Site-specific chemical protein conjugation using genetically encoded aldehyde tags

Rabuka, David; Rush, Jason S.; deHart, Gregory W.; Wu, Peng; Bertozzi, Carolyn R.

doi:10.1038/nprot.2012.045

Cited by 226 publications

(196 citation statements)

References 21 publications

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“…FGly generating enzymes (FGEs), found in eukaryotes or aerobically living prokaryotes, generate FGly by oxidizing a cysteine residue on the target sulfatase using molecular oxygen (6,7), whereas anaerobic sulfatase maturating enzymes (anSMEs) generate FGly from either cysteine or serine residues on their target sulfatases using S-adenosyl-L-methionine (AdoMet) radical chemistry (8)(9)(10)(11). In addition to their importance for sulfatase chemistry, FGEs have commercial applications for generating site-specific "aldehyde tags" to use in proteinlabeling technology (12). While FGEs have been characterized in terms of structure and mechanism (6, 7), far less is known about their anaerobic cousins, the anSMEs.…”

mentioning

confidence: 99%

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

Goldman¹,

Grove²,

Sites³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

112

197

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Arylsulfatases require a maturating enzyme to perform a co-or posttranslational modification to form a catalytically essential formylglycine (FGly) residue. In organisms that live aerobically, molecular oxygen is used enzymatically to oxidize cysteine to FGly. Under anaerobic conditions, S-adenosylmethionine (AdoMet) radical chemistry is used. Here we present the structures of an anaerobic sulfatase maturating enzyme (anSME), both with and without peptidyl-substrates, at 1.6-1.8 Å resolution. We find that anSMEs differ from their aerobic counterparts in using backbone-based hydrogen-bonding patterns to interact with their peptidylsubstrates, leading to decreased sequence specificity. These anSME structures from Clostridium perfringens are also the first of an AdoMet radical enzyme that performs dehydrogenase chemistry. Together with accompanying mutagenesis data, a mechanistic proposal is put forth for how AdoMet radical chemistry is coopted to perform a dehydrogenation reaction. In the oxidation of cysteine or serine to FGly by anSME, we identify D277 and an auxiliary [4Fe-4S] cluster as the likely acceptor of the final proton and electron, respectively. D277 and both auxiliary clusters are housed in a cysteinerich C-terminal domain, termed SPASM domain, that contains homology to ∼1,400 other unique AdoMet radical enzymes proposed to use [4Fe-4S] clusters to ligate peptidyl-substrates for subsequent modification. In contrast to this proposal, we find that neither auxiliary cluster in anSME bind substrate, and both are fully ligated by cysteine residues. Instead, our structural data suggest that the placement of these auxiliary clusters creates a conduit for electrons to travel from the buried substrate to the protein surface.iron-sulfur cluster fold | radical SAM dehydrogenase

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mentioning

confidence: 99%

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

Goldman¹,

Grove²,

Sites³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

112

197

View full text Add to dashboard Cite

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“…Fifth, it affords a practical alternative to genetic fusions for the installation of protein-sized fusion partners that may affect the conformation and secretion of one or both fusion partners, as well as heterooligomeric proteins, provided at least one of them has an exposed stretch of glycine residues. The sortase-mediated installation of "click" handles or aldehyde tags further extends the range of possibilities (9,25). Whether there is an upper limit to the size of incoming nucleophiles that can be used in a sortase-mediated transpeptidation reaction remains to be determined, but we have successfully used polypeptides of as much as ∼100 kDa in reactions that effectuate head-to-tail protein-protein fusions (26).…”

Section: Discussionmentioning

confidence: 99%

Sortase-mediated modification of αDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes

Swee

Guimarães

Sehrawat

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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A monoclonal antibody against the C-type lectin DEC205 (αDEC205) is an effective vehicle for delivery of antigens to dendritic cells through creation of covalent αDEC205-antigen adducts. These adducts can induce antigen-specific T-cell immune responses or tolerance. We exploit the transpeptidase activity of sortase to install modified peptides and protein-sized antigens onto the heavy chain of αDEC205, including linkers that contain nonnatural amino acids. We demonstrate stoichiometric site-specific labeling on a scale not easily achievable by genetic fusions (49 distinct fusions in this report). We conjugated a biotinylated version of a class I MHCrestricted epitope to unlabeled αDEC205 and monitored epitope generation upon binding of the adduct to dendritic cells. Our results show transfer of αDEC205 heavy chain to the cytoplasm, followed by proteasomal degradation. Introduction of a labile dipeptide linker at the N terminus of a T-cell epitope improves proteasome-dependent class I MHC-restricted peptide cross-presentation when delivered by αDEC205 in vitro and in vivo. We also conjugated αDEC205 with a linker-optimized peptide library of known CD8 T-cell epitopes from the mouse γ-herpes virus 68. Animals immunized with such conjugates displayed a 10-fold reduction in viral load.antibody conjugates | vaccines | vaccine development | protein engineering A monoclonal antibody against the C-type lectin DEC205, αDEC205, recognizes DEC205, a ∼250-kDa surface protein on dendritic cells (DCs) (1, 2). Attachment of an antigenic moiety to αDEC205, either through chemical conjugation (3) or as a genetic fusion (4), allows its targeted delivery to DCs in vivo, and elicits potent immune responses even with very small amounts of the αDEC205 adduct. This response includes CD4 and CD8 T cells specific for the antigen attached to the antibody (5). Equipping αDEC205 with a suitable payload, as well as similar modifications of other antibodies directed against yet other surface proteins on professional antigen presenting cells (6), holds promise as a purely protein-based vaccine strategy that does not require replicating or infectious agents.Most chemical conjugation methods one might consider for modification of αDEC205 lack precision, as they predominantly target exposed lysine or cysteine residues. Even the introduction of a single suitably exposed cysteine by mutagenesis, although it affords good site-specificity of labeling, still limits the range of possibilities for covalent attachment of peptides and proteins. Fusion of αDEC205 and the payload of interest can be attained genetically, but expression and purification of each new individual adduct needs to be explored and optimized. These limitations impelled us to develop an alternative method to install payloads of interest onto αDEC205 in a more flexible manner. An efficient and straightforward chemoenzymatic alternative with the use of sortase A (7) should allow not only the facile installation of a large diversity of antigens, but also an analysis of the mechanisms th...

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“…The conjugations between these proteins and their substrates exhibit high selectivity and high affinity, even within cells and cell extracts [24]. The second technology is a series of 'enzyme-mediated' labeling strategies such as biotin ligase-mediated [31], phosphopantetheine transferase-mediated [32], lipoic acid ligase-mediated [33], and formylglycine-generating enzyme-mediated [34,35] approaches. These techniques involve the recognition of specific amino-acid motifs of cellular proteins that are then modified with remarkably high specificity.…”

Section: Box 1 Labeling Proteins With Organic Fluorophores Within Celmentioning

confidence: 99%

Bringing single-molecule spectroscopy to macromolecular protein complexes

Joo

Fareh

Kim

2013

Trends in Biochemical Sciences

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Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. Single-molecule protein studiesSingle-molecule techniques have become potent tools for the discovery of novel protein mechanisms by allowing high spatiotemporal resolution. Sub-nanometer resolution, the ultimate scale of biological systems, has been reached with single-molecule fluorescence microscopy[1] and spectroscopy [2]; single-molecule force and torque spectroscopy [3,4]; atomic force microscopy [5] and spectroscopy [6]; and nanopores [7]. Measurements can be carried out on the biologically relevant time scales of microseconds to minutes.Among these techniques, single-molecule fluorescence spectroscopy has enabled researchers to unveil the action mechanism of a protein by imaging protein activity in real time. Benefitting from advances in general microscopy[1], detection devices [8], and fluorophore physics [9,10], single-molecule fluorescence spectroscopy has reached sub-nanometer spatial resolution [11] and microsecond temporal resolution [12,13]. Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17] Single-molecule observations using cell extractsUnlike purified recombinant proteins, crude cell extracts provide more biologically relevant environment in which molecules of interest can interact not only with their partners but also with other cellular components. It was anticipated to combine this approach with single molecule techniques and observe the assembly and function of macromolecular complexes in real time. However, technical hurdles related to the specificity and efficiency of labeling molecules of interest within crude cell extracts had been reported [22][23][24]. In order to overcome this limitation, several groups recently developed original approaches. Hoskins et al. used protein complexes specifically tagged with fluorophores [14,19], and Yardimci et al. immunostained reaction products using specific fluorescent antibodies [15,20]. Real-time observation of spliceosome assemblyDespite the relatively small number of genes in the human genome, we have extraordin...

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Site-specific chemical protein conjugation using genetically encoded aldehyde tags

Cited by 226 publications

References 21 publications

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

Sortase-mediated modification of αDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes

Bringing single-molecule spectroscopy to macromolecular protein complexes

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