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The rational synthesis of trinuclear emissive organometallic complexes with two equivalent platinum(II) centres appended to the ancillary substituted 2,2'-bipyridyl ligand of the cyclometalated iridium(III) centre is reported here. The alkynyl-platinum moiety and cyclometalated iridium(III) centres have been separated through a non-conjugated CH2 -O-CH2 linkage. The emission titration with amino acids reveals that the complexes sense free amino acids. The luminescence sensing of BSA is thus attributed to the amino acid sensing ability of the complexes and confirmed by emission anisotropy and Far-UV CD spectral study. The decrease in α-helix in the CD spectra signifies the changes in the secondary structure of protein in presence of the complexes.

This Letter reports a counterintuitive observation that methylation of the glycine-iodide cluster leads to fewer conformations and spectroscopic simplicity. Cryogenic "iodide-tagging" negative ion photoelectron spectroscopy (NIPES) is used to probe specific binding sites of three N-methylated glycine derivatives, i.e., N-methylglycine (sarcosine), N,N-dimethylglycine, and N,N,N-trimethylglycine (glycine betaine). NIPES reveals a progressive spectral simplification of the iodide clusters with increasing methylation due to fewer contributing structures. Low energy conformers and tautomers of each cluster are computationally identified, and those observed in the experiments are assigned based on excellent agreement between the NIPE spectra and theoretical simulations. Zwitterionic cluster structures are found to be less stable than their canonical forms and do not contribute to the observed spectra. This work demonstrates the power of iodide-tagging NIPES in probing conformations of amino acid-iodide clusters and provides a molecular level understanding on the effect of methyl substitution on amino acid binding sites.

Chalcone derivatives (7a–k) have been synthesized and characterized by 1H-NMR, 13C-NMR, mass, and elemental analysis. The synthesized compounds 7a, 7d, and 7g have been examined and it is confirmed that the most promising cytotoxicity and also cell morphology analysis exhibited good apoptotic activity against lung cancer A549 cell. The free energy binding of compound 7d exhibits − 8.96 kcal/mol with five hydrogen bonding Asn1254, Arg1253, Asp1249, and Gly1123 amino acids of ALK receptors. The LUMO of the electron density present in α, β-double bond and influence the anticancer activity. The Mulliken atomic charges and MEPs are scrutinizing the ligand interaction with the amino acid binding of ALK receptors. UV-visible and photoluminescence spectra showed that the properties of chalcone derivatives have a significant effect on the visible absorption and emission maxima (531–535 nm) red shift in the emission spectra which also systematically investigated electrochemical in various solvents with increasing solvent polarity.

Dehydrins, a family of hydrophilic and intrinsically disordered proteins, are a subgroup of late embryogenesis abundant proteins that perform different protective roles in plants. Although the transition from a disordered to an ordered state has been associated with dehydrin function or interactions with specific partner molecules, the question of how the primary and secondary dehydrin protein structure is related to specific functions or target molecule preferences remains unresolved. This work addresses the in silico sequencing analysis and in vitro functional characterization of two dehydrin isoforms, VviDHN2 and VviDHN4, from Vitis vinifera. Conformational changes suggest potential interactions with a broad range of molecules and could point to more than one function. The in silico analysis showed differences in conserved segments, specific amino acid binding sequences, heterogeneity of structural properties and predicted sites accessible for various post-translational modifications between the sequence of both dehydrins. Moreover, in vitro functional analysis revealed that although they both showed slight antifungal activity, only VviDHN4 acts as a molecular shield that protects proteins from freezing and dehydration. VviDHN4 also demonstrated high potential as a chaperone and reactive oxygen species scavenger, in addition to presenting antifreeze activity, all of which confirms its multifunctional nature. Our findings highlight the significant role of Y-segments and the differential and specific amino acid composition of less conserved segments that are rich in polar/charged residues between S- and K-segments, coupled with post-translational modifications, in modulating and switching dehydrin biological function., This research was financed by the CICYT Spanish Projects AGL 2014-53081-R (MINECO) and AGL 2017-85291-R (MINECO/AEI/FEDER, UE). M. Vazquez-Hernandez acknowledges the support of the FPI program financed by the MICINN.

The current library of synthetic molecular sensors for small polar molecules is limited. In this work, we describe the synthesis of two diastereomeric mono-phosphonate calix[4]pyrrole cavitands, 1in and 1out, acting as fluorescent sensors for amino acids. The two isomeric cavitands differ in the relative orientation, in (1in) and out (1out), of their PO bridging group and the N-phenyl-naphthalamine fluorophore directly attached to it, with respect to their polar aromatic cavities. Using 1H and 31P NMR spectroscopy and non-fluorescent cavitand analogues (5in and 5out), we demonstrate the formation of 1 : 1 complexes with L-proline (L-Pro) and the relevance of the inwardly directed PO group in the binding of the amino acid. Only the L-Pro⊂5in complex establishes a charged hydrogen bonding interaction between the receptor PO group and the protonated amine of the bound zwitterionic amino acid. This interaction is responsible for an increase in the thermodynamic stability of the complex compared to the L-Pro⊂5out counterpart. We investigate the binding properties of the fluorescent cavitands, 1in and 1out, with L-Pro and L-pipecolic acid (L-Pip) at micromolar concentration using emission spectroscopy (direct binding-based sensing, BBS). The observed emission changes in the BBS experiments were small but evidenced the role of the cavitands as fluorescent sensors. In agreement with the millimolar concentration results (1H NMR experiments), the fluorescent 1in sensor displays a larger binding affinity for L-Pro than the 1out isomer. Conversely, the 1out isomer experienced larger emission changes upon amino acid binding. We developed FRET-based indicator displacement assays (IDA) owing to the small emission changes observed in the direct BBS experiments. At micromolar concentration, the competitive displacement of the quencher N-oxide 6 from the cavity of the 1 : 1 supramolecular ensemble (6⊂1in and 6⊂1out), by L-Pro, L-Pip, and L-phenylalanine (L-Phe) produced fluorescence “turn-on”. The results of the BBS and IDA experiments assigned a binding selectivity to the 1in isomer for L-Pro.

Dietary restriction (DR) promotes healthy aging in diverse species. Essential amino acids play a key role, but the molecular mechanisms are unknown. The evolutionarily conserved Sestrin protein, an inhibitor of activity of the target of rapamycin complex 1 (TORC1), has recently been discovered as a sensor of amino acids in vitro. Here, we show that Sestrin null mutant flies have a blunted response of lifespan to DR. A mutant Sestrin fly line, with blocked amino acid binding and TORC1 activation, showed delayed development, reduced fecundity, extended lifespan and protection against lifespan-shortening, high-protein diets. Sestrin mediated reduced intestinal stem cell activity and gut cell turnover from DR, and stem cell proliferation in response to dietary amino acids, by regulating the TOR pathway and autophagy. Sestrin expression in intestinal stem cells was sufficient to maintain gut homeostasis and extend lifespan. Sestrin is thus a molecular link between dietary amino acids, stem cell function and longevity. The authors identify the evolutionarily conserved amino acid-sensing protein Sestrin as a key mediator of dietary restriction benefits in flies. Sestrin regulates gut stem cell activity via the TOR pathway, resulting in improved gut health and longevity.

