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.

Strong anion-exchange fast performance liquid chromatography as a versatile tool for preparation and purification of RNA produced by in vitro transcription.

Here we demonstrate the use of strong anion-exchange fast performance liquid chromatography (FPLC) as a simple, fast, and robust method for RNA production by in vitro transcription. With this technique, we have purified different transcription templates from unreacted reagents in large quantities. The same buffer system could be used to readily remove nuclease contamination from the overexpressed pyrophosphatase, the important reagent for in vitro transcription. In addition, the method can be used to monitor in vitro transcription reactions to enable facile optimization of reaction conditions, and we have compared the separation performance between strong and weak anion-exchange FPLC for various transcribed RNAs, including the Diels-Alder ribozyme, the hammerhead ribozyme tRNA, and 4.5S RNA. The functionality of the purified tRNA(Cys) has been confirmed by the aminoacylation assay. Only the purification by strong anion-exchange FPLC has led to the enrichment of the functional tRNA from run-off transcripts as revealed by both enzymatic and electrophoretic analysis.

Inhibition of TDP-43 aggregation by nucleic acid binding.

The aggregation of TAR DNA-binding protein (TDP-43) has been shown as a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) since 2006. While evidence has suggested that mutation or truncation in TDP-43 influences its aggregation process, nevertheless, the correlation between the TDP-43 aggregation propensity and its binding substrates has not been fully established in TDP-43 proteinopathy. To address this question, we have established a platform based on the in vitro protein expression system to evaluate the solubility change of TDP-43 in response to factors such as nucleotide binding and temperature. Our results suggest that the solubility of TDP-43 is largely influenced by its cognate single-strand DNA (ssDNA) or RNA (ssRNA) rather than hnRNP, which is known to associate with TDP-43 C-terminus. The direct interaction between the refolded TDP-43, purified from E.coli, and ssDNA were further characterized by Circular Dichroism (CD) as well as turbidity and filter binding assay. In addition, ssDNA or ssRNA failed to prevent the aggregation of the F147L/F149L double mutant or truncated TDP-43 (TDP208-414). Consistently, these two mutants form aggregates, in contrast with the wild-type TDP-43, when expressed in Neuro2a cells. Our results demonstrate an intimate relationship between the solubility of TDP-43 and its DNA or RNA binding affinity, which may shed light on the role of TDP-43 in ALS and FTLD.

The N-terminus of TDP-43 promotes its oligomerization and enhances DNA binding affinity.

TDP-43 is a DNA/RNA-binding protein associated with different neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U). Here, the structural and physical properties of the N-terminus on TDP-43 have been carefully characterized through a combination of nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence anisotropy studies. We demonstrate for the first time the importance of the N-terminus in promoting TDP-43 oligomerization and enhancing its DNA-binding affinity. An unidentified structural domain in the N-terminus is also disclosed. Our findings provide insights into the N-terminal domain function of TDP-43.

Cotranslational protein folding within the ribosome tunnel influences trigger-factor recruitment.

In recent years, various folding zones within the ribosome tunnel have been identified and explored through x-ray, cryo-electron microscopy (cryo-EM), and molecular biology studies. Here, we generated ribosome-bound nascent polypeptide complexes (RNCs) with different polyalanine (poly-A) inserts or signal peptides from membrane/secretory proteins to explore the influence of nascent chain compaction in the Escherichia coli ribosome tunnel on chaperone recruitment. By employing time-resolved fluorescence resonance energy transfer and immunoblotting, we were able to show that the poly-A inserts embedded in the passage tunnel can form a compacted structure (presumably helix) and reduce the recruitment of Trigger Factor (TF) when the helical motif is located in the region near the tunnel exit. Similar experiments on nascent chains containing signal sequences that may form compacted structural motifs within the ribosome tunnel and lure the signal recognition particle (SRP) to the ribosome, provided additional evidence that short, compacted nascent chains interfere with TF binding. These findings shed light on the possible controlling mechanism of nascent chains within the tunnel that leads to chaperone recruitment, as well as the function of L23, the ribosomal protein that serves as docking sites for both TF and SRP, in cotranslational protein targeting.

Mutagenesis study of rice nonspecific lipid transfer protein 2 reveals residues that contribute to structure and ligand binding.

