Abstract
Protein splicing catalyzed by inteins utilizes many different combinations of amino-acid types at active sites. Inteins have been classified into three classes based on their characteristic sequences. We investigated the structural basis of the protein splicing mechanism of class 3 inteins by determining crystal structures of variants of a class 3 intein from Mycobacterium chimaera and molecular dynamics simulations, which suggested that the class 3 intein utilizes a different splicing mechanism from that of class 1 and 2 inteins. The class 3 intein uses a bond cleavage strategy reminiscent of proteases but share the same Hedgehog/INTein (HINT) fold of other intein classes. Engineering of class 3 inteins from a class 1 intein indicated that a class 3 intein would unlikely evolve directly from a class 1 or 2 intein. The HINT fold appears as structural and functional solution for trans-peptidyl and trans-esterification reactions commonly exploited by diverse mechanisms using different combinations of amino-acid types for the active-site residues.
Highlights
Protein splicing is catalyzed by intervening protein sequences termed inteins
We originally attempted the crystallization of the class 3 DnaB1 intein from Mycobacterium smegmatis (MsmDnaB1) but failed, presumably because the purified MsmDnaB1 intein was not well-folded as judged from the HSQC spectrum (Supplemental Figure S1b)
This observation was in line with our tests for protein cis-splicing activity of class 3 inteins from Deinococcus radiodurans (Dra), Mycobacterium smegmatis (Msm), and Mycobacterium chimaera (Mch) using a model protein system
Summary
Protein splicing is catalyzed by intervening protein sequences termed inteins. The protein-splicing reaction involves the self-removal of the intein and concomitant joining of the two flanking sequences (exteins) (Figure 1) [1,2]. The biological function of protein splicing is still enigmatic despite several proposals for eventual regulatory functions [3]. Inteins are often considered merely as selfish gene elements because they can be generally removed without any fitness cost for their host organisms. Inteins commonly insert in conserved sequences close to the active sites of essential proteins. Any mutations within inteins detrimental to the protein splicing activity could be lethal or strongly affect the fitness of their host, likely ensures intein persistence and protection from functional degeneration during evolution [4]
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