Conformation Of Residues Research Articles

Molecular profiling of ginsenoside metabolites to identify estrogen receptor alpha activity.

20(S)-Protopanaxadiol (PPD) and 20(S)-Protopanaxatriol (PPT) are major metabolites of ginseng in humans and are considered to have estrogenic activity in cellular bioassays. In this study, we conducted in silico analyses to determine whether PPD and PPT interact with estrogen receptor alpha (ERα) and compared them with ERα agonists, partial agonists, and antagonists to identify their ERα activity. The transcriptome profile of 17β-estradiol (E2), PPD, and PPT in MCF-7 cells expressing ERα was further compared to understand the ERα activity of ginsenoside metabolites. The results showed that PPD and PPT interacted with the 1ERE, 1GWR, and 3UUD ERα proteins in the E2 interaction model, the 3ERD protein in the diethylstilbestrol (DES) interaction model, and the 1X7R protein in the genistein (GEN) interaction model. Conversely, neither the 4PP6 protein of the interaction model with the antagonist resveratrol (RES) nor the 1ERR protein of the interaction model with the antagonist raloxifene (RAL) showed the conformation of amino acid residues. When E2, PPD, and PPT were exposed to MCF-7 cells, cell proliferation and gene expression were observed. The transcriptomic profiles of E2, PPD, and PPT were compared using a knowledge-based pathway. PPD-induced transcription profiling was similar to that of E2, and the neural transmission pathway was detected in both compounds. In contrast, PPT-induced transcription profiling displayed characteristics of gene expression associated with systemic lupus erythematosus. These results suggest that ginsenoside metabolites have ERα agonist activity and exhibit neuroprotective effects and anti-inflammatory actions. However, a meta-analysis using public microarray data showed that the mother compounds GRb1 and GRg1 of PPD and PPT showed metabolic functions in insulin signaling pathways, condensed DNA repair and cell cycle pathways, and immune response and synaptogenesis. These results suggest that the ginsenoside metabolites have potent ERα agonist activity; however, their gene expression profiles may differ from those of E2.

Structures and implications of the nuclease domain of human parvovirus B19 NS1 protein

Infection of human parvovirus B19 (B19V) can cause a variety of diseases, such as hydrops fetalis, erythema infectiosum in children and acute arthropathy in women. Although B19V infection mainly occurs during childhood, about 50 % of adults are still susceptible to B19V infection. As the major replication protein of B19V, deletion of NS1 completely abolishes the infectivity of the virus. The nuclease domain of NS1 (NS1_Nuc) is responsible for DNA Ori binding and nicking that is critical for B19V viral DNA replication. NS1 has various variants, the structure and function for the majority of the variants are poorly studied. Here, we report two high-resolution crystal structures of NS1_Nuc, revealed the detailed conformations of many key residues. Structural comparison indicates that these residues are important for ssDNA or dsDNA binding by NS1. NS1 belongs to the HUH-endonuclease superfamily and it shares conserved ssDNA cleavage mechanism with other HUH-endonuclease members. However, our structural analyses, mutagenesis and in vitro assay results all suggested that NS1_Nuc utilizes one unique model in ssDNA binding.

Effect of a cis-4-aminopiperidine-3-carboxylic acid (cis-APiC) residue on mixed-helical folding of unnatural peptides.

The α/β-peptide 11/9-helix and the β-peptide 12/10-helix belong to "mixed" helices, in which two types of hydrogen bonds with opposite directionality alternate along the helical axis. cis-2-Aminocyclohexanecarboxylic acid (cis-ACHC) is known to promote these mixed helices and stabilize the helical propensity more than other acyclic β-residues. Application of a mixed-helical backbone still requires sufficient solubility in aqueous solution. In this regard, we chose cis-4-aminopiperidine-3-carboxylic acid (cis-APiC) as a foldamer building block that can provide both sufficient aqueous solubility and mixed-helical propensity. Conformational analyses of α/β- and β-peptides containing a cis-APiC residue by circular dichroism spectroscopy and single-crystal X-ray crystallography suggest that the incorporation of cis-APiC instead of cis-ACHC can enhance the aqueous solubility of the mixed-helical peptides without any adverse effect on helical folding. In addition, the ratio between right- and left-handed 12/10-helices of β-peptides can be rationalized by relative energies between the local conformations of the cis-APiC residue.

