Asp Residues Research Articles

Catalase-peroxidase StKatG is a bacterial manganese oxidase from endophytic Salinicola tamaricis

Manganese (Mn) oxides in iron/manganese plaques are widely distributed in the rhizosphere of wetland plants and contribute significantly to elemental cycling and pollutant removal. Mn oxides are primarily produced by bacterial processes using Mn oxidases. However, the molecular mechanism underlying the formation of rhizosphere Mn oxides is still largely unknown. This study identified a manganese-oxidizing enzyme, the catalase-peroxidase StKatG, from an endophytic bacterium Salinicola tamaricis from the wetland plant. The gene encoding StKatG was cloned and overexpressed in Escherichia coli. The recombinant StKatG displayed different structure and enzymatic properties from the previously reported Mn oxidases. The enzyme activity of StKatG yielded Mn oxides with the mixed-valent state: Mn(II), Mn(III), and Mn(IV). The optimum pH and temperature for StKatG are 7.5 and 50 °C, respectively. Structurally, StKatG is organized into two domains, whereas the reported Mn oxidases are mainly single-domain proteins. Based on the site-directed mutagenesis studies, the presence of aspartic acid (Asp) residues in the loop of StKatG are critical to Mn-oxidizing activity. These findings identified a novel bacterial Mn oxidase and provided insights into the molecular mechanism of Mn oxidation in the plant rhizosphere.

Crystal Structures of Epstein-Barr Virus Bcl-2 Homolog BHRF1 Bound to Bid and Puma BH3 Motif Peptides.

Apoptosis is a powerful defense mechanism used by multicellular organisms to counteract viral infection. In response to premature host cell suicide, viruses have evolved numerous countermeasures to ensure cell viability to optimize their replication by encoding proteins homologous in structure and function to cellular pro-survival Bcl-2 proteins. Epstein–Barr virus (EBV), a member of the Gammaherpesviridae, encodes the Bcl-2 homolog BHRF1, a potent inhibitor of Bcl-2-mediated apoptosis. BHRF1 acts by directly targeting Bid and Puma, two proapoptotic proteins of the Bcl-2 family. Here, we determined the crystal structures of BHRF1 bound to peptides spanning the Bcl-2 binding motifs (Bcl-2 homology 3 motif, BH3) of Bid and Puma. BHRF1 engages BH3 peptides using the canonical ligand-binding groove of its Bcl-2 fold and maintains a salt bridge between an Arg residue with a conserved Asp residue in the BH3 motif mimicking the canonical ionic interaction seen in host Bcl-2:BH3 motif complexes. Furthermore, both Bid and Puma utilize a fifth binding pocket in the canonical ligand binding groove of BHRF1 to provide an additional hydrophobic interaction distinct from the interactions previously seen with Bak and Bim. These findings provide a structural basis for EBV-mediated suppression of host cell apoptosis and reveal the flexibility of virus encoded Bcl-2 proteins in mimicking key interactions from the endogenous host signaling pathways.

Differential N-terminal processing of beta and gamma actin

SummaryCytoplasmic beta- and gamma-actin are ubiquitously expressed in every eukaryotic cell. They are encoded by different genes, but their amino acid sequences differ only by four conservative substitutions at the N-termini, making it difficult to dissect their individual regulation. Here, we analyzed actin from cultured cells and tissues by mass spectrometry and found that beta, unlike gamma actin, undergoes sequential removal of N-terminal Asp residues, leading to truncated actin species found in both F- and G-actin preparations. This processing affects up to ∼3% of beta actin in different cell types. We used CRISPR/Cas-9 in cultured cells to delete two candidate enzymes capable of mediating this type of processing. This deletion abolishes most of the beta actin N-terminal processing and results in changes in F-actin levels, cell spreading, filopodia formation, and cell migration. Our results demonstrate previously unknown isoform-specific actin regulation that can potentially affect actin functions in cells.

Site Specific Preparation of N‐Glycosylated Peptides: Thioamide‐Directed Activation of Aspartate

AbstractA site‐specific method for the preparation of N‐glycosylated peptides is described. Incorporation of a peptide backbone thioamide linkage adjacent to an Asp residue facilitates a AgI‐promoted, site‐specific conversion to N‐glycosylated Asn residues in peptides.

Site Specific Preparation of N-Glycosylated Peptides: Thioamide-Directed Activation of Aspartate.

A site-specific method for the preparation of N-glycosylated peptides is described. Incorporation of a peptide backbone thioamide linkage adjacent to an Asp residue facilitates a AgI -promoted, site-specific conversion to N-glycosylated Asn residues in peptides.

Highly effective peptide-calcium chelate prepared from aquatic products processing wastes: Stickwater and oyster shells

Stickwater and oyster shells can be used as cost-effective sources to produce high-value peptide-calcium chelate. In this study, the optimal preparation process of chelate from peptides of stickwater hydrolysate and calcium of oyster shells was determined by response surface methodology, under which a high calcium chelation rate of 87.62% was achieved. The analysis of peptides sequence showed that all chelated peptides were from the myosin light chain, and the molecular weight of most of them was lower than 1500 Da. There were 19, 20, and 19 of the 26 chelated peptides containing Glu, Asp, and Gly residues, respectively, which have been proved to exhibit high calcium-binding ability in previous studies. Structural characterizations revealed that –NH2, COO-, N–H, CO, C–H, and –OH groups might participate in the formation of peptide-calcium chelate, and new interaction forces of crystal structure were generated in the chelate. Besides, morphology analysis showed that folded, aggregated, and compact structures formed in the peptide-calcium chelate. Moreover, the peptide-calcium chelate was relatively stable under high temperature and digestion conditions in the gastrointestinal tract, indicating that it could be used as a highly effective calcium supplement.

Real-Time 1H NMR reveals position and sequence dependences of amino acid isomerization in amyloid beta fragments in situ

Amino acid isomers and their biologically relevant functions have been found in a variety of peptides and proteins in a living system. It is challenging, however, to specify how such isomerization proceeds at each amino acid independently in situ. In this work, the amino acid isomerization was quantified simultaneously at multiple residues in real time, by using solution-state 1H NMR in situ. The ability of NMR to distinguish isomerization product and reactant was demonstrated at different positions of aspartyl (Asp) residues in amyloid beta (Aβ) 1–40 fragments, as well as the glutamyl (Glu) residue. This enabled us to compare the isomerization of Asp1, Asp7, and Asp23, and Glu3 systematically in various Aβ fragments, in terms of the kinetics of isomerization in real time. It was found that Asp1 and Asp7 were prone to convert to iso-aspartyl (isoAsp) form, although Asp23 and Glu3 showed limited reactivity. The analysis demonstrated the role of histidine (His) in regulating Asp isomerization; His6 and His13/14 showed an inhibitory effect on the progress of isomerization at Asp1 and Asp7, respectively, by forming Asp-His complex that serves to slow down the conversion from Asp to isoAsp.

Bacterial hemerythrin domain-containing oxygen and redox sensors: Versatile roles for oxygen and redox signaling.

Hemerythrin is an oxygen-binding protein originally found in certain marine invertebrates. Oxygen reversibly binds at its non-heme diiron center, which consists of two oxo-bridged iron atoms bound to a characteristic conserved set of five His residues, one Glu residue, and one Asp residue. It was recently discovered that several bacteria utilize hemerythrin as an oxygen- and redox-sensing domain in responding to changes in cellular oxygen concentration or redox status, and immediately adapt to these environmental changes in order to maintain important physiological processes, including chemotaxis and c-di-GMP synthesis and degradation. This Mini Review focuses on the recent progress made on structural and functional aspects of these emerging bacterial hemerythrin domain-containing oxygen and redox sensors, revealing characteristic features of this family of proteins.

B-Box Transcription Factor FaBBX22 Promotes Light-Induced Anthocyanin Accumulation in Strawberry (Fragaria × ananassa).

B-box transcription factors (TFs) play a vital role in light-induced anthocyanin accumulation. Here, the FaBBX22 gene encoding 287 amino acids B-box TF was isolated from the cultivated strawberry variety ‘Benihoppe’ and characterized functionally. The expression analysis showed that FaBBX22 was expressed in the roots, stems, leaves, flowers and fruits, and its transcription level was upregulated under the red- or blue-light irradiation. FaBBX22 was localized in the nucleus and showed trans-acting activity in yeast cells. Ectopic overexpression of FaBBX22 in Arabidopsis enhanced the accumulation of anthocyanin. Additionally, we obtained transgenic strawberry calli that overexpressed the FaBBX22 gene, and strawberry calli coloration assays showed that FaBBX22 increased anthocyanin accumulation by upregulating the expression of anthocyanin biosynthetic genes (FaPAL, FaANS, FaF3′H, FaUFGT1) and transport gene FaRAP in a light-dependent manner. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation assays indicated that FaBBX22 interacted with FaHY5. Furthermore, mutation of the 70th Asp residue in FaBBX22 protein to an Ala residue disrupted the interaction between FaBBX22 and FaHY5. Further, a transient expression assay demonstrated that the co-expression of FaBBX22 and FaHY5 could strongly promote anthocyanin accumulation in strawberry fruits. Collectively, these results revealed the positive regulatory role of FaBBX22 in light-induced anthocyanin accumulation.

Searching conformational analysis of Asp residues through theoretical 3J vicinal coupling constants and Karplus equations

AbstractThe dependence of the vicinal spin–spin coupling constants on the torsion side chain angle χ1 through the Karplus equations is considered for the study of the structure of Asp amino acid residues. Experimental and theoretical, obtained with density functional theory methods, vicinal coupling constants combined with extended Karplus equations, which include six coefficients, are applied to a dipeptide model of the amino acid Asp. To find out the empirical χ1 angles of the side chain, a statistical analysis procedure is developed to compute the rmsd values and find the χ1 as the minimum of those values. The χ1 values obtained in this work for nine Asp residues of the flavodoxin protein Desulfovibrio vulgaris are successfully compared with those derived by nuclear magnetic resonance and X‐rays.

Inhibition of the carnitine acylcarnitine carrier by carbon monoxide reveals a novel mechanism of action with non-metal-containing proteins

Both toxic and physiological effects of CO are mostly caused by well described interactions with heme-groups of proteins. Interactions of CO with non-heme proteins have also been unveiled. Besides interaction of CO with mitochondrial heme containing respiratory complexes, a BK channel and the phosphate carrier which do not contain metal cofactors, have been identified as CO targets. However, the molecular mechanisms of interaction with non-metal-containing proteins are not understood. We show in this work the effect of CO on the mitochondrial carnitine carrier (SLC25A20) using CORM-3, a widely recognized CO releasing compound. CO exerts an inhibitory effect at the micromolar concentration on the transport function of the transporter extracted from treated mitochondria. The effect is due to a single Cys residue, C136 as revealed by mass spectrometry analysis. A computational approach predicted the need for vicinal Asp and Lys residues for the C136 carbonylation to occur. These data demonstrate a novel mechanism of interaction of CO with a protein not containing metal atoms and will enable the prediction of CO targets.

Abstract 3596: RM-036, a first-in-class, orally-bioavailable, Tri-Complex covalent KRASG12D(ON) inhibitor, drives profound anti-tumor activity in KRASG12D mutant tumor models

Abstract KRASG12D mutant cancers represent a significant unmet medical need with 55,000 new diagnoses annually in the US. The KRASG12D mutation occurs commonly in multiple tumor histotypes, including about 20%, 29% and 17% of KRAS mutant colorectal, pancreatic, and non-small cell lung cancers, respectively. However, there are no directly targeted inhibitors of KRASG12D in clinical development. Mutant RAS proteins exist predominantly in the GTP-bound RAS(ON) state, leading to excessive downstream signaling via interaction with effectors such as RAF kinases. Highly selective inhibitors that covalently target the KRASG12C(OFF) state and inhibit conversion to KRASG12C(ON) have been shown to be active and well tolerated in the clinic, and we have described a novel, covalent tri-complex KRASG12C(ON) inhibitor that has robust anti-tumor activity and is well tolerated in preclinical studies. Developing a covalent inhibitor of KRASG12D presents several challenges. The Asp residue in KRASG12D is significantly less reactive toward electrophiles than is Cys. And most reported covalent chemical warheads capable of reacting with Asp exhibit high nonspecific reactivity. The intrinsic GTP hydrolysis rate of KRASG12D is about 2.6-fold lower than that of KRASG12C, further biasing the cellular KRASG12D pool to the RAS(ON) state and emphasizing the importance of targeting the KRASG12D(ON) state for maximal suppression of this oncogenic driver. Based on these considerations we designed RM-036, a potent, selective, orally bioavailable, covalent KRASG12D(ON) inhibitor with attractive drug-like properties. It forms a tri-complex between the abundant intracellular chaperone cyclophilin A (CypA) and the active state of KRASG12D, positioning the warhead relative to KRASG12D and enabling selective covalent engagement of Asp. In cellular models, RM-036 exhibited efficient and selective covalent binding to KRASG12D, with no detectable reactivity toward the adjacent Asp residue in cells with the KRASG13D mutation. RM-036 showed low off-target reactivity in cell-based proteomic screens. RM-036 potently inhibited growth and induced apoptosis in KRASG12D mutant cancer cells in vitro but not in BRAFV600E-dependent cells. RM-036, as a single agent, produced deep, durable, and dose-dependent suppression of tumor RAS pathway activation with covalent target engagement in vivo following repeat oral administration. Profound tumor regressions, including complete regressions, were observed in various KRASG12D mutant xenograft models upon treatment with RM-036. All treatments regimens tested with RM-036 were well tolerated in vivo. These preclinical findings provide a strong foundation for advancing RM-036 toward clinical evaluation in patients with tumors bearing the KRASG12D mutation. Citation Format: John E. Knox, Jingjing Jiang, G. Leslie Burnett, Yang Liu, Caroline E. Weller, Zhican Wang, Laura McDowell, Shelby L. Steele, Shook Chin, Kang Jye Chou, Fang Wang, Mengqi Zhong, Elena S. Koltun, David Wildes, Mallika Singh, Adrian L. Gill, Jacqueline A. Smith. RM-036, a first-in-class, orally-bioavailable, Tri-Complex covalent KRASG12D(ON) inhibitor, drives profound anti-tumor activity in KRASG12D mutant tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3596.

Toward Rapid Aspartic Acid Isomer Localization in Therapeutic Peptides Using Cyclic Ion Mobility Mass Spectrometry

There is an increasing emphasis on the critical evaluation of interbatch purity and physical stability of therapeutic peptides. This is due to concerns over the impact that product- and process-related impurities may have on safety and efficacy of this class of drug. Aspartic acid isomerization to isoaspartic acid is a common isobaric impurity that can be very difficult to identify without first synthesizing isoAsp peptide standards for comparison by chromatography. As such, analytical tools that can determine if an Asp residue has isomerized, as well as the site of isomerization within the peptide sequence, are highly sought after. Ion mobility-mass spectrometry is a conformation-selective method that has developed rapidly in recent years particularly with the commercialization of traveling wave ion mobility instruments. This study employed a cyclic ion mobility (cIMS) mass spectrometry system to investigate the conformational characteristics of a therapeutic peptide and three synthetic isomeric forms, each with a single Asp residue isomerized to isoAsp. cIMS was able to not only show distinct conformational differences between each peptide but crucially, in conjunction with a simple workflow for comparing ion mobility data, it correctly located which Asp residue in each peptide had isomerized to isoAsp. This work highlights the value of cIMS as a potential screening tool in the analysis of therapeutic peptides prone to the formation of isoAsp impurities.

Protonation states of hen egg-white lysozyme observed using D/H contrast neutron crystallography.

Hen egg-white lysozyme (HEWL) is an enzymatic protein with two acidic amino acids, Glu35 and Asp52, in its active site. Glu35 acts as a proton donor to the substrate and Asp52 interacts with the positively charged substrate, suggesting different protonation states of these residues. However, neutron crystallographic studies thus far have not provided a consistent picture of the protonation states of these residues. Only one study succeeded in observing the active protonation states of Glu35 and Asp52 in the triclinic crystal system. However, their active states in the most widely studied tetragonal crystal system are still unknown. The application of the D/H contrast technique in neutron crystallography improves the ability to locate exchangeable D/H atoms in proteins. In the present study, D2O and H2O solvent crystals were prepared. Each neutron data set was collected for only five days by combining a time-of-flight diffractometer (iBIX) and the spallation neutron source at the Japan Proton Accelerator Research Complex. The D/H contrast map provided better visualization of the D/H atoms in HEWL than the conventional neutron scattering length density map. The neutron D/H contrast map demonstrated the alternative protonation of the OE1 and OE2 atoms in the carboxyl group of Glu35. This alternative protonation occurs in the absence of a substrate, where high selectivity of the protonation site does not occur. In this case, only the OE1-HE1 bond attacks the substrate in an equilibrium between OE1-HE1 and OE2-HE2, or the H+ ion of the OE2-HE2 bond moves to the OE1 atom just before or after substrate binding to initiate the catalytic reaction. In contrast, the carboxyl group of Asp52 is not protonated. Protonation of the carboxyl group was not observed for other Asp and Glu residues. These results are consistent with results from NMR spectroscopy and explain the protonation states at the active site in the apo form of HEWL.

Expression, Characterization, and Analysis of Type II Metacaspases from the Fungus Schizophyllum commune

Programmed cell death (PCD), otherwise known as apoptosis, is implicit across all kingdoms of life especially in the processes of development, aging, reproduction, responses to stress, and maintaining homeostasis. In humans and other animals, apoptosis is mediated by caspases, proteases that cleave other proteins after Asp residues. Plants, fungi, and protozoa do not have caspases, but rather produce homologous proteins known as metascapases. In these organisms, metacaspases are the proteins primarily responsible for programmed cell death. Studying their mechanisms can provide insight into opportunistic fungal infections. In our research, Schizophyllum commune is the model organism for studying metacaspase proteolytic function in fungi. There are two types of metacaspases in S. commune with varying structures and organization (Type I and Type II). Both types contain a larger p20 catalytic region with a Cys‐His catalytic dyad similar to casapses, and a smaller p10 region with a conserved sequence. The Type I metacaspases cleave preferentially after Lys and Arg and are activated by calcium ions. The focus of this research project is the characterization and analysis of Type II metacaspases (ScMCA‐II) in S. commune. Type II metacaspases have a large linker region between the p20 and p10 subunits, and are hypothesized to be the chief enzyme involved in apoptosis. Previous literature has focused primarily on Type I metacaspases, making the studies of Type II metacaspase especially crucial. There are six Type II metacaspases that have been identified in the fungus S. commune (labelled ScMCAIIa‐f). Full‐length constructs of ScMCAIIa and ScMCAIIf are expressed at low levels in E. coli and expression is not improved by decreasing the growth temperature. Preliminary results with small amounts of partially‐purified protein suggest that S. commune Type II metacaspases are cleaved during expression and are active against the metacaspase substrate Z‐GGR‐AMC in both the presence and absence of calcium.

Oncohistone Mutations Occur at Functional Sites of Regulatory ADP-Ribosylation.

This study identifies cancer-driving mutations in histones as sites of PARP1-mediated ADP-ribosylation in breast and ovarian cancers, providing a molecular pathway by which cancers may subvert the growth-regulating effects of PARP1.

Enhanced ion mobility resolution of Abeta isomers from human brain using high-resolution demultiplexing software.

Isomerization of aspartic acid (Asp) residues in long-lived proteins is a key feature associated with neurodegenerative proteinopathies such as Alzheimer's disease (AD). Recently, using ultra high-performance liquid chromatography (UHPLC) coupled with drift tube ion mobility mass spectrometry (DTIMS-MS), we documented the extensive Asp isomerization in amyloid-beta (Aβ) peptides depositing in the extracellular cortical plaques (senile plaques) of the AD brain. Aβ1-15 was estimated to be ~ 85% isomerized, while Aβ4-15 another major constituent of these senile plaques was ~ 50% isomerized in AD brain. Low resolution on the standard demultiplexed ion mobility resulted in poor separation of these N-truncated Aβ isomers in the ion mobility domain. Here, using the same ion multiplexed dataset, we applied new post-acquisition data reconstruction technique, high-resolution demultiplexing (HRdm), to improve the resolution of these Aβ isomers in the ion mobility dimension. We demonstrate that for the complex proteomic AD brain digests, HRdm could successfully resolve three out of four major Asp isomers of Aβ1-15. For Aβ2-15 and Aβ4-15, the significant resolution enhancement in the HRdm data resulted in baseline peak separation of the respective Asp isomers. An analysis of two-peak resolution (Rpp) and peak-to-peak separation (ΔP) indicated twofold enhancement for the Asp-isomerized Aβ species. HRdm performed with an effective resolving power (Rp) of between 150 and 160 for the highest deconvolution settings in comparison to ~ 40 to 65 in the standard settings. These major resolution improvements in the ion mobility domain for the endogenous Aβ isomers demonstrate the feasibility of in situ measurement of peptide isomers and their role in the mechanism of amyloid plaque formation in AD.

