Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A–ssDNA complex defines the 5′–3′ directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics.

Many viruses package their genomes into procapsids using an ATPase machine that is among the most powerful known biological motors. However, how this motor couples ATP hydrolysis to DNA translocation is still unknown. Here, we introduce a model system with unique properties for studying motor structure and mechanism. We describe crystal structures of the packaging motor ATPase domain that exhibit nucleotide-dependent conformational changes involving a large rotation of an entire subdomain. We also identify the arginine finger residue that catalyzes ATP hydrolysis in a neighboring motor subunit, illustrating that previous models for motor structure need revision. Our findings allow us to derive a structural model for the motor ring, which we validate using small-angle X-ray scattering and comparisons with previously published data. We illustrate the model's predictive power by identifying the motor's DNA-binding and assembly motifs. Finally, we integrate our results to propose a mechanistic model for DNA translocation by this molecular machine.ouble-stranded DNA (dsDNA) viruses ranging from bacteriophages to the human pathogens of the herpesvirus family form infectious virions by packaging their genomes into preformed procapsids using a powerful ATPase machine (1). The viral genome packaging motor is a multicomponent molecular machine that must complete several tasks in sequential order, the foremost of which is the ATP-dependent pumping of viral DNA into the procapsid (Fig. 1A). Because the DNA progresses from a flexible state to a semicrystalline state as it fills the capsid interior, the motor pumps against a tremendous force. The pressures inside the filled capsid are estimated to reach 60-70 atm (2, 3), equivalent to 10-fold higher than a bottle of champagne. Thus, the viral packaging motor represents one of the most powerful biological motors known (2, 4).The central component of the packaging motor is the ATPase subunit, which drives DNA translocation. The ATPase subunit is a member of the additional strand, conserved glutamate (ASCE) superfamily of ATPases (5). In herpesviruses, as well as many bacteriophages, this ATPase is from a specific clade of the ASCE family called the terminase family (1, 6). In viruses that use a terminase-type motor for genome packaging, the motor consists of several proteins that assemble into homomeric rings (7) (Fig. 1A). The large terminase (TerL) protein harbors the motor's two enzymatic activities (7): the ATPase activity that pumps DNA into the capsid and an endonuclease domain that cleaves packaged DNA from the remaining concatemeric DNA when the capsid is full. Cryoelectron microscopy (cryo-EM) studies indicate that a pentamer of TerL subunits attaches to the capsid by binding to a dodecameric assembly called portal (8). However, there are conflicting reports as to the orientation of TerL relative to portal during packaging (8-10).A structural model for the bacteriophage T4 TerL ring has been previously proposed (8) with these salient features: (i) the nuclease domains...

The oncogenic corepressors C-terminal Binding Protein (CtBP) 1 and 2 harbor regulatory D-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domains. 4-Methylthio 2-oxobutyric acid (MTOB) exhibits substrate inhibition and can interfere with CtBP oncogenic activity in cell culture and mice. Crystal structures of human CtBP1 and CtBP2 in complex with MTOB and NAD+ revealed two key features: a conserved tryptophan that likely contributes to substrate specificity and a hydrophilic cavity that links MTOB with an NAD+ phosphate. Neither feature is present in other D2-HDH enzymes. These structures thus offer key opportunities for the development of highly selective anti-neoplastic CtBP inhibitors.

Oncogenic transcriptional coregulators C-terminal Binding Protein (CtBP) 1 and 2 possess regulatory D-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domains that provide an attractive target for small molecule intervention. Findings that the CtBP substrate 4-methylthio 2-oxobutyric acid (MTOB) can interfere with CtBP oncogenic activity in cell culture and in mice confirm that such inhibitors could have therapeutic benefit. Recent crystal structures of CtBP 1 and 2 revealed that MTOB binds in an active site containing a dominant tryptophan and a hydrophilic cavity, neither of which are present in other D2-HDH family members. Here we demonstrate the effectiveness of exploiting these active site features for design of high affinity inhibitors. Crystal structures of two such compounds, phenylpyruvate (PPy) and 2-hydroxyimino-3-phenylpropanoic acid (HIPP), show binding with favorable ring stacking against the CtBP active site tryptophan and alternate modes of stabilizing the carboxylic acid moiety. Moreover, ITC experiments show that HIPP binds to CtBP with an affinity greater than 1000-fold over that of MTOB and enzymatic assays confirm that HIPP substantially inhibits CtBP catalysis. These results, thus, provide an important step, and additional insights, for the development of highly selective antineoplastic CtBP inhibitors.

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