RT Journal Article
SR Electronic
T1 Molecular mimicry in deoxy-nucleotide catalysis: the structure of <em>Escherichia coli</em> dGTPase reveals the molecular basis of dGTP selectivity: New structural methods offer insight on dGTPases
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 385401
DO 10.1101/385401
A1 Christopher O. Barnes
A1 Ying Wu
A1 Jinhu Song
A1 Guowu Lin
A1 Elizabeth L. Baxter
A1 Aaron S. Brewster
A1 Veeranagu Nagarajan
A1 Andrew Holmes
A1 Michael Soltis
A1 Nicholas K. Sauter
A1 Jinwoo Ahn
A1 Aina E. Cohen
A1 Guillermo Calero
YR 2018
UL http://biorxiv.org/content/early/2018/11/29/385401.abstract
AB Deoxynucleotide triphosphate triphosphyohydrolyases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools by hydrolyzing dNTPs into deoxynucleosides and inorganic triphosphate (PPPi). While the vast majority of these enzymes display broad activity towards canonical dNTPs, exemplified by Sterile Alpha Motif (SAM) and Histidine-aspartate (HD) domain-containing protein 1 (SAMHD1), which blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric E. coli dGTPase. To obtain these structures, we applied UV-fluorescence microscopy, video analysis and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly-located on fixed target holders, resulting in the highest indexing-rates observed for a serial femtosecond crystallography (SFX) experiment. The structure features a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo form. Moreover, despite no sequence homology, dGTPase and SAMHD1 share similar active site and HD motif architectures; however, dGTPase residues at the end of the substrate-binding pocket mimic Watson Crick interactions providing Guanine base specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures sheds light into the mechanism by which long distance binding (25 Å) of single stranded DNA in an allosteric site primes the active site by conformationally “opening” a tyrosine gate allowing enhanced substrate binding.Significance Statement dNTPases play a critical role in cellular survival through maintenance of cellular dNTP. While dNTPases display activity towards dNTPs, such as SAMHD1 –which blocks reverse transcription of HIV-1 in macrophages– Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. Here we use novel free electron laser data collection to shed light into the mechanisms of (Ec)-dGTPase selectivity. The structure features a dynamic active site where conformational changes are coupled to dGTP binding. Moreover, despite no sequence homology between (Ec)-dGTPase and SAMHD1, both enzymes share similar active site architectures; however, dGTPase residues at the end of the substrate-binding pocket provide dGTP specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP.