MINI REVIEW published: 06 April 2017 doi: 10.3389/fimmu.2017.00351 A Novel Regulator of Activation- Induced Cytidine Deaminase/ APOBECs in Immunity and Cancer: Schrödinger’s CATalytic Pocket Justin J. King and Mani Larijani* Immunology and Infectious Diseases Program, Division of Biomedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, NL, Canada Activation-induced cytidine deaminase (AID) and its relative APOBEC3 cytidine deam- inases boost immune response by mutating immune or viral genes. Because of their genome-mutating activities, AID/APOBECs are also drivers of tumorigenesis. Due to highly charged surfaces, extensive non-specific protein–protein/nucleic acid interactions, formation of polydisperse oligomers, and general insolubility, structure elucidation of these proteins by X-ray crystallography and NMR has been challenging. Hence, almost all available AID/APOBEC structures are of mutated and/or truncated versions. In 2015, Edited by: Harry W. Schroeder, we reported a functional structure for AID using a combined computational–biochemical University of Alabama at approach. In so doing, we described a new regulatory mechanism that is a first for Birmingham, USA human DNA/RNA-editing enzymes. This mechanism involves dynamic closure of the Reviewed by: catalytic pocket. Subsequent X-ray and NMR studies confirmed our discovery by show- Linda Chelico, University of Saskatchewan, ing that other APOBEC3s also close their catalytic pockets. Here, we highlight catalytic Canada pocket closure as an emerging and important regulatory mechanism of AID/APOBEC3s. Silvo G. Conticello, Istituto Toscano Tumori, Italy We focus on three sub-topics: first, we propose that variable pocket closure rates across AID/APOBEC3s underlie differential activity in immunity and cancer and review *Correspondence: Mani Larijani supporting evidence. Second, we discuss dynamic pocket closure as an ever-present [email protected] internal regulator, in contrast to other proposed regulatory mechanisms that involve extrinsic binding partners. Third, we compare the merits of classical approaches of X-ray Specialty section: This article was submitted to and NMR, with that of emerging computational–biochemical approaches, for structural B Cell Biology, elucidation specifically for AID/APOBEC3s. a section of the journal Frontiers in Immunology Keywords: lymphoma, antibodies, DNA mutations, leukocytes, enzymes and coenzymes, HIV infections Received: 07 November 2016 Accepted: 10 March 2017 Published: 06 April 2017 IMPORTANCE AND CHALLENGES OF SOLVING AID/APOBEC Citation: STRUCTURES King JJ and Larijani M (2017) A Novel Regulator of Activation- Activation-induced cytidine deaminase (AID) is a 198 amino acid DNA-editing enzyme that deami- Induced Cytidine Deaminase/ APOBECs in Immunity and Cancer: nates deoxycytidine (dC) to deoxyuridine (dU) in single-stranded DNA (ssDNA) (1–6). It acts on Schrödinger’s CATalytic Pocket. immunoglobulin (Ig) loci initiating hypermutation and recombination events that lead to improved Front. Immunol. 8:351. and class-switched antibodies (1, 7–9). However, AID is loosely targeted to Ig loci, and hence, it doi: 10.3389/fimmu.2017.00351 induces genome-wide mutations and double-strand breaks which can lead to tumors (10–16). In Frontiers in Immunology | www.frontiersin.org 1 April 2017 | Volume 8 | Article 351 King and Larijani Novel Regulatory Mechanism for AID/APOBECs addition, continued AID expression increases genetic plasticity of discovery in 1999, despite intense efforts. Thus, we posited that tumors thereby accelerating disease progression (17, 18). even if AID’s structure were to be solved by traditional methods Activation-induced cytidine deaminase is a member of of X-ray or NMR, it would most likely be of a truncated and/ the apolipoprotein B RNA-editing catalytic component (AID/ or heavily mutated version. We proposed an alternative meth- APOBEC) family of cytidine deaminases, a Zn-dependent fam- odology to gain insight into the functional and native structure ily with 11 members in humans: AID, APOBEC1, APOBEC2, of AID. We utilized eight recently solved structures of AID’s the APOBEC3 sub-branch (A-H, excluding E), and APOBEC4 APOBEC relatives as templates to generate thousands of AID (9, 19, 20). The APOBEC3 (A3) sub-branch members are anti- predicted model structures followed by identification of the low- retroviral/retroelement restriction factors thereby also playing an est energy clusters (58) (Figure 1A). Concurrently and guided by immune function (21, 22); however, in the last few years, a role in the computational predictions, we generated a library of 400 AID cancer initiation has also emerged for the A3 sub-branch of the variants and carried out extensive biochemical characterization