An Atypical Mechanism of Split Intein Molecular Recognition and Folding

Abstract

Split inteins associate to trigger protein splicing in trans, a post-translational modification in which protein sequences fused to the intein pair are ligated together in a traceless manner. Recently, a family of naturally split inteins has been identified that is split at a noncanonical location in the primary sequence. These atypically split inteins show considerable promise in protein engineering applications; however, the mechanism by which they associate is unclear and must be different from that of previously characterized canonically split inteins due to unique topological restrictions. Here, we use a consensus design strategy to generate an atypical split intein pair (Cat) that has greatly improved activity and is amenable to detailed biochemical and biophysical analysis. Guided by the solution structure of Cat, we show that the association of the fragments involves a disorder-to-order structural transition driven by hydrophobic interactions. This molecular recognition mechanism satisfies the topological constraints of the intein fold and, importantly, ensures that premature chemistry does not occur prior to fragment complementation. Our data lead a common blueprint for split intein complementation in which localized structural rearrangements are used to drive folding and regulate protein-splicing activity.

Live-cell protein engineering with an ultra-short split intein

“…The resultant 10-fold increase in K d despite a near 1,000-fold increase in NaCl concentration (38 ± 12 nM at 5 mM NaCl; 416 ± 86 nM at 4 M NaCl) supports an association and folding mechanism with little contribution from electrostatic interactions. This finding is similar to a recent report for another family of atypical inteins with significantly longer N-intein fragments (16), but stands in contrast to the well-studied "capture and collapse" mechanism of the DnaE family of canonically split inteins (4).…”

“…Atypically split inteins, which feature much shorter Int N fragments compared to their canonically split counterparts, hold great promise for studying challenging protein substrates (13)(14)(15)(16). In principle, the small size of the Int N fragment expands the range of N-extein cargoes that can be appended through synthetic means, opening new possibilities for protein semisynthesis, including potential live-cell applications.…”

Chemoenzymatic Semisynthesis of Proteins

“…This basic paradigm is also true for the atypically split intein, Cat TerL, even though the details of the folding pathway differ. 392…”

Chemical methods for studying the crosstalk between histone H2B ubiquitylation and H3 methylation

“…More importantly, techniques suitable in in vivo applications remain one of the key challenges in studying histone PTMs. [97][98][99][100][101][102][103][104] Therefore, we anticipate that more chemical methods would be developed to constitute a versatile toolbox that can facilitate the mechanistic understanding of histone PTM crosstalk.…”

“…Besides, the methods summarized in this review are unable to stabilize a weak or transient enzyme–substrate complex for structure determination. More importantly, techniques suitable in in vivo applications remain one of the key challenges in studying histone PTMs 97–104 . Therefore, we anticipate that more chemical methods would be developed to constitute a versatile toolbox that can facilitate the mechanistic understanding of histone PTM crosstalk.…”