Facing worldwide challenges associated with multifactorial etiology of breast cancer, designing of combinatorial therapies using natural compounds is currently the emergent way of treating several cancers including breast cancer in a synergistic way, which may mitigate several problems associated with multiple receptor targeting. In this research, Estrogen receptor positive breast cancer was taken as prototype and several key receptors associated with this particular disease were targeted by virtual screening of natural compounds found in Indian originated medicinal plants using Computer aided Drug Designing (CADD) strategies. We found the combination of Carpusin, Paulownin Cornigerine, Nororientaline, Oryzalexin B, Romucosine H and Colchicine as effective against six potential receptors i.e. FGFR2, ESR1, PIK3CA, PIK3CB, PIK3CD and AR in Estrogen receptor positive breast cancer with their binding energies in the range of ∆G ≤ -8.0 Kcal/mol as well as significant number of common amino acid binding residues as compared with binding sites of receptors. Thus this research holds significant implications for the designing of combinatorial therapeutic agents against breast cancer which can be further tested in-vitro and in-vivo to prove their synergistic efficiency.

Calcaium sensing receptors (CaSRs) play a central role in regulating extracellular calcium (Ca(2+)) homeostasis and many (patho)physiological processes. This regulation is primarily orchestrated in response to extracellular stimuli via the extracellular domain (ECD). This paper first reviews the modeled structure of the CaSR ECD and the prediction and investigation of the Ca(2+) and amino acid binding sites. Several recently solved X-ray structures are then compared to support a proposed CaSR activation model involving functional cooperativity. The review also discusses recent implications for drug development. These studies provide new insights into the molecular basis of diseases and the design of therapeutic agents that target CaSR and other family C G protein-coupled receptors (cGPCRs).

Dipeptides have attracted much attention as post-amino acids with physical properties and functions different from those of amino acids. However, a given dipeptide cannot be distinguished by mass spectrometry from its structural isomer with an opposite amino acid binding order unless these isomers are separated before introduction, which complicates the comprehensive analysis of dipeptides. Herein, a novel analytical platform for dipeptide analysis by capillary electrophoresis tandem mass spectrometry and liquid chromatography tandem mass spectrometry is developed. This method is used to quantitate 335 dipeptides and achieves excellent separation of structural isomers with opposite binding orders, high correlation coefficients, and low instrumental detection limits (0.088-83.1 nM). Moreover, acceptable recoveries (70-135%) are observed for most tested dipeptides in chicken liver samples spiked both before and after preparation. The developed method is also applied to the quantitation of dipeptides in the livers of mice fed different diets to detect 236 dipeptides, and the shift from a normal diet to a high-fat diet is shown to increase/decrease (p < 0.05, fold-change < 0.5) the contents of 0/29 dipeptides, respectively. The developed method is expected to facilitate the search for new dipeptide applications such as novel functional components of foods and biomarkers of diseases.

The provenance of the aminoacyl-tRNA synthetases (aaRS) poses unusually challenging questions because of their role in the emergence and evolution of genetic coding. We investigate evidence about their ancestry from highly curated structure-based multiple sequence alignments of a small “scaffold” that is structurally invariant in all 10 canonical Class I aaRS. Statistically different values of two uncorrelated phylogenetic metrics—residue by residue conservation derived from Clustal and row-by-row cladistic congruence derived from BEAST2—suggest that the Class I scaffold is a mosaic assembled from distinct, successive genetic sources. These data are especially significant in light of: (i) experimental fragmentations of the Class I scaffold into three partitions that retain catalytic activities in proportion to their length; and (ii) multiple sources of evidence that two of these partitions arose from an ancestral Class I aaRS gene encoding a Class II ancestor in frame on the opposite strand. Two additional metrics output by BEAST2 vary in accordance with the presumed functionality endowed by the various modules. The new evidence supplements previous aaRS phylogenies. It identifies a previously characterized 46-residue Class I “protozyme” as preceding the adaptive radiation of the superfamily containing variations of the Rossmann dinucleotide binding fold related to amino acid discrimination, and thus as root of that molecular tree. Such a rooting is consistent with near simultaneous emergence of genetic coding and the origin of the proteome, resolving a conundrum posed by previous inferences that Class I aaRS evolved long after the genetic code had been implemented in an RNA world. Further, it establishes a timeline for the growth of coding from a binary amino acid alphabet by pinpointing discontinuous enhancements of aaRS fidelity.Author SummaryPhylogenetic analysis uncovers evolutionary connections between different protein superfamily members. We describe complementary, uncorrelated, phylogenetic metrics that support multiple evolutionary histories for different segments within members of the Class I aminoacyl-tRNA synthetase superfamily. Using a carefully curated 3D crystal structure superposition as the primary source of the multiple sequence alignment substantially reduced dependence of these metrics on empirical amino acid substitution matrices. Two metrics are derived from the amino acid distribution observed in each successive position. A third depends on how individual sequences distribute into phylogenetic tree branches for each of the ten amino acids activated by the superfamily. All metrics confirm that a segment previously identified as an inserted element is, indeed, a more recent acquisition, despite its structural conservation. The residue-by-residue conservation metrics reveal significant co-variation of mutational frequencies between a core segment that forms the amino acid binding site and a neighboring segment derived from the more recent insertion element. We attribute that covariation to the differentiation of superfamily members as evolutionary divergence enhanced amino acid specificity. Finally, evidence that the insertion element is a recent acquisition implies a new branching order for much of the proteome.

Protein post-translational modifications regulate protein structure and function. Lysine benzoylation is a new type of histone mark with unique physiological relevance. To construct proteins with this modification site-specifically introduced, here we generated orthogonal tRNA Pyl -MaBzKRS pairs by engineering Methanomethylophilus alvus pyrrolysyl-tRNA synthetase, allowing the genetic incorporation of e-N-benzoyllysine (BzK) into proteins with high efficiency in E. coli and mammalian cells. Two types of MaBzKRS were identified to incorporate BzK using mutations located at different positions of the amino acid binding pocket. These MaBzKRS are small in size and highly expressed, which will afford broad utilities in studying the biological effects of lysine benzoylation.

