Background: Protein arginine methyltransferase 7 (PRMT7) is associated with various functions and diseases, but its substrate specificity is poorly defined. Results: Insect cell-expressed PRMT7 forms -monomethylarginine residues at basic RXR sequences in peptides and histone H2B. Conclusion: PRMT7 is a type III PRMT with a unique substrate specificity. Significance: Novel post-translational modification sites generated by PRMT7 may regulate biological function.

Elucidating biological and pathological functions of protein lysine acetyltransferases (KATs) greatly depends on the knowledge of the dynamic and spatial localization of their enzymatic targets in the cellular proteome. Here we report the design and application of chemical probes for facile labeling and detection of substrates of the three major human KAT enzymes. In this approach, we attempted to create engineered KATs in junction with synthetic Ac-CoA surrogates to effectively label KAT substrates even in the presence of competitive nascent cofactor acetyl-CoA. The functionalized and transferable acyl moiety of the Ac-CoA analogs further allowed the labeled substrates to be probed with alkynyl or azido-tagged fluorescent reporters by the copper-catalyzed azide–alkyne cycloaddition. The synthetic co-factors, in combination with either native or rationally engineered KAT enzymes, provide a versatile chemical biology strategy to label and profile cellular targets of KATs at the proteomic level.

Although the structure of insulin has been well studied, the formation pathway of the three disulfide bridges during the refolding of insulin precursor is ambiguous. Here, we reported the in vitro disulfide-forming pathway of a recombinant porcine insulin precursor (PIP). In redox buffer containing L-arginine, the yield of native PIP from fully reduced/denatured PIP can reach 85%. The refolding process was quenched at different time points, and three distinct intermediates, including one with one disulfide linkage and two with two disulfide bridges, have been captured and characterized. An intra-A disulfide bridge was found in the former but not in the latter. The two intermediates with two disulfide bridges contain the common A20-B19 disulfide linkage and another inter-AB one. Based on the time-dependent formation and distribution of disulfide pairs in the trapped intermediates, two different forming pathways of disulfide bonds in the refolding process of PIP in vitro have been proposed. The first one involves the rapid formation of the intra-A disulfide bond, followed by the slower formation of one of the inter-AB disulfide bonds and then the pairing of the remaining cysteines to complete the refolding of PIP. The second pathway begins first with the formation of the A20-B19 disulfide bridge, followed immediately by another inter-AB one, possibly nonnative. The nonnative two-disulfide intermediates may then slowly rearrange between CysA6, CysA7, CysA11, and CysB7, until the native disulfide bond A6-A11 or A7-B7 is formed to complete the refolding of PIP. The proposed refolding behavior of PIP is compared with that of IGF-I and discussed.

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.crystal structure | enzyme catalysis | PRMT | histone methylation | epigenetics P osttranslational modifications of proteins can affect their structure, catalytic activity, and molecular interactions (1). Methylation of the guanidino group of arginine residues represents a prominent subset of these reactions (2). Histone arginine methylation is associated with gene silencing and activation (3); the modification of arginine residues in a variety of nonhistone proteins, including splicing and transcription factors, can regulate their activity (4, 5).Most of the enzymes that catalyze arginine methylation are designated protein arginine methyltransferases (PRMTs) and require the cofactor S-adenosyl-L-methionine (AdoMet) as the methyl donor (6). Four types of arginine methylation products havedimethylarginine (SDMA), and δ-N G -monomethylarginine (6, 7). Accordingly, PRMTs can be categorized into four groups: Type I PRMTs catalyze ADMA formation, type II PRMTs catalyze SDMA formation, type III PRMTs catalyze MMA formation, and type IV PRMTs catalyze δ-N G -monomethylarginine formation. Type I, II, and III PRMTs are widely distributed in nature whereas type IV PRMTs seem to be limited to yeasts and plants (8). Interestingly, whereas type I and II enzymes catalyze MMA production in addition ...