Aspartic Acid Residues Research Articles

Evidence for a high pK a of an aspartic acid residue in the active site of CALB by a fully atomistic multiscale approach

Candida antarctica Lipase B (CALB) is a paradigm for the family of lipases. At pH 7, the optimal pH for catalysis, the protonation state of an aspartic acid of the active site (Asp134) could not be conclusively assigned. In fact, the pK a estimate provided by a widely used computational tool, namely PropKa, that predicts pKa values of ionizable groups in proteins based on the crystallographic structure, is only slightly above 7 (pKa = 7.25). This, along with the lack of an experimental evaluation, makes the assignment of its protonation state at neutral pH challenging. Here, we calculate the pK a of Asp134 by means of a fully atomistic multiscale computational approach based on classical molecular dynamics (MD) simulation and the perturbed matrix method (PMM), namely the MD-PMM approach. MD-PMM is able to take into account the dynamics of the system and, at the same time, to treat the deprotonation step at the quantum level. The calculations provide a pK a value of 8.9 ± 1.1, hence suggesting that Asp134 in CALB should be protonated at neutral, and even at slightly basic, pH. Communicated by Ramaswamy H. Sarma

Specificity Determinants of Lysine Deacetylases

Lysine acetylation is a reversible post‐translational modification that has been found on thousands of nuclear and cytoplasmic proteins. A family of enzymes, known as lysine deacetylases (KDACs), catalyzes the removal of acetyl groups. This reversible modification is a regulatory mechanism for proteins involved in numerous cellular processes; however, few KDAC‐substrate pairs have been identified, and there is a lack of information regarding how individual KDACs interact with substrates. We hypothesized that differences between KDACs near the active site would influence substrate specificity, allowing members of the family to preferentially react with a unique subset of acetylated proteins. Using a peptide library derived from a previously‐identified substrate of KDAC8, we demonstrated that KDAC1, KDAC6, and KDAC8 have distinct preferences regarding the residues surrounding the acetylated lysine. A combination of activity data using an in vitro assay and molecular dynamics simulations revealed discrete features of the KDACs that contribute to substrate specificity. In KDAC8, but not in other KDACs, an aspartic acid residue (D101) forms an ionic interaction with positively charged residues in the ‐1 position of the substrate relative to the acetyllysine, promoting deacetylation. Additionally, nearby hydrophobic residues further influence substrate preferences in a KDAC‐specific manner. Using interaction models based on these data, we were able to predict biologically relevant peptide substrates for individual KDACs. While the identified residues are undoubtedly not the only KDAC specificity determinants, understanding the role of these close contacts on enzyme preferences has greatly increased our understanding of KDAC specificity and has given us a handle to identify potential protein substrates from cells.

Skeletal muscle alpha actin (ACTA1) acetylation negatively regulates muscle function in response to obesity

Post‐translational modifications (PTMs) of proteins are an increasingly important area of study that contributes to our understanding for protein function in the control of health and disease. Acetylation, for example, has long been associated with histone modifications to control gene expression, yet emerging omics data demonstrates that acetylation occurs across the proteome. Our most recent work further shows global shifts in non‐histone protein acetylation within the left ventricle (LV) in response to diet‐induced obesity. Despite these advances in omics technologies, our understanding for how non‐histone protein acetylation regulates cardiac biology remains a ‘black box’. In this study, we examined the functional importance for skeletal muscle alpha actin (ACTA1) acetylation in the control of muscle function. For this, we used actin‐motility assays to examine the biophysical changes between acetylated ACTA1 and the other myofibrillar proteins involved in cardiac contraction and relaxation (e.g. myosin, tropomyosin). Here, we show that ACTA1 acetylation increased actin‐myosin binding, decreased actin sliding velocity and increased Ca2+ sensitivity. These data suggest that ACTA1 acetylation significantly alters muscle function. Of note, we previously published that lysine (K) residue 50 (K50) of ACTA1 was acetylated, similar to other proteomics reports. Interestingly, the positively charged K50 on ACTA1 sits juxtaposed to the negatively charged aspartic acid (D) residue 391 of the cardiac myosin binding protein (cMy‐BP). cMy‐BP is essential for actin‐myosin interactions, and this suggests an important role for ACTA1 K50 acetylation in the control of muscle contraction. Thus, we knocked out endogenous actin (Act88F) from the indirect flight muscles (IFMs) of D. melanogaster and inserted an Act88F K50Q mutant, which mimics acetylation specifically at this site. Previous reports have shown that IFMs behave similarly to cardiac muscle. Phenotypically, the K50Q flies behave similar to their wild‐type counterparts. However, current studies are underway to examine changes in flight as well as purify the K50Q actin for actin‐motility assays to examine biophysical changes in myofibrillar proteins. Combined, our data suggests that ACTA1 acetylation negatively impacts muscle function in response to obesity, current work will determine the implications for K50 actin acetylation in this phenotype.