The NKT cell TCR repertoire can accommodate structural modifications to the lipid and orientation of the terminal carbohydrate of iGb3

Isoglobotrihexosylceramide (iGb3) is a known NKT cell agonist, however the specific interactions required to trigger NKT cell TCR activation in response to this mammalian glycolipid are not fully understood. Here we report the synthesis of 1,3-β-Gal-LacCer (βG-iGb3) that displays a β-linked terminal sugar. βG-iGb3 activated NKT cells to a similar extent as iGb3 with a terminal α-linkage, indicating that the conformation of the terminal sugar residue of iGb3 is not essential to facilitate NKT cell TCR recognition. In addition, the immunological activity of four recently described iGb3 analogues with modifications to their terminal sugar or lipid backbone were also investigated. These iGb3 analogues all induced NKT cell proliferation, with IL-13 the predominate cytokine detected. This highlights the ability of the NKT cell TCR to accommodate variations in iGb3-based glycolipids and suggests that undiscovered NKT cell ligands may exist within the lacto-series of mammalian glycosphingolipids.

Symmetric activation and modulation of the human calcium-sensing receptor

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca2+ levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near-full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.

Fragment-Based Ligand Discovery Applied to the Mycolic Acid Methyltransferase Hma (MmaA4) from Mycobacterium tuberculosis: A Crystallographic and Molecular Modelling Study.

The mycolic acid biosynthetic pathway represents a promising source of pharmacological targets in the fight against tuberculosis. In Mycobacterium tuberculosis, mycolic acids are subject to specific chemical modifications introduced by a set of eight S-adenosylmethionine dependent methyltransferases. Among these, Hma (MmaA4) is responsible for the introduction of oxygenated modifications. Crystallographic screening of a library of fragments allowed the identification of seven ligands of Hma. Two mutually exclusive binding modes were identified, depending on the conformation of residues 147–154. These residues are disordered in apo-Hma but fold upon binding of the S-adenosylmethionine (SAM) cofactor as well as of analogues, resulting in the formation of the short η1-helix. One of the observed conformations would be incompatible with the presence of the cofactor, suggesting that allosteric inhibitors could be designed against Hma. Chimeric compounds were designed by fusing some of the bound fragments, and the relative binding affinities of initial fragments and evolved compounds were investigated using molecular dynamics simulation and generalised Born and Poisson–Boltzmann calculations coupled to the surface area continuum solvation method. Molecular dynamics simulations were also performed on apo-Hma to assess the structural plasticity of the unliganded protein. Our results indicate a significant improvement in the binding properties of the designed compounds, suggesting that they could be further optimised to inhibit Hma activity.

Structural basis of constitutive c-Src kinase activity due to R175L and W118A mutations

