NTPases that have a G-X-X-X-X-G-K-[S/T] consensus sequence (where X is any amino acid), known as the Walker A or P-loop motif, constitute a superfamily of P-loop NTPases. A subset of ATPases within this superfamily contains a modified Walker A motif, X-K-G-G-X-G-K-[S/T], wherein the first invariant lysine residue is essential to stimulate nucleotide hydrolysis. Although the proteins in this subset have vastly differing functions, ranging from electron transport during nitrogen fixation to targeting of integral membrane proteins to their correct membranes, they have evolved from a shared ancestor and have thus retained common structural features that affect their functions. These commonalities have only been disparately characterized in the context of their individual proteins systems, but have not been generally annotated as features that unite the members of this family. In this review, we report an analysis based on the sequences, structures, and functions of several members in this family that highlight their remarkable similarities. A principal feature of these proteins is their dependence on homodimerization. Since their functionalities are heavily influenced by changes that happen in conserved elements at the dimer interface, we refer to the members of this subclass as intradimeric Walker A ATPases.

Structures of Get3d reveal a distinct architecture associated with the emergence of photosynthesis.

Homologs of the protein Get3 have been identified in all domains yet remain to be fully characterized. In the eukaryotic cytoplasm, Get3 delivers tail-anchored (TA) integral membrane proteins, defined by a single transmembrane helix at their C terminus, to the endoplasmic reticulum. While most eukaryotes have a single Get3 gene, plants are notable for having multiple Get3 paralogs. Get3d is conserved across land plants and photosynthetic bacteria and includes a distinctive C-terminal α-crystallin domain. After tracing the evolutionary origin of Get3d, we solve the Arabidopsis thaliana Get3d crystal structure, identify its localization to the chloroplast, and provide evidence for a role in TA protein binding. The structure is identical to that of a cyanobacterial Get3 homolog, which is further refined here. Distinct features of Get3d include an incomplete active site, a "closed" conformation in the apo-state, and a hydrophobic chamber. Both homologs have ATPase activity and are capable of binding TA proteins, supporting a potential role in TA protein targeting. Get3d is first found with the development of photosynthesis and conserved across 1.2 billion years into the chloroplasts of higher plants across the evolution of photosynthesis suggesting a role in the homeostasis of photosynthetic machinery.

Structurally derived universal mechanism for the catalytic cycle of the tail-anchored targeting factor Get3.

Tail-anchored (TA) membrane proteins, accounting for roughly 2% of proteomes, are primarily targeted posttranslationally to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. For this complicated process, it remains unknown how the central targeting factor Get3 uses nucleotide to facilitate large conformational changes to recognize then bind clients while also preventing exposure of hydrophobic surfaces. Here, we identify the GET pathway in Giardia intestinalis and present the structure of the Get3-client complex in the critical postnucleotide-hydrolysis state, demonstrating that Get3 reorganizes the client-binding domain (CBD) to accommodate and shield the client transmembrane helix. Four additional structures of GiGet3, spanning the nucleotide-free (apo) open to closed transition and the ATP-bound state, reveal the details of nucleotide stabilization and occluded CBD. This work resolves key conundrums and allows for a complete model of the dramatic conformational landscape of Get3.

Concise Synthesis of Tunicamycin†V and Discovery of a Cytostatic DPAGT1 Inhibitor.

A short total synthesis of tunicamycin V (1), a non-selective phosphotransferase inhibitor, is achieved via a Büchner-Curtius-Schlotterbeck type reaction. Tunicamycin V can be synthesized in 15 chemical steps from D-galactal with 21 % overall yield. The established synthetic scheme is operationally very simple and flexible to introduce building blocks of interest. The inhibitory activity of one of the designed analogues 28 against human dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1) is 12.5 times greater than 1. While tunicamycins are cytotoxic molecules with a low selectivity, the novel analogue 28 displays selective cytostatic activity against breast cancer cell lines including a triple-negative breast cancer.

Sequence-based features that are determinant for tail-anchored membrane protein sorting in eukaryotes.

The correct targeting and insertion of tail-anchored (TA) integral membrane proteins is critical for cellular homeostasis. TA proteins are defined by a hydrophobic transmembrane domain (TMD) at their C-terminus and are targeted to either the ER or mitochondria. Derived from experimental measurements of a few TA proteins, there has been little examination of the TMD features that determine localization. As a result, the localization of many TA proteins are misclassified by the simple heuristic of overall hydrophobicity. Because ER-directed TMDs favor arrangement of hydrophobic residues to one side, we sought to explore the role of geometric hydrophobic properties. By curating TA proteins with experimentally determined localizations and assessing hypotheses for recognition, we bioinformatically and experimentally verify that a hydrophobic face is the most accurate singular metric for separating ER and mitochondria-destined yeast TA proteins. A metric focusing on an 11 residue segment of the TMD performs well when classifying human TA proteins. The most inclusive predictor uses both hydrophobicity and C-terminal charge in tandem. This work provides context for previous observations and opens the door for more detailed mechanistic experiments to determine the molecular factors driving this recognition.

The STI1-domain is a flexible alpha-helical fold with a hydrophobic groove.

STI1-domains are present in a variety of co-chaperone proteins and are required for the transfer of hydrophobic clients in various cellular processes. The domains were first identified in the yeast Sti1 protein where they were referred to as DP1 and DP2. Based on hidden Markov model searches, this domain had previously been found in other proteins including the mammalian co-chaperone SGTA, the DNA damage response protein Rad23, and the chloroplast import protein Tic40. Here, we refine the domain definition and carry out structure-based sequence alignment of STI1-domains showing conservation of five amphipathic helices. Upon examinations of these identified domains, we identify a preceding helix 0 and unifying sequence properties, determine new molecular models, and recognize that STI1-domains nearly always occur in pairs. The similarity at the sequence, structure, and molecular levels likely supports a unified functional role.

Molecular basis of tail-anchored integral membrane protein recognition by the cochaperone Sgt2.

The targeting and insertion of tail-anchored (TA) integral membrane proteins (IMPs) into the correct membrane is critical for cellular homeostasis. The fungal protein Sgt2, and its human homolog SGTA, is the entry point for clients to the guided entry of tail-anchored protein (GET) pathway, which targets endoplasmic reticulum-bound TA IMPs. Consisting of three structurally independent domains, the C terminus of Sgt2 binds to the hydrophobic transmembrane domain (TMD) of clients. However, the exact binding interface within Sgt2 and molecular details that underlie its binding mechanism and client preference are not known. Here, we reveal the mechanism of Sgt2 binding to hydrophobic clients, including TA IMPs. Through sequence analysis, biophysical characterization, and a series of capture assays, we establish that the Sgt2 C-terminal domain is flexible but conserved and sufficient for client binding. A molecular model for this domain reveals a helical hand forming a hydrophobic groove approximately 15 Å long that is consistent with our observed higher affinity for client TMDs with a hydrophobic face and a minimal length of 11 residues. This work places Sgt2 into a broader family of TPR-containing cochaperone proteins, demonstrating structural and sequence-based similarities to the DP domains in the yeast Hsp90 and Hsp70 coordinating protein, Sti1.

DPAGT1 Inhibitors of Capuramycin Analogues and Their Antimigratory Activities of Solid Tumors.

Capuramycin displays a narrow spectrum of antibacterial activity by targeting bacterial translocase I (MraY). In our program of development of new N-acetylglucosaminephosphotransferase1 (DPAGT1) inhibitors, we have identified that a capuramycin phenoxypiperidinylbenzylamide analogue (CPPB) inhibits DPAGT1 enzyme with an IC50 value of 200 nM. Despite a strong DPAGT1 inhibitory activity, CPPB does not show cytotoxicity against normal cells and a series of cancer cell lines. However, CPPB inhibits migrations of several solid cancers including pancreatic cancers that require high DPAGT1 expression in order for tumor progression. DPAGT1 inhibition by CPPB leads to a reduced expression level of Snail but does not reduce E-cadherin expression level at the IC50 (DPAGT1) concentration. CPPB displays a strong synergistic effect with paclitaxel against growth-inhibitory action of a patient-derived pancreatic adenocarcinoma, PD002: paclitaxel (IC50: 1.25 µM) inhibits growth of PD002 at 0.0024-0.16 µM in combination with 0.10-2.0 µM CPPB (IC50: 35 µM).