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.

How Do Point Mutations Enhancing the Basic Character of the RBDs of SARS-CoV-2 Variants Affect Their Transmissibility and Infectivity Capacities?

The spread of SARS-CoV-2 variants in the population depends on their ability to anchor the ACE2 receptor in the host cells. Differences in the electrostatic potentials of the spike protein RBD (electropositive/basic) and ACE2 receptor (electronegative/acidic) play a key role in both the rapprochement and the recognition of the coronavirus by the cell receptors. Accordingly, point mutations that result in an increase in electropositively charged residues, e.g., arginine and lysine, especially in the RBD of spike proteins in the SARS-CoV-2 variants, could contribute to their spreading capacity by favoring their recognition by the electronegatively charged ACE2 receptors. All SARS-CoV-2 variants that have been recognized as being highly transmissible, such as the kappa (κ), delta (δ) and omicron (o) variants, which display an enhanced electropositive character in their RBDs associated with a higher number of lysine- or arginine-generating point mutations. Lysine and arginine residues also participate in the enhanced RBD–ACE2 binding affinity of the omicron variant, by creating additional salt bridges with aspartic and glutamic acid residues from ACE2. However, the effects of lysine- and arginine-generating point mutations on infectivity is more contrasted, since the overall binding affinity of omicron RBD for ACE2 apparently results from some epistasis among the whole set of point mutations.

Structural basis for the catalytic activity of filamentous human serine beta-lactamase-like protein LACTB

Serine beta-lactamase-like protein (LACTB) is a mammalian mitochondrial serine protease that can specifically hydrolyze peptide bonds adjacent to aspartic acid residues and is structurally related to prokaryotic penicillin-binding proteins. Here, we determined the cryoelectron microscopy structures of human LACTB (hLACTB) filaments from wild-type protein, a middle region deletion mutant, and in complex with the inhibitor Z-AAD-CMK at 3.0-, 3.1-, and 2.8-Å resolution, respectively. Structural analysis and activity assays revealed that three interfaces are required for the assembly of hLACTB filaments and that the formation of higher order helical structures facilitates its cleavage activity. Further structural and enzymatic analyses of middle region deletion constructs indicated that, while this region is necessary for substrate hydrolysis, it is not required for filament formation. Moreover, the inhibitor-bound structure showed that hLACTB may cleave peptide bonds adjacent to aspartic acid residues. These findings provide the structural basis underlying hLACTB catalytic activity.

Verticillium dahliae CFEM proteins manipulate host immunity and differentially contribute to virulence

BackgroundVerticillium dahliae is a fungal pathogen that causes a vascular wilt on many economically important crops. Common fungal extracellular membrane (CFEM) domain proteins including secreted types have been implicated in virulence, but their roles in this pathogen are still unknown.ResultsNine secreted small cysteine-rich proteins (VdSCPs) with CFEM domains were identified by bioinformatic analyses and their differential suppression of host immune responses were evaluated. Two of these proteins, VdSCP76 and VdSCP77, localized to the plant plasma membrane owing to their signal peptides and mediated broad-spectrum suppression of all immune responses induced by typical effectors. Deletion of either VdSCP76 or VdSCP77 significantly reduced the virulence of V. dahliae on cotton. Furthermore, VdSCP76 and VdSCP77 suppressed host immunity through the potential iron binding site conserved in CFEM family members, characterized by an aspartic acid residue in seven VdSCPs (Asp-type) in contrast with an asparagine residue (Asn-type) in VdSCP76 and VdSCP77. V. dahliae isolates carrying the Asn-type CFEM members were more virulent on cotton than those carrying the Asp-type.ConclusionsIn the iron-insufficient xylem, V. dahliae is likely to employ the Asp-type CFEM members to chelate iron, and Asn-type CFEM members to suppress immunity, for successful colonization and propagation in host plants.

Non-Canonical Roles of Apoptotic Caspases in the Nervous System.

