Discovery, characterization and engineering of ligases for amide synthesis.

Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl-L-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways1-4. Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown1,2. Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and L-Ile in plants5. This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions-Jar1 producing a compound essential for plant development4,5, and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.

The role of leucine and isoleucine in tuning the hydropathy of class A GPCRs.

Leucine and Isoleucine are two amino acids that differ only by the positioning of one methyl group. This small difference can have important consequences in α-helices, as the ß-branching of Ile results in helix destabilization. We set out to investigate whether there are general trends for the occurrences of Leu and Ile residues in the structures and sequences of class A GPCRs (G protein-coupled receptors). GPCRs are integral membrane proteins in which α-helices span the plasma membrane seven times and which play a crucial role in signal transmission. We found that Leu side chains are generally more exposed at the protein surface than Ile side chains. We explored whether this difference might be attributed to different functions of the two amino acids and tested if Leu tunes the hydrophobicity of the transmembrane domain based on the Wimley-White whole-residue hydrophobicity scales. Leu content decreases the variation in hydropathy between receptors and correlates with the non-Leu receptor hydropathy. Both measures indicate that hydropathy is tuned by Leu. To test this idea further, we generated protein sequences with random amino acid compositions using a simple numerical model, in which hydropathy was tuned by adjusting the number of Leu residues. The model was able to replicate the observations made with class A GPCR sequences. We speculate that the hydropathy of transmembrane domains of class A GPCRs is tuned by Leu (and to some lesser degree by Lys and Val) to facilitate correct insertion into membranes and/or to stably anchor the receptors within membranes.

Study on solubility and solvation thermodynamics for the advancement of biorelevant activities of l-isoleucine and l-serine in aqueous ammonium chloride solutions in the temperature range of 288.15-308.15 K.

This study investigated the dissolution behavior of l-isoleucine and l-serine in an aqueous salt solution (ammonium chloride), examining how variations in temperature and electrolyte concentration affect their solubility. We conducted careful experiments and used mathematical calculations to explore interactions at a molecular level. We observed that the structure of these amino acids and salt concentration in the aqueous medium influence their interactions, which affects dissolution. In the presence of electrolytes, l-isoleucine demonstrated a salting-out effect whereas l-serine showed a salting-in effect. This work examines the solute-solvent interactions of these solutes in aqueous ammonium chloride solutions. l-isoleucine exhibits a nonspontaneous reaction with increasing salt concentrations whereas l-serine shows spontaneous behavior. Gibbs free energy analysis revealed greater stability of l-serine. The pH and conductance measurements showed how these factors influence solution properties. This insight helps us comprehend the nature and behavior of these molecules in different situations, which could be helpful in drug formulation or protein purification in the future.

Synthesis of (2S,3R,4R)-Dihydroxyisoleucine for Use in Amatoxin Synthesis.

We report a streamlined synthesis of (2S,3R,4R)-4,5-dihydroxy isoleucine (DHIle), an amino acid found in α-amanitin, which appears to be critical for toxicity. This synthetic route is transition metal-free and enables the production of significant quantities of DHIle with suitable protection for use in peptide synthesis. Its incorporation into a cytotoxic amatoxin analog is reported.

Importance of isoleucine residue in ion channel formation ability of 11-residue peptaibols.

Peptaibols are a class of short peptides, typically 7 to 20 amino acids long, characterized by noncanonical amino acid residues such as aminoisobutyric acid (Aib). Although the helix length is shorter than the membrane thickness, the 11-residue peptaibol trichorovin-XII (TV-XII) can form ion channels in membranes. Assuming that a higher proportion of isoleucine (Ile) relative to leucine (Leu) residues is crucial for maintaining the ion channel activity of TV-XII, peptide analogs of TV-XII with varying Ile content were designed, synthesized, and evaluated. The secondary structure of all derivatives under hydrophobic conditions was confirmed by CD measurement as an α-helix-like ß-bend ribbon spiral structure. The most stable ion channel activity was found in compound 4a with maximum Ile. Furthermore, the C-terminal Ile analog showed greater ion channel activity compared to the Leu analog. This suggests that the choice between Leu and Ile can influence the expression of ion channel activity, which will be crucial for the de novo designed functional peptides.

Phosphorylation of a chronic pain mutation in the voltage-gated sodium channel Nav1.7 increases voltage sensitivity.