Substrate Tolerance of Bacterial Glycosyltransferase MurG: Novel Fluorescence-Based Assays.

MurG (uridine diphosphate-N-acetylglucosamine/N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase) is an essential bacterial glycosyltransferase that catalyzes the N-acetylglucosamine (GlcNAc) transformation of lipid I to lipid II during peptidoglycan biosynthesis. Park's nucleotide has been a convenient biochemical tool to study the function of MraY (phospho-MurNAc-(pentapeptide) translocase) and MurG; however, no fluorescent probe has been developed to differentiate individual processes in the biotransformation of Park's nucleotide to lipid II via lipid I. Herein, we report a robust assay of MurG using either the membrane fraction of a M. smegmatis strain or a thermostable MraY and MurG of Hydrogenivirga sp. as enzyme sources, along with Park's nucleotide or Park's nucleotide-Nε-C6-dansylthiourea and uridine diphosphate (UDP)-GlcN-C6-FITC as acceptor and donor substrates. Identification of both the MraY and MurG products can be performed simultaneously by HPLC in dual UV mode. Conveniently, the generated lipid II fluorescent analogue can also be quantitated via UV-Vis spectrometry without the separation of the unreacted lipid I derivative. The microplate-based assay reported here is amenable to high-throughput MurG screening. A preliminary screening of a collection of small molecules has demonstrated the robustness of the assays and resulted in rediscovery of ristocetin A as a strong antimycobacterial MurG and MraY inhibitor.

A practical synthesis of a novel DPAGT1 inhibitor, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) for in vivo studies.

Immunotherapy that targets N-linked glycans has not yet been developed due in large part to the lack of specificity of N-linked glycans between normal and malignant cells. N-Glycan chains are synthesized by the sequential action of glycosyl transferases in the Golgi apparatus. It is an overwhelming task to discover drug-like inhibitors of glycosyl transferases that block the synthesis of specific branching processes in cancer cells, killing tumor cells selectively. It has long been known that N-glycan biosynthesis can be inhibited by disruption of the first committed enzyme, dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1). Selective DPAGT1 inhibitors have the promising therapeutic potential for certain solid cancers that require increased branching of N-linked glycans in their growth progressions. Recently, we discovered that an anti-Clostridium difficile molecule, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) showed DPAGT1 inhibitory activity with the IC50 value of 0.25 µM. It was confirmed that APPB inhibits N-glycosylation of ß-catenin at 2.5 nM concentration. A sharp difference between APPB and tunicamycin was that the hemolytic activity of APPB is significantly attenuated (IC50 > 200 µM RBC). Water solubility of APPB is >350-times greater than that of tunicamycin (78.8 mg/mL for APPB, <0.2 mg/mL for tunicamycin). A novel DPAGT1 inhibitor, APPB selectively inhibits growth of the solid tumors (e.g. KB, LoVo, SK-OV-3, MDA-MB-432S, HCT116, Panc-1, and AsPC-1) at low µM concentrations, but does not inhibit growth of a leukemia cell (L1210) and the healthy cells (Vero and HPNE) at these concentrations. In vitro metabolic stability using rat liver microsomes indicated that a half-life (t 1/2) of APPB is sufficiently long (>60 min) for in vivo studies (PK/PD, safety profiles, and in vivo efficacy) using animal models. We have refined all steps in the previously reported synthesis for APPB for larger-scale. This article summarizes protocols of gram-scale synthesis of APPB and its physicochemical data, and a convenient DPAGT1 assay. •Remember that the abstract is what readers see first in electronic abstracting & indexing services.•This is the advertisement of your article. Make it interesting, and easy to be understood.•Be accurate and specific, keep it as brief as possible.

Semisynthesis of an Anticancer DPAGT1 Inhibitor from a Muraymycin Biosynthetic Intermediate.