Cancer cells regulate key enzymes in the glycolytic pathway to control the glycolytic flux, which is necessary for their growth and proliferation. One of the enzymes is pyruvate kinase muscle isoform 2 (PKM2), which is allosterically regulated by various small molecules. Using detailed biochemical and kinetic studies, we demonstrate that cysteine inhibits wild-type (wt) PKM2 by shifting from an active tetramer to a mixture of a tetramer and a less active dimer/monomer equilibrium and that the inhibition is dependent on cysteine concentration. The cysteine-mediated PKM2 inhibition is reversed by fructose 1,6-bisphosphate, an allosteric activator of PKM2. Furthermore, kinetic studies using two dimeric PKM2 variants, S437Y PKM2 and G415R PKM2, show that the reversal is caused by the tetramerization of wtPKM2. The crystal structure of the wtPKM2-Cys complex was determined at 2.25 Å, which showed that cysteine is held to the amino acid binding site via its main chain groups, similar to that observed for phenylalanine, alanine, serine, and tryptophan. Notably, ligand binding studies using fluorescence and isothermal titration calorimetry show that the presence of phosphoenolpyruvate alters the binding affinities of amino acids for wtPKM2 and vice versa, thereby unravelling the existence of a functionally bidirectional coupling between the amino acid binding site and the active site of wtPKM2.

Haemoglobin A1c (HbA1c) is a significant glycaemic marker for diabetes mellitus. The level of HbA1c reflects the mean blood glucose level over the prior 2–3 months and it is useful for the assessment of therapeutic effectiveness and for diagnosis. In this study, we report the label-free affinity sensor for HbA1c based on the chemiresistor-type field-effect transistor, which has a simple sensor configuration. Single-walled carbon nanotubes (SWNTs) were used as the transducing element. The fructosyl amino acid binding protein from Rhizobium radiobacter (SocA), which binds to α-fructosyl amino acid specifically, was used as the biorecognition element for fructosyl valine (FV), the product of the proteolytic hydrolysis of HbA1c. The developed sensor shows the ability to measure as low as 1.2 nM FV, which is 14-fold more sensitive compared to the previously reported fluorescence-based sensor using SocA. This sensor also exhibits high specificity where no significant response is observed from either fructosyl lysine (FK) or glucose, which are potential interferents. FK is the e-fructosyl amino acid from glycated albumin, another glycated protein, whereas glucose is naturally present at very high concentration in the blood. We propose that the modulation of the surface charges on the SWNTs caused by the conformational change in SocA upon ligand binding leads to the proportionate changes in the number of carriers in the SWNT channel.

ASCT2 is a transporter that exchanges neutral amino acids such as glutamine across the cell membrane, an essential process for rapidly growing cells, including cancerous ones. Ndaru et al. report the synthesis and characterization of a novel class of inhibitors useful for studying this transporter., The neutral amino acid transporter alanine serine cysteine transporter 2 (ASCT2) belongs to the solute carrier 1 (SLC1) family of transport proteins and transports neutral amino acids, such as alanine and glutamine, into the cell in exchange with intracellular amino acids. This amino acid transport is sodium dependent, but not driven by the transmembrane Na+ concentration gradient. Glutamine transport by ASCT2 is proposed to be important for glutamine homoeostasis in rapidly growing cancer cells to fulfill the energy and nitrogen demands of these cells. Thus, ASCT2 is thought to be a potential anticancer drug target. However, the pharmacology of the amino acid binding site is not well established. Here, we report on the synthesis and characterization of a novel class of ASCT2 inhibitors based on an amino acid scaffold with a sulfonamide/sulfonic acid ester linker to a hydrophobic group. The compounds were designed based on an improved ASCT2 homology model using the human glutamate transporter hEAAT1 crystal structure as a modeling template. The compounds were shown to inhibit with a competitive mechanism and a potency that scales with the hydrophobicity of the side chain. The most potent compound binds with an apparent affinity, Ki, of 8 ± 4 µM and can block the alanine response with a Ki of 40 ± 23 µM at 200 µM alanine concentration. Computational analysis predicts inhibitor interactions with the binding site through molecular docking. In conclusion, the sulfonamide/sulfonic acid ester scaffold provides facile synthetic access to ASCT2 inhibitors with a potentially large variability in chemical space of the hydrophobic side chain. These inhibitors will be useful chemical tools to further characterize the role of ASCT2 in disease as well as improve our understanding of inhibition mechanisms of this transporter.

One of the central challenges in the development of single-molecule protein sequencing technologies is achieving high-fidelity sequential recognition and detection of specific amino acids that comprise the peptide sequence. An approach towards achieving this goal is to leverage naturally occurring proteins that function through recognition of amino (N)-terminal amino acids (NAAs). One such protein, the N-end rule pathway adaptor protein ClpS, natively recognizes NAAs on a peptide chain. The native ClpS protein has a high specificity albeit modest affinity for the amino acid Phe at the N-terminus but also recognizes the residues Trp, Tyr, and Leu at the N-terminal position. Here, we employed directed evolution methods to select for ClpS variants with enhanced affinity and selectivity for two NAAs (Phe and Trp). Using this approach, we identified two promising variants of the Agrobacterium tumefaciens ClpS protein with native residues 34-36 ProArgGlu mutated to ProMetSer and CysProSer. In vitro surface binding assays indicate that the ProMetSer variant has enhanced affinity for Phe at the N-terminus with sevenfold tighter binding relative to wild-type ClpS, and that the CysProSer variant binds selectively to Trp over Phe at the N-terminus while having a greater affinity for both Trp and Phe. Taken together, this work demonstrates the utility of engineering ClpS to make it more effective for potential use in peptide sequencing applications.

In this study, the Zodo gum exudated by Amygdalus scoparia spach underwent the periodate oxidation process for chemical modification and the formation of dialdehyde groups. Modification of the Zodo gum properties was done using the periodate oxidation method, response surface methodology (RSM) and central composite design (CCD), with 4 factors of sodium periodate volume (6.4–19.2 mL), temperature (35–55 °C), pH (3–5) and time (2–4 h). Dialdehyde Zodo gum (DZG) was produced by controlling test variables and measuring some responses including dialdehyde content and efficacy, in addition to evaluating the rheological parameters. Quadratic, linear polynomial equations were then fitted with the insignificant Lack of fit and high R2 to address the relationship between the mentioned variables and responses. Optimal test conditions, including pH = 3.9, T = 43 °C and Time = 3.5 h, were also determined for the production of DZG10, DZG20 and DZG30 samples. The results of 1H-13C NMR, FTIR and determination of the aldehyde content indicated the formation of dialdehyde groups in equilibrium with the dominant hemiacetal form. The AFM study of the DZG30 sample also showed over-oxidation and depolymerization, which could be associated with increased hydrophobic properties and the reduced viscosity. Although the DZG30 sample had the highest amount of the dialdehyde factor group with the tendency to combine with the amino group of proteins, DZG10 and DZG20 samples could be recommended for industrial applications due to the nonoccurrence of overoxidation.

Selective binding of aqueous-phase amino acids to mineral surfaces is regarded as a plausible first step in oligopeptide formation on early Earth. To clarify the strength and underlying mechanism of amino acid binding to pyrite surfaces, we measured the unbinding (pull-off) force of ten amino acids and two oligo-peptides from water-pyrite interfaces using atomic force microscopy (AFM). The most probable unbinding force could be described by a linearly increasing function with the size of the amino acid and a characteristic offset. A good correlation was obtained between the most probable unbinding force and the residue volume, surface area and polarizability of samples suggesting at least a partial contribution of van der Waals (vdW) forces, especially the London dispersion force. These results are useful in analysis of adhesion phenomena of amino acids in the given environmental settings such as in this work.

Single-molecule detection of transport turnovers shows a distribution of rates with fast transporters favored by mutations., Kinetic properties of membrane transporters are typically poorly defined because high-resolution functional assays analogous to single-channel recordings are lacking. Here, we measure single-molecule transport kinetics of a glutamate transporter homolog from Pyrococcus horikoshii, GltPh, using fluorescently labeled periplasmic amino acid binding protein as a fluorescence resonance energy transfer–based sensor. We show that individual transporters can function at rates varying by at least two orders of magnitude that persist for multiple turnovers. A gain-of-function mutant shows increased population of the fast-acting transporters, leading to a 10-fold increase in the mean transport rate. These findings, which are broadly consistent with earlier single-molecule measurements of GltPh conformational dynamics, suggest that GltPh transport is defined by kinetically distinct populations that exhibit long-lasting “molecular memory.”

Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron–electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity., Cyclohexadienyl dehydratase (CDT) evolved from a cationic amino acid binding protein ancestor without enzymatic activity (AncCDT-1) via a series of intermediates. Here, the authors combine EPR, X-ray crystallography and MD simulations to study the structural dynamics of these evolutionary intermediates and observe that they predominantly populate catalytically unproductive conformations, while CDT exclusively samples catalytically relevant compact states, and which reveals how the conformational landscape changes along the evolutionary trajectory.

Density functional theory (DFT) calculations are employed to examine the adsorption behaviors of cysteine on the gold surface using Au6 and Au8 species as model reactants. Computed results show that cysteine molecules prefer to bind with gold clusters via the S-atom of the thiol group in vacuum and thiolate group in water. The gas-phase adsorption energies are around 20.2 kcal/mol for Au6 and 24.4 kcal/mol for Au8. In water environment, such values are slightly reduced for Au6 (19.6 kcal/mol), but increased a little more for Au8 (25.6 kcal/mol). As a result, if a visible light with a frequency of ν ≈ 6 × 1014 Hz (500 nm) is applied, the time for the recovery of Au6 and Au8 from the most stable complexes will be about 0.38 and 9.3 × 103 s, respectively, at 298 K in water. The Au6 is in addition found to benefit from a larger change of energy gap that could be converted to an electrical signal for detection of cysteine.

Genetic code and translation are key to all life. As a consequence, all kingdoms and species share the enzymes known as aminoacyl-tRNA synthetases, which link amino acids to their codons. For life to flourish, it is vital that these enzymes correctly implement the genetic code and hence correctly recognize amino acids. There are many theories on the emergence of aminoacyl-tRNA synthetases and their amino acid binding sites. While many insights have been gained from sequence analysis, the accurate amino acid recognition remains elusive. Here we use a novel approach to analyze all currently available aminoacyl-tRNA synthetase structures, which cover the recognition of all proteinogenic amino acids across all kingdoms of life. For the first time, we extensively characterize and quantify the interactions between aminoacyl-tRNA synthetases and bound amino acids. Our results show how different interaction features are used to delineate between the two major enzyme classes and the individual amino acids. Furthermore, we show that these features are conserved across a wide variety of species. The quantification of the similarity between the recognition of individual amino acids, allows to pinpoint where the genetic code is vulnerable to encoding errors and additional correction mechanisms had to evolve.

In this chapter, an overview is provided of the different concepts of cucurbiturils directly interacting with epitopes on proteins. This overview also serves as an illustration of the potential of this highly efficient supramolecular chemistry on proteins. Cucurbiturils are ideally suited for the recognition of protein elements, because of their combined hydrophobic and polar recognition motifs and hand-in-glove steric match with selected amino acid residues. Especially, the diverse chemical characteristics of cucurbit[6]uril, cucurbit[7]uril, and cucurbit[8]uril have been shown to be ideally suited to bind different molecular epitopes on proteins, including two-fold epitope binding by cucurbit[8]uril, as illustrated via examples on N-terminal and mid-chain amino acid binding as well as multiple amino acid epitope recognition. Clever molecular recognition concepts and protein engineering allows for functional modulation and study of protein activity or controled protein assembly by virtue of the cucurbituril recognition. Enzyme activity regulation, inhibition of amyloid aggregation, directed protein-wire assembly, and generation of supramolecular biopharmaceuticals are highlights of the potential of the concept of molecular recognition of proteins by cucurbiturils.

Divalent d-block metal cations (DDMCs), such as Fe, Zn and Mn, participate in many biological processes. Understanding how specific DDMCs are transported to and within the cell and what controls their binding selectivity to different proteins is crucial for defining the mechanisms of metalloproteins. To better understand such processes, we scanned the RCSB Protein Data Bank, performed a de novo structural-based comprehensive analysis of seven DDMCs and found their amino acid binding and coordination geometry propensities. We then utilized these results to characterize the correlation between metal selectivity, specific binding site composition and phylogenetic classification of the cation diffusion facilitator (CDF) protein family, a family of DDMC transporters found throughout evolution and sharing a conserved structure, yet with different members displaying distinct metal selectivity. Our analysis shows that DDMCs differ, at times significantly, in terms of their binding propensities, and that in each CDF clade, the metal selectivity-related binding site has a unique and conserved sequence signature. However, only limited correlation exists between the composition of the DDMC binding site in each clade and the metal selectivity shown by its proteins.

Auxin plays a central role in growth and plant development. To maintain auxin homeostasis, biological processes such as biosynthesis, transport, degradation, and reversible conjugation are essential. The Gretchen Hagen 3 (GH3) family genes codify for the enzymes that esterify indole-3-acetic acid (IAA) to various amino acids, which is a key process in the induction of somatic embryogenesis (SE). The GH3 family is one of the principal families of early response to auxin genes, exhibiting IAA-amido synthetase activity to maintain optimal levels of free auxin in the cell. In this study, we carried out a systematic identification of the GH3 gene family in the genome of Coffea canephora, determining a total of 18 CcGH3 genes. Analysis of the genetic structures and phylogenetic relationships of CcGH3 genes with GH3 genes from other plant species revealed that they could be clustered in two major categories with groups 1 and 2 of the GH3 family of Arabidopsis. We analyzed the transcriptome expression profiles of the 18 CcGH3 genes using RNA-Seq analysis-based data and qRT-PCR during the different points of somatic embryogenesis induction. Furthermore, the endogenous quantification of free and conjugated indole-3-acetic acid (IAA) suggests that the various members of the CcGH3 genes play a crucial role during the embryogenic process of C. canephora. Three-dimensional modeling of the selected CcGH3 proteins showed that they consist of two domains: an extensive N-terminal domain and a smaller C-terminal domain. All proteins analyzed in the present study shared a unique conserved structural topology. Additionally, we identified conserved regions that could function to bind nucleotides and specific amino acids for the conjugation of IAA during SE in C. canephora. These results provide a better understanding of the C. canephora GH3 gene family for further exploration and possible genetic manipulation.

The UBR-box is a 70-residue zinc finger domain present in the UBR family of E3 ubiquitin ligases that directly binds N-terminal degradation signals in substrate proteins. UBR6, also called FBXO11, is an UBR-box containing E3 ubiquitin ligase that does not bind N-terminal signals. Here, we present the crystal structure of the UBR-box domain from human UBR6. The dimeric crystal structure reveals a unique form of domain swapping mediated by zinc coordination, where three independent protein chains come together to regenerate the topology of the monomeric UBR-box fold. Analysis of the structure suggests that the absence of N-terminal residue binding arises from the lack of an amino acid binding pocket.

Water soluble amphiphilic anion receptors based on urea-substituted β-cyclodextrin were synthesized via a copper(I) mediated azide-alkyne coupling reaction. The synthetic route was designed to minimize the number of operations of cyclodextrins. Stable products were obtained in 90% yield. They were successfully tested as amino acid receptors, showing excellent affinity constants (103-104M-1) in a highly competitive environment (pH 8 phosphate-buffered water solution). Isothermal titration calorimetry indicated that complex formation strongly depends on the hydrophobic nature of the guest and that the urea moiety of the receptor is necessary to efficiently bind amino acids.

Key message Among the qhir11 and qhir12 sub-regions of a major QTL qhir1, only qhir11 has significant effect on maternal haploid induction, segregation distortion and kernel abortion. Abstract In vivo haploid induction in maize can be triggered in high frequencies by pollination with special genetic stocks called haploid inducers. Several genetic studies with segregating populations from non-inducer x inducer crosses identified a major QTL, qhir1, on chromosome 1.04 contributing to in vivo haploid induction. A recent Genome Wide Association Study using 51 inducers and 1482 non-inducers also identified two sub-regions within the qhir1 QTL region, named qhir11 and qhir12; qhir12 was proposed to be mandatory for haploid induction because the haplotype of qhir11 was also present in some non-inducers and putative candidate genes coding for DNA and amino acid binding proteins were identified in the qhir12 region. To characterize the effects of each sub-region of qhir1 on haploid induction rate, F2 recombinants segregating for one of the sub-regions and fixed for the other were identified in a cross between CML269 (non-inducer) and a tropicalized haploid inducer TAIL8. To quantify the haploid induction effects of qhir11 and qhir12, selfed progenies of recombinants between these sub-regions were genotyped. F3 plants homozygous for qhir11 and/or qhir12 were identified, and crossed to a ligueless tester to determine their haploid induction rates. The study revealed that only the qhir11 sub-region has a significant effect on haploid induction ability, besides causing significant segregation distortion and kernel abortion, traits that are strongly associated with maternal haploid induction. The results presented in this study can guide fine mapping efforts of qhir1 and in developing new inducers efficiently using marker assisted selection. Electronic supplementary material The online version of this article (doi:10.1007/s00122-017-2873-9) contains supplementary material, which is available to authorized users.

We have designed and synthesized two new achiral hexa-peri-hexabenzocoronene (HBC) derivatives, HBCCE and HBCTEG-CE , which bear the crown ether as the pendant for the amino acid binding site. The HBCCE self-assembled into a racemic mixture of P- and M-handed helical nanocoils, however, in the presence of chiral amino acid guests, it formed helical nanocoils with one-handed screw sense. The effects of the concentration, type and configuration of the guests on the induced circular dichroism (ICD) during the co-assembly of HBCCE with chiral amino acids were also investigated. Additionally, after complete removal of the chiral guests, the optically active nanocoils did not racemize, even in the presence of excess amino acids with the opposite configuration. In contrast, HBCTEG-CE with a long triethylene glycol (TEG) chain between the crown ether group and the HBC unit did not exhibit ICD during the co-assembly with chiral amino acids.

Lon is a widely-conserved housekeeping protease found in all domains of life. Bacterial Lon is involved in the recovery from various types of stress, including tolerance to fluoroquinolone antibiotics, and is linked to pathogenesis in a number of organisms. However, detailed functional studies of Lon have been limited by the lack of selective, cell-permeable inhibitors. Here we describe the use of positional scanning libraries of hybrid peptide substrates to profile the primary sequence specificity of bacterial Lon. In addition to identifying optimal natural amino acid binding preferences, we identified several non-natural residues that were leveraged to develop optimal peptide substrates as well as a potent peptidic boronic acid inhibitor of Lon. Treatment ofE. coliwith this inhibitor promotes UV-induced filamentation and reduces tolerance to ciprofloxacin, phenocopying establishedlon-deletion phenotypes. It is also non-toxic to mammalian cells due to its increased selectivity for Lon over the proteasome. Our results provide new insight into the primary substrate specificity of Lon and identify substrates and an inhibitor that will serve as useful tools for dissecting the diverse cellular functions of Lon.

Earlier reported crystal structure of CLasTcyA revealed unique features like relatively a larger substrate binding pocket, an extended C-terminal loop restricted by a disulfide bond and involvement of residues from hinge region in substrate binding. In present study, CLasTcyA mutants were created to evaluate the importance of these unique features through biophysical characterization. The Val58 in CLasTcyA was replaced by Trp, conserved in most cystine binding proteins, to reduce the size of the binding pocket. All other mutations were created in CLasTcyAV58W mutant as the presence of Trp could be used for intrinsic fluorescence studies. The CLasTcyAV58W showed a noticeable increase in binding affinity and thermal stability as compared to the native form. The mutation of two cysteines in triple mutant CLasTcyAV58W/C212S/C239S, removal of C-terminal extended loop in truncated CLasTcyAV58W/C212S and mutation of His95 from hinge region in the double mutant CLasTcyAV58W/H95A showed a marked decrease in stability-indicating the importance of the unique features in structure of CLasTcyA. The bioinformatics-based virtual screening was employed to screen the potential inhibitor molecules for detailed future studies. The results clearly establish the importance of unique features in structure-function relationship of CLasTcyA.

Purpose: Pancreatic ductal adenocarcinoma (PDA) is rarely cured, and single-agent immune checkpoint inhibition has not demonstrated clinical benefit despite the presence of large numbers of CD8+ T cells. We hypothesized that tumor-infiltrating CD8+ T cells harbor latent antitumor activity that can be reactivated using combination immunotherapy. Experimental Design: Preserved human PDA specimens were analyzed using multiplex IHC (mIHC) and T-cell receptor (TCR) sequencing. Fresh tumor was treated in organotypic slice culture to test the effects of combination PD-1 and CXCR4 blockade. Slices were analyzed using IHC, flow cytometry, and live fluorescent microscopy to assess tumor kill, in addition to T-cell expansion and mobilization. Results: mIHC demonstrated fewer CD8+ T cells in juxtatumoral stroma containing carcinoma cells than in stroma devoid of them. Using TCR sequencing, we found clonal expansion in each tumor; high-frequency clones had multiple DNA rearrangements coding for the same amino acid binding sequence, which suggests response to common tumor antigens. Treatment of fresh human PDA slices with combination PD-1 and CXCR4 blockade led to increased tumor cell death concomitant with lymphocyte expansion. Live microscopy after combination therapy demonstrated CD8+ T-cell migration into the juxtatumoral compartment and rapid increase in tumor cell apoptosis. Conclusions: Endogenous tumor-reactive T cells are present within the human PDA tumor microenvironment and can be reactivated by combined blockade of PD-1 and CXCR4. This provides a new basis for the rational selection of combination immunotherapy for PDA. See related commentary by Medina and Miller, p. 3747

Cadaverine is produced in organisms from the amino acid lysine in a decarboxylation reaction catalyzed by lysine decarboxylase (EC 4.1.1.18). The inducible lysine decarboxylase CadA plays a vital role in acid stress response for enteric bacteria. Vibrio vulnificus is an extremely virulent human pathogen causing gastroenteritis when the acid conditions that prevent survival of V. vulnificus in the stomach or small intestine are overcome. A gene encoding CadA was identified from V. vulnificus. Subsequent analyses showed that CadA from V. vulnificus (VvCadA) is a decamer with a 82-kDa subunit. Homogenous VvCadA was purified from Escherichia coli and used for lysine decarboxylation with an optimal pH of 6.0 and optimal temperature of 37°C. The apparent Vmax and Km for lysine were 9.45 ± 0.24 μM/min and 0.45 ± 0.05 mM, respectively. Mutation analysis suggested that the amino-acid-binding pyridoxal phosphate, the cofactor of the enzyme, plays a vital role in the reaction. Mutation of the negatively charged residues interacting with lysine also affected the activity of the enzyme to some extent. Quantitative RT-PCR showed that expression of VvcadA was up-regulated under low pH, low salinity, and oxidative stresses. Furthermore, the concentration of cadaverine released to the cell exterior also increased under these stresses. Protein sequence similarity network (SSN) analysis indicated that lysine decarboxylases with ornithine decarboxylases and arginine decarboxylases shared a common ancestor, and that lysine decarboxylases are more conserved during evolution. Our data provide evidence for the biochemical characteristics and important roles of VvCadA under stress conditions.

SARS-CoV-2 virus is the serious health concern throughout the world. A comprehensive investigation of binding of SARS-CoV-2 active site with host receptor protein hACE2 is important in designing effective drugs. In the present work, the major amino acid binding partners between the virus CTD and host receptor have been studied and are compared with SARS-CoV RBD binding with hACE2. Our investigation show that some unique hydrogen bond pairs which were not reported in previous work. Along with hydrogen bonding, salt-bridges, hydrophobic interactions and contributions of electrostatic and van der Waals contacts play significant role in binding mechanism. The binding affinity of SARS-CoV-2 CTD/hACE2 is greater than SARS-CoV RBD/hACE2. This outcome is also verified from the free energy estimation by using umbrella sampling.

Quantitative analysis of the L-tryptophan content in commercial products by chemosensor has not been reported yet. In this study, the chiral carbazole based sensor (CCS), bearing a chiral “urea-carboxylic acid” binding site was prepared. CCS was proved to be effective in the consecutive detection of Fe3+, L-tryptophan (Trp), and L-histidine (His) by naked eyes. CCS exhibits different color changes and fluorescent ‘turn-off’ response toward Fe3+ and Fe2+. The discrimination of these two oxidation states of iron by CCS is important to understand the iron-related biological reactions. The limit of detection for Fe3+ by CCS is 84 nM in a high water-content solution (99 % water). CCS sensor can act as a reversible fluorescence quenching sensor for Fe3+. The results of Job’s plot analysis and mass spectra reveal that Fe3+ binds to CCS with a 1:1 stoichiometry. The CCS-Fe3+ complex can sense tryptophan, or histidine by a dose-dependent turn-on fluorescent response in a high water-content solution (99 % water). The mass spectra suggest the sensing of L-histidine or L-tryptophan by CCS-Fe3+ was performed with a CCS-Fe3+: amino acid binding ratio of 1:1. In comparison with other chemosensors reported in the literature, CCS can detect the L-Trp and L-His with a low limit of detection (0.31 μM L-Trp, 7.64 μM L-His) in a high water content environment. Furthermore, the CCS - Fe3+ complex based chemosensor was successfully applied to quantify the L-tryptophan content in commercial sleep-improving capsules containing various ingredients. The measured L-tryptophan contents in two samples are close to the announced values.