Mutations in voltage-gated sodium channels (Navs) can cause alterations in pain sensation, such as chronic pain diseases like inherited erythromelalgia. The mutation causing inherited erythromelalgia, Nav1.7 p.I848T, is known to induce a hyperpolarized shift in the voltage dependence of activation in Nav1.7. So far, however, the mechanism to explain this increase in voltage sensitivity remains unknown. In the present study, we show that phosphorylation of the newly introduced Thr residue explains the functional change. We expressed wildtype human Nav1.7, the I848T mutant, or other mutations in HEK293T cells and performed whole-cell patch-clamp electrophysiology. As the insertion of a Thr residue potentially creates a novel phosphorylation site for Ser/Thr kinases and because Nav1.7 had been shown in Xenopus oocytes to be affected by protein kinases C and A, we used different nonselective and selective kinase inhibitors and activators to test the effect of phosphorylation on Nav1.7 in a human system. We identify protein kinase C, but not protein kinase A, to be responsible for the phosphorylation of T848 and thereby for the shift in voltage sensitivity. Introducing a negatively charged amino acid instead of the putative phosphorylation site mimics the effect on voltage gating to a lesser extent. 3D modeling using the published cryo-EM structure of human Nav1.7 showed that introduction of this negatively charged site seems to alter the interaction of this residue with the surrounding amino acids and thus to influence channel function. These results could provide new opportunities for the development of novel treatment options for patients with chronic pain.

DR7dA, a Novel Antioxidant Peptide Analog, Demonstrates Antifibrotic Activity in Pulmonary Fibrosis In Vivo and In Vitro.

Pulmonary fibrosis (PF), which is characterized by enhanced extracellular matrix (ECM) deposition, is an interstitial lung disease that lacks an ideal clinical treatment strategy. It has an extremely poor prognosis, with an average survival of 3-5 years after diagnosis. Our previous studies have shown that the antioxidant peptide DR8 (DHNNPQIR-NH2), which is extracted and purified from rapeseed, can alleviate PF and renal fibrosis. However, natural peptides are easily degraded by proteases in vivo, which limits their potency. We have since synthesized a series of DR8 analogs based on amino acid scanning substitution. DR7dA [DHNNPQ (D-alanine) R-NH2] is an analog of DR8 in which L-isoleucine (L-Ile) is replaced with D-alanine (D-Ala), and its half-life is better than that of DR8. In the current study, we verified that DR7dA ameliorated tumor growth factor (TGF)-ß1-induced fibrogenesis and bleomycin-induced PF. The results indicated that DR7dA reduced the protein and mRNA levels of TGF-ß1 target genes in TGF-ß1-induced models. Surprisingly, DR7dA blocked fibrosis in a lower concentration range than DR8 in cells. In addition, DR7dA ameliorated tissue pathologic changes and ECM accumulation in mice. BLM caused severe oxidative damage, but administration of DR7dA reduced oxidative stress and restored antioxidant defense. Mechanistic studies suggested that DR7dA inhibits ERK, P38, and JNK phosphorylation in vivo and in vitro All results indicated that DR7dA attenuated PF by inhibiting ECM deposition and oxidative stress via blockade of the mitogen-activated protein kinase (MAPK) pathway. Hence, compared with its parent peptide, DR7dA has higher druggability and could be a candidate compound for PF treatment in the future. SIGNIFICANCE STATEMENT: In order to improve druggability of DR8, we investigated the structure-activity relationship of it and replaced the L-isoleucine with D-alanine. We found that the stability and antifibrotic activity of DR7dA were significantly improved than DR8, as well as DR7dA significantly attenuated tumor growth factor (TGF)-ß1-induced fibrogenesis and ameliorated bleomycin-induced fibrosis by inhibiting extracellular matrix deposition and oxidative stress via blockade of the MAPK pathway, suggesting DR7dA may be a promising candidate compound for the treatment of PF.

Differentiation of leucine and isoleucine residues in peptides using charge transfer dissociation mass spectrometry (CTD-MS).

RATIONALE: The function of a protein or the binding affinity of an antibody can be substantially altered by the replacement of leucine (Leu) with isoleucine (Ile), and vice versa, so the ability to identify the correct isomer using mass spectrometry can help resolve important biological questions. Tandem mass spectrometry approaches for Leu/Ile (Xle) discrimination have been developed, but they all have certain limitations. METHODS: Four model peptides and two wild-type peptide sequences containing either Leu or Ile residues were subjected to charge transfer dissociation (CTD) mass spectrometry on a modified three-dimensional ion trap. The peptides were analyzed in both the 1+ and 2+ charge states, and the results were compared to conventional collision-induced dissociation spectra of the same peptides obtained using the same instrument. RESULTS: CTD resulted in 100% sequence coverage for each of the studied peptides and provided a variety of side-chain cleavages, including d, w and v ions. Using CTD, reliable d and w ions of Xle residues were observed more than 80% of the time. When present, d ions are typically greater than 10% of the abundance of the corresponding a ions from which they derive, and w ions are typically more abundant than the z ions from which they derive. CONCLUSIONS: CTD has the benefit of being applicable to both 1+ and 2+ precursor ions, and the overall performance is comparable to that of other high-energy activation techniques like hot electron capture dissociation and UV photodissociation. CTD does not require chemical modifications of the precursor peptides, nor does it require additional levels of isolation and fragmentation.