We have explored a method to convert a muraymycin biosynthetic intermediate 3 to an anticancer drug lead 2 for in vivo and thorough preclinical studies. Cu(OAc)2 forms a stable complex with the amide 4 and prevents electrophilic reactions at the 2-((3-aminopropyl)amino)acetamide moiety. Under the present conditions, the desired 5″-primary amine was selectively protected with (Boc)2O to yield 6. The intermediate 6 was converted to 2 in two steps with 90% yield.

Novel FR-900493 Analogues That Inhibit the Outgrowth of Clostridium difficile Spores.

The spectrum of antibacterial activity for the nucleoside antibiotic FR-900493 (1) can be extended by chemical modifications. We have generated a small focused library based on the structure of 1 and identified UT-17415 (9), UT-17455 (10), UT-17460 (11), and UT-17465 (12), which exhibit anti-Clostridium difficile growth inhibitory activity. These analogues also inhibit the outgrowth of C. difficile spores at 2× minimum inhibitory concentration. One of these analogues, 11, relative to 1 exhibits over 180-fold and 15-fold greater activity against the enzymes, phospho-MurNAc-pentapeptide translocase (MraY) and polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA), respectively. The phosphotransferase inhibitor 11 displays antimicrobial activity against several tested bacteria including Bacillus subtilis, Clostridium spp., and Mycobacterium smegmatis, but no growth inhibitory activity is observed against the other Gram-positive and Gram-negative bacteria. The selectivity index (Vero cell cytotoxicity/C. difficileantimicrobial activity) of 11 is approximately 17, and 11 does not induce hemolysis even at a 100 µM concentration.

A statistical model for improved membrane protein expression using sequence-derived features.

The heterologous expression of integral membrane proteins (IMPs) remains a major bottleneck in the characterization of this important protein class. IMP expression levels are currently unpredictable, which renders the pursuit of IMPs for structural and biophysical characterization challenging and inefficient. Experimental evidence demonstrates that changes within the nucleotide or amino acid sequence for a given IMP can dramatically affect expression levels, yet these observations have not resulted in generalizable approaches to improve expression levels. Here, we develop a data-driven statistical predictor named IMProve that, using only sequence information, increases the likelihood of selecting an IMP that expresses in Escherichia coli The IMProve model, trained on experimental data, combines a set of sequence-derived features resulting in an IMProve score, where higher values have a higher probability of success. The model is rigorously validated against a variety of independent data sets that contain a wide range of experimental outcomes from various IMP expression trials. The results demonstrate that use of the model can more than double the number of successfully expressed targets at any experimental scale. IMProve can immediately be used to identify favorable targets for characterization. Most notably, IMProve demonstrates for the first time that IMP expression levels can be predicted directly from sequence.

Structural basis for regulation of the nucleo-cytoplasmic distribution of Bag6 by TRC35.

The metazoan protein BCL2-associated athanogene cochaperone 6 (Bag6) forms a hetero-trimeric complex with ubiquitin-like 4A and transmembrane domain recognition complex 35 (TRC35). This Bag6 complex is involved in tail-anchored protein targeting and various protein quality-control pathways in the cytosol as well as regulating transcription and histone methylation in the nucleus. Here we present a crystal structure of Bag6 and its cytoplasmic retention factor TRC35, revealing that TRC35 is remarkably conserved throughout the opisthokont lineage except at the C-terminal Bag6-binding groove, which evolved to accommodate Bag6, a unique metazoan factor. While TRC35 and its fungal homolog, guided entry of tail-anchored protein 4 (Get4), utilize a conserved hydrophobic patch to bind their respective partners, Bag6 wraps around TRC35 on the opposite face relative to the Get4-5 interface. We further demonstrate that TRC35 binding is critical not only for occluding the Bag6 nuclear localization sequence from karyopherin α to retain Bag6 in the cytosol but also for preventing TRC35 from succumbing to RNF126-mediated ubiquitylation and degradation. The results provide a mechanism for regulation of Bag6 nuclear localization and the functional integrity of the Bag6 complex in the cytosol.

Improving membrane protein expression by optimizing integration efficiency.

The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies.