N-terminal Truncation Research Articles

Solution structure and assembly of β-amylase 2 from Arabidopsis thaliana.

Starch is a key energy-storage molecule in plants that requires controlled synthesis and breakdown for effective plant growth. β-Amylases (BAMs) hydrolyze starch into maltose to help to meet the metabolic needs of the plant. In the model plant Arabidopsis thaliana there are nine BAMs, which have apparently distinct functional and domain structures, although the functions of only a few of the BAMs are known and there are no 3D structures of BAMs from this organism. Recently, AtBAM2 was proposed to form a tetramer based on chromatography and activity assays of mutants; however, there was no direct observation of this tetramer. Here, small-angle X-ray scattering data were collected from AtBAM2 and its N-terminal truncations to describe the structure and assembly of the tetramer. Comparison of the scattering of the AtBAM2 tetramer with data collected from sweet potato (Ipomoea batatas) BAM5, which is also reported to form a tetramer, showed there were differences in the overall assembly. Analysis of the N-terminal truncations of AtBAM2 identified a loop sequence found only in BAM2 orthologs that appears to be critical for AtBAM2 tetramer assembly as well as for activity.

Integrative Model to Coordinate the Oligomerization and Aggregation Mechanisms of CCL5

CC-type chemokine ligand 5 (CCL5) is involved in the pathogenesis of many inflammatory conditions. Under physiological conditions, CCL5 oligomerization and aggregation are considered to be responsible for its inflammatory properties. The structural basis of CCL5 oligomerization remains controversial because the current oligomer models contain no consensus interactions. In this study, NMR and biophysical analyses proposed evidence that the CC-type CCL5 dimer acts as the basic unit to constitute the oligomer and that CCL5 oligomerizes alternatively through E66-K25 and E66-R44/K45 interactions. In addition, a newly determined trimer structure, constituted by CCL5 and the E66S mutant, reported an interfacial interaction through the N-terminal 12FAY14 sequence. The interaction contributes to CCL5 aggregation and precipitation but not to oligomerization. In accordance with the observations, an integrative model explains the CCL5 oligomerization and aggregation mechanism in which CCL5 assembly consists of two types of dimer-dimer interactions and one aggregation mechanism. For full-length CCL5, the molecular accumulation triggers oligomerization through the E66-K25 and E66-R44/K45 interactions, and the 12FAY14 interaction acts as a secondary effect to derive aggregation and precipitation. In contrast, the E66-R44/K45 interaction might dominate in CCL5 N-terminal truncations, and the interaction would lead to the filament-like formation in solution.

Polarity of the ATP binding site of the Na+,K+-ATPase, gastric H+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase

A fluorescence ratiometric method utilizing the probe eosin Y is presented for estimating the ATP binding site polarity of P-type ATPases in different conformational states. The method has been calibrated by measurements in a series of alcohols and tested using complexation of eosin Y with methyl-β-cyclodextrin. The results obtained with the Na+,K+-, H+,K+- and sarcoplasmic reticulum Ca2+-ATPases indicate that the ATP binding site, to which eosin is known to bind, is significantly more polar in the case of the Na+,K+- and H+,K+-ATPases compared to the Ca2+-ATPase. This result was found to be consistent with docking calculations of eosin with the E2 conformational state of the Na+,K+-ATPase and the Ca2+-ATPase. Fluorescence experiments showed that eosin binds significantly more strongly to the E1 conformation of the Na+,K+-ATPase than the E2 conformation, but in the case of the Ca2+-ATPase both fluorescence experiments and docking calculations showed no significant difference in binding affinity between the two conformations. This result could be due to the fact that, in contrast to the Na+,K+- and H+,K+-ATPases, the E2-E1 transition of the Ca2+-ATPase does not involve the movement of a lysine-rich N-terminal tail which may affect the overall enzyme conformation. Consistent with this hypothesis, the eosin affinity of the E1 conformation of the Na+,K+-ATPase was significantly reduced after N-terminal truncation. It is suggested that changes in conformational entropy of the N-terminal tail of the Na+, K+- and the H+,K+-ATPases during the E2-E1 transition could affect the thermodynamic stability of the E1 conformation and hence its ATP binding affinity.