family, in particular, A3A, A3B (23–32), and more recently A3H of catalytic function and DNA binding to rigorously test key pre- haplotype I (33). dictions of models. This library included different point mutants Given their intimate links to immunity and cancer, much for each residue along the length of AID, orthologous AIDs, and effort has been placed on understanding the molecular structures chimeras involving regions of other deaminases exchanged into of AID/APOBECs over the last decade. A major hurdle in this the AID scaffold, or vice-versa. Our rationale for including AID effort has been the isolation and purification of native AID/ orthologs was that divergent AID from distantly evolved species APBOEC proteins to absolute purity. To different measures may have distinct biochemical properties and characterizing for each individual family member, the challenges include cel- these through a combination of homology structural modeling lular genotoxicity, highly charged surfaces mediating extensive and functional analysis of mutated and chimeric enzymes would non-specific protein–protein, protein–DNA/RNA interactions generate structure:function insights. Differences among ortholo- (9, 34), and polydisperse oligomerization (35, 36). Consequently, gous AIDs included catalytic rates, substrate preferences, DNA- in most cases, structure resolution by the traditional method- binding affinities, and thermosensitivity profiles (63–65). Since ologies of X-ray crystallography and NMR has necessitated these differences are typically due to structural features, being substantial alterations to stabilize protein charge and enhance reflective of catalytic motifs, surface composition, and overall solubility or crystallization. Therefore, the vast majority (22 of protein flexibility, respectively, characterizing the basis of these 24) of APOBEC structures solved by X-ray or NMR are of trun- differences among orthologs proved a valuable tool to gain insight cated and/or mutated versions, necessary to enhance solubility into AID’s structure:function relationships. This computational– (Table 1) (37–57). Nevertheless, it has become clear that AID/ biochemical approach led to the first relatively detailed 3D maps APOBEC family member enzymes share the core structure of a of AID’s functional structure with special focus on catalytic central β-sheet with 4 or 5 β strands sandwiched between 6 and pocket architecture and ssDNA-binding motifs (9, 58, 63, 64). 7 α-helices, connected by 12–13 flexible loops of variable lengths The architecture and dynamics of an enzyme’s catalytic (Figure 1A). Second, they all have highly charged DNA-binding pocket are important determinants of its activity. In addition to grooves, necessary to bind negatively charged polynucleotides. the core catalytic pocket composed of the aforementioned triad The arrangement of core catalytic residues in the catalytic pocket Zinc-coordinating residues and a Glutamic acid, we identified is also conserved, consisting of a Zn-coordinating triad of two an additional 21 amino acids that are not directly involved in the cysteines and a histidine, atop a catalytic proton-donor glutamic deamination reaction, but compose the pocket’s physiochemi- acid (C87, C90, H56, and E58 in AID) (9, 19, 20, 58). cal microenvironment (58). These residues termed secondary catalytic residues form the “walls” and “floors” of the pocket and stabilize dC binding. We noted that the conformations of these SOLVING THE STRUCTURE OF AID AND secondary catalytic residues exhibited more variability than that DISCOVERY OF CATALYTIC POCKET of the primary catalytic residues among predictions, because these CLOSURE IN AID/APOBECs residues reside on several highly flexible connecting loops without secondary structures of their own, that surround the catalytic The biochemical properties of AID have been previously pocket (Figure 1B). Because of this placement, we observed that described. AID has an exceptionally high affinity (nM range) the catalytic pocket of AID appeared to be only marginally stable for binding ssDNA and an unusually slow catalytic rate of one such that ~75% of conformations exhibit a occluded pocket unable reaction in several minutes (5, 60), ~2,000 times slower than a to accommodate dC. Thus, we hypothesized that dynamic catalytic typical enzyme (61). We postulated that this catalytic lethargy pocket closure is a built-in mechanism that limits AID activity. and high-binding affinity to DNA had evolved to protect genomes There are several lines of indirect but strong evidence for the from rampant AID activity (9). In direct support of this notion, existence of catalytic pocket closure in AID/APOBEC3s. First, mutants of AID with higher catalytic rates were shown to mediate