Brendan J. Hilbert

Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity.

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.

Structure and mechanism of the ATPase that powers viral genome packaging

Significance Many viruses use a molecular motor to pump DNA into a preformed protein shell called the capsid, a process that is essential for the formation of infectious virus particles. The ATPase machine powering this process is the strongest known biological motor. However, the structure and mechanism of this motor are unknown. Here, we derive a structural model of the ATPase assembly using a combination of X-ray crystallography, small-angle X-ray scattering, molecular modeling, and biochemical data. We identify residues critical for ATP hydrolysis and DNA binding, and derive a mechanistic model for the translocation of DNA into the viral capsid. Our studies introduce a model for ATPase assembly and illustrate how DNA is pumped with high force. 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.

Crystal structures of human CtBP in complex with substrate MTOB reveal active site features useful for inhibitor design

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.

Structure-guided design of a high affinity inhibitor to human CtBP

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 the 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.

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain.

Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA.

A Disease-Causing Variant in PCNA Disrupts a Promiscuous Protein Binding Site.

The eukaryotic DNA polymerase sliding clamp, proliferating cell nuclear antigen or PCNA, is a ring-shaped protein complex that surrounds DNA to act as a sliding platform for increasing processivity of cellular replicases and for coordinating various cellular pathways with DNA replication. A single point mutation, Ser228Ile, in the human PCNA gene was recently identified to cause a disease whose symptoms resemble those of DNA damage and repair disorders. The mutation lies near the binding site for most PCNA-interacting proteins. However, the structural consequences of the S228I mutation are unknown. Here, we describe the structure of the disease-causing variant, which reveals a large conformational change that dramatically transforms the binding pocket for PCNA client proteins. We show that the mutation markedly alters the binding energetics for some client proteins, while another, p21(CIP1), is only mildly affected. Structures of the disease variant bound to peptides derived from two PCNA partner proteins reveal that the binding pocket can adjust conformation to accommodate some ligands, indicating that the binding site is dynamic and pliable. Our work has implications for the plasticity of the binding site in PCNA and reveals how a disease mutation selectively alters interactions to a promiscuous binding site that is critical for DNA metabolism.

Design, synthesis, and biological evaluation of substrate-competitive inhibitors of C-terminal Binding Protein (CtBP).

C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24μM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18μM) and 3-chloro- (IC50=0.17μM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer.

Abstract 2003: C-terminal binding protein (CtBP): An emerging oncogene and small molecule drug target in solid tumors

C-terminal binding proteins 1 and 2 (CtBP) are transcriptional coregulators whose overexpression has been linked to poor prognosis and/or chemoresistance in most common solid tumor types. CtBP exerts oncogenic activities through modulation of gene expression programs governing cell survival, epithelial/mesenchymal transitions, migration/invasion, and cell cycle, and is also required for colon cancer tumor initiating cell function. CtBP, however, has never been formally characterized as an oncogene. We now show that Ctbp2 exhibits transforming activity in immortalized NIH 3T3 as well as mouse and human primary cells. Ctbp2 alone, or in cooperation with SV40 large T antigen (LT) transformed NIH 3T3 cells and MEF’s, respectively, to anchorage independence with similar efficiency to mutant H-Ras. Human BJ foreskin fibroblasts were also transformed to anchorage independence by CtBP2 in cooperation with LT, SV40 small T antigen, and h-TERT with efficiency similar to H-Ras, indicating that CtBP overexpression, as found in the majority of human colon, ovary, breast, and prostate cancers, may be a key oncogenic driver gene. To confirm the physiologic role of Ctbp2 in a mouse tumor model with Ctbp overexpression, we bred Apcmin/+ mice to Ctbp2 hemizygote (Ctbp2+/-) mice, which are otherwise healthy. CtBP is a known target of the APC E3 ligase and is thus stabilized in APC mutated human colon cancers and is found in high levels in APCmin polyps. Remarkably, survival to humane endpoint at 37 weeks for Apcmin/+ vs. Apcmin/+-Ctbp2+/- mice was 0% vs. 100% (p Citation Format: Evan T. Sumner, Sudha Korwar, Benjamin L. Morris, Martin M. Dcona, Brendan J. Hilbert, William E. Royer, Keith C. Ellis, Steven Grossman. C-terminal binding protein (CtBP): An emerging oncogene and small molecule drug target in solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2003.

Abstract 1633: C-terminal binding proteins are novel drug targets

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The CtBP transcriptional corepressors have been identified as proto-oncogenes. CtBP represses pro-apoptotic genes and promotes cancer cell migration and invasion in a redox sensitive manner through a regulatory dehydrogenase domain. The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and induces apoptosis in the human colorectal cancer cell line HCT116. MTOB induced apoptosis is dependent upon expression of the BH3 only protein BIK, a previously established CtBP target. Treatment of both native and transformed Mouse Embryonic Fibroblasts revealed a wide therapeutic index for CtBP inhibition. In human colon cancer cell peritoneal xenografts, MTOB treatment reduced tumor growth and induced apoptosis in vivo without notable toxicity. To verify the potential utility of CtBP as a therapeutic target in human cancer, the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Thus, CtBP is a viable therapeutic target in human cancer. In order to identify novel CtBP inhibitors, a simple dehydrogenase assay was developed that utilizes the spectrophotometric measurement of enzymatic conversion of NADH to NAD+ by the CtBP dehydrogenase activity. This assay successfully detects MTOB substrate and inhibitor activity, as well as inhibition of CtBP activity by the related compound 4-methylthio-2-hydroxybutyric acid (MTHB). This assay will be adapted for high throughput screening of small molecule libraries for novel CtBP inhibitors that can be further characterized in pre-clinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1633. doi:10.1158/1538-7445.AM2011-1633