Structural and functional characterization of the role of acetylation on the interactions of the human Atg8-family proteins with the autophagy receptor TP53INP2/DOR.

The Atg8-family proteins (MAP1LC3/LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2) play a pivotal role in macroautophagy/autophagy through their ability to help form autophagosomes. Although autophagosomes form in the cytoplasm, nuclear levels of the Atg8-family proteins are significant. Recently, the nuclear/cytoplasmic shuttling of LC3B was shown to require deacetylation of two Lys residues (K49 and K51 in LC3B), which are conserved in Atg8-family proteins. To exit the nucleus, deacetylated LC3B must bind TP53INP2/DOR (tumor protein p53 inducible nuclear protein 2) through interaction with the LC3-interacting region (LIR) of TP53INP2 (TP53INP2LIR). To examine their selectivity for TP53INP2 and the role of the conserved Lys residues in Atg8-family proteins, we prepared the six human Atg8-family proteins and acetylated variants of LC3A and GABARAP for biophysical and structural characterization of their interactions with the TP53INP2LIR. Isothermal titration calorimetry (ITC) experiments demonstrate that this LIR binds preferentially to GABARAP subfamily proteins, and that only acetylation of the second Lys residue reduces binding to GABARAP and LC3A. Crystal structures of complexes with GABARAP and LC3A (acetylated and deacetylated) define a ß-sheet in the TP53INP2LIR that determines the GABARAP selectivity and establishes the importance of acetylation at the second Lys. The in vitro results were confirmed in cells using acetyl-mimetic variants of GABARAP and LC3A to examine nuclear/cytoplasmic shuttling and colocalization with TP53INP2. Together, the results demonstrate that TP53INP2 shows selectivity to the GABARAP subfamily and acetylation at the second Lys of GABARAP and LC3A disrupts key interactions with TP53INP2 required for their nuclear/cytoplasmic shuttling.

An LIR motif in the Rift Valley fever virus NSs protein is critical for the interaction with LC3 family members and inhibition of autophagy.

Rift Valley fever virus (RVFV) is a viral zoonosis that causes severe disease in ruminants and humans. The nonstructural small (NSs) protein is the primary virulence factor of RVFV that suppresses the host's antiviral innate immune response. Bioinformatic analysis and AlphaFold structural modeling identified four putative LC3-interacting regions (LIR) motifs (NSs 1-4) in the RVFV NSs protein, which suggest that NSs interacts with the host LC3-family proteins. Using, isothermal titration calorimetry, X-ray crystallography, co-immunoprecipitation, and co-localization experiments, the C-terminal LIR motif (NSs4) was confirmed to interact with all six human LC3 proteins. Phenylalanine at position 261 (F261) within NSs4 was found to be critical for the interaction of NSs with LC3, retention of LC3 in the nucleus, as well as the inhibition of autophagy in RVFV infected cells. These results provide mechanistic insights into the ability of RVFV to overcome antiviral autophagy through the interaction of NSs with LC3 proteins.

Unorthodox PCNA Binding by Chromatin Assembly Factor 1.

The eukaryotic DNA replication fork is a hub of enzymes that continuously act to synthesize DNA, propagate DNA methylation and other epigenetic marks, perform quality control, repair nascent DNA, and package this DNA into chromatin. Many of the enzymes involved in these spatiotemporally correlated processes perform their functions by binding to proliferating cell nuclear antigen (PCNA). A long-standing question has been how the plethora of PCNA-binding enzymes exert their activities without interfering with each other. As a first step towards deciphering this complex regulation, we studied how Chromatin Assembly Factor 1 (CAF-1) binds to PCNA. We demonstrate that CAF-1 binds to PCNA in a heretofore uncharacterized manner that depends upon a cation-pi (π) interaction. An arginine residue, conserved among CAF-1 homologs but absent from other PCNA-binding proteins, inserts into the hydrophobic pocket normally occupied by proteins that contain canonical PCNA interaction peptides (PIPs). Mutation of this arginine disrupts the ability of CAF-1 to bind PCNA and to assemble chromatin. The PIP of the CAF-1 p150 subunit resides at the extreme C-terminus of an apparent long α-helix (119 amino acids) that has been reported to bind DNA. The length of that helix and the presence of a PIP at the C-terminus are evolutionarily conserved among numerous species, ranging from yeast to humans. This arrangement of a very long DNA-binding coiled-coil that terminates in PIPs may serve to coordinate DNA and PCNA binding by CAF-1.

