Functional elucidation of TfuA in peptide backbone thioamidation.
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ABSTRACT:
SUBMITTER: Liu A
PROVIDER: S-EPMC8084935 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.
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Project description:Thioamides are found in a few natural products and two known protein assemblies: the Escherichia coli ribosome and methyl-coenzyme M reductase (MCR) from methane-metabolizing archaea. Compared to an amide, thioamides alter the physical and chemical properties of peptide backbones, including the conformation dynamics, proteolytic stability, hydrogen-bonding capabilities, and possibly reactivity of a protein when installed. Recently, there has been significant progress in elucidating enzymatic post-translational thioamide installation, with most work leveraging the archaeal MCR-modifying enzymes. This chapter describes the protocols used for the in vitro enzymatic thioamidation of MCR-derived peptides, including polypeptide overexpression, purification, reaction reconstitution, and mass spectrometry-based product analysis. In addition, we highlight the protocols used for the biochemical, kinetics, and binding studies using recombinant enzymes obtained heterologously from E. coli. We anticipate that these methods will serve to guide future studies on peptide post-translational thioamidation, as well as other peptide backbone modifications using a parallel workflow.
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Project description:Amidines are a structural surrogate for peptide bonds, yet have received considerably little attention in peptides due to limitations in existing methods to access them. The synthetic strategy developed in this study represents the first robust and general procedure for the introduction of amidines into the peptide backbone. We exploit and further develop the utility and efficiency of thioimidate protecting groups as a means to side-step reactivity that ultimately renders existing methods unsuitable for the installation of amidines along the main-chain of peptides. This work is significant because it describes a generally applicable path to access unexplored peptide designs and architectures for new therapeutics made possible by the unique properties of amidines.
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Project description:Thioamide-containing nonribosomal peptides (NRPs) are exceedingly rare. Recently the biosynthetic gene cluster for the thioamidated NRP antibiotic closthioamide (CTA) was reported, however, the enzyme responsible for and the timing of thioamide formation remained enigmatic. Here, genome editing, biochemical assays, and mutational studies are used to demonstrate that an Fe-S cluster containing member of the adenine nucleotide ?-hydrolase protein superfamily (CtaC) is responsible for sulfur incorporation during CTA biosynthesis. However, unlike all previously characterized members, CtaC functions in a thiotemplated manner. In addition to prompting a revision of the CTA biosynthetic pathway, the reconstitution of CtaC provides the first example of a NRP thioamide synthetase. Finally, CtaC is used as a bioinformatic handle to demonstrate that thioamidated NRP biosynthetic gene clusters are more widespread than previously appreciated.
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Project description:The N-dicyclopropylmethyl (Dcpm) residue, introduced into amino acids via reaction of dicyclopropylmethanimine hydrochloride with an amino acid ester followed by sodium cyanoborohydride or triacetoxyborohydride reduction, can be used as an amide bond protectant for peptide synthesis. Examples which demonstrate the amelioration of aggregation effects include syntheses of the alanine decapeptide and the prion peptide (106-126). Avoidance of cyclization to the aminosuccinimide followed substitution of Fmoc-(Dcpm)Gly-OH for Fmoc-Gly-OH in the assembly of sequences containing the sensitive Asp-Gly unit.
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Project description:Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting approximately 1-2% of the world population. One of the characteristic features of RA is the presence of autoantibodies. Especially the highly specific anti-citrullinated peptide antibodies (ACPAs), which have been found in up to 70% of RA patients' sera, have received much attention. Several citrullinated proteins are associated with RA, suggesting that ACPAs may react with different sequence patterns, separating them from traditional antibodies, whose reactivity usually is specific towards a single target. As ACPAs have been suggested to be involved in the development of RA, knowledge about these antibodies may be crucial. In this study, we examined the influence of peptide backbone for ACPA reactivity in immunoassays. The antibodies were found to be reactive with a central Cit-Gly motif being essential for ACPA reactivity and to be cross-reactive between the selected citrullinated peptides. The remaining amino acids within the citrullinated peptides were found to be of less importance for antibody reactivity. Moreover, these findings indicated that the Cit-Gly motif in combination with peptide backbone is essential for antibody reactivity. Based on these findings it was speculated that any amino acid sequence, which brings the peptide into a properly folded structure for antibody recognition is sufficient for antibody reactivity. These findings are in accordance with the current hypothesis that structural homology rather than sequence homology are favored between citrullinated epitopes. These findings are important in relation to clarifying the etiology of RA and to determine the nature of ACPAs, e.g., why some Cit-Gly-containing sequences are not targeted by ACPAs.
