Protein–Nucleic Acid Conjugation with Sterol Linkers Using Hedgehog Autoprocessing

• Published in: Bioconjugate chemistry 2019-11, Vol.30 (11), p.2799-2804
• Main Authors: Zhang, Xiaoyu, Xu, Zihan, Moumin, Dina S, Ciulla, Daniel A, Owen, Timothy S, Mancusi, Rebecca A, Giner, José-Luis, Wang, Chunyu, Callahan, Brian P
• Format: Article
• Language: English
• Subjects: Animals; Chimeras; Conjugates; Conjugation; Deoxyribonucleic acid; DNA; Drosophila; Drosophila Proteins - chemistry; Drosophila Proteins - metabolism; Enzymatic activity; Glycine; Hedgehog protein; Hedgehog Proteins - chemistry; Hedgehog Proteins - metabolism; Kinetics; Modularity; Nucleic acids; Nucleic Acids - chemistry; Nucleic Acids - metabolism; Organic chemistry; Precursors; Proteins; Reaction kinetics; Reagents; Sterols; Sterols - chemistry; Sterols - metabolism; Substrates
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/acs.bioconjchem.9b00550

Hedgehog (Hh) precursor proteins contain an autoprocessing domain called HhC whose native function is protein cleavage and C-terminal glycine sterylation. The transformation catalyzed by HhC occurs in cis from a precursor protein and exhibits wide tolerance toward both sterol and protein substrates. Here, we repurpose HhC as a 1:1 protein–nucleic acid ligase, with the sterol serving as a molecular linker. A procedure is described for preparing HhC-active sterylated DNA, called steramers, using aqueous compatible chemistry and commercial reagents. Steramers have K M values of 7–11 μM and reaction t 1/2 values of ∼10 min. Modularity of the HhC/steramer method is demonstrated using four different proteins along with structured and unstructured sterylated nucleic acids. The resulting protein–DNA conjugates retain the native solution properties and biochemical function. Unlike self-tagging domains, HhC does not remain fused to the conjugate; rather, enzymatic activity is mechanistically coupled to conjugate release. That unique feature of HhC, coupled with efficient kinetics and substrate tolerance, may ease access and open new applications for these suprabiological chimeras.