Structural Flexibility Of An Essential Viral Enzyme Measured By NMR
RNA-directed RNA polymerase (RdRp) is encoded in the genome of all RNA-containing viruses (except retroviruses), such as the polio, hepatitis A, C and E, measles, rabies, Dengue, West Nile, influenza and many others viruses. It is an essential protein that catalyzes the replication of RNA from an RNA template and its sequence is among the most conserved from these viruses. Researchers from NYSBC and the City College of New York (CCNY) recently characterized the fast (pico to nanosecond) dynamics of this enzyme, using the cystovirus φ12 RdRp protein (P2) as a model (Alphonse et al. 2015).
Location on RdRp of I, L, V and M amino acids with the highest (red) and lowest (blue) mobility relative to all amino acids analyzed. Highly mobile sites cluster around the mouth of the template RNA entry tunnel (arrow) and at the entry channel for substrate NTPs (at the back, orthogonal to template tunnel).
The findings indicate that the entry portals for template RNA and substrate nucleotides are relatively disordered, while conserved motifs involved in metal ion binding (Mg2+, used as catalyst), nucleotide selection, and catalysis display greater rigidity. Binding of Mg2+ leads to an overall decrease in flexibility, while binding of nucleotides reverses this effect. Perturbations at the active site through binding of metal ions, nucleotides or functional mutation affect dynamics not only in the immediate vicinity but also at remote regions. Comparison with homologous proteins suggests similar pattern of dynamics in RdRps from other viruses.
These dynamics likely play a major role in determining the affinity and specificity of template or substrate binding. While they are too fast to directly influence catalysis, an indirect role could occur through their hierarchical influence on slower, catalytic time scales. Interestingly, the observed dynamic coupling of remote regions to the catalytic site could lead to the design of unique antiviral therapies that target these remote regions to disrupt enzymatic activity.
Alphonse et a. 2015, Biochemistry Sep 29;54(38):5828-38
Alphonse et al. 2014, J Mol Biol Jul 15;426(14):2580-93.