Protein Thioesters Research Articles

Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display

Intein-mediated expressed protein ligation (EPL) permits the site-specific chemical customization of proteins. While traditional techniques have used purified, soluble proteins, we have extended these methods to release and modify intein fusion proteins expressed on the yeast surface, thereby eliminating the need for soluble protein expression and purification. To this end, we sought to simultaneously release yeast surface-displayed proteins and selectively conjugate with chemical functionalities compatible with EPL and click chemistry. Single-chain antibodies (scFv) and green fluorescent protein (GFP) were displayed on the yeast surface as fusions to the N-terminus of the Mxe GyrA intein. ScFv and GFP were released from the yeast surface with either a sulfur nucleophile (MESNA) or a nitrogen nucleophile (hydrazine) linked to an azido group. The hydrazine azide permitted the simultaneous release and azido functionalization of displayed proteins, but nonspecific reactions with other yeast proteins were detected, and cleavage efficiency was limited. In contrast, MESNA released significantly more protein from the yeast surface while also generating a unique thioester at the carboxy-terminus of the released protein. These protein thioesters were subsequently reacted with a cysteine alkyne in an EPL reaction and then employed in an azide-alkyne cycloaddition to immobilize the scFv and GFP on an azide-decorated surface with >90% site-specificity. Importantly, the immobilized proteins retained their activity. Since yeast surface display is also a protein engineering platform, these approaches provide a particularly powerful tool for the rapid assessment of engineered proteins.

Protein thioester synthesis enabled by sortase.

Proteins containing a C-terminal thioester are important intermediates in semisynthesis. Currently there is one main method for the synthesis of protein thioesters that relies upon the use of engineered inteins. Here we report a simple strategy, utilizing sortase A, for routine preparation of recombinant proteins containing a C-terminal (α)thioester. We used our method to prepare two different anthrax toxin cargo proteins: one containing an (α)thioester and another containing a D-polypeptide segment situated between two protein domains. We show that both variants can translocate through protective antigen pore. This new method to synthesize a protein thioester allows for interfacing of sortase-mediated ligation and native chemical ligation.

Adaptor Protein Self-Assembly Drives the Control of a Cullin-RING Ubiquitin Ligase

The E3 ligases recruit substrate proteins for targeted ubiquitylation. Recent insights into the mechanisms of ubiquitylation demonstrate that E3 ligases can possess active regulatory properties beyond those of a simple assembly scaffold. Here, we describe the dimeric structure of the E3 ligase adaptor protein SPOP (speckle-type POZ protein) in complex with the N-terminal domain of Cul3 at 2.4Å resolution. We find that SPOP forms large oligomers that can form heteromeric species with the closely related paralog SPOPL. In combination, SPOP and SPOPL (SPOP-like) form a molecular rheostat that can fine-tune E3 ubiquitin ligase activity by affecting the oligomeric state of the E3 complex. We propose that adaptor protein self-assembly provides a graded level of regulation of the SPOP/Cul3 E3 ligase toward its multiple protein substrates.

The Burkholderia cenocepacia BDSF quorum sensing fatty acid is synthesized by a bifunctional crotonase homologue having both dehydratase and thioesterase activities

Signal molecules of the diffusible signal factor (DSF) family have been shown recently to be involved in regulation of pathogenesis and biofilm formation in diverse Gram-negative bacteria. DSF signals are reported to be active not only on their cognate bacteria, but also on unrelated bacteria and the pathogenic yeast, Candida albicans. DSFs are monounsaturated fatty acids of medium chain length containing an unusual cis-2 double bond. Although genetic analyses had identified genes involved in DSF synthesis, the pathway of DSF synthesis was unknown. The DSF of the important human pathogen Burkholderia cenocepacia (called BDSF) is cis-2-dodecenoic acid. We report that BDSF is synthesized from a fatty acid synthetic intermediate, the acyl carrier protein (ACP) thioester of 3-hydroxydodecanoic acid. This intermediate is intercepted by protein Bcam0581 and converted to cis-2-dodecenoyl-ACP. Bcam0581 is annotated as a homologue of crotonase, the first enzyme of the fatty acid degradation pathway. We demonstrated Bcam0581to be a bifunctional protein that not only catalysed dehydration of 3-hydroxydodecanoyl-ACP to cis-2-dodecenoyl-ACP, but also cleaved the thioester bond to give the free acid. Both activities required the same set of active-site residues. Although dehydratase and thioesterase activities are known activities of the crotonase superfamily, Bcam0581 is the first protein shown to have both activities.