Importance of Ile71 in ß-actin on histidine methyltransferase SETD3 catalysis.

SETD3-catalysed N3-methylation of His73 in ß-actin plays a key role in stabilisation of actin filaments in the metazoan cells. Overexpression and/or dysregulation of SETD3 is associated with several human pathologies, including cancer. Here, we examined the role of the Ile71 residue in ß-actin on human SETD3 catalysis. Substitution of Ile71 in ß-actin peptides by its natural and unnatural mimics reveals that the 'secondary' Ile71 binding pocket modulates the substrate efficiency of ß-actin. Our enzymatic work demonstrates that human SETD3 can accommodate structurally diverse hydrophobic side chains in its Ile71 binding pocket, providing clear limits of the size and shape of Ile analogues. Water thermodynamics calculations reveal that the Ile71 pocket is occupied by high-energy water molecules, that are released upon the Ile71 binding, contributing favourably to the SETD3-ßA complex formation. The work highlights that the hydrophobic Ile71 binding site plays an essential role in SETD3 catalysis, contributing to an ongoing effort in the design and development of chemical probes targeting SETD3.

How wide is the window opened by high-resolution relaxometry on the internal dynamics of proteins in solution?

The dynamics of molecules in solution is usually quantified by the determination of timescale-specific amplitudes of motions. High-resolution nuclear magnetic resonance (NMR) relaxometry experiments-where the sample is transferred to low fields for longitudinal (T1) relaxation, and back to high field for detection with residue-specific resolution-seeks to increase the ability to distinguish the contributions from motion on timescales slower than a few nanoseconds. However, tumbling of a molecule in solution masks some of these motions. Therefore, we investigate to what extent relaxometry improves timescale resolution, using the "detector" analysis of dynamics. Here, we demonstrate improvements in the characterization of internal dynamics of methyl-bearing side chains by carbon-13 relaxometry in the small protein ubiquitin. We show that relaxometry data leads to better information about nanosecond motions as compared to high-field relaxation data only. Our calculations show that gains from relaxometry are greater with increasing correlation time of rotational diffusion.

Molecular dynamic simulations, GIAO-NMR and TD-DFT spectroscopy analyze for zwitterionic isoleucine (ILE)N , 1 ≤ N ≤ 6, in water solution.

In this article, we investigate the effects of the isoleucine (ILE)N amino acid chain growth, N = 1.0.6, the ILE conformational effect as well as the solvent presence on the electrical and magnetic spectroscopic properties when these compounds are in aqueous solution. Computational molecular dynamics simulations were performed to include the solvent medium and generate uncorrelated configurations involving solute-solvent structures. The charge point model for solvent was used to obtain the results for quantum mechanical calculation, in special DFT calculations, for (ILE)N structures. Our results for the magnetic shielding constant obtained via GIAO-DFT-NMR calculations show that there is evidence of a magnetic behavior that characterizes the number of peptide bonds and, therefore, how the N isoleucine polypeptide chain is composed. TD-DFT results also show an absorption band shift to larger wavelengths indicating a dependence on N growth.

Lactoyl leucine and isoleucine are bioavailable alternatives for canonical amino acids in cell culture media.

Increasing demands for protein-based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules, and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine, and isoleucine, in particular at physiological pH. This study sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed-batch processes. N-lactoyl-leucine and N-lactoyl-isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N-lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.

5-Aryl-furan derivatives bearing a phenylalanine- or isoleucine-derived rhodanine moiety as potential PTP1B inhibitors.

Two series of 5-aryl-furan derivatives bearing a phenylalanine- or isoleucine-derived rhodanine moiety were identified as competitive protein tyrosine phosphatase 1B (PTP1B) inhibitors. Among the compounds studied, 5g was found to have the best PTP1B inhibitory potency (IC50 = 2.66 ± 0.16 µM) and the best cell division cycle 25 homolog B (CDC25B) inhibitory potency (IC50 = 0.25 ± 0.02 µM). Enzymatic data together with molecular modeling results demonstrated that the introduction of a sec-butyl group at the 2-position of the carboxyl group remarkably improved the PTP1B inhibitory activity.

Reversible Oligomerization and Reverse Hydrophobic Effect Induced by Isoleucine Tags Attached at the C-Terminus of a Simplified BPTI Variant.

Protein amorphous aggregation has become the focus of great attention, as it can impair the ability of cells to function properly. Here, we evaluated the effects of three peptide tags, consisting of one, three, and five consecutive isoleucines attached at the C-terminus end of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, BPTI-19A, on the thermal stability and oligomerization by circular dichroism spectrometry and differential scanning calorimetry in detail. All of the BPTI-19A variants exhibited a reversible and apparently two-state thermal transition like BPTI-19A at pH 4.7. The thermal transition of the five-isoleucine-tagged variant showed clear protein-concentration dependence, where the apparent denaturation temperature decreased as the protein concentration increased. Quantitative analysis indicated that this phenomenon originated from the presence of reversibly oligomerized (RO) states at high temperatures. The results also illustrated that the thermodynamic stability difference between the native and the monomeric denatured state in all the proteins was destabilized by the hydrophobic tags and was well explained by the reverse hydrophobic effect due to the tags. The existence of the RO states was confirmed by both analytical ultracentrifugation and dynamic light scattering. This indicated that the five-isoleucine hydrophobic tag is strong enough to induce intermolecular hydrophobic contact among the denatured molecules leading to oligomerization, and even one- or three-isoleucine tags are effective enough to generate intramolecular hydrophobic contact, thus provoking denaturation through the reverse hydrophobic effect.

Ligand-receptor co-evolution shaped the jasmonate pathway in land plants.

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates defense, growth and developmental responses in vascular plants. Bryophytes have conserved sequences for all JA-Ile signaling pathway components but lack JA-Ile. We show that, in spite of 450 million years of independent evolution, the JA-Ile receptor COI1 is functionally conserved between the bryophyte Marchantia polymorpha and the eudicot Arabidopsis thaliana but COI1 responds to different ligands in each species. We identified the ligand of Marchantia MpCOI1 as two isomeric forms of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA). We demonstrate that AtCOI1 functionally complements Mpcoi1 mutation and confers JA-Ile responsiveness and that a single-residue substitution in MpCOI1 is responsible for the evolutionary switch in ligand specificity. Our results identify the ancestral bioactive jasmonate and clarify its biosynthetic pathway, demonstrate the functional conservation of its signaling pathway, and show that JA-Ile and COI1 emergence in vascular plants required co-evolution of hormone biosynthetic complexity and receptor specificity.

Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.

Despite recent advances in structural definition of GPCR-G protein complexes, the basis of receptor selectivity between G proteins remains unclear. The Gα12 and Gα13 subtypes together form the least studied group of heterotrimeric G proteins. G protein-coupled receptor 35 (GPR35) has been suggested to couple efficiently to Gα13 but weakly to Gα12. Using combinations of cells genome-edited to not express G proteins and bioluminescence resonance energy transfer-based sensors, we confirmed marked selectivity of GPR35 for Gα13. Incorporating Gα12/Gα13 chimeras and individual residue swap mutations into these sensors defined that selectivity between Gα13 and Gα12 was imbued largely by a single leucine-to-isoleucine variation at position G.H5.23. Indeed, leucine could not be substituted by other amino acids in Gα13 without almost complete loss of GPR35 coupling. The critical importance of leucine at G.H5.23 for GPR35-G protein interaction was further demonstrated by introduction of this leucine into Gαq, resulting in the gain of coupling to GPR35. These studies demonstrate that Gα13 is markedly the most effective G protein for interaction with GPR35 and that selection between Gα13 and Gα12 is dictated largely by a single conservative amino acid variation.-Mackenzie, A. E., Quon, T., Lin, L.-C., Hauser, A. S., Jenkins, L., Inoue, A., Tobin, A. B., Gloriam, D. E., Hudson, B. D., Milligan, G. Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.

Identification and quantification of leucine and isoleucine residues in peptides using photoexcited tryptophan.