GATA1 and cooperating mutations in myeloid leukaemia of Down syndrome.

Myeloid leukaemia of Down syndrome (ML-DS) is an acute megakaryoblastic/erythroid leukaemia uniquely found in children with Down syndrome (constitutive trisomy 21). It has a unique clinical course, being preceded by a pre-leukaemic condition known as transient abnormal myelopoiesis (TAM), and provides an excellent model to study multistep leukaemogenesis. Both TAM and ML-DS blasts carry acquired N-terminal truncating mutations in the erythro-megakaryocytic transcription factor GATA1. These result in exclusive production of a shorter isoform (GATA1s). The majority of TAM cases resolve spontaneously without the need for treatment; however, around 10% acquire additional cooperating mutations and transform to leukaemia, with differentiation block and clinically significant cytopenias. Transformation is driven by the acquisition of additional mutation(s), which cooperate with GATA1s to perturb normal haematopoiesis.

Protein knots provide mechano-resilience to an AAA+ protease-mediated proteolysis with profound ATP energy expenses

Knotted proteins are some of the most fascinating examples of how linear polypeptide chains can achieve intricate topological arrangements efficiently and spontaneously. The entanglements of polypeptide chains could potentially enhance their folding stabilities. We recently reported the unprecedented mechanostability of the Gordian (52) knotted family of human ubiquitin C-terminal hydrolases (UCHs) in the context of withstanding the mechanical unfolding of the bacterial AAA+ proteasome, ClpXP; a green fluorescence protein (GFP) was fused to the N-terminus of various UCHs as a reporter of the unfolding and degradation of these topologically knotted substrates, but it also limited the ability to examine the effect of untying the knotted topology via N-terminal truncation. In this study, we directly monitored the ClpXP-mediated degradation of UCH variants by electrophoresis and quantitative imaging analyses. We demonstrated that untying of the 52 knot in UCHL1 via N-terminal truncation (UCHL1Δ11) significantly reduces its mechanostability. We further quantified the ATP expenditures of degrading different UCH variants by ClpXP. The unknotted UCHL1Δ11 underwent accelerated ClpXP-dependent proteolysis, with a 30-fold reduction in ATP consumption compared to the knotted wild type. Unlike all other known ClpXP substrates, UCHL5, which is the most resilient substrate known to date, significantly slowed down the ATP turnover rate by ClpXP. Furthermore, UCHL5 required 1000-fold more ATP to be fully degraded by ClpXP compared to GFP. Our results underscored how the complex, knotted folding topology in UCHs may interfere with the mechano-unfolding processes of the AAA+ unfoldase, ClpX.

Molecular dissection of the replication system of plasmid pIGRK encoding two in-frame Rep proteins with antagonistic functions

BackgroundGene overlapping is a frequent phenomenon in microbial genomes. Excluding so-called “trivial overlapping”, there are significant implications of such genetic arrangements, including regulation of gene expression and modification of protein activity. It is also postulated that, besides gene duplication, the appearance of overlapping genes (OGs) is one of the most important factors promoting a genome’s novelty and evolution. OGs coding for in-frame proteins with different functions are a particularly interesting case. In this study we identified and characterized two in-frame proteins encoded by OGs on plasmid pIGRK from Klebsiella pneumoniae, a representative of the newly distinguished pHW126 plasmid family.ResultsA single repR locus located within the replication system of plasmid pIGRK encodes, in the same frame, two functional polypeptides: a full-length RepR protein and a RepR’ protein (with N-terminal truncation) translated from an internal START codon. Both proteins form homodimers, and interact with diverse DNA regions within the plasmid replication origin and repR promoter operator. Interestingly, RepR and RepR’ have opposing functions – RepR is crucial for initiation of pIGRK replication, while RepR’ is a negative regulator of this process. Nevertheless, both proteins act cooperatively as negative transcriptional regulators of their own expression.ConclusionsRegulation of the initiation of pIGRK replication is a complex process in which a major role is played by two in-frame proteins with antagonistic functions. In-frame encoded Rep proteins are uncommon, having been described in only a few plasmids. This is the first description of such proteins in a plasmid of the pHW126 family.