Protein immobilization on liposomes and lipid‐coated nanoparticles by protein trans‐splicing

A plethora of methods exist to link proteins to surfaces in order to generate functionalized materials. However, general tools that lead to functional immobilization of recombinantly expressed proteins on membranes such as liposomes or lipid-coated nanoparticles are rare. Here we present an approach that takes advantage of a double-palmitoylated peptide that mediates stable membrane anchoring in combination with protein trans-splicing for efficient immobilization of recombinant proteins fused to split intein segments. Two different DnaE split inteins from Synechocystis and Nostoc punctiforme are tested and compared to immobilization via direct native chemical ligation using a protein thioester. Protein trans-splicing proceeds at low protein concentrations and leads to functionalized vesicles and membrane-coated silica nanoparticles.

ChemInform Abstract: Peptide and Protein Thioester Synthesis via N→S Acyl Transfer

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Structural and Enzymatic Analysis of Orf6, a Novel Dehydratase from a Deep‐Sea Polyunsaturated Fatty Acid Synthase

The polyketide synthase (PKS) genes responsible for eicosapentanoic acid (EPA) biosynthesis in the deep‐sea bacterium, Photobacterium profundum, have been cloned and sequenced, being a total of five polyunsaturated fatty acid (pfa) genes (pfaA, pfaB, pfaC, pfaD and Orf6) enough for EPA production. One of these enzymes is Orf6, a putative thioesterase (TE) domain believed to perform the last step in the fatty acid biosynthesis, catalyzing the hydrolysis and release of the final product from the carrier protein. The purpose of our research is to make a structure‐function analysis of Orf6 TE using its three dimensional structure determined by X‐ray crystallography to guide the re‐engineering of the enzyme. In parallel, we are also performing in vitro enzymatic assays with its likely substrates loaded onto coenzyme A (CoA). The Orf6 structure determined at 1.0 Å resolution reveals a novel hot‐dog fold containing a conserved amino acid, Asp17, in the active site. Results from activity assays indicate that Orf6 does not hydrolyze the CoA thioesters of long‐chain fatty acids. Additional efforts include in vitro assays using CoA thioesters of short‐chain carboxylic acids or acyl carrier protein (ACP) thioesters as substrates for Orf6. Structural and mechanistic knowledge of this seemingly novel enzyme will be important for the design of lipid‐based drugs or the development of industrial applications like biofuels.

Chemical Approaches for Studying Histone Modifications

Histones form the protein core around which genomic DNA is wrapped in eukaryotic chromatin. Numerous genetic studies have established that the structure and transcriptional state of chromatin are closely related to histone post-translational modifications. Further elucidation of the precise mechanistic roles for individual histone modifications requires the ability to isolate and study homogeneously modified histones. However, the highly heterogeneous nature of histone modifications in vivo poses a significant challenge for such studies. Chemical tools that have enabled biochemical and biophysical studies of site-specifically modified histones are the focus of this minireview.

Expression of Vibrio harveyi Acyl-ACP Synthetase Allows Efficient Entry of Exogenous Fatty Acids into the Escherichia coli Fatty Acid and Lipid A Synthetic Pathways

Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function.

Peptide and protein thioester synthesis via N→S acyl transfer

Peptide and protein thioesters are playing an increasingly prominent role in the chemical toolbox for protein assembly and modification through Native Chemical Ligation (NCL). In this Emerging Area we highlight recent developments in a somewhat surprising route to thioesters: selective disruption of amides, the more stable carboxylic acid derivatives.

ChemInform Abstract: 3‐Mercaptopropionic Acid‐Mediated Synthesis of Peptide and Protein Thioesters.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

3-Mercaptopropionic acid-mediated synthesis of peptide and protein thioesters.

Peptides and proteins fragment sequence-specifically in the presence of 3-mercaptopropionic acid to afford thioesters which can be used in native chemical ligation reactions.

Expressed Protein Ligation (EPL) in the Study of Signal Transduction, Ion Conduction, And Chromatin Biology

Expressed protein ligation (EPL) is a semisynthetic technique in which a recombinant protein thioester, generated by thiolysis of an intein fusion protein, is reacted with a synthetic or recombinant peptide with an N-terminal cysteine to produce a native peptide bond. This method has been used extensively for the incorporation of biophysical probes, unnatural amino acids, and post-translational modifications in proteins. In the 10 years since this technique was developed, the applications of EPL to studying protein structure and function have grown ever more sophisticated. In this Account, we review the use of EPL in selected systems in which substantial mechanistic insights have recently been gained through the use of the semisynthetic protein derivatives. EPL has been used in many studies to unravel the complexity of signaling networks and subcellular trafficking. Herein, we highlight this application to two different systems. First, we describe how phosphorylated or otherwise modified proteins in the TGF-beta signaling network were prepared and how they were applied to understanding the complexities of this pathway, from receptor activation to nuclear import. Second, Rab-GTPases are multiply modified with lipid derivatives, and EPL-based techniques were used to incorporate these modifications, allowing for the elucidation of the biophysical basis of membrane association and dissociation. We also review the use of EPL to understand the biology of two other systems, the potassium channel KcsA and histones. EPL was used to incorporate d-alanine and an amide-to-ester backbone modification in the selectivity filter of the KcsA potassium channel, providing insight into the mechanism of selectivity in ion conduction. In the case of histones, which are among the most heavily post-translationally modified proteins, the modifications play a key role in the regulation of gene transcription and chromatin structure. We describe how native chemical ligation and EPL were used to generate acetylated, phosphorylated, methylated, and ubiquitylated histones and how these modified histones were used to interrogate chromatin biology. Collectively, these studies demonstrate the utility of EPL in protein science. These techniques and concepts are applicable to many other systems, and ongoing advances promise to extend this semisynthetic technique to increasingly complex biological problems.

One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

BackgroundExpression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.ResultsA novel redox buffer consisting of MESNA and diMESNA showed a refolding efficiency comparable to that of GSH/GSSG and prevented loss of the protein's thioester functionality. Moreover, introduction of the MESNA/diMESNA redox couple in the cleavage buffer allowed simultaneous on-column refolding of Ribonuclease A and intein-mediated cleavage to yield Ribonuclease A with a C-terminal MESNA-thioester. The C-terminal thioester was shown to be active in native chemical ligation.ConclusionAn efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters. Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.

The Intrinsic Affinity between E2 and the Cys Domain of E1 in Ubiquitin-like Modifications

Ubiquitin-like modifications, which are carried out by similar biochemical mechanisms, regulate nearly every aspect of cellular function. Despite the recent advancements in characterizing their enzymology, our knowledge about the dynamic processes of these modifications is still fragmentary. In this study, we have uncovered an intrinsic affinity between the SUMO E2 and the Cys domain of SUMO E1. NMR studies in combination with paramagnetic spin labeling demonstrate that this interaction is mediated by previously unknown interfaces on both E1 and E2 and places the two catalytic Cys residues of the two enzymes in close proximity. Site-directed mutagenesis and enzymatic assays indicate that the interaction is fundamentally important for the transfer of SUMO from E1 to E2. Results from this study suggest that the interaction between E2 and the Cys domain of E1 participates in guiding the E2's translocation to E1's enzymatic active site in ubiquitin-like modifications.

Structure and reactivity of LpxD, the N -acyltransferase of lipid A biosynthesis

The external layer of the Gram-negative bacterial outer membrane is primarily composed of a protective, selectively permeable LPS. The biosynthesis of LPS relies on UDP-3-O-acyl-glucosamine N-acyltransferase (LpxD), which transfers 3-hydroxy-arachidic acid from acyl carrier protein to the 2' amine of UDP-3-O-myristoyl glucosamine in Chlamydia trachomatis. Our crystallographic study reveals that LpxD is a homotrimer, each subunit of which is constructed from a novel combination of an N-terminal uridine-binding domain, a core lipid-binding domain, and a C-terminal helical extension. Highly conserved residues dominate nucleotide binding. Phe-43 and Tyr-49 form pi-stacking interactions with uracil, and Asn-46 and His-284 form hydrogen bonds with the phosphate groups. These interactions place the glucosamine moiety at the catalytic center formed by two adjacent subunits. Here His-247 and His-284 contribute to a mechanism involving nucleophilic attack by the amine of one substrate on the carbonyl carbon of an acyl carrier protein thioester conjugate. Serendipitously, our study reveals a fatty acid (FA) binding groove near the catalytic center. MS elucidated the presence of a FA mixture binding to LpxD, with palmitic acid the most prevalent. The placement of UDP-N-acetylglucosamine and the FA provides details of N-acyltransferase ligand interactions and allows for a description of structure and reactivity at an early stage of LPS assembly.

Site-specific labeling of DNA–protein conjugates by means of expressed protein ligation

Site-specific bioconjugation of protein thioesters with a DNA oligonucleotide was achieved by Expressed Protein Ligation (EPL) and the new thiol group formed upon EPL in the conjugate was selectively coupled with small molecule labels using maleimide chemistry.

Microtiter Plate‐Based Screening for the Optimization of DNA–Protein Conjugate Synthesis by Means of Expressed Protein Ligation

We report a rapid microtiter plate screening assay for the optimization of the synthesis of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter containing a C-terminal thioester that enables a mild and highly specific reaction with N-terminal cysteine compounds. To screen for optimal reaction conditions, we developed a microtiter plate-based assay that utilizes DNA-directed immobilization of the products formed in the ligation reaction of cysteine-modified DNA oligonucleotides with the model protein thioester of the maltose-binding protein (MBP), recombinantly expressed as an intein-fusion protein in E. coli. The screening assay allowed the rapid quantitative monitoring of various reaction parameters, such as the ratio of the reactants, reaction times, pH and ion strength of the buffer, the influence of various thiol additives and the nature of the chemical linker within the cysteine-bearing DNA oligonucleotide. As the consequence of the assay-based optimization, the ligation of MBP with the oligonucleotide was improved to near quantitative yields.

Expressed enzymatic ligation for the semisynthesis of chemically modified proteins.

Enzymatic synthesis and expressed protein ligation were combined in a method for protein synthesis in which protein thioesters with specific leaving groups could be used by protease V8 from Staphylococcus aureus to catalyze the formation of peptide bonds. This method was applied to the synthesis of fluorescence-labeled (Fl) analogues of proneuropeptide Y (proNPY; see scheme). CBD=chitin binding domain.

The Escherichia coli lipB gene encodes lipoyl (octanoyl)-acyl carrier protein:protein transferase.

In an earlier study (S. W. Jordan and J. E. Cronan, Jr., J. Biol. Chem. 272:17903-17906, 1997) we reported a new enzyme, lipoyl-[acyl carrier protein]-protein N-lipoyltransferase, in Escherichia coli and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase. It was also shown that E. coli lipB mutants lack this enzyme activity, a finding consistent with lipB being the gene that encoded the lipoyltransferase. However, it remained possible that lipB encoded a positive regulator required for lipoyltransferase expression or action. We now report genetic and biochemical evidence demonstrating that lipB encodes the lipoyltransferase. A lipB temperature-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the lipB-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. In the course of these experiments the ATG initiation codon commonly assigned to lipB genes in genomic databases was shown to produce a nonfunctional E. coli LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymatically active protein. Prior genetic results (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Bacteriol. 177:1-10, 1995) suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addition to its normal substrates lipoic acid plus ATP. We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. A nonhydrolyzable lipoyl-AMP analogue was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chemically diverse reactions.