Stabilization of poliovirus polymerase by NTP binding and fingers-thumb interactions.
The viral RNA-dependent RNA polymerases show a conserved structure where the fingers domain interacts with the top of the thumb domain to create a tunnel through which nucleotide triphosphates reach the active site. We have solved the crystal structures of poliovirus polymerase (3D(pol)) in complex with all four NTPs, showing that they all bind in a common pre-insertion site where the phosphate groups are not yet positioned over the active site. The NTPs interact with both the fingers and palm domains, forming bridging interactions that explain the increased thermal stability of 3D(pol) in the presence of NTPs. We have also examined the importance of the fingers-thumb domain interaction for the function and structural stability of 3D(pol). Results from thermal denaturation experiments using circular dichroism and 2-anilino-6-napthaline-sulfonate (ANS) fluorescence show that 3D(pol) has a melting temperature of only approximately 40 degrees C. NTP binding stabilizes the protein and increases the melting by 5-6 degrees C while mutations in the fingers-thumb domain interface destabilize the protein and reduce the melting point by as much as 6 degrees C. In particular, the burial of Phe30 and Phe34 from the tip of the index finger into a pocket at the top of the thumb and the presence of Trp403 on the thumb domain are key interactions required to maintain the structural integrity of the polymerase. The data suggest the fingers domain has significant conformational flexibility and exists in a highly dynamic molten globule state at physiological temperature. The role of the enclosed active site motif as a structural scaffold for constraining the fingers domain and accommodating conformational changes in 3D(pol) and other viral polymerases during the catalytic cycle is discussed.
Thompson
Aaron A
AA
Program in Cellular and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA.
Albertini
Rebecca A
RA
Peersen
Olve B
OB
D020816:Amino Acid Motifs; D024322:Amino Acids, Aromatic / Q000737:chemistry; D002413:Cations, Divalent; D002942:Circular Dichroism; D004795:Enzyme Stability; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D017955:Poliovirus / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship; D013696:Temperature; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*
D024322:Amino Acids, Aromatic; D002413:Cations, Divalent; D012265:Ribonucleotides; D017361:Viral Nonstructural Proteins; D008345:Manganese; D012313:RNA; D012324:RNA-Dependent RNA Polymerase; D008274:Magnesium