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Project description:Antifreeze proteins (AFPs) are a class of ice-binding proteins that promote survival of a variety of cold-adapted organisms by decreasing the freezing temperature of bodily fluids. A growing number of biomedical, agricultural, and commercial products, such as organs, foods, and industrial fluids, have benefited from the ability of AFPs to control ice crystal growth and prevent ice recrystallization at subzero temperatures. One limitation of AFP use in these latter contexts is their tendency to denature and irreversibly lose activity at the elevated temperatures of certain industrial processing or large-scale AFP production. Using the small, thermolabile type III AFP as a model system, we demonstrate that AFP thermostability is dramatically enhanced via split intein-mediated N- and C-terminal end ligation. To engineer this circular protein, computational modeling and molecular dynamics simulations were applied to identify an extein sequence that would fill the 20-Å gap separating the free ends of the AFP, yet impose little impact on the structure and entropic properties of its ice-binding surface. The top candidate was then expressed in bacteria, and the circularized protein was isolated from the intein domains by ice-affinity purification. This circularized AFP induced bipyramidal ice crystals during ice growth in the hysteresis gap and retained 40% of this activity even after incubation at 100°C for 30 min. NMR analysis implicated enhanced thermostability or refolding capacity of this protein compared to the noncyclized wild-type AFP. These studies support protein backbone circularization as a means to expand the thermostability and practical applications of AFPs.
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Project description:YcaO enzymes are known to catalyze the ATP-dependent formation of azoline heterocycles, thioamides, and (macro)lactamidines on peptide substrates. These enzymes are found in multiple biosynthetic pathways, including those for several different classes of ribosomally synthesized and post-translationally modified peptides (RiPPs). However, there are major knowledge gaps in the mechanistic and structural underpinnings that govern each of the known YcaO-mediated modifications. Here, we present the first structure of any YcaO enzyme bound to its peptide substrate in the active site, specifically that from Methanocaldococcus jannaschii which is involved in the thioamidation of the α-subunit of methyl-coenzyme M reductase (McrA). The structural data are leveraged to identify and test the residues involved in substrate binding and catalysis by site-directed mutagenesis. We also show that thioamide-forming YcaOs can carry out the cyclodehydration of a related peptide substrate, which underscores the mechanistic conservation across the YcaO family and allows for the extrapolation of mechanistic details to azoline-forming YcaOs involved in RiPP biosynthesis. A bioinformatic survey of all YcaOs highlights the diverse sequence space in azoline-forming YcaOs and suggests their early divergence from a common ancestor. The data presented within provide a detailed molecular framework for understanding this family of enzymes, which reconcile several decades of prior data on RiPP cyclodehydratases. These studies also provide the foundational knowledge to impact our mechanistic understanding of additional RiPP biosynthetic classes.
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Project description:We calculate the solubility limit of pentapeptides in water by simulating the phase separation in an oversaturated aqueous solution. The solubility limit order followed by our model peptides (GGRGG > GGDGG > GGGGG > GGVGG > GGQGG > GGNGG > GGFGG) is found to be the same as that reported for amino acid monomers from experiment (R > D > G > V > Q > N > F). Investigation of dynamical properties of peptides shows that the higher the solubility of a peptide is, the lower the time spent by the peptide in the aggregated cluster is. We also demonstrate that fluctuations in conformation and hydration number of peptide in monomeric form are correlated with the solubility of the peptide. We considered energetic mechanisms and dynamical properties of interbackbone CO-CO and CO···HN interactions. Our results confirm that CO-CO interactions more than the interbackbone H-bonds are important in peptide self-assembly and association. Further, we find that the stability of H-bonded peptide pairs arises mainly from coexisting CO-CO and CO···HN interactions.
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