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EV71 is responsible for several major epidemics worldwide;however, the effective antiviral drug is unavailable to date.The 2A proteinase (2Apro) of EV71 presents a drug target due to its multiple roles in virus replication, inhibition of host protein synthesis and evasion of innate immunity.We determined the crystal structure of EV71 2Apro at 1.85 A resolution, revealing that the proteinase maintains a chymotrypsin-like fold.The active site is comprised of the catalytic triads C116A, H21 and D39 with the geometry similar to that found in other picomaviral 2Apro, 3Cpro and serine proteinases.The cl-to-el2 loop at the N-terminal domain of EV71 2Apro adopts a highly stable conformation and contributes to the hydrophilic surface, which are strikingly different in HRV2 2Apro, but similar in CVB4 2Apro, suggesting that the enterovirus 2Apro may share the mechanism in host target recognition, which is different in rhinovirus 2Apro.We identified a hydrophobic motif "LLWL" followed by an acidic motif "DEE" at the C-terminus of EV71 2Apro.The "LLWL" motif is folded into a double β-turn that is essential for the positioning of the acidic motif.Our mutagenesis study demonstrated that both the negative charging and the correct positioning of the acidic motif are essential for EV71 replication.Deletion of the "LLWL" motif abrogates the proteolytic activity and viral production, indicating that the "LLWL" motif is critical for maintaining the active proteinase conformation.Our findings provide the structural and functional insights to EV71 2Apro and establish a framework for the structure-guided inhibitor design.