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Site-selective photo crosslink to methyllysine readers via single electron transfer between sulfonium and tryptophan

ABSTRACT: Reader proteins play significant roles in cell signaling from site-specific lysine methylation, but the progress of new reader investigations is rather slow. Photoreactive crosslinkers enable reader identifications, but it requires laborious chemical synthesis for optimization of the photoreactive group placement. In addition, active intermediate such as nitrene and carbene may cause significant non-specific crosslinking. Here we report dimethylsulfonium as methyllysine mimic binds to specific readers and subsequently crosslinks to conserved tryptophan inside binding pocket via single electron transfer under UV irradiation. Because the σ-π electron-donor-acceptor interaction between sulfonium and indole is a key step, the crosslinking exhibits excellent tryptophan site-selectivity and orthogonality to distinct methyllysine readers. Moreover, excited tryptophan without proximate sulfonium leads to minimal non-specific crosslinking from relaxation. Consequently, this method could escalate discovery of methyllysine readers from complicated cell samples. Furthermore, this photo crosslinking concept could be expanded to develop more types of microenvironment-dependent conjugations to site-specific tryptophan.

ORGANISM(S): Homo Sapiens

SUBMITTER: Lihua Zhang

PROVIDER: PXD049149 | iProX | Sun Feb 04 00:00:00 GMT 2024

REPOSITORIES: iProX

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Project description:The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco containing compartments known as pyrenoids in diverse algal lineages and carboxysomes in prokaryotes. Inside these compartments actively transported bicarbonate is converted into CO2 gas, which saturates the carboxylase with its substrate. Using co-immunoprecipitation experiments in Phaeodactylum tricornutum we have identified the Rubisco linker protein PYCO1. Similar to the green algal Rubisco linker protein EPYC1, PYCO1 is intrinsically disordered, possesses repeats and is positively charged at physiological pH. However, it possesses no sequence similarity to EPYC1, as expected for convergent evolution of a red Rubisco containing pyrenoid. Fluorescent PYCO1 fusion proteins localize as a rod shaped structure in the diatom chloroplast, consistent with the shape of the pyrenoid defined by transmission electron microscopy. To test the hypothesis that PYCO1 is the diatom pyrenoid scaffold we produced pure protein in Escherichia coli. Recombinant PYCO1 protein undergoes homotypic liquid liquid phase separation in a salt dependent manner. Diatom Rubisco specifically partitions into PYCO1 condensates. Heterotypic PYCO1-Rubisco condensates can bind up to three Rubisco hexadecamers per PYCO1 protein. Rubisco carboxylase function is unaffected in the condensates. PYCO1 is highly mobile in homotypic condensates. In contrast PYCO1 condensates saturated with diatom Rubisco have greatly reduced dynamics, with both PYCO1 and Rubisco becoming immobile. Consistently, FRAP experiments indicate that PYCO1 is not mobile in vivo. A combination of Cryo-electron microscopy and site-directed mutagenesis data show that the KWSP motif found in PYCO1 repeats binds to small subunits at the entrance of the Rubisco hexadecamer’s solvent channel. Analysis of mutant PYCO1 proteins show that both the “KWSP” tryptophan and another repeating tyrosine are essential for homotypic phase separation. We speculate that the unusual material properties of the PYCO1-Rubisco condensate are necessary to support the unusual non-spherical shape of the Phaeodactylum pyrenoid. Careful characterization of multiple diverse Rubisco condensates will strengthen translational approaches aiming to introduce pyrenoids and other metabolic condensates into new host organisms.

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Site-selective photo crosslink to methyllysine readers via single electron transfer between sulfonium and tryptophan

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