Cellular Src (c-Src) belongs to a non-receptor membrane-associated tyrosine kinase family that plays essential roles in cellular processes. Growing evidence suggests that R175L and W118A mutations in SH2/SH3 domains of c-Src functionally inactivate these domains leading to constitutive activation of kinase domain (KD). Here we modeled c-SrcR175L, c-SrcW118A and c-SrcW118A+R175L structures by inducing phosphorylation at Y416 or Y527, respectively to characterize the comparative dynamics in the active versus inactive states through molecular dynamics simulation assay. We observed more conformational readjustments in c-Srcopen than its close variants. In particular, C-terminal tail residues of c-SrcW118A-open and c-SrcW118A+R175L-open demonstrate significantly higher transitions. The cross-correlation analysis revealed an anticorrelation behavior in the motion of KD with respect to SH2, SH3 and the linker region of SrcW118A+R175L-open, while in c-SrcWT-open, SH2 and SH3 domains were anticorrelated, while KD and C-terminal tail motions were correlated. Due to these conformational differences, c-Src open forms exhibited lower interaction between pY527 and SH2 domain. Through detailed structural analysis, we observed a uniform myristate binding cavity in c-SrcWT-open, while the myristoyl pockets of mutant forms were deformed. We propose that constitutive activation of mutant Src forms may presumably be achieved by the prolonged membrane binding due to unusual conformations of C-terminal and myristoyl switch residues that may result in a higher dephosphorylation rate at pY527 in the myristoylated c-Src. Thus, our study establishes novel clues to decipher the constitutive activation status of c-Src in response to known mutations that may help in devising novel therapeutic strategies for cancer metastasis treatment. Communicated by Ramaswamy H. Sarma

Arabidopsis N-acetyltransferase activity 2 preferentially acetylates 1,3-diaminopropane and thialysine

Polyamine acetylation has an important regulatory role in polyamine metabolism. It is catalysed by GCN5-related N-acetyltransferases, which transfer acetyl groups from acetyl-coenzyme A to the primary amino groups of spermidine, spermine (Spm), or other polyamines and diamines, as was shown for the human Spermidine/Spermine N1-acetyltransferase 1 (HsSSAT1). SSAT homologues specific for thialysine, a cysteine-derived lysine analogue, were also identified (e.g., HsSSAT2). Two HsSSAT1 homologues are present in Arabidopsis, namely N-acetyltransferase activity (AtNATA) 1 and 2. AtNATA1 was previously shown to be specific for 1,3-diaminopropane, ornithine, putrescine and thialysine, rather than Spm and spermidine. In the present study, in an attempt to find a plant Spm-specific SSAT, AtNATA2 was expressed in a heterologous bacterial system and catalytic properties of the recombinant protein were determined. Data indicate that recombinant AtNATA2 preferentially acetylates 1,3-diaminopropane and thialysine, throwing further light on AtNATA1 substrate specificity. Structural analyses evidenced that the preference of AtNATA1, AtNATA2 and HsSSAT2 for short amine substrates can be ascribed to different main-chain conformation or substitution of HsSSAT1 residues interacting with Spm distal regions. Moreover, gene expression studies evidenced that AtNATA1 gene, but not AtNATA2, is up-regulated by cytokinins, thermospermine and Spm, suggesting the existence of a link between AtNATAs and N1-acetyl-Spm metabolism. This study provides insights into polyamine metabolism and structural determinants of substrate specificity of non Spm-specific SSAT homologues.

Bioinformatics-Guided Expansion and Discovery of Graspetides.

Graspetides are a class of ribosomally synthesized and post-translationally modified peptide natural products featuring ATP-grasp ligase-dependent formation of macrolactones/macrolactams. These modifications arise from serine, threonine, or lysine donor residues linked to aspartate or glutamate acceptor residues. Characterized graspetides include serine protease inhibitors such as the microviridins and plesiocin. Here, we report an update to Rapid ORF Description and Evaluation Online (RODEO) for the automated detection of graspetides, which identified 3,923 high-confidence graspetide biosynthetic gene clusters. Sequence and co-occurrence analyses doubled the number of graspetide groups from 12 to 24, defined based on core consensus sequence and putative secondary modification. Bioinformatic analyses of the ATP-grasp ligase superfamily suggest that extant graspetide synthetases diverged once from an ancestral ATP-grasp ligase and later evolved to introduce a variety of ring connectivities. Furthermore, we characterized thatisin and iso-thatisin, two graspetides related by conformational stereoisomerism from Lysobacter antibioticus. Derived from a newly identified graspetide group, thatisin and iso-thatisin feature two interlocking macrolactones with identical ring connectivity, as determined by a combination of tandem mass spectrometry (MS/MS), methanolytic, and mutational analyses. NMR spectroscopy of thatisin revealed a cis conformation for a key proline residue, while molecular dynamics simulations, solvent-accessible surface area calculations, and partial methanolytic analysis coupled with MS/MS support a trans conformation for iso-thatisin at the same position. Overall, this work provides a comprehensive overview of the graspetide landscape, and the improved RODEO algorithm will accelerate future graspetide discoveries by enabling open-access analysis of existing and emerging genomes.