Selective Allosteric Modulation of N-Terminally Cleaved, but Not Full Length CCL3 in CCR1

Chemokines undergo post-translational modification such as N-terminal truncations. Here, we describe how N-terminal truncation of full length CCL3(1-70) affects its activity at CCR1. Truncated CCL3(5-70) has 10-fold higher potency and enhanced efficacy in β-arrestin recruitment, but less than 2-fold increased potencies in G protein signaling determined by calcium release, cAMP and IP3 formation. Small positive ago-allosteric ligands modulate the two CCL3 variants differently as the metal ion chelator bipyridine in complex with zinc (ZnBip) enhances the binding of truncated, but not full length CCL3, while a size-increase of the chelator to a chloro-substituted terpyridine (ZnClTerp), eliminates its allosteric, but not agonistic action. By employing a series of receptor mutants and in silico modeling we describe residues of importance for chemokine and small molecule binding. Notably, the chemokine receptor-conserved Glu2877.39 interacts with the N-terminal amine of truncated CCL3(5-70) and with Zn2+ of ZnBip, thereby bridging their binding sites and enabling the positive allosteric effect. Our study emphasizes that small allosteric molecules may act differently toward chemokine variants and thus selectively modulate interactions of specific chemokine subsets with their cognate receptors.

The N-terminal region of RTP1S plays important roles in dimer formation and odorant receptor-trafficking

Receptor-transporting protein 1S (RTP1S) is an accessory protein that mediates the transport of mammalian odorant receptors (ORs) into the plasma membrane. Although most ORs fail to localize to the cell surface when expressed alone in nonolfactory cells, functional expression of ORs is achieved with the coexpression of RTP1S. However, the mechanism for RTP1S-mediated OR trafficking remains unclear. In this study, we attempted to reveal the mode of action and critical residues of RTP1S in OR trafficking. Experiments using N-terminal truncation and Ala substitution mutants of RTP1S demonstrated that four N-terminal amino acids have essential roles in OR trafficking. Additionally, using recombinant proteins and split luciferase assays in mammalian cells, we provided evidence for the dimer formation of RTP1S. Furthermore, we determined that the 2nd Cys residue is required for the efficient dimerization of RTP1S. Altogether, these findings provide insights into the mechanism for plasma membrane transport of ORs by RTP1S.

Effect of Post-Translational Modifications and Mutations on Amyloid-β Fibrils Dynamics at N Terminus

We investigate the variability in the dynamics of the disordered N-terminal domain of amyloid-β fibrils (Aβ), comprising residues 1–16 of Aβ1–40, due to post-translational modifications and mutations in the β-bend regions known to modulate aggregation properties. Using 2H static solid-state NMR approaches, we compare the dynamics in the wild-type Aβ fibrils in the threefold symmetric polymorph with the fibrils from three post-translational modification sequences: isoaspartate-D7, the phosphorylation of S8, and an N-terminal truncation ΔE3. Additional comparisons are made with the mutants in the β-bend region (residues 21–23) corresponding to the familial Osaka E22Δ deletion and D23N Iowa mutation. We also include the aggregates induced by Zn2+ ions. The dynamics are probed at the F4 and G9 positions. The main motional model involves two free states undergoing diffusion and conformational exchanges with the bound state in which the diffusion is quenched because of transient interactions involving fibril core and other intrastrand contacts. The fraction of the bound state increases in a sigmoidal fashion with a decrease in temperature. There is clear variability in the dynamics: the phosphorylation of S8 variant is the most rigid at the G9 site in line with structural studies, the ΔE3 fibrils are more flexible at the G9 site in line with the morphological fragmentation pattern, the Zn-induced aggregates are the most mobile, and the two β-bend mutants have the strongest changes at the F4 site toward higher rigidity. Overall, the changes underlie the potential role of conformational ensembles in setting the stage for aggregation-prone states.

Differing Membrane Interactions of Two Highly Similar Drug-Metabolizing Cytochrome P450 Isoforms: CYP 2C9 and CYP 2C19.