Structural analyses of the Group A flavin-dependent monooxygenase PieE reveal a sliding FAD cofactor conformation bridging OUT and IN conformations.

Group A flavin-dependent monooxygenases catalyze the cleavage of the oxygen-oxygen bond of dioxygen, followed by the incorporation of one oxygen atom into the substrate molecule with the aid of NADPH and FAD. These flavoenzymes play an important role in many biological processes, and their most distinct structural feature is the choreographed motions of flavin, which typically adopts two distinct conformations (OUT and IN) to fulfill its function. Notably, these enzymes seem to have evolved a delicate control system to avoid the futile cycle of NADPH oxidation and FAD reduction in the absence of substrate, but the molecular basis of this system remains elusive. Using protein crystallography, size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), and small-angle X-ray scattering (SEC-SAXS) and activity assay, we report here a structural and biochemical characterization of PieE, a member of the Group A flavin-dependent monooxygenases involved in the biosynthesis of the antibiotic piericidin A1. This analysis revealed that PieE forms a unique hexamer. Moreover, we found, to the best of our knowledge for the first time, that in addition to the classical OUT and IN conformations, FAD possesses a "sliding" conformation that exists in between the OUT and IN conformations. This observation sheds light on the underlying mechanism of how the signal of substrate binding is transmitted to the FAD-binding site to efficiently initiate NADPH binding and FAD reduction. Our findings bridge a gap currently missing in the orchestrated order of chemical events catalyzed by this important class of enzymes.

Piercing the lipid raft: the case of Vibrio cholerae cytolysin.

In a recent issue of Biochemical Journal, Kathuria et al. [Biochem. J. (2018) 475, 3039-3055] report that membrane binding of the pore-forming toxin Vibrio cholerae cytolysin (VCC) is facilitated by the presence of cholesterol, and the presence of this sterol within the lipid bilayer is key for the formation of a functional pore. Yet, in the presence of accessory non-lipid components, VCC retains its membrane-binding capability likely through membrane lipid raft structures. In light of their results, the authors provide new insights into the roles of cholesterol and of membrane microstructures in the binding, the oligomeric assembly and the cytolytic pore formation of VCC which all take place following infection by V. cholerae.

The hydrophobic region of the Leishmania peroxin 14: requirements for association with a glycosome mimetic membrane.

Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.

Structural characterization of interactions between transactivation domain 1 of the p65 subunit of NF-κB and transcription regulatory factors.

p65 is a member of the NF-κB family of transcriptional regulatory proteins that functions as the activating component of the p65-p50 heterodimer. Through its acidic transactivation domain (TAD), p65 has the capacity to form interactions with several different transcriptional regulatory proteins, including TFIIB, TFIIH, CREB-binding protein (CBP)/p300 and TAFII31. Like other acidic TADs, the p65 TAD contains two subdomains (p65TA1 and p65TA2) that interact with different regulatory factors depending on the target gene. Despite its role in controlling numerous NF-κB target genes, there are no high-resolution structures of p65TA1 bound to a target transcriptional regulatory factor. In this work, we characterize the interaction of p65TA1 with two factors, the Tfb1/p62 subunit of TFIIH and the KIX domain of CBP. In these complexes, p65TA1 transitions into a helical conformation that includes its characteristic ΦXXΦΦ motif (Φ = hydrophobic amino acid). Structural and functional studies demonstrate that the two binding interfaces are primarily stabilized by three hydrophobic amino acids within the ΦXXΦΦ motif and these residues are also crucial to its ability to activate transcription. Taken together, the results provide an atomic level description of how p65TA1 is able to bind different transcriptional regulatory factors needed to activate NF-κB target genes.