Activation of Abl1 Kinase Explored Using Well-Tempered Metadynamics Simulations on an Essential Dynamics Sampled Path.

Well-tempered metadynamics (wT-metaD) simulations using path collective variables (CVs) have been successfully applied in recent years to explore conformational transitions in protein kinases and other biomolecular systems. While this methodology has the advantage of describing the transitions with a limited number of predefined path CVs, it requires as an input a reference path connecting the initial and target states of the system. It is desirable to automate the path generation using approaches that do not rely on the choice of geometric CVs to describe the transition of interest. To this end, we developed an approach that couples essential dynamics sampling with wT-metaD simulations. We used this newly developed procedure to explore the activation mechanism of Abl1 kinase and compute the associated free energy barriers. Through these simulations, we identified a three-step mechanism for the activation that involved two metastable intermediates that possessed a partially open activation loop and differed primarily in the “in” or “out” conformation of the aspartate residue of the DFG motif. One of these states is similar to a conformation that was detected in previous spectroscopic studies of Abl1 kinase, albeit its mechanistic role in the activation was hitherto not well understood. The present study establishes its intermediary role in the activation and predicts a rate-determining free energy barrier of 13.8 kcal/mol that is in good agreement with previous experimental and computational estimates. Overall, our study demonstrates the usability of essential dynamics sampling as a path CV in wT-metaD to conveniently study conformational transitions and accurately calculate the associated barriers.

Structural Comparisons of Cefotaximase (CTX-M-ase) Sub Family 1.

The cefotaximase or CTX-M, family of serine-β-lactamases represents a significant clinical concern due to the ability for these enzymes to confer resistance to a broad array of β-lactam antibiotics an inhibitors. This behavior lends CTX-M-ases to be classified as extended spectrum β-lactamases (ESBL). Across the family of CTX-M-ases most closely related to CTX-M-1, the structures of CTX-M-15 with a library of different ligands have been solved and serve as the basis of comparison within this review. Herein we focus on the structural changes apparent in structures of CTX-M-15 in complex with diazabicyclooctane (DABCO) and boronic acid transition state analog inhibitors. Interactions between a positive surface patch near the active site and complementary functional groups of the bound inhibitor play key roles in the dictating the conformations of active site residues. The insights provided by analyzing structures of CTX-M-15 in complex with DABCO and boronic acid transition state analog inhibitors and analyzing existing structures of CTX-M-64 offer opportunities to move closer to making predictions as to how CTX-M-ases may interact with potential drug candidates, setting the stage for the further development of new antibiotics and β-lactamase inhibitors.

Intermolecular insights into allosteric inhibition of histone lysine-specific demethylase 1

BackgroundHistone lysine-specific demethylase 1 (LSD1) has become a potential anticancer target for the novel drug discovery. Recent reports have shown that SP2509 and its derivatives strongly inhibit LSD1 as allosteric inhibitors. However, the binding mechanism of these allosteric inhibitors in the allosteric site of LSD1 is not known yet. MethodsThe stability and binding mechanism of allosteric inhibitors in the binding site of LSD1 were evaluated by molecular docking, ligand-based pharmacophore, molecular dynamics (MD) simulations, molecular mechanics generalized born surface area (MM/GBSA) analysis, quantum mechanics/molecular mechanics (QM/MM) calculation and Hirshfeld surface analysis. ResultsThe conformational geometry and the intermolecular interactions of allosteric inhibitors showed high binding affinity towards allosteric site of LSD1 with the neighboring amino acids (Gly358, Cys360, Leu362, Asp375 and Glu379). Meanwhile, MD simulations and MM/GBSA analysis were performed on selected allosteric inhibitors in complex with LSD1 protein, which confirmed the high stability and binding affinity of these inhibitors in the allosteric site of LSD1. ConclusionThe simulation results revealed the crucial factors accounting for allosteric inhibitors of LSD1, including different protein–ligand interactions, the positions and conformations of key residues, and the ligands flexibilities. Meanwhile, a halogen bond interaction between chlorine atom of ligand and key residues Trp531 and His532 was recurrent in our analysis confirming its importance. General significanceOverall, our research analyzed in depth the binding modes of allosteric inhibitors with LSD1 and could provide useful information for the design of novel allosteric inhibitors.

