# A Unique THN Motif Is Critical for Enabling Efficient C‐Terminal Traceless Cleavage

## TL;DR

Researchers found that a specific THN motif is crucial for efficient traceless protein cleavage in inteins, which could improve their use in biotechnology.

## Contribution

The study identifies the THN motif as essential for high-efficiency traceless C-terminal cleavage in inteins.

## Key findings

- The THN motif is critical for separating Asp107 from Asn125 and C-extein residues, enabling efficient cleavage.
- Mutating Thr123 disrupts the THN motif and leads to inactivity in traceless cleavage.
- A flexible block F enhances the speed of C-cleavage in inteins.

## Abstract

Traceless protein cleavage is a significant challenge in intein application, as most common inteins studied today are not both active and promiscuous. In this study, the intein gp41‐1 is engineered, which demonstrates the most efficient traceless cleavage reported to date and shows high compatibility to 1st amino acid. The evidence provided for the first time is that the unique THN motif, which is prevalent in class 3 inteins, is essential for achieving high‐efficiency traceless C‐terminal cleavage. The hydrogen bond between the hydroxyl group of Thr123 and the main chain of His124 is suggested to be indispensable for stabilizing the THN motif to separate Asp107 (the limiting factor for C‐cleavage) from Asn125 and the C‐extein residues from the active sites, which jointly lead to the highest traceless C‐cleavage activity. Both cleavage data and molecular dynamics (MD) simulations results demonstrate that mutating Thr123 greatly disturbed the THN motif, leading to inactivity. These findings reveal a pivotal motif for intein traceless cleavage efficiency, providing valuable insights for designing inteins with enhanced traceless C‐terminal cleavage capabilities in future applications.

THN is identified as the key motif to facilitate the rapid traceless cleavage of gp41‐1. The deflection of THN motif not only avoids the interaction between C‐exteins and active sites, but also separates Asp107 away from Asn125. In addition, a flexible block F contributes to faster C‐cleavage. The unique THN offers a new insight for identifying high active inteins.