Caspases are a family of cysteine proteases that predominantly cleave their substrates after aspartic acid residues. Much of what we know of caspases emerged from investigation a highly conserved form of programmed cell death called apoptosis. This form of cell death is regulated by several caspases, including caspase-2, caspase-3, caspase-7, caspase-8 and caspase-9. However, these “killer” apoptotic caspases have emerged as versatile enzymes that play key roles in a wide range of non-apoptotic processes. Much of what we understand about these non-apoptotic roles is built on work investigating how “killer” caspases control a range of neuronal cell behaviors. This review will attempt to provide an up to date synopsis of these roles.

Delineating the interactions between the cannabinoid CB2 receptor and its regulatory effectors; β-arrestins and GPCR kinases.

The cannabinoid CB2 receptor (CB2 ) is a promising therapeutic target for modulating inflammation. However, little is known surrounding the mechanisms underpinning CB2 desensitisation and regulation, particularly the role of GPCR kinases (GRKs). Here, we evaluated the role of six GRK isoforms in β-arrestin recruitment to CB2 . Mutagenesis of several distal C-terminal aspartic acid residues was also performed in an attempt to delineate additional structural elements involved in the regulation of CB2 . In CB2 -expressing HEK 293 cells, β-arrestin translocation was measured using real-time BRET assays. G protein dissociation BRET assays were performed to assess the activation and desensitisation of CB2 in the presence of β-arrestin 2. Overexpression of GRK isoforms 1-6 failed to considerably improve translocation of either β-arrestin 1 or β-arrestin 2 to CB2 . Consistent with this, inhibition of endogenous GRK2/3 did not substantially reduce β-arrestin 2 translocation. Mutagenesis of C-terminal aspartic acid residues resulted in attenuation of β-arrestin 2 translocation, which translated to a reduction in desensitisation of G protein activation. Our findings suggest that CB2 does not adhere to the classical GPCR regulatory paradigm, entailing GRK-mediated and β-arrestin-mediated desensitisation. Instead, C-terminal aspartic acid residues may act as phospho-mimics to induce β-arrestin activation. This study provides novel insights into the regulatory mechanisms of CB2 , which may aid in our understanding of drug tolerance and dependence.

Detailed Analyses of Molecular Interactions between Favipiravir and RNA Viruses In Silico.

There are currently no antiviral agents for human metapneumovirus (HMPV), respiratory syncytial virus (RSV), mumps virus (MuV), or measles virus (MeV). Favipiravir has been developed as an anti-influenza agent, and this agent may be effective against these viruses in vitro. However, the molecular mechanisms through which the agent affects virus replication remain to be fully elucidated. Thus, to clarify the detailed molecular interactions between favipiravir and the RNA-dependent RNA polymerase (RdRp) of HMPV, RSV, MuV, MeV, and influenza virus, we performed in silico studies using authentic bioinformatics technologies. As a result, we found that the active form of favipiravir (favipiravir ribofuranosyl-5′-triphosphate [F-RTP]) can bind to the RdRp active sites of HMPV, RSV, MuV, and MeV. The aspartic acid residue of RdRp active sites was involved in the interaction. Moreover, F-RTP was incorporated into the growing viral RNA chain in the presence of nucleotide triphosphate and magnesium ions. The results suggested that favipiravir shows two distinct mechanisms in various viruses: RdRp active site inhibition and/or genome replication inhibition.

PIMT-Mediated Labeling of l-Isoaspartic Acid with Tris Facilitates Identification of Isomerization Sites in Long-Lived Proteins.

Isomerization of individual residues in long-lived proteins (LLPs) is a subject of growing interest in connection with many age-related human diseases. When isomerization occurs in LLPs, it can lead to deleterious changes in protein structure, function, and proteolytic degradation. Herein, we present a novel labeling technique for rapid identification of l-isoAsp using the enzyme protein l-isoaspartyl methyltransferase (PIMT) and Tris. The succinimide intermediate formed during reaction of l-isoAsp-containing peptides with PIMT and S-adenosyl methionine (SAM) is reactive with Tris base and results in a Tris-modified aspartic acid residue with a mass shift of +103 Da. Tris-modified aspartic acid exhibits prominent and repeated neutral loss of water when subjected to collisional activation. In addition, another dissociation pathway regenerates the original peptide following loss of a characteristic mass shift. Furthermore, it is demonstrated that Tris modification can be used to identify sites of isomerization in LLPs from biological samples such as the lens of the eye. This approach simplifies identification by labeling isomerization sites with a tag that causes a mass shift and provides characteristic loss during collisional activation.