Plant nonspecific lipid transfer protein 2 (nsLTP2) is a small (7 kDa) protein that binds lipid-like ligands. An inner hydrophobic cavity surrounded by alpha-helices is the defining structural feature of nsLTP2. Although nsLTP2 structures have been reported earlier, the detailed mechanisms of ligand binding and lipid transfer remain unclear. In this study, we used site-directed mutagenesis to determine the role of various hydrophobic residues (L8, I15, F36, F39, Y45, Y48, and V49) in the structure, stability, ligand binding, and lipid transfer activity of rice nsLTP2. Three single mutations (L8A, F36A, and V49A) drastically alter the native tertiary structure and perturb ligand binding and lipid transfer activity. Therefore, these three residues are structurally important. The Y45A mutant, however, retains a native-like structure but has decreased lipid binding affinity and lipid transfer activity, implying that this aromatic residue is critical for these biological functions. The mutants, I15A and Y48A, exhibit quite different ligand binding affinities. Y48 is involved in planar sterol binding but not linear lysophospholipid association. As for I15A, it had the highest dehydroergosterol binding affinity in spite of the lower lipid binding and transfer abilities. Our results suggest that the long alkyl side chain of I15 would restrict the flexibility of loop I (G13-A19) for sterol entry. Finally, F39A can markedly increase the exposed hydrophobic surface to maintain its transfer efficiency despite reduced ligand binding affinity. These findings suggest that the residues forming the hydrophobic cavity play various important roles in the structure and function of rice nsLTP2.

Structure-based protein engineering for alpha-amylase inhibitory activity of plant defensin.

The structure of a novel plant defensin isolated from the seeds of the mung bean, Vigna radiate, has been determined by (1)H nuclear magnetic resonance spectroscopy. The three-dimensional structure of VrD2, the V. radiate plant defensin 2 protein, comprises an alpha-helix and one triple-stranded anti-parallel beta-sheet stabilized by four disulfide bonds. This protein exhibits neither insecticidal activity nor alpha-amylase inhibitory activity in spite of showing a similar global fold to that of VrD1, an insecticidal plant defensin that has been suggested to function by inhibiting insect alpha-amylase. Our previous study proposed that loop L3 of plant defensins is important for this inhibition. Structural analyses and surface charge comparisons of VrD1 and VrD2 revealed that the charged residues of L3 correlate with the observed difference in inhibitory activities of these proteins. A VrD2 chimera that was produced by transferring the proposed functional loop of VrD1 onto the structurally equivalent loop of VrD2 supported this hypothesis. The VrD2 chimera, which differs by only five residues compared with VrD2, showed obvious activity against Tenebrio molitor alpha-amylase. These results clarify the mode of alpha-amylase inhibition of plant defensins and also represent a possible approach for engineering novel alpha-amylase inhibitors. Plant defensins are important constituents of the innate immune system of plants, and thus the application of protein engineering to this protein family may provide an efficient method for protecting against crop losses.

Characterization and structural analyses of nonspecific lipid transfer protein 1 from mung bean.

Plant nonspecific lipid transfer proteins (nsLTPs) are thermal stable proteins that are capable of transferring lipid molecules between bilayers in vitro. This family of proteins, abundant in plants, is proposed to be involved in defense, pollination, and germination; the in vivo biological function remains, however, elusive. Here we report the purification and sequencing of an nsLTP1 from mung bean sprouts. We have also determined the solution structure of this nsLTP1, which represents the first 3D structure of the dicotyledonous nsLTP1 family. The global fold of mung bean nsLTP1 is similar to those of the monocotyledonous nsLTP1 structures and consists of four alpha-helices stabilized by four disulfide bonds. There are, however, some notable differences in the C-terminal tails and internal hydrophobic cavities. Circular dichroism and fluorescence spectroscopy were used to compare the thermodynamics and lipid transfer properties of mung bean nsLTP1 with those of rice nsLTP1. Docking of a lipid molecule into the solution structure of mung bean nsLTP1 reveals similar binding cavities and hydrophobic interactions as in rice nsLTP1, consistent with their comparable lipid transfer properties measured experimentally.