Structural differences in the FAD-binding pockets and lid loops of mammalian CRY1 and CRY2 for isoform-selective regulation

The circadian clock is a biological timekeeper that operates through transcription-translation feedback loops in mammals. Cryptochrome 1 (CRY1) and Cryptochrome 2 (CRY2) are highly conserved core clock components having redundant and distinct functions. We recently identified the CRY1- and CRY2-selective compounds KL101 and TH301, respectively, which provide useful tools for the exploration of isoform-selective CRY regulation. However, intrinsic differences in the compound-binding FAD (flavin adenine dinucleotide) pockets between CRY1 and CRY2 are not well understood, partly because of nonoptimal properties of previously reported apo form structures in this particular region constituted by almost identical sequences. Here, we show unliganded CRY1 and CRY2 crystal structures with well-defined electron densities that are largely free of crystal contacts at the FAD pocket and nearby lid loop. We revealed conformational isomerism in key residues. In particular, CRY1 W399 and corresponding CRY2 W417 in the FAD pocket had distinct conformations ("out" for CRY1 and "in" for CRY2) by interacting with the lid loop residues CRY1 Q407 and CRY2 F424, respectively, resulting in different overall lid loop structures. Molecular dynamics simulations supported that these conformations were energetically favorable to each isoform. Isoform-selective compounds KL101 and TH301 preferred intrinsic "out" and "in" conformations of the tryptophan residue in CRY1 and CRY2, respectively, while the nonselective compound KL001 fit to both conformations. Mutations of lid loop residues designed to perturb their isoform-specific interaction with the tryptophan resulted in reversed responses of CRY1 and CRY2 to KL101 and TH301. We propose that these intrinsic structural differences of CRY1 and CRY2 can be targeted for isoform-selective regulation.

Characteristic chemical probing patterns of loop motifs improve prediction accuracy of RNA secondary structures.

RNA structures play a fundamental role in nearly every aspect of cellular physiology and pathology. Gaining insights into the functions of RNA molecules requires accurate predictions of RNA secondary structures. However, the existing thermodynamic folding models remain less accurate than desired, even when chemical probing data, such as selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reactivities, are used as restraints. Unlike most SHAPE-directed algorithms that only consider SHAPE restraints for base pairing, we extract two-dimensional structural features encoded in SHAPE data and establish robust relationships between characteristic SHAPE patterns and loop motifs of various types (hairpin, internal, and bulge) and lengths (2–11 nucleotides). Such characteristic SHAPE patterns are closely related to the sugar pucker conformations of loop residues. Based on these patterns, we propose a computational method, SHAPELoop, which refines the predicted results of the existing methods, thereby further improving their prediction accuracy. In addition, SHAPELoop can provide information about local or global structural rearrangements (including pseudoknots) and help researchers to easily test their hypothesized secondary structures.