α,ß-Dicarbonyl reduction is mediated by the Saccharomyces Old Yellow Enzyme.

The undesirable flavor compounds diacetyl and 2,3-pentanedione are vicinal diketones (VDKs) formed by extracellular oxidative decarboxylation of intermediate metabolites of the isoleucine, leucine and valine (ILV) biosynthetic pathway. These VDKs are taken up by Saccharomyces and enzymatically converted to acetoin and 3-hydroxy-2-pentanone, respectively. Purification of a highly enriched diacetyl reductase fraction from Saccharomyces cerevisiae in conjunction with mass spectrometry identified Old Yellow Enzyme (Oye) as an enzyme capable of catalyzing VDK reduction. Kinetic analysis of recombinant Oye1p, Oye2p and Oye3p isoforms confirmed that all three isoforms reduced diacetyl and 2,3-pentanedione in an NADPH-dependent reaction. Transcriptomic analysis of S. cerevisiae (ale) and S. pastorianus (lager) yeast during industrial fermentations showed that the transcripts for OYE1, OYE2, arabinose dehydrogenase (ARA1), α-acetolactate synthase (ILV2) and α-acetohydroxyacid reductoisomerase (ILV5) were differentially regulated in a manner that correlated with changes in extracellular levels of VDKs. These studies provide insights into the mechanism for reducing VDKs and decreasing maturation times of beer which are of commercial importance.

A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence.

Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH.

Analysis of the Leishmania peroxin 7 interactions with peroxin 5, peroxin 14 and PTS2 ligands.

LPEX7 (Leishmania peroxin 7) is essential for targeting newly synthesized proteins with a PTS2 (peroxisome-targeting signal type 2) import signal into the glycosome. In the present paper, we describe the biophysical characterization of a functional LPEX7 isolated from Escherichia coli inclusion bodies. Pull-down assays showed that LPEX7 binds the interacting partners LdPEX5 (Leishmania donovani peroxin 5) and LdPEX14, but, more importantly, this receptor can specifically bind PTS2 cargo proteins in the monomeric and dimeric states. However, in the absence of interacting partners, LPEX7 preferentially adopts a tetrameric structure. Mapping studies localized the LdPEX5- and LdPEX14-binding sites to the N-terminal portion of LPEX7. Deletion of the first 52 residues abolished LdPEX14 association without altering the LdPEX5 interaction. Intrinsic fluorescence techniques suggested that each LPEX7 subunit has a single unique binding site for each of the respective interacting partners LdPEX5, LdPEX14 and PTS2 cargo proteins. Extrinsic fluorescence studies with ANS (8-anilinonaphthalene-1-sulfonic acid) demonstrated that LPEX7 contains a surface-exposed hydrophobic region(s) that was not altered by the binding of a PTS2 protein or LdPEX5. However, in the presence of these ligands, the accessibility of the hydrophobic domain was dramatically restricted, suggesting that both ligands are necessary to induce notable conformational changes in LPEX7. In contrast, binding of LdPEX14 did not alter the hydrophobic domain on LPEX7. It is possible that the hydrophobic surfaces on LPEX7 may be a crucial characteristic for the shuttling of this receptor in and out of the glycosome.

The N-terminal amphipathic region of the Escherichia coli type III secretion system protein EspD is required for membrane insertion and function.

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. These pathogenic E. coli express a syringe-like protein machine, known as the type III secretion system (T3SS), used for the injection of virulence factors into the cytosol of the host epithelial cell. Breaching the epithelial plasma membrane requires formation of a translocation pore that contains the secreted protein EspD. Here we demonstrate that the N-terminal segment of EspD, encompassing residues 1-171, contains two amphipathic domains spanning residues 24-41 and 66-83, with the latter of these helices being critical for EspD function. Fluorescence and circular dichroism analysis revealed that, in solution, His6-EspD1₋171 adopts a native disordered structure; however, on binding anionic small unilamellar vesicles composed of phosphatidylserine, His6-EspD1₋171 undergoes a pH depended conformational change that increases the α-helix content of this protein approximately sevenfold. This change coincides with insertion of the region circumscribing Trp47 into the hydrophobic core of the lipid bilayer. On the HeLa cell plasma membrane, His6-EspD1₋171 forms a homodimer that is postulated to promote EspD-EspD oligomerization and pore formation. Complementation of ΔespD null mutant bacteria with an espDΔ66-83 gene showed that this protein was secreted but non-functional.

