The crystal structure of dGTPase reveals the molecular basis of dGTP selectivity

Journal Article · Wed Apr 24 00:00:00 EDT 2019 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1814999116· OSTI ID:1577155

Barnes, Christopher O.; Wu, Ying; Song, Jinhu; Lin, Guowu; Baxter, Elizabeth L.; Brewster, Aaron S.; Nagarajan, V.; Holmes, Andrew; Soltis, S. Michael; Sauter, Nicholas K.; Ahn, Jinwoo; Cohen, Aina E.; Calero, Guillermo

Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that 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 Ec-dGTPase, including an X-ray free-electron laser structure of the free Ec--dGTPase enzyme to 3.2 Å. To obtain this structure, we developed a method that 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 experiment. Our structures show 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, Ec-dGTPase and SAMHD1 share similar active-site and HD motif architectures; however, Ec-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 shed light on 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.

• Research Organization: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
• Sponsoring Organization: DOE - BASIC ENERGY SCIENCESNIH
• OSTI ID: 1577155
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America, Vol. 116, Issue (19) ; 05, 2019; ISSN 0027-8424
• Publisher: National Academy of Sciences
• Country of Publication: United States
• Language: ENGLISH