TanP: A Multifunctional Anionic Peptide From Tityus stigmurus Scorpion Venom.

Anionic peptides of scorpions are molecules rich in aspartic and/or glutamic acid residues and correspond to a class of peptides without disulfide bonds that are still little explored. TanP is a linear anionic peptide (50 amino acid residues and net charge −20) present in the venom gland of the scorpion, Tityus stigmurus, with chelating properties for Cu2+ ion and immunomodulatory properties. The therapeutic application of chelating molecules is related to cases of acute or chronic intoxication by metals, neurodegenerative diseases, hematological diseases, healing of skin wounds, cardiovascular diseases, and cancer. In this approach, the chelating activity of TanP was evaluated in relation to new metal ions (Fe2+ and Zn2+) of biological importance, as well as its antioxidant, hemostatic, immunomodulatory, and healing potential, aiming to expand the biological and biotechnological potential of this peptide. TanP (25 µM) was able to form stable complexes with Fe2+ in a ratio of 1:5 (TanP: Fe2+). Theoretical results suggest that TanP can work as a sensor to identify and quantify Fe2+ ions. The fluorescence intensity of TanP (1.12 µM) decreased significantly after the addition of Fe2+, obtaining the highest ratio 1: 7.4 (TanP: Fe2+) that led to the lowest fluorescence intensity. For Zn2+, no relevant spectral change was noted. TanP (50 µM) showed a maximum of 3% of hemolytic activity, demonstrating biocompatibility, as well as exhibiting a 1,1-diphenyl-2-picrylhydrazyl radical–scavenging activity of above 70% at all the concentrations tested (1–25 μM), and 89.7% iron-chelating activity at 25 μM and 96% hydroxyl radical–scavenging activity at 73.6 μM. In addition, TanP (12.5 and 25 µM) revealed an anticoagulant effect, prolonging the clotting time in prothrombin time and activated partial thromboplastin time assays, with no fibrinogenolytic activity. TanP (12.5 and 25 µM) induced the release of TNF-α by murine macrophages, in the absence of lipopolysaccharides, with a concentration-dependent increase and also stimulated the migration of 3T3 cells in the in vitro healing assay. Thus, TanP revealed a multifunctional potential, being useful as a prototype for the development of new therapeutic and biotechnological agents.

A negative charge at position D+5 of Motif A is critical for function of the major facilitator superfamily multidrug/H+antiporter MdtM

Abstract The phenomenon of antimicrobial resistance represents a major public health risk. The activity of integral membrane transporter proteins contributes to antimicrobial resistance in pathogenic bacteria and proton gradient-driven multidrug efflux representatives of the major facilitator superfamily (MFS) of secondary transporters are the dominant antimicrobial efflux proteins in Escherichia coli. In many, but not all, of the characterized MFS multidrug transporters, an aspartic acid residue at position D+5 of the conserved signature Motif A is essential for transport activity. The present work extends those studies to the E. coli MFS multidrug/H+ antiporter MdtM and used a combination of mutagenesis, expression studies, antimicrobial resistance assays, and transport activity measurements to reveal that a negatively charged residue at position D+5 is critical for MdtM transport function.

Dual enzymatic inhibitory mechanism of WM382 on plasmepsin IX and X: Atomistic perspectives from dynamic analysis

The unprecedented occurrence of malaria and the increase rate of parasite resistance to current therapeutics have over the past decade elicited researchers’ interest in probing novel drug targets and unabatedly searching for competitive inhibitors. Plasmepsin (PM) family of aspartic proteases has recently been at the forefront in this pursuit to augment malaria treatment. In malaria infections engineered by P. falciparum, PMIX and PMX are highlighted as mediators of egress, invasion and spread of the infection in humans. Despite these proteases' druggability, there is currently no authorized inhibitor. WM382, a small molecule inhibitor, has been demonstrated to have dual inhibitory properties against both proteases, which is promising. However, the structural dynamics associated with the dual inhibition mechanism remain unclear. As such, we employed integrative computer-assisted atomistic techniques to provide thorough structural dynamic insights. The binding WM382 confers structural stability on both proteases through a mesh of hydrogen and hydrophobic interactions with binding site residues. The overall binding energies of PMIX and PMX were estimated to be −27.27 kcal/mol and −23.95 kcal/mol respectively, however, the trademark aspartic acid residues in the binding pocket of these proteases contributed minimally to this effect. WM382 increased structural flexibility creating excessively loose conformation that distorts the characteristic “twist motion” of the members of the plasmepsin family. These assimilated conformations further affect the biological role of both proteases. Energy decomposition analysis proved that Phe77/Phe75 contributed significantly to the overall binding of WM382 within the binding pockets of both PMIX and PMX eliciting strong intermolecular interactions, thus favouring binding affinity. The stable orientations of WM382 within the respective hydrophobic pockets allowed favorable interactions with binding site residues which accounted for its high binding free energy.

