1.92 Angstrom Zinc-Free APOBEC3F Catalytic Domain Crystal Structure

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Abstract

The APOBEC3 family of DNA cytosine deaminases is capable of restricting the replication of HIV-1 and other pathogens. Here, we report a 1.92 Å resolution crystal structure of the Vif-binding and catalytic domain of APOBEC3F (A3F). This structure is distinct from the previously published APOBEC and phylogenetically related deaminase structures, as it is the first without zinc in the active site. We determined an additional structure containing zinc in the same crystal form that allows direct comparison with the zinc-free structure. In the absence of zinc, the conserved active site residues that normally participate in zinc coordination show unique conformations, including a 90 degree rotation of His249 and disulfide bond formation between Cys280 and Cys283. We found that zinc coordination is influenced by pH, and treating the protein at low pH in crystallization buffer is sufficient to remove zinc. Zinc coordination and catalytic activity are reconstituted with the addition of zinc only in a reduced environment likely due to the two active site cysteines readily forming a disulfide bond when not coordinating zinc. We show that the enzyme is active in the presence of zinc and cobalt but not with other divalent metals. These results unexpectedly demonstrate that zinc is not required for the structural integrity of A3F and suggest that metal coordination may be a strategy for regulating the activity of A3F and related deaminases.

Original languageEnglish (US)
Pages (from-to)2307-2316
Number of pages10
JournalJournal of Molecular Biology
Volume428
Issue number11
DOIs
StatePublished - Jun 5 2016

Bibliographical note

Funding Information:
The Harris lab is supported by NIH NIAID AI064046 , NIGMS GM091743 , and HHMI . The Aihara lab is supported by NIH NIGMS grants GM095558 and GM109770 . This work is also supported by NIGMS P41 GM103403 (NE-CAT beamlines) and NIH-ORIP HEI S10 RR029205 (Pilatus Detector) to the Advanced Photon Source operated for the DOE Office by Argonne National Laboratory (DE-AC02-06CH11357).

Publisher Copyright:
© 2016 Elsevier Ltd. All rights reserved.

Keywords

  • APOBEC3 regulation
  • HIV-1 restriction factor
  • X-ray crystallography
  • cytosine deaminase
  • zinc-free and zinc-bound APOBEC3 structures

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