Rational Control of Poliovirus RNA-Dependent RNA Polymerase Fidelity by Modulating Motif-D Loop Conformational Dynamics

Abstract

The conserved structural motif D is an important determinant of the speed and fidelity of viral RNA-dependent RNA polymerases (RdRps). Structural and computational studies have suggested that conformational changes in the motif-D loop that help to reposition the catalytic lysine represent critical steps in nucleotide selection and incorporation. Conformations of the motif-D loop in the poliovirus RdRp are likely controlled in part by noncovalent interactions involving the motif-D residue Glu364. This residue swivels between making interactions with Lys228 and Asn370 to stabilize the open and closed loop conformations, respectively. We show here that we can rationally control the motif-D loop conformation by breaking these interactions. The K228A variant favors a more active closed conformation, leading to increased nucleotide incorporation rates and decreased nucleotide selectivity, and the N370A variant favors a less active open conformation, leading to decreased nucleotide incorporation rates and increased nucleotide selectivity. Similar competing interactions likely control nucleotide incorporation rates and fidelity in other viral RdRps. Rational engineering of these interactions may be important in the generation of live, attenuated vaccine strains, considering the established relationships between RdRp function and viral pathogenesis.

Open Gate of Corynebacterium glutamicum Threonine Deaminase for Efficient Synthesis of Bulky α-Keto Acids

“…This approach is compatible with a recent trend in protein engineering that stresses the construction of small and smart libraries, such as the triple-code saturation mutagenesis − and hydroclassified combinatorial saturation mutagenesis libraries . Furthermore, by using different tunnel-identification methods, such as X-ray crystallography, state kinetics, and molecular modeling, , as well as appropriate hotspot-identification methods such as volume scanning, , hydroclassified scanning, and conformational dynamics, − the open-gate strategy can be modified to engineer other gated enzymes while reducing the size of the mutation library and improving evolution efficiency. Overall, this strategy falls within the general trend of protein engineering and exhibits potential for universal application.…”

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“…It is now appreciated that protein conformational dynamics play important roles in determining protein function. Specifically, conformational dynamics have been previously been shown to be important for protease activity and substrate specificity for many other systems [ 35 , 36 , 37 , 38 , 39 ] and the conformational dynamics of viral RdRps have likewise been shown to play critical roles in replication speed and fidelity [ 40 , 41 , 42 , 43 , 44 ]. Conformational dynamics and protein flexibility are also important in structure-based drug design [ 45 , 46 , 47 , 48 , 49 ].…”

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“…RdRP is subsequently used to complete the transcriptional synthesis of the negative-strand subgenomic RNA, the synthesis of various structural protein-related mRNA, and the replication of viral genomic RNA [ [57] , [58] , [59] , [60] ]. RdRP can accurately and efficiently synthesize tens of thousands of nucleotides and thus facilitates all other biological activities after the virus invades the host cell [ [61] , [62] , [63] ]. RdRP is an effective target of broad-spectrum antiviral drugs, and at present, most of the anti-coronavirus drugs targeting RdRP are nucleoside (Nuc) analogues or RNA interferons [ 64 ].…”