The human cytochrome P450 (CYP) 2C9 and 2C19 enzymes are two highly similar isoforms with key roles in drug metabolism. They are anchored to the endoplasmic reticulum membrane by their N-terminal transmembrane helix and interactions of their cytoplasmic globular domain with the membrane. However, their crystal structures were determined after N-terminal truncation and mutating residues in the globular domain that contact the membrane. Therefore, the CYP-membrane interactions are not structurally well-characterized and their dynamics and the influence of membrane interactions on CYP function are not well understood. We describe herein the modeling and simulation of CYP 2C9 and CYP 2C19 in a phospholipid bilayer. The simulations revealed that, despite high sequence conservation, the small sequence and structural differences between the two isoforms altered the interactions and orientations of the CYPs in the membrane bilayer. We identified residues (including K72, P73, and I99 in CYP 2C9 and E72, R73, and H99 in CYP 2C19) at the protein-membrane interface that contribute not only to the differing orientations adopted by the two isoforms in the membrane, but also to their differing substrate specificities by affecting the substrate access tunnels. Our findings provide a mechanistic interpretation of experimentally observed effects of mutagenesis on substrate selectivity.

Catalytic Ability Improvement of Phenylalanine Hydroxylase from Chromobacterium violaceum by N-Terminal Truncation and Proline Introduction.

Phenylalanine hydroxylase from Chromobacterium violaceum (CvPAH) is a monomeric enzyme that converts phenylalanine to tyrosine. It shares high amino acid identity and similar structure with a subunit of human phenylalanine hydroxylase that is a tetramer, resulting in the latent application in medications. In this study, semirational design was applied to CvPAH to improve the catalytic ability based on molecular dynamics simulation analyses. Four Nterminal truncated variants and one single point variant were constructed and characterized. The D267P variant showed a 2.1-fold increased thermal stability compared to the wild type, but lower specific activity was noted compared with the wild type. The specific activity of all truncated variants was a greater than 25% increase compared to the wild type, and these variants showed similar or slightly decreased thermostability with the exception of the N-Δ9 variant. Notably, the N-Δ9 variant exhibited a 1.2-fold increased specific activity, a 1.3-fold increased thermostability and considerably increased catalytic activity under the neutral environment compared with the wild type. These properties of the N-Δ9 variant could advance medical and pharmaceutical applications of CvPAH. Our findings indicate that the N-terminus might modulate substrate binding, and are directives for further modification and functional research of PAH and other enzymes.

Thyrotropin Receptor: Allosteric Modulators Illuminate Intramolecular Signaling Mechanisms at the Interface of Ecto- and Transmembrane Domain.

The large TSH-bound ectodomain of the thyrotropin receptor (TSHR) activates the transmembrane domain (TMD) indirectly via an internal agonist (IA). The ectodomain/TMD interface consists of a converging helix, a Cys-Cys-bridge-linked IA, and extracellular loops (ECL). To investigate the intramolecular course of molecular activation, especially details of the indirect activation, we narrowed down allosteric inhibition sites of negative allosteric modulator (NAM) by mutagenesis, homology modeling, and competition studies with positive allosteric modulator (PAM). From the inhibitory effects of NAM S37a on: 1) chimeras with swapped ectodomain, 2) stepwise N-terminal truncations, 3) distinct constitutively active mutations distributed across the hinge region and ECL, but not across the TMD, we conclude that S37a binds at the ectodomain/TMD interface, between the converging helix, ECL1, and the IA. This is also supported by the noncompetitive inhibition of PAM-C2-activation by S37a in the TSHR-TMD construct lacking the ectodomain. Mutagenesis studies on the IA and ECL were guided by our refined model of the ectodomain/TMD interface and indicate an interaction with the TSHR-specific residues E404 (preceding IA) and H478 (ECL1). At this new allosteric interaction site, NAM S37a blocks both TSH- and PAM-induced activation of the TSHR. Our refined models, mutations, and new allosteric binding pocket helped us to gain more detailed insights into the intramolecular course of TSHR activation at the ectodomain/TMD interface, including the delocalization of the converging helix and rearrangement of the conformation of IA. These changes are embedded between the ECL and cooperatively trigger active conformations of TMD. SIGNIFICANCE STATEMENT: The intramolecular activation mechanisms of the TSHR appear to be distinct from those of other G protein-coupled receptors, as the TSHR has a uniquely large N-terminal ectodomain that includes the hormone binding site and an internal agonist sequence. We present new molecular and structural insights into the interface between ectodomain and transmembrane domain in the TSHR, as well as the transfer of activation to the transmembrane domain. This knowledge is critical for understanding activation or inhibition of the receptor by allosteric ligands. We have identified a new allosteric antagonist binding pocket that is located exactly at this interface and possesses specific features that may allow the generation of potent highly TSHR-selective drugs, of potential value for the treatment of Graves' orbitopathy.