Crystal structure of breast regression protein 39 (BRP39), a signaling glycoprotein expressed during mammary gland apoptosis, at 2.6 Å resolution

Breast regression protein 39 (BRP39) is a 39 kDa protein that is a member of chitolectin class of glycosyl hydrolase family 18 (GH18). High expression levels of BRP39 have been detected in breast carcinoma. It helps in proliferation of cells during the progression of this disease and may act as a signaling factor. BRP39 may act as a potential candidate for rational structure-based drug design against breast carcinoma. In this study, we report the crystal structure of mouse recombinant BRP39 expressed in E. coli. The structure was solved by molecular replacement and refined to 2.6 Å resolution. The overall structure of BRP39 consisted of two globular domains: a large (β/α)8 triosephosphate isomerase (TIM) barrel domain and a small (α + β) domain. Three non-proline cis-peptides were detected in the sugar-binding cleft of BRP39, including Ser57-Phe58, Leu141-Tyr142, and Trp353-Ala354. The latter residues were conserved in other GH18 family members. It was notable that the conformation of critical Trp100 residue within the sugar-binding cleft was oriented away from the barrel. The side-chain conformation was found to be similar to that observed in chitinases, however, it was oriented into the barrel in other chitinase-like proteins (CLPs). The conformation of this critical residue may have significant implications in sugar binding. Further, two amino acid substitutions were observed in the sugar-binding groove of BRP39. The conserved Asn100 and Arg263 in Hcgp39 and other CLPs proteins (SPX-40 structures) were substituted by Lys101 and Lys264 in BRP39 which may have a significant impact on the sugar-binding properties.

CS-Annotate: A Tool for Using NMR Chemical Shifts to Annotate RNA Structure.

Here, we introduce CS-Annotate, a tool that uses assigned NMR chemical shifts to annotate structural features in RNA. At its core, CS-Annotate is a deployment of a multitask deep learning model that simultaneously classifies the solvent exposure, base-stacking and -pairing status, and conformation of individual RNA residues from their chemical shift fingerprint. Here, we briefly describe how we trained and tested the classifier and demonstrate its application to a model RNA system. CS-Annotate can be accessed via the SMALTR (Structure-based MAchine Learning Tools for RNA) Science Gateway (smaltr.org).

Recapitulating the Binding Affinity of Nrf2 for KEAP1 in a Cyclic Heptapeptide, Guided by NMR, X-ray Crystallography, and Machine Learning.

Macrocycles, including macrocyclic peptides, have shown promise for targeting challenging protein-protein interactions (PPIs). One PPI of high interest is between Kelch-like ECH-Associated Protein-1 (KEAP1) and Nuclear Factor (Erythroid-derived 2)-like 2 (Nrf2). Guided by X-ray crystallography, NMR, modeling, and machine learning, we show that the full 20 nM binding affinity of Nrf2 for KEAP1 can be recapitulated in a cyclic 7-mer peptide, c[(D)-β-homoAla-DPETGE]. This compound was identified from the Nrf2-derived linear peptide GDEETGE (KD = 4.3 μM) solely by optimizing the conformation of the cyclic compound, without changing any KEAP1 interacting residue. X-ray crystal structures were determined for each linear and cyclic peptide variant bound to KEAP1. Despite large variations in affinity, no obvious differences in the conformation of the peptide binding residues or in the interactions they made with KEAP1 were observed. However, analysis of the X-ray structures by machine learning showed that locations of strain in the bound ligand could be identified through patterns of subangstrom distortions from the geometry observed for unstrained linear peptides. We show that optimizing the cyclic peptide affinity was driven partly through conformational preorganization associated with a proline substitution at position 78 and with the geometry of the noninteracting residue Asp77 and partly by decreasing strain in the ETGE motif itself. This approach may have utility in dissecting the trade-off between conformational preorganization and strain in other ligand-receptor systems. We also identify a pair of conserved hydrophobic residues flanking the core DxETGE motif which play a conformational role in facilitating the high-affinity binding of Nrf2 to KEAP1.

