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The recently elucidated Get proteins are responsible for the targeted delivery of the majority of tail-anchored (TA) proteins to the endoplasmic reticulum. Get4 and Get5 have been identified in the early steps of the pathway mediating TA substrate delivery to the cytoplasmic targeting factor Get3. Here we report a crystal structure of Get4 and an N-terminal fragment of Get5 from Saccharomyces cerevisae. We show Get4 and Get5 (Get4/5) form an intimate complex that exists as a dimer (two copies of Get4/ 5) mediated by the C-terminus of Get5. We further demonstrate that Get3 specifically binds to a conserved surface on Get4 in a nucleotide dependent manner. This work provides further evidence for a model in which Get4/5 operates upstream of Get3 and mediates the specific delivery of a TA substrate. crystallography | Get pathway | Mdy2 | Ubl4|a I tail-anchor doi/10.1073/pnas.1006036107

The Get3 ATPase directs the delivery of tail-anchored (TA) proteins to the endoplasmic reticulum (ER). TA-proteins are characterized by having a single transmembrane helix (TM) at their extreme C terminus and include many essential proteins, such as SNAREs, apoptosis factors, and protein translocation components. These proteins cannot follow the SRP-dependent co-translational pathway that typifies most integral membrane proteins; instead, post-translationally, these proteins are recognized and bound by Get3 then delivered to the ER in the ATP dependent Get pathway. To elucidate a molecular mechanism for TA protein binding by Get3 we have determined three crystal structures in apo and ADP forms from Saccharomyces cerevisae (ScGet3-apo) and Aspergillus fumigatus (AfGet3-apo and AfGet3-ADP). Using structural information, we generated mutants to confirm important interfaces and essential residues. These results point to a model of how Get3 couples ATP hydrolysis to the binding and release of TA-proteins. ArsA | crystallography | Deviant Walker A | Get pathway | protein transport

The apparatus responsible for translocation of proteins across bacterial membranes is the conserved SecY complex, consisting of SecY, SecE, and SecG. Prior genetic analysis provided insight into the mechanisms of protein export, as well as the interactions between the component proteins. In particular, the prl suppressor alleles of secE and secY, which allow export of secretory proteins with defective signal sequences, have proven particularly useful. Here, we report the isolation of novel mutations in secE and secY, as well as the phenotypic effects of combinations of prl mutations. These new alleles, as well as previously characterized prl mutations, were analyzed in light of the recently published crystal structure of the archaeal SecY complex. Our results support and expand a model of Prl suppressor activity that proposes that all of the prlA and prlG alleles either destabilize the closed state of the channel or stabilize the open form. These mutants thus allow channel opening to occur without the triggering event of signal sequence binding that is required in a wild-type complex.

During their biosynthesis, many proteins pass through the membrane via a hydrophilic channel formed by the heterotrimeric Sec61/SecY complex. Whether this channel forms at the interface of multiple copies of Sec61/SecY or is intrinsic to a monomeric complex, as suggested by the recently solved X-ray structure of the Methanococcus jannaschii SecY complex, is a matter of contention. By introducing a single cysteine at various positions in Escherichia coli SecY and testing its ability to form a disulfide bond with a single cysteine in a translocating chain, we provide evidence that translocating polypeptides pass through the center of the SecY complex. The strongest cross-links were observed with residues that would form a constriction in an hourglass-shaped pore. This suggests that the channel makes only limited contact with a translocating polypeptide, thus minimizing the energy required for translocation.

The ATPase SecA mediates the posttranslational translocation of a wide range of polypeptide substrates through the SecY channel in the cytoplasmic membrane of bacteria. We have determined the crystal structure of a monomeric form of Bacillus subtilis SecA at a 2.2-[Angstrom] resolution. A comparison with the previously determined structures of SecA reveals a nudeotide-independent, large conformational change that opens a deep groove similar to that in other proteins that interact with diverse polypeptides. We propose that the open form of SecA represents an activated state.

Results describe the three-dimensional X-ray crystallographic structures of the small 30S ribosomal subunit of Thermus thermophilus complexed with antibiotics tetracycline, pactamycin, and hygromycin B. Data indicate that the antibiotics bind to different regions of the ribosome subunit and often exhibiting dissimilarities with those of previously determined antibiotic complexes.

The structure of the 30S ribosomal subunit is presented, based on an electron-density map at 5.5 A resolution. Double-helical RNA and alpha-helices and Beta sheets of proteins can be identified at this resolution and seven 30S-subunit ribosomal proteins and a large portion of double-stranded RNA helices were fitted into density.

The location of translational initiation factor IF3 bound to the 30S subunit of the Thermus thermophilus ribosome has been determined by cryoelectron microscopy. Both the 30S-IF3 complex and control 30S subunit structures were determined to 27-[angstrom] resolution. The difference map calculated from the two reconstructions reveals three prominent lobes of positive density. The previously solved crystal structure of IF3 fits very well into two of these lobes, whereas the third lobe probably arises from conformational changes induced in the 30S subunit as a result of IF3 binding. Our placement of IF3 on the 30S subunit allows an understanding in structural terms of the biochemical functions of this initiation factor, namely its ability to dissociate 70S ribosomes into 30S and 50S subunits and the preferential selection of initiator tRNA by IF3 during initiation.

Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 165 RNA. These bases interact intimately with the minor groove of the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA. The third, or 'wobble,' position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs., During protein synthesis, the ribosome catalyzes the sequential addition of amino acids to a growing polypeptide chain, using mRNA as a template and aminoacylated tRNAs (aa-tRNAs) as substrates. Correct base [...]

Initiation of translation at the correct position on messenger RNA is essential for accurate protein synthesis. In prokaryotes, this process requires three initiation factors: IF1, IF2, and IF3. Here we report the crystal structure of a complex of IF1 and the 30S ribosomal subunit. Binding of IF1 occludes the ribosomal A site and flips out the functionally important bases A1492 and A1493 from helix 44 of 16S RNA, burying them in pockets in IF1. The binding of IF1 causes long-range changes in the conformation of H44 and leads to movement of the domains of 30S with respect to each other. The structure explains how localized changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit., The synthesis of functional polypeptides requires initiation of translation to occur at the correct mRNA codon. In prokaryotes, selection of the start codon involves formation of a 30S initiation complex [...]

The article offers information on the secretory (SEC) pathway, which provides a role for a conserved membrane protein complex. It mentions that the integral membrane proteins (IMPs) are rapidly degraded in the cytoplasm to prevent aggregation, which can lead to broad disruptions in cellular homeostasis. It states that in IMPs the signal-recognition particle (SRP) binds the N-terminal signal as it emerges from a ribosome.

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., (© 2024. The Author(s).)

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.

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., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., (© 2022 Wiley-VCH GmbH.)

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., (© 2021 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

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., (© 2021 The Protein Society.)

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 IC 50 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 IC 50 (DPAGT1) concentration. CPPB displays a strong synergistic effect with paclitaxel against growth-inhibitory action of a patient-derived pancreatic adenocarcinoma, PD002: paclitaxel (IC 50 : 1.25 μM) inhibits growth of PD002 at 0.0024-0.16 μM in combination with 0.10-2.0 μM CPPB (IC 50 : 35 μM).

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- N ε -C 6 -dansylthiourea and uridine diphosphate (UDP)-GlcN-C 6 -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.

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) 2 O to yield 6. The intermediate 6 was converted to 2 in two steps with 90% yield.