Comparative studies of the capsid precursor polypeptide P1 and the capsid protein VP1 cDNA vectors for DNA vaccination against foot-and-mouth disease virus.

BACKGROUND: Foot-and-mouth disease virus (FMDV) causes a severe livestock disease, and the virus is an interesting target for virology and vaccine studies. MATERIALS AND METHODS: Here we evaluated comparatively three different viral antigen-encoding DNA sequences, delivered via two physical means (i.e., gene gun delivery into skin and electroporation delivery into muscle), for naked DNA-mediated vaccination in a mouse system. RESULTS: Both methods gave similar results, demonstrating commonality of the observed DNA vaccine effects. Immunization with a cDNA vector expressing the major viral antigen (VP1) alone routinely failed to induce the production of anti-VP1 or neutralizing antibodies in test mice. As a second approach, the plasmid L-VP1 that produces a transgenic membrane-anchored VP1 protein elicited a strong antibody response, but all test mice failed in the FMDV challenge experiment. In contrast, for mice immunized with the viral capsid precursor protein (P1) cDNA expression vector, both neutralizing antibodies and 80-100% protection in test mice were detected. CONCLUSIONS: This strategy of using the whole capsid precursor protein P1 cDNA for vaccination, intentionally without the use of virus-specific protease or other encoding genes for safety reasons, may thus be employed as a relevant experimental system for induction or upgrading of effective neutralizing antibody response, and as a convenient surrogate test system for DNA vaccination studies of FMDV and presumably other viral diseases.

Binding mechanism of nonspecific lipid transfer proteins and their role in plant defense.

Plant nonspecific lipid transfer proteins (nsLTPs) are small basic proteins that transport phospholipids between membranes. On the basis of molecular mass, nsLTPs are subdivided into nsLTP1 and nsLTP2. NsLTPs are all helical proteins stabilized by four conserved disulfide bonds. The existence of an internal hydrophobic cavity, running through the molecule, is a typical characteristic of nsLTPs that serves as the binding site for lipid-like substrates. NsLTPs are known to participate in plant defense, but the exact mechanism of their antimicrobial action against fungi or bacteria is still unclear. To trigger plant defense responses, a receptor at the plant surface needs to recognize the complex of a fungal protein (elicitin) and ergosterol. NsLTPs share high structural similarities with elicitin and need to be associated with a hydrophobic ligand to stimulate a defense response. In this study, binding of sterol molecules with rice nsLTPs is analyzed using various biophysical methods. NsLTP2 can accommodate a planar sterol molecule, but nsLTP1 binds only linear lipid molecules. Although the hydrophobic cavity of rice nsLTP2 is smaller than that of rice nsLTP1, it is flexible enough to accommodate the voluminous sterol molecule. The dissociation constant for the nsLTP2/cholesterol complex is approximately 71.21 microM as measured by H/D exchange and mass spectroscopic detection. Schematic models of the nsLTP complex structure give interesting clues about the reason for differential binding modes. Comparisons of NMR spectra of the sterol/rice nsLTP2 complex and free nsLTP2 revealed the residues involved in binding.

Profiling and characterization antioxidant activities in Anoectochilus formosanus hayata.

Phytochemical characteristics and antioxidant activities of the crude and fractionated plant extracts of Anoectochilus formosanus were evaluated using five different assay systems. An acid-treatment (2 N HCl in 95% ethanol) was employed to treat a butanol fraction (BuOH), creating an acid-hydrolyzed BuOH fraction. The IC(50) values for DPPH radicals in the BuOH and acid-hydrolyzed BuOH fractions were 0.521 and 0.021 mg/mL, respectively. The acid-hydrolyzed BuOH exhibited approximately 5-fold higher activity in scavenging superoxide anion than catechin. The acid-hydrolyzed BuOH fraction also effectively protected phi x174 supercoiled DNA against strand cleavage induced by H(2)O(2) and reduced oxidative stress in HL-60 cells. Metabolite profiling showed that the aglycones of flavonoid glycosides in BuOH were produced after acid hydrolytic treatment, and this resulted in a significant increase in antioxidant activities of acid-hydrolyzed BuOH. One new diarylpentanoid, kinsenone, and three known flavonoid glycosides and their derivatives were identified for the first time from A. formosanus, with strong antioxidant properties.