ПРОСТРАНСТВЕННАЯ СТРУКТУРА НЕЙРОПЕПТИДА MET-CALLATOSTATIN

In work by the method of molecular mechanics in approximation of atom-atom potential functions is studied the spatial structure and conformational properties of the molecules from allatostatin family, got the name of Met-callatostatin. The researches were based on minimizing of the energy of intramolecular interactions (non-bonded, electrostatic, torsion) in the vicinity of the internal rotation angles corresponding to all combinations of stable conformations of monopeptide residues forming a molecule. There determined ten energetically stable conformations of molecules, implemented under the conditions of implicitly given aqueous environment, the relative conformational energy of which varies in the range of 0-10 kcal / mol. It was shown that the system of hydrogen bonds, despite a small contribution to the total energy of the molecule is essential for the preservation of a stable structure and restriction of Met-callatostatin. Key words: neuropeptides, structure, conformational analysis, peptides, by molecular mechanics

Differential lysine-mediated allosteric regulation of plant dihydrodipicolinate synthase isoforms.

Lysine biosynthesis in plants occurs via the diaminopimelate pathway. The first committed and rate-limiting step of this pathway is catalysed by dihydrodipicolinate synthase (DHDPS), which is allosterically regulated by the end product, l-lysine (lysine). Given that lysine is a common nutritionally limiting amino acid in cereal crops, there has been much interest in probing the regulation of DHDPS. Interestingly, knockouts in Arabidopsisthaliana of each isoform (AtDHDPS1 and AtDHDPS2) result in different phenotypes, despite the enzymes sharing >85% protein sequence identity. Accordingly, in this study, we compared the catalytic activity, lysine-mediated inhibition and structures of both A.thaliana DHDPS isoforms. We found that although the recombinantly produced enzymes have similar kinetic properties, AtDHDPS1 is 10-fold more sensitive to lysine. We subsequently used X-ray crystallography to probe for structural differences between the apo- and lysine-bound isoforms that could account for the differential allosteric inhibition. Despite no significant changes in the overall structures of the active or allosteric sites, we noted differences in the rotamer conformation of a key allosteric site residue (Trp116) and proposed that this could result in differences in lysine dissociation. Microscale thermophoresis studies supported our hypothesis, with AtDHDPS1 having a ~6-fold tighter lysine dissociation constant compared to AtDHDPS2, which agrees with the lower half minimal inhibitory concentration for lysine observed. Thus, we highlight that subtle differences in protein structures, which could not have been predicted from the primary sequences, can have profound effects on the allostery of a key enzyme involved in lysine biosynthesis in plants. DATABASES: Structures described are available in the Protein Data Bank under the accession numbers 6VVH and 6VVI.

A Single-Point Mutation in d-Arginine Dehydrogenase Unlocks a Transient Conformational State Resulting in Altered Cofactor Reactivity.

Proteins are inherently dynamic, and proper enzyme function relies on conformational flexibility. In this study, we demonstrated how an active site residue changes an enzyme's reactivity by modulating fluctuations between conformational states. Replacement of tyrosine 249 (Y249) with phenylalanine in the active site of the flavin-dependent d-arginine dehydrogenase yielded an enzyme with both an active yellow FAD (Y249F-y) and an inactive chemically modified green FAD, identified as 6-OH-FAD (Y249F-g) through various spectroscopic techniques. Structural investigation of Y249F-g and Y249F-y variants by comparison to the wild-type enzyme showed no differences in the overall protein structure and fold. A closer observation of the active site of the Y249F-y enzyme revealed an alternative conformation for some active site residues and the flavin cofactor. Molecular dynamics simulations probed the alternate conformations observed in the Y249F-y enzyme structure and showed that the enzyme variant with FAD samples a metastable conformational state, not available to the wild-type enzyme. Hybrid quantum/molecular mechanical calculations identified differences in flavin electronics between the wild type and the alternate conformation of the Y249F-y enzyme. The computational studies further indicated that the alternate conformation in the Y249F-y enzyme is responsible for the higher spin density at the C6 atom of flavin, which is consistent with the formation of 6-OH-FAD in the variant enzyme. The observations in this study are consistent with an alternate conformational space that results in fine-tuning the microenvironment around a versatile cofactor playing a critical role in enzyme function.