The P-site A76 2′-OH acts as a peptidyl shuttle in a stepwise peptidyl transfer mechanism

Abstract

Notwithstanding various unsolved questions regarding the structure and function of the ribosome, the process of peptide bond formation is of particular importance, being the heart of protein synthesis. Several experimental studies have been carried out on the pre- and post-peptidyl transfer structures in the ribosomal active site. Based on these structures, different reaction mechanisms have been proposed and further investigated using computational techniques. However, the detailed mechanism of peptidyl transfer, as well as the atoms and functional groups involved in this process are still in limbo. Although it was suggested that the A2451 is present in the active site of the ribosome in the previous crystallographic structures, the details of its participation have not been fully investigated. Furthermore, despite the highlighted importance of the P-site A76 2′-OH group in previous studies, its actual role during the process is still unclear. Finally, whether the process of peptidyl transfer is a stepwise mechanism or a concerted one is still under debate. Several computational mechanistic studies have been carried out to investigate the catalytic power of the ribosome, yet, they do not cover all the three concerns mentioned above. Therefore, as well as re-investigating the previously proposed reaction mechanisms with a higher level of theory and basis set (i.e. M06-2X/6-31++G(d,p)//M06-2X/6-311++G(d,p)) in this study, we propose three new reaction mechanisms based on three different pre- and post-peptidyl transfer structures obtained from previous experimental studies. The results of this study highlights the important role played by the P-site A76 2′-OH group in catalyzing the reaction. However, instead of acting as a so-called proton shuttle, this group acts as a polypeptidyl shuttle, transferring the growing polypeptide chain from the leaving 3′-O to the attacking nucleophile through a mechanism known as transesterification. This reaction is aided by the A2451 3′-OH rRNA base, acting as a proton shuttle between the P-site 3′-O and the 2′-O and further stabilizing the transition structure.