Top-down proteomic identification of plasmid and host proteins produced by pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry.

Fourteen proteins produced by three pathogenic Escherichia coli strains were identified using antibiotic induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis using software developed in-house. Host proteins as well as plasmid proteins were identified. Mature, intact protein ions were fragmented by post-source decay (PSD), and prominent fragment ions resulted from the aspartic acid effect fragmentation mechanism wherein polypeptide backbone cleavage (PBC) occurs on the C-terminal side of aspartic acid (D), glutamic acid (E) and asparagine (N) residues. These highly specific MS/MS-PSD fragment ions were compared to b- and y-type fragment ions on the C-terminal side of D-, E- and N-residues of in silico protein sequences derived from whole genome sequencing. Nine proteins were found to be post-translationally modified with either removal of an N-terminal methionine or a signal peptide. The protein sequence truncation algorithm of our software correctly identified all full and truncated protein sequences. Truncated sequences were compared to those predicted by SignalP. Nearly complete concurrence was obtained except for one protein where SignalP mis-identified the cleavage site by one residue. Two proteins had intramolecular disulfide bonds that were inferred by the absence of PBC on the C-terminal side of a D-residue located within the disulfide loop. These results demonstrate the utility of MALDI-TOF-TOF for identification of full and truncated bacterial proteins.

Aspartate-phobia of thermophiles as a reaction to deleterious chemical transformations.

Prokaryotes growing at high temperatures have a high proportion of charged residues in their proteins to stabilize their 3D structure. By mining 175 disparate bacterial and archaeal proteomes we found that, against the general trend for charged residues, the frequency of aspartic acid residues decreases strongly as natural growth temperature increases. In search of the explanation, we hypothesized that the reason for such unusual correlation is the deleterious consequences of spontaneous chemical transformations of aspartate at high temperatures. Our subsequent statistical analysis supported this hypothesis. This finding reveals that organisms have likely adapted to high temperatures by minimizing the harmful consequences of spontaneous chemical transformations.

A molecular dynamics-guided mutagenesis identifies two aspartic acid residues involved in the pH-dependent activity of OG-OXIDASE 1

During the infection, plant cells secrete different OG-oxidase (OGOX) paralogs, defense flavoproteins that oxidize the oligogalacturonides (OGs), homogalacturonan fragments released from the plant cell wall that act as Damage Associated Molecular Patterns. OGOX-mediated oxidation inactivates their elicitor nature, but on the other hand makes OGs less hydrolysable by microbial endo-polygalacturonases (PGs). Among the different plant defense responses, apoplastic alkalinization can further reduce the degrading potential of PGs by boosting the oxidizing activity of OGOXs. Accordingly, the different OGOXs so far characterized showed an optimal activity at pH values greater than 8. Here, an approach of molecular dynamics (MD)-guided mutagenesis succeeded in identifying the amino acids responsible for the pH dependent activity of OGOX1 from Arabidopsis thaliana. MD simulations indicated that in alkaline conditions (pH 8.5), the residues Asp325 and Asp344 are engaged in the formation of two salt bridges with Arg327 and Lys415, respectively, at the rim of enzyme active site. According to MD analysis, the presence of such ionic bonds modulates the size and flexibility of the cavity used to accommodate the OGs, in turn affecting the activity of OGOX1. Based on functional properties of the site-directed mutants OGOX1.D325A and OGOX.D344A, we demonstrated that Asp325 and Asp344 are major determinants of the alkaline-dependent activity of OGOX1.

Functional Characterisation of Bile Metagenome: Study of Metagenomic Dark Matter.