Alzheimer’s disease (AD), a devastating neurodegenerative disease, is associated with the abnormal accumulation and aggregation of β-amyloid proteins (Aβ) along with the deposition of high levels of Cu, Fe and Zn ions in the brain, causing neuronal cell deaths to lead the cognitive disabilities and even death. As there is a direct relationship between AD and Aβ aggregation, an intense research activity has been made to develop drug materials that serve as probes and inhibitors for controlling the pathways of Aβ peptide aggregation. However, their relatively instability in aqueous medium, tedious sample treatment, multistep syntheses, or low detection ability limit their potential applications. Therefore, the development of photoactive metal complexes for the selective detection and inhibition effects of Aβ aggregation is a thrust area in biomedical research. In this review, the use of non-conventional photoactive metal complexes including Ru(II), Re(I), Ir(III) and Pt(II) has the potential advantages of probes for monitoring and inhibiting the fibrillation as well as the toxicity of Aβ over conventional dyes such as Thioflavin T (ThT). The geometry, multiple electronic/spin states and redox nature of metal centres have made them tunable properties. Upon binding to the Aβ peptide aggregates, they exhibit promising potential as anti-AD agents due to their fascinating photophysical properties include red emissions, large Stokes shifts, and long lifetimes, which differentiate the competitive binding of other short-lived fluorescent molecules via photoluminescence, and time-resolved measurements. In addition, metal complexes display their remarkable selectivity and superiority over ThT. Competition study between photoactive metal complexes and ThT on fibrillation process show their effective binding of metal complex with Aβ42 fibrils by hindering the ThT binding to give higher binding constants than that of ThT. Computational studies predicted a hydrophobic domain between amino acid binding sites and the functional group of photoactive metal complexes via different noncovalent interactions. Thus, attractive characteristics of photoactive metal complexes could influence remarkable evolutions in new dimensions, which in turn address current challenges in the clinical use of the detection and inhibition of Aβ fibrils.

Molecular Dynamics (MD) simulations seek to provide atomic-level insights into conformationally dynamic biological systems at experimentally relevant time resolutions, such as those afforded by single-molecule fluorescence measurements. However, limitations in the time scales of MD simulations and the time resolution of single-molecule measurements have challenged efforts to obtain overlapping temporal regimes required for close quantitative comparisons. Achieving such overlap has the potential to provide novel theories, hypotheses, and interpretations that can inform idealized experimental designs that maximize the detection of the desired reaction coordinate. Here, we report MD simulations at time scales overlapping with in vitro single-molecule Förster (fluorescence) resonance energy transfer (smFRET) measurements of the amino acid binding protein LIV-BPSS at sub-millisecond resolution. Computationally efficient all-atom structure-based simulations, calibrated against explicit solvent simulations, were employed for sampling multiple cycles of LIV-BPSS clamshell-like conformational changes on the time scale of seconds, examining the relationship between these events and those observed by smFRET. The MD simulations agree with the smFRET measurements and provide valuable information on local dynamics of fluorophores at their sites of attachment on LIV-BPSS and the correlations between fluorophore motions and large-scale conformational changes between LIV-BPSS domains. We further utilize the MD simulations to inform the interpretation of smFRET data, including Förster radius (R0) and fluorophore orientation factor (κ2) determinations. The approach we describe can be readily extended to distinct biochemical systems, allowing for the interpretation of any FRET system conjugated to protein or ribonucleoprotein complexes, including those with more conformational processes, as well as those implementing multi-color smFRET., Author summary Förster (fluorescence) resonance energy transfer (FRET) has been used extensively by biophysicists as a molecular-scale ruler that yields fundamental structural and kinetic insights into transient processes including complex formation and conformational rearrangements required for biological function. FRET techniques require the identification of informative fluorophore labeling sites, spaced at defined distances to inform on a reaction coordinate of interest and consideration of noise sources that have the potential to obscure quantitative interpretations. Here, we describe an approach to leverage advancements in computationally efficient all-atom structure-based molecular dynamics simulations in which structural dynamics observed via FRET can be interpreted in full atomistic detail on commensurate time scales. We demonstrate the potential of this approach using a model FRET system, the amino acid binding protein LIV-BPSS conjugated to self-healing organic fluorophores. LIV-BPSS exhibits large scale, sub-millisecond clamshell-like conformational changes between open and closed conformations associated with ligand unbinding and binding, respectively. Our findings inform on the molecular basis of the dynamics observed by smFRET and on strategies to optimize fluorophore labeling sites, the manner of fluorophore attachment, and fluorophore composition.

The Fenna–Matthews–Olson (FMO) pigment–protein complex in green sulfur bacteria transfers excitation energy from the chlorosome antenna complex to the reaction center. In understanding energy transfer in the FMO protein, the individual contributions of the bacteriochlorophyll pigments to the FMO complex's absorption spectrum could provide detailed information with which molecular and energetic models can be constructed. The absorption properties of the pigments, however, are such that their spectra overlap significantly. To overcome this, we used site-directed mutagenesis to construct a series of mutant FMO complexes in the model green sulfur bacterium Chlorobaculum tepidum (formerly Chlorobium tepidum). Two cysteines at positions 49 and 353 in the C. tepidum FMO complex, which reside near hydrogen bonds between BChls 2 and 3, and their amino acid binding partner serine 73 and tyrosine 15, respectively, were changed to alanine residues. The resulting C49A, C353A, and C49A C353A double mutants were analyzed with a combination of optical absorption and circular dichroism (CD) spectroscopies. Our results revealed changes in the absorption properties of several underlying spectral components in the FMO complex, as well as the redox behavior of the complex in response to the reductant sodium dithionite. A high-resolution X-ray structure of the C49A C353A double mutant reveals that these spectral changes appear to be independent of any major structural rearrangements in the FMO mutants. Our findings provide important tests for theoretical calculations of the C. tepidum FMO absorption spectrum, and additionally highlight a possible role for cysteine residues in the redox activity of the pigment–protein complex.

DNA-binding proteins play critical roles in biological processes including gene expression, DNA packaging and DNA repair. They bind to DNA target sequences with different degrees of binding specificity, ranging from highly specific to non-specific. Alterations of DNA-binding specificity, due to either genetic variation or somatic mutations, can lead to various diseases. In this study, a comparative analysis of protein-DNA complex structures was carried out to investigate the structural features that contribute to binding specificity. Protein-DNA complexes were grouped into three general classes based on degrees of binding specificity: highly specific (HS), multi-specific (MS), and non-specific (NS). Our results show a clear trend of structural features among the three classes, including amino acid binding propensities, simple and complex hydrogen bonds, major/minor groove and base contacts, and DNA shape. We found that aspartate is enriched in highly specific DNA binding proteins and predominately binds to a cytosine through a single hydrogen bond or two consecutive cytosines through bidentate hydrogen bonds. Aromatic residues, histidine and tyrosine, are highly enriched in the HS and MS groups and may contribute to specific binding through different mechanisms. To further investigate the role of protein flexibility in specific protein-DNA recognition, we analysed the conformational changes between the bound and unbound states of DNA-binding proteins and structural variations. The results indicate that highly specific and multi-specific DNA-binding domains have larger conformational changes upon DNA-binding and larger degree of flexibility in both bound and unbound states.

In vivo haploid induction (HI) triggered by pollination with special intraspecific genotypes, called inducers, is unique to Zea mays L. within the plant kingdom and has revolutionized maize breeding during the last decade. However, the molecular mechanisms underlying HI in maize are still unclear. To investigate the genetic basis of HI, we developed a new approach for genome-wide association studies (GWAS), termed conditional haplotype extension (CHE) test that allows detection of selective sweeps even under almost perfect confounding of population structure and trait expression. Here, we applied this test to identify genomic regions required for HI expression and dissected the combined support interval (50.34 Mb) of the QTL qhir1, detected in a previous study, into two closely linked genomic segments relevant for HI expression. The first, termed qhir11 (0.54 Mb), comprises an already fine-mapped region but was not diagnostic for differentiating inducers and noninducers. The second segment, termed qhir12 (3.97 Mb), had a haplotype allele common to all 53 inducer lines but not found in any of the 1482 noninducers. By comparing resequencing data of one inducer with 14 noninducers, we detected in the qhir12 region three candidate genes involved in DNA or amino acid binding, however, none for qhir11. We propose that the CHE test can be utilized in introgression breeding and different fields of genetics to detect selective sweeps in heterogeneous genetic backgrounds.

Realization of binding reagents that can perform high-fidelity, sequential recognition and detection of amino acids is important for the success of many proposed approaches for single-molecule protein sequencing. Towards this purpose, a variant of the Agrobacterium tumefaciens protein ClpS was previously engineered with improved binding affinity for phenylalanine at the N-terminus of a peptide. In this study, this variant was further engineered for attributes necessary for robust application as a biotechnology reagent such as increased thermostability and improved selectivity under different buffer conditions. The stabilized variant was further characterized in terms of selectivity for the N-terminal residue in the context of different peptides with varying residues at positions 2 (P2) and 3 (P3). The implication of the study for the use of ClpS based reagents in peptide sequencing technologies are described.

Taste receptor type 1 (T1r) is responsible for the perception of essential nutrients, such as sugars and amino acids, and evoking sweet and umami (savory) taste sensations. T1r receptors recognize many of the taste substances at their extracellular ligand-binding domains (LBDs). In order to detect a wide array of taste substances in the environment, T1r receptors often possess broad ligand specificities. However, the entire ranges of chemical spaces and their binding characteristics to any T1rLBDs have not been extensively analyzed. In this study, we exploited the differential scanning fluorimetry (DSF) to medaka T1r2a/T1r3LBD, a current sole T1rLBD heterodimer amenable for recombinant preparation, and analyzed their thermal stabilization by adding various amino acids. The assay showed that the agonist amino acids induced thermal stabilization and shifted the melting temperatures (Tm) of the protein. An agreement between the DSF results and the previous biophysical assay was observed, suggesting that DSF can detect ligand binding at the orthosteric-binding site in T1r2a/T1r3LBD. The assay further demonstrated that most of the tested L-amino acids, but no D-amino acid, inducedTmshifts of T1r2a/T1r3LBD, indicating the broad L-amino acid specificities of the proteins probably with several different manners of recognition. TheTmshifts by each amino acid also showed a fair correlation with the responses exhibited by the full-length receptor, verifying the broad amino-acid binding profiles at the orthosteric site in LBD observed by DSF.

The members of the neurotransmitter transporter family SLC6A exhibit a high degree of structural homology; however differences arise in many aspects of their transport mechanisms. In this study we report that mouse B(0)AT1 (mouse Slc6a19) mediates the electrogenic transport of a broad range of neutral amino acids but not of the chemically similar substrates transported by other SLC6A family members. Cotransport of L: -Leu and Na(+) generates a saturable, reversible, inward current with Michaelis-Menten kinetics (Hill coefficient approximately 1) yielding a K(0.5) for L: -Leu of 1.16 mM and for Na(+) of 16 mM at a holding potential of -50 mV. Changing the membrane voltage influences both substrate binding and substrate translocation. Li(+) can substitute partially for Na(+) in the generation of L: -Leu-evoked inward currents, whereas both Cl(-) and H(+) concentrations influence its magnitude. The simultaneous measurement of charge translocation and L: -Leu uptake in the same cell indicates that B(0)AT1 transports one Na(+) per neutral amino acid. This appears to be accomplished by an ordered, simultaneous mechanism, with the amino acid binding prior to the Na(+), followed by the simultaneous translocation of both co-substrates across the plasma membrane. From this kinetic analysis, we conclude that the relatively constant [Na(+)] along the renal proximal tubule both drives the uptake of neutral amino acids via B(0)AT1 thermodynamically and ensures that, upon binding, these are translocated efficiently into the cell.

One hundred and fifty GCN5-like acetyltransferases with amino acid-binding (ACT)-GCN5-related N-acetyltransferase (GNAT) domain organization have been identified in actinobacteria. The ACT domain is fused to the GNAT domain, conferring amino acid-induced allosteric regulation to these protein acetyltransferases (Pat) (amino acid sensing acetyltransferase, (AAPatA)). Members of the AAPatA family share similar secondary structure and are divided into two groups based on the allosteric ligands of the ACT domain: the asparagine (Asn)-activated PatA and the cysteine (Cys)-activated PatA. The former are mainly found in Streptomyces; the latter are distributed in other actinobacteria. We investigated the effect of Asn and Cys on the acetylation activity of Sven_0867 (SvePatA, from Streptomyces venezuelae DSM 40230) and Amir_5672 (AmiPatA, from Actinosynnema mirum strain DSM 43827), respectively, as well as the relationship between the structure and function of these enzymes. These findings indicate that the activity of PatA and acetylation level of proteins may be closely correlated with intracellular concentrations of Asn and Cys in actinobacteria. Amino acid-sensing signal transduction in acetyltransferases may be a mechanism that regulates protein acetylation in response to nutrient availability. Future work examining the relationship between protein acetylation and amino acid metabolism will broaden our understanding of post-translational modifications (PTMs) in feedback regulation.

Transporters in the SLC6 family play key roles in regulating neurotransmission and are the targets for a wide range of therapeutics. Important insights into the transport mechanisms and the specificity of drug interactions of SLC6 transporters have been obtained from the crystal structures of a bacterial homologue of the family, LeuTAa, and more recently the Drosophila dopamine transporter and the human serotonin transporter. However, there is disputed evidence that the bacterial leucine transporter, LeuTAa, contains two substrate binding sites that work cooperatively in the mechanism of transport, with the binding of a second substrate being required for the release of the substrate from the primary site. An alternate proposal is that there may be low affinity binding sites that serve to direct the flow of substrates to the primary site. We have used a combination of molecular dynamics simulations of substrate interactions with a homology model of GlyT2, together with radiolabeled amino acid uptake assays and electrophysiological analysis of wild-type and mutant transporters, to provide evidence that substrate selectivity of GlyT2 is determined entirely by the primary substrate binding site and, furthermore, if a secondary site exists then it is a low affinity nonselective amino acid binding site.