Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B

Abstract

The single-stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but it is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3-family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high-resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2-pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution. K E Y W O R D Sactive site plasticity, APOBEC3B, cytosine deaminase, DNA mutation, enzymes, molecular dynamics simulations, protein structure, small molecule, x-ray crystallography, zinc ion 50 | SHI et al

Structural basis of substrate specificity in human cytidine deaminase family APOBEC3s

“…Therefore, changes surrounding the active site including variations in the arrangements of these loops are likely largely responsible for distinctions in substrate specificity, binding affinity, and deamination activity for ssDNA, as well as the physiological functions of A3s. Structural analysis using molecular modeling and dynamics simulations have been used for investigating the molecular mechanisms underlying biological functions of A3s ( 55 , 56 , 57 , 58 , 59 , 60 ). These studies have revealed the importance of active site loops in substrate binding/activity and specificity.…”

Structural plasticity of substrate selection by activation‐induced cytidine deaminase as a regulator of its genome‐wide mutagenic activity

“…Though numerous other enzymes involved in various cellular and metabolic pathways have been shown to undergo such loop dynamics that can impact the accessibility of the active site [30,31], the discovery of such a state in AID represents a novel mode of regulation in DNA/RNA‐editing enzymes. This catalytic pocket duality, termed ‘Schrodinger's CATalytic pocket’, has also been reported in crystal structures and molecular dynamic simulations of APOBEC3B and has emerged as an important activity regulator across the AID/APOBEC family [32–35].…”

Structural Characterization of a Minimal Antibody against Human APOBEC3B

“…The previously crystallized A3Bctd-QMΔloop3 and its Arg-to-Glu or Arg-to-Ala mutants were expressed with a C-terminal 6xHis tag in E. coli strain C41(DE3)pLysS and purified as described [ 8 , 11 , 12 ]. A3A-E72A was also produced as described [ 13 ].…”