Mass spectrometric studies of the variety of beta-amyloid proteoforms in Alzheimer's disease.

This review covers the results of the application of mass spectrometric (MS) techniques to study the diversity of beta-amyloid (Aβ) peptides in human samples. Since Aβ is an important hallmark of Alzheimer's disease (AD), which is a socially significant neurodegenerative disorder of the elderly worldwide, analysis of its endogenous variations is of particular importance for elucidating the pathogenesis of AD, predicting increased risks of the disease onset, and developing effective therapy. MS approaches have no alternative for the study of complex samples, including a wide variety of Aβ proteoforms, differing in length and modifications. Approaches based on matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography with electrospray ionization tandem MS are most common in Aβ studies. However, Aβ forms with isomerized and/or racemized Asp and Ser residues require the use of special methods for separation and extra sensitive and selective methods for detection. Overall, this review summarizes current knowledge of Aβ species found in human brain, cerebrospinal fluid, and blood plasma; focuses on application of different MS approaches for Aβ studies; and considers the potential of MS techniques for further studies of Aβ-peptides.

Spliced isoforms of the cardiac Nav1.5 channel modify channel activation by distinct structural mechanisms.

Alternative splicing is an important cellular mechanism that fine tunes the gating properties of both voltage- and ligand-gated ion-channels. The cardiac voltage-gated sodium channel, Nav1.5, is subject to alternative splicing of the DI S3-S4 linker, which generates two types of channels with different activation properties. Here, we show that the gating differences between the adult (mH1) and neonatal (Nav1.5e) isoforms of Nav1.5 are mediated by two amino acid residues: Thr/Ser at position 207 and Asp/Lys at position 211. Electrophysiological experiments, in conjunction with molecular dynamics simulations, revealed that each residue contributes equally to the overall gating shifts in activation, but that the underlying structural mechanisms are different. Asp/Lys at position 211 acts through electrostatic interactions, whereas Thr/Ser at position 207 is predicted to alter the hydrogen bond network at the top of the S3 helix. These distinct structural mechanisms work together to modify movement of the voltage-sensitive S4 helix to bring about channel activation. Interestingly, mutation of the homologous Asp and Thr residues of the skeletal muscle isoform, Nav1.4, to Lys and Ser, respectively, confers a similar gating shift in channel activation, suggesting that these residues may fulfill a conserved role across other Nav channel family members.

Structural Basis for the Prenylation Reaction of Carbazole-Containing Natural Products Catalyzed by Squalene Synthase-Like Enzymes.

Some enzymes annotated as squalene synthase catalyze the prenylation of carbazole-3,4-quinone-containing substrates in bacterial secondary metabolism. Their reaction mechanisms remain unclear because of their low sequence similarity to well-characterized aromatic substrate prenyltransferases (PTs). We determined the crystal structures of the carbazole PTs, and these revealed that the overall structure is well superposed on those of squalene synthases. In contrast, the stacking interaction between the prenyl donor and acceptor substrates resembles those observed in aromatic substrate PTs. Structural and mutational analyses suggest that the Ile and Asp residues are essential for the hydrophobic and hydrophilic interactions with the carbazole-3,4-quinone moiety of the prenyl acceptor, respectively, and a deprotonation mechanism of an intermediary σ-complex involving a catalytic triad is proposed. Our results provide a structural basis for a new subclass of aromatic substrate PTs.