Gallbladder metagenome involves a wide range of unidentified sequences comprising the so-called metagenomic dark matter. Therefore, this study aimed to characterise three gallbladder metagenomes and a fosmid library with an emphasis on metagenomic dark matter fraction. For this purpose, a novel data analysis strategy based on the combination of remote homology and molecular modelling has been proposed. According to the results obtained, several protein functional domains were annotated in the metagenomic dark matter fraction including acetyltransferases, outer membrane transporter proteins, membrane assembly factors, DNA repair and recombination proteins and response regulator phosphatases. In addition, one deacetylase involved in mycothiol biosynthesis was found in the metagenomic dark matter fraction of the fosmid library. This enzyme may exert a protective effect in Actinobacteria against bile components exposure, in agreement with the presence of multiple antibiotic and multidrug resistance genes. Potential mechanisms of action of this novel deacetylase were elucidated by molecular simulations, highlighting the role of histidine and aspartic acid residues. Computational pipelines presented in this work may be of special interest to discover novel microbial enzymes which had not been previously characterised.

Role of aspartic acid residues D87 and D89 in APS kinase domain of human 3′-phosphoadenosine 5′-phosphosulfate synthase 1 and 2b: A commonality with phosphatases/kinases

3′-phosphoadenosine 5′-phosphosulfate (PAPS) is synthesized in two steps by PAPS synthase (PAPSS). PAPSS is comprised of ATP sulfurylase (ATPS) and APS kinase (APSK) domain activities. ATPS combines inorganic sulfate with α-phosphoryl of ATP to form adenosine 5′-phosphosulfate (APS) and PPi. In the second step APS is phosphorylated at 3′-OH using another mole of ATP to form PAPS and ADP catalyzed by APSK. The transfer of gamma-phosphoryl from ATP onto 3′-OH requires Mg2+ and purported to involve residues D87GD89N. We report that mutation of either aspartic residue to alanine completely abolishes APSK activity in PAPS formation. PAPSS is an, unique enzyme that binds to four different nucleotides: ATP and APS on both ATPS and APSK domains and ADP and PAPS exclusively on the APSK domain. The thermodynamic binding and the catalytic interplay must be very tightly controlled to form the end-product PAPS in the forward direction. Though APS binds to ATPS and APSK, in ATPS domain, the APS is a product and for APSK it is a substrate. DGDN motif is absent in ATPS and present in APSK. Mutation of D87 and D89 did not hamper ATPS activity however abolished APSK activity severely. Thus, D87GD89N region is required for stabilization of Mg2+-ATP, in the process of splitting the γ-phosphoryl from ATP and transfer of γ-phosphoryl onto 3′-OH of APS to form PAPS a process that cannot be achieved by ATPS domain. In addition, gamma32P-ATP, trapped phosphoryl enzyme intermediate more with PAPSS2 than with PAPSS1. This suggests inherent active site residues could control novel catalytic differences. Molecular docking studies of hPAPSS1with ATP + Mg2+ and APS of wild type and mutants supports the experimental results.

Repeating Aspartic Acid Residues Prefer Turn-like Conformations in the Unfolded State: Implications for Early Protein Folding.

Protein folding can be described as a motion of the polypeptide chain in a potential energy funnel, where the conformational manifold is narrowed as the chain traverses from a completely unfolded state until it reaches the folded (native) state. The initial folding stages set the tone for this process by substantially narrowing the manifold of accessible conformations. In an ideally unfolded state with no long-range stabilizing forces, local conformations (i.e., residual structures) are likely to drive the folding process. While most amino acid residues tend to predominantly adopt extended structures in unfolded proteins and peptides, aspartic acid exhibits a relatively high intrinsic preference for turn-forming conformations. Regions in an unfolded polypeptide or protein that are rich in aspartic acid residues may therefore be crucial sites for protein folding steps. By combining NMR and vibrational spectroscopies, we observed that the conformational sampling of multiple sequentially neighbored aspartic acid residues in the model peptides GDDG and GDDDG even show an on average higher propensity for turn-forming structures than the intrinsic reference system D in GDG, which suggests that nearest neighbor interactions between adjacent aspartic acid residues stabilize local turn-forming structures. In the presence of the unlike neighbor phenylalanine, nearest neighbor interactions are of a totally different nature in that it they decrease the turn-forming propensities and mutually increase the sampling of polyproline II (pPII) conformations. We hypothesize the structural role of aspartic residues in intrinsically disordered proteins in general, and particularly in small linear motifs, that are very much determined by their respective neighbors.