Abstract 864: Mitochondrial Morphology During Stress is Regulated by GJA1-20k Interaction With Actin

Connexin (Cx) 43 is encoded by the GJA1 gene and GJA1-20k has been identified as an N-terminal truncation of Cx43 generated endogenously by internal translation. Previously we have found GJA1-20k increases with ischemic stress and induces actin organization and, in mice, mitochondrial biogenesis. In this study we explored the acute effect of GJA1-20k on mitochondria. We transfected GFP-tagged GJA1-20k plasmid into HEK293T cells and analyzed mitochondrial morphology by fluorescent microscopy. We found GJA1-20k results in mitochondria that are associated with actin and also are smaller and more circular than in GST-transfected control cells. In addition, quantitative analysis indicated preservation of mitochondrial area and thus enhanced mitochondrial fission in the presence of GJA1-20k. Disruption of actin polymerization inhibited the effect of GJA1-20k on mitochondrial fission as did use of the actin stabilizer phalloidin, indicating the need for robust actin dynamics for GJA1-20k to be effective. These data suggest that the interaction between GJA1-20k and actin cytoskeleton is necessary for stress related mitochondrial fission. The results indicate that, during stress, upregulated GJA1-20k recruits the actin cytoskeleton to directly affect mitochondrial dynamics and cellular metabolic function.

Absence of photosynthetic state transitions in alien chloroplasts

Main conclusionThe absence of state transitions in a Nt(Hn) cybrid is due to a cleavage of the threonine residue from the misprocessed N-terminus of the LHCII polypeptides.The cooperation between the nucleus and chloroplast genomes is essential for plant photosynthetic fitness. The rapid and specific interactions between nucleus-encoded and chloroplast-encoded proteins are under intense investigation with potential for applications in agriculture and renewable energy technology. Here, we present a novel model for photosynthesis research in which alien henbane (Hyoscyamus niger) chloroplasts function on the nuclear background of a tobacco (Nicotiana tabacum). The result of this coupling is a cytoplasmic hybrid (cybrid) with inhibited state transitions—a mechanism responsible for balancing energy absorption between photosystems. Protein analysis showed differences in the LHCII composition of the cybrid plants. SDS-PAGE analysis revealed a novel banding pattern in the cybrids with at least one additional ‘LHCII’ band compared to the wild-type parental species. Proteomic work suggested that the N-terminus of at least some of the cybrid Lhcb proteins was missing. These findings provide a mechanistic explanation for the lack of state transitions—the N-terminal truncation of the Lhcb proteins in the cybrid included the threonine residue that is phosphorylated/dephosphorylated in order to trigger state transitions and therefore crucial energy balancing mechanism in plants.

Biophysical analyses and functional implications of the interaction between Heat Shock Protein 27 and antibodies to HSP27

Heat Shock Protein 27 (HSP27) is a small molecular chaperone that reduces the development of atherosclerosis by lowering plasma cholesterol levels as well as inflammation. Human studies show an inverse correlation between atherosclerotic burden and HSP27 expression, and are supported by murine models in which augmenting HSP27 levels curbs experimental atherogenesis. Natural HSP27 auto-antibodies (AAb) are found in human plasma, however their role in modulating the athero-protective effects of HSP27 is unknown. The purpose of this study is to characterize the biophysical interaction between human recombinant HSP27 and AAb. A validated polyclonal anti-HSP27 IgG antibody (PAb) was used to mimic natural AAb. Homology modeling and secondary structure prediction tools facilitated the design of HSP27 truncation and phosphorylation mutants. Secondary structural changes were identified using Circular Dichroism (CD) and Dynamic Light Scattering (DLS). Similar to prior structural investigations of HSP27, there was a predominance of α-helical content in the N-terminal truncation and dephosphorylation (“AA”) mutants. The α-crystallin domain (ACD) predominantly consists of β-strands, with the addition of the N-terminal increasing helical content and the C-terminal maintaining β structure. With increasing ratios of PAb to HSP27 β structure abundance and particle size increased, with a similar trend observed with the N-terminus, C-terminus and ACD peptides but an opposite trend with the phosphorylation peptides. Taken together, these studies provide insights into the interaction of HSP27 and its AAb that ultimately may aid in optimizing the design of HSP27 peptidomimetics with anti-atherogenic potential.

Influence of Chemokine N-Terminal Modification on Biased Agonism at the Chemokine Receptor CCR1.

Leukocyte migration, a hallmark of the inflammatory response, is stimulated by the interactions between chemokines, which are expressed in injured or infected tissues, and chemokine receptors, which are G protein-coupled receptors (GPCRs) expressed in the leukocyte plasma membrane. One mechanism for the regulation of chemokine receptor signaling is biased agonism, the ability of different chemokine ligands to preferentially activate different intracellular signaling pathways via the same receptor. To identify features of chemokines that give rise to biased agonism, we studied the activation of the receptor CCR1 by the chemokines CCL7, CCL8, and CCL15(Δ26). We found that, compared to CCL15(Δ26), CCL7 and CCL8 exhibited biased agonism towards cAMP inhibition and away from β-Arrestin 2 recruitment. Moreover, N-terminal substitution of the CCL15(Δ26) N-terminus with that of CCL7 resulted in a chimera with similar biased agonism to CCL7. Similarly, N-terminal truncation of CCL15(Δ26) also resulted in signaling bias between cAMP inhibition and β-Arrestin 2 recruitment signals. These results show that the interactions of the chemokine N-terminal region with the receptor transmembrane region play a key role in selecting receptor conformations coupled to specific signaling pathways.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry with Activated Ion Electron Transfer Dissociation for Large-scale Top-down Proteomics.

Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for top-down proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AI-ETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E values) of proteoforms than CZE-HCD and CZE-ETD, indicating a higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n = 3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal, and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected.

Structure and characterization of Aspergillus fumigatus lipase B with a unique, oversized regulatory subdomain.

Fungal lipases are efficient and environment-friendly biocatalysts for many industrially relevant processes. One of the most widely applied lipases in the manufacturing industry is Candida antarctica lipase B (CALB). Here, we report the biochemical and structural characterization of a novel CALB-like lipase from an important human pathogen-Aspergillus fumigatus (AFLB), which has high sn-1,3-specificity toward triolein. AFLB crystal structure displays a CALB-like catalytic domain and hosts a unique tightly closed 'lid' domain that contains a disulfide bridge, as well as an extra N-terminal subdomain composed of residues 1-128 (including the helix α1-α5 located above the active site). To gain insight into the function of this novel lid and N-terminal subdomain, we constructed and characterized a series of mutants in these two domains. Deleting the protruding bulk lid's residues, replacing the bulk and tight lid with a small and loose lid from CALB, or breaking the disulfide bridge increased the affinity of CALB for glyceride substrates and improved its catalytic activity, along with the loss of enzyme fold stability and thermostability. N-terminal truncation mutants revealed that the N-terminal peptide (residues 1-59) is a strong inhibitor of AFLB binding to lipid films. This peptide thus limits AFLB's penetration power and specific activity, revealing a unique enzyme activity regulatory mechanism. Our findings on the functional and structural properties of AFLB provide a better understanding of the functions of the CALB-like lipases and pave the way for its future protein engineering. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6IDY.

Properties of a cryptic lysyl oxidase from haloarchaeon Haloterrigena turkmenica.

BackgroundLysyl oxidases (LOX) have been extensively studied in mammals, whereas properties and functions of recently found homologues in prokaryotic genomes remain enigmatic.MethodsLOX open reading frame was cloned from Haloterrigena turkmenica in an E. coli expression vector. Recombinant Haloterrigena turkmenica lysyl oxidase (HTU-LOX) proteins were purified using metal affinity chromatography under denaturing conditions followed by refolding. Amine oxidase activity has been measured fluorometrically as hydrogen peroxide release coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting.ResultsCultured H. turkmenica has no detectable amine oxidase activity. HTU-LOX may be expressed in E. coli with a high protein yield. The full-length protein gives no catalytic activity. For this reason, we hypothesized that the hydrophobic N-terminal region may interfere with proper folding and its removal may be beneficial. Indeed, truncated His-tagged HTU-LOX lacking the N-terminal hydrophobic signal peptide purified under denaturing conditions can be successfully refolded into an active enzyme, and a larger N-terminal truncation further increases the amine oxidase activity. Refolding is optimal in the presence of Cu2+ at pH 6.2 and is not sensitive to salt. HTU-LOX is sensitive to LOX inhibitor 3-aminopropionitrile. HTU-LOX deaminates usual substrates of mammalian LOX such as lysine-containing polypeptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed substrate specificity of the former. HTU-LOX readily oxidizes various primary amines including such compounds as taurine and glycine, benzylamine being a poor substrate. Of note, HTU-LOX is also active towards several aminoglycoside antibiotics and polymyxin. Western blotting indicates that epitopes for the anti-HTU-LOX polyclonal antibodies coincide with a high molecular weight protein in H. turkmenica cells.ConclusionH. turkmenica contains a lysyl oxidase gene that was heterologously expressed yielding an active recombinant enzyme with important biochemical features conserved between all known LOXes, for example, the sensitivity to 3-aminopropionitrile. However, the native function in the host appears to be cryptic.SignificanceThis is the first report on some properties of a lysyl oxidase from Archaea and an interesting example of evolution of enzymatic properties after hypothetical horizontal transfers between distant taxa.

The Crg1 N-Terminus Is Essential for Methyltransferase Activity and Cantharidin Resistance in Saccharomyces cerevisiae.

Crg1 is an S-adenosylmethionine (SAM)-dependent methyltransferase required for cantharidin resistance in yeast. Crg1 has a well-characterized methyltransferase domain that inactivates cantharidin by methylation. However, the remaining part of the Crg1 protein is yet to be functionally characterized. In this study, we identified an essential role of the N-terminus of Crg1 in methyltransferase activity and cantharidin resistance. Yeast cells lacking 41 residues of the N-terminus of Crg1 ( crg1ΔN) showed hypersensitivity to cantharidin as same as the null mutant, crg1. The mass spectrometry-based biochemical enzyme assay revealed a loss of methyltransferase activity in Crg1ΔN, which justifies the loss of cantharidin resistance, as well. The subcellular distribution of Crg1ΔN-daGFP showed cytoplasmic aggregates, whereas wild-type Crg1-daGFP was distributed normally in the cytoplasm. Interestingly, the Crg1-methyltransferase domain point mutants (D44A, D67A, and E105A/D108A) also showed the same cytoplasmic aggregates as Crg1ΔN-daGFP. In silico prediction of the tertiary structures of these mutants indicated an altered protein conformation. Altogether, these observations suggest that the N-terminal truncation, as well as the point mutations in the methyltransferase domain, alters the native folding of Crg1 methyltransferase, resulting in a loss of enzyme activity. Furthermore, the crg1ΔN mutant showed the same phenotypes as the crg1 null mutant in the presence of cantharidin, i.e., lethal cell growth, PE auxotrophy, temperature sensitivity, endoplasmic reticulum stress, GPI anchor missorting, and cell wall damage. Overall, this study identifies an essential role of the N-terminus of Crg1 in methyltransferase activity and cantharidin resistance.