The molecular recognition ability of tryptophan (Trp) for isomeric amino acids, such as leucine (Leu) and isoleucine (Ile), and isomeric amino acid-containing dipeptides, such as Leu-Gly, Ile-Gly, Gly-Leu, and Gly-Ile (where Gly denotes glycine), was investigated using a tandem mass spectrometer equipped with an electrospray ionization source and cold ion trap. The ultraviolet photodissociation spectra of the cold gas-phase clusters of Leu and Ile with Na+Trp in the wavelength range of 265-290 nm revealed that the relative intensities of Leu and Ile were only different in the wavelength range of 265-273 nm; however, no differences in the relative intensities were observed when the wavelength exceeded 274 nm. The molecular recognition ability of photoexcited Trp was used for the identification and quantification of Leu and Ile in dipeptides in solution. The mole fractions of Leu and Ile in dipeptides could be determined from the abundances observed in a single product ion spectrum of the cold gas-phase clusters of dipeptides with Na+Trp.

Why do eukaryotic proteins contain more intrinsically disordered regions?

Intrinsic disorder is more abundant in eukaryotic than prokaryotic proteins. Methods predicting intrinsic disorder are based on the amino acid sequence of a protein. Therefore, there must exist an underlying difference in the sequences between eukaryotic and prokaryotic proteins causing the (predicted) difference in intrinsic disorder. By comparing proteins, from complete eukaryotic and prokaryotic proteomes, we show that the difference in intrinsic disorder emerges from the linker regions connecting Pfam domains. Eukaryotic proteins have more extended linker regions, and in addition, the eukaryotic linkers are significantly more disordered, 38% vs. 12-16% disordered residues. Next, we examined the underlying reason for the increase in disorder in eukaryotic linkers, and we found that the changes in abundance of only three amino acids cause the increase. Eukaryotic proteins contain 8.6% serine; while prokaryotic proteins have 6.5%, eukaryotic proteins also contain 5.4% proline and 5.3% isoleucine compared with 4.0% proline and ≈ 7.5% isoleucine in the prokaryotes. All these three differences contribute to the increased disorder in eukaryotic proteins. It is tempting to speculate that the increase in serine frequencies in eukaryotes is related to regulation by kinases, but direct evidence for this is lacking. The differences are observed in all phyla, protein families, structural regions and type of protein but are most pronounced in disordered and linker regions. The observation that differences in the abundance of three amino acids cause the difference in disorder between eukaryotic and prokaryotic proteins raises the question: Are amino acid frequencies different in eukaryotic linkers because the linkers are more disordered or do the differences cause the increased disorder?

Synthesis and activity of isoleucine sulfonamide derivatives as novel botulinum neurotoxin serotype A light chain inhibitors.

The botulinum neurotoxin (BoNT) is the most lethal protein known to man causing the deadly disease botulinum. The neurotoxin, composed of a heavy (HC) and light (LC) chain, work in concert to cause muscle paralysis. A therapeutic strategy to treat individuals infected with the neurotoxin is inhibiting the catalytic activity of the BoNT LC. We report the synthesis, inhibition study and computational docking analysis of novel small molecule BoNT/A LC inhibitors. A structure activity relationship study resulted in the discovery of d-isoleucine functionalized with a hydroxamic acid on the C-terminal and a biphenyl with chlorine at C- 2 connected by a sulfonamide linker at the N-terminus. This compound has a measured IC50 of 0.587 µM for the BoNT/A LC. Computational docking analysis indicates the sulfonamide linker adopts a geometry that is advantageous for binding to the BoNT LC active site. In addition, Arg363 is predicted to be involved in key binding interactions with the scaffold in this study.

Sequence-Dependent Nanofiber Structures of Phenylalanine and Isoleucine Tripeptides.

The molecular design of short peptides to achieve a tailor-made functional architecture has attracted attention during the past decade but remains challenging as a result of insufficient understanding of the relationship between peptide sequence and assembled supramolecular structures. We report a hybrid-resolution model to computationally explore the sequence-structure relationship of self-assembly for tripeptides containing only phenylalanine and isoleucine. We found that all these tripeptides have a tendency to assemble into nanofibers composed of laterally associated filaments. Molecular arrangements within the assemblies are diverse and vary depending on the sequences. This structural diversity originates from (1) distinct conformations of peptide building blocks that lead to different surface geometries of the filaments and (2) unique sidechain arrangements at the filament interfaces for each sequence. Many conformations are available for tripeptides in solution, but only an extended ß-strand and another resembling a right-handed turn are observed in assemblies. It was found that the sequence dependence of these conformations and the packing of resulting filaments are determined by multiple competing noncovalent forces, with hydrophobic interactions involving Phe being particularly important. The sequence pattern for each type of assembly conformation and packing has been identified. These results highlight the importance of the interplay between conformation, molecular packing, and sequences for determining detailed nanostructures of peptides and provide a detailed insight to support a more precise design of peptide-based nanomaterials.