Acidic residues in the purine binding site govern the 6-oxopurine specificity of the Leishmania donovani xanthine phosphoribosyltransferase.

Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase enzymes that mediate purine salvage, an obligatory nutritional function for these pathogenic parasites. The xanthine phosphoribosyltransferase preferentially uses xanthine as a substrate, while the hypoxanthine-guanine phosphoribosyltransferase phosphoribosylates only hypoxanthine and guanine. These related phosphoribosyltransferases were used as model system to investigate the molecular determinants regulating the 6-oxopurine specificity of these enzymes. Analysis of the purine binding domains showed two conserved acidic amino acids; glutamate residues in the xanthine phosphoribosyltransferase (E198 and E215) and aspartate residues in the hypoxanthine-guanine phosphoribosyltransferase (D168 and D185). Genetic and biochemical analysis established that the single E198D and E215D mutations increased the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity. However, the E215Q and E198,215D mutations converted the Leishmania xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Similarly, the D168,185E double mutation transformed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases.

Leishmania donovani peroxin 14 undergoes a marked conformational change following association with peroxin 5.

The import of PTS1 proteins into the glycosome or peroxisome requires binding of a PTS1-laden PEX5 receptor to the membrane-associated protein PEX14 to facilitate translocation of PTS1 proteins into the lumen of these organelles. Quaternary structure analysis of protozoan parasite Leishmania donovani PEX14 (LdPEX14) revealed that this protein forms a homomeric complex with a size > 670 kDa. Moreover, deletion mapping indicated that disruption of LdPEX14 oligomerization correlated with the elimination of the hydrophobic region and coiled-coil motif present in LdPEX14. Analysis of the LdPEX5-LdPEX14 interaction by isothermal titration calorimetry revealed a molar binding stoichiometry of 1:4 (LdPEX5: LdPEX14) and an in-solution dissociation constant (K(d)) of approximately 74 nm. Calorimetry, circular dichroism, intrinsic fluorescence, and analytical ultracentrifugation experiments showed that binding of LdPEX5 resulted in a dramatic conformational change in the LdPEX14 oligomeric complex that involved the reorganization of the hydrophobic segment in LdPEX14. Finally, limited tryptic proteolysis assays established that in the presence of LdPEX5, LdPEX14 became more susceptible to proteolytic degradation consistent with this protein interaction triggering a significant conformational change in the recombinant and native LdPEX14 structures. These structural changes provide essential clues to how LdPEX14 functions in the translocation of folded proteins across the glycosomal membrane.

Alpha,beta-dicarbonyl reduction by Saccharomyces D-arabinose dehydrogenase.

An alpha,beta-dicarbonyl reductase activity was purified from Saccharomyces cerevisiae and identified as the cytosolic enzyme D-Arabinose dehydrogenase (ARA1) by MALDI-TOF/TOF. Size exclusion chromatography analysis of recombinant Ara1p revealed that this protein formed a homodimer. Ara1p catalyzed the reduction of the reactive alpha,beta-dicarbonyl compounds methylglyoxal, diacetyl, and pentanedione in a NADPH dependant manner. Ara1p had apparent Km values of approximately 14 mM, 7 mM and 4 mM for methylglyoxal, diacetyl and pentanedione respectively, with corresponding turnover rates of 4.4, 6.9 and 5.9 s(-1) at pH 7.0. pH profiling showed that Ara1p had a pH optimum of 4.5 for the diacetyl reduction reaction. Ara1p also catalyzed the NADP+ dependant oxidation of acetoin; however this back reaction only occurred at alkaline pH values. That Ara1p was important for degradation of alpha,beta-dicarbonyl substrates was further supported by the observation that ara1-Delta knockout yeast mutants exhibited a decreased growth rate phenotype in media containing diacetyl.