Our preclinical data show APE1 could be a synthetic lethality (SL) target in BRCA deficient ovarian cancers. BRCA1- and BRCA2-associated hereditary breast and ovarian cancer syndrome (HBOC) is characterized by increased risk for breast cancer, ovarian cancer (includes fallopian tube and primary peritoneal cancers) and other solid tumours (including pancreatic cancers and prostate cancers). About 10-20% of TNBC patients harbor germline BRCA mutations resulting in homologous recombination deficiency (HRD), and even amongst wild-type BRCA patients, somatic mutations occur more frequently producing a range of HRD phenotypes collectively termed as “BRCAness”. In epithelial ovarian cancers, germ-line BRCA mutations can be detected in up to 20% of high serous ovarian cancers, and up to 50% of somatic tumours can also manifest a HRD phenotype. PARP1 inhibitor (Niraparib, Olaparib, Rucaparib) maintenance therapy improves progression-free survival in BRCA germline deficient ovarian cancer or platinum sensitive ovarian cancers. Olaparib and Talazoparib are approved for use in BRCA germ-line deficient breast cancers. A response rate to PARPi of up to 60% has been described in clinical studies. Routine clinical use has shown that not all patients with BRCA deficient cancers respond to PARPi either due to intrinsic or acquired resistance. The search is on for other SL targets in BRCA/HRD deficient cancers that either bypass PARPi resistance or are more effective than PARP blockade. We evaluated APE1 protein expression in human epithelial ovarian cancers, correlated with clinicopathological features and survival outcomes and compared APE1 transcript levels in a panel of solid tumours and normal tissue. APE1 transcripts were evaluated in publicly available ovarian cancer clinical datasets (n=1257) for clinical outcomes. Highest levels of transcripts were observed in ovarian cancers compared to other solid tumours. When compared to normal ovarian tissue, there was striking elevation of transcripts in ovarian cancer tissues. High mRNA expression was associated with poor progression free survival (PFS) (p=0.00019). High nuclear APE1 protein expression was seen in 64% of tumours, a feature associated with high grade serous ovarian cancer, poor PFS and poor overall survival. XPTx-91 and XPTx-387 have biochemical IC50s of 250-500 nM. Optimized compounds were screened for selective toxicity in PEO1 (BRCA2 deficient, platinum sensitive, PARPi sensitive) and PEO4 (BRCA2 proficient, platinum resistant, PARPi resistant) cell lines. Several compounds screened in MTS cell proliferation assay showed preferential cytotoxicity in PEO1 cells compared to PEO4 cells. Further cell based (clonogenics, 3D spheroids) and functional assays (DNA double strand break accumulation, cell cycle progression and apoptosis) are ongoing. Debanu Das, Ashley Deacon, Matthew Duncton, David M. Wilson III, Mashael Algethami, Ayat Leshan, Emad A. Rakha, Çağla Tosun, Srinivasan Madhusudan. Novel APE1 endonuclease inhibitors as precision oncology therapeutics in high grade serous ovarian cancers discovered and developed from crystallography-based fragment library screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4193.

The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM (“SAR by X-ray Poses Quickly”) platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or “undruggable” targets, allows for (i) hit generation; (ii) the mapping of protein–ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.

Quickly generating reliable hits from library screening and rapidly converting them into lead compounds, which can be verified in functional assays, is critical in early preclinical drug discovery. The expedited validation of “druggability” of targets and identification of modulators to advance to preclinical studies can significantly increase the success of drug discovery and development. Our SaXPy (“SAR by X-ray Poses Quickly”) platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or “undruggable” targets, allows (i) hit generation; (ii) mapping of protein-ligand interactions; (iii) assessment of target ligandability; (iv) discovery of novel and potential allosteric binding sites; and (v) hit-to-lead progression. These advances inform chemical tractability, downstream biology, and intellectual property. We describe here the application of SaXPy in the discovery and development of DNA Damage Response (DDR) inhibitors against DNA polymerase eta (Pol 𝝶 or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our studies resulted in the first crystal structures of these targets bound to small molecules and the discovery of novel, multiple binding sites, which we could quickly progress from hit-to-lead development and demonstrate functional activity in bioassays. Citation Format: Debanu Das, Matthew Duncton, Ashley Deacon, David Wilson III. Applying a new drug discovery platform for the discovery and development of inhibitors of DDR proteins APE1 and Pol eta [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B053.

e15036 Background: Cancer cells respond to increases in DNA damage by deploying their DNA damage response (DDR) pathways. We are building a platform for the discovery and development of target-specific DDR therapeutics, including small molecule inhibitors and targeted protein degradation warheads, founded on fragment- and structure-based drug discovery. Methods: Our DDR platform, which includes hit-to-lead, lead optimization and candidate selection, starts with hit generation from a new technology that uses high-throughput protein X-ray crystallography to directly screen compound libraries. Our hit generation process produces empirical evidence of direct target engagement. The elucidation of high-quality ligand-bound 3D structures reveals the location and pose of the ligand and details of the protein-ligand interactions. Thus we can predict the structure-function consequences of the hit molecule engagement, which sets the stage for rapid assessment of synthetic tractability and intellectual property. After hit identification, we apply a multi-pronged approach in hit-to-lead conversion and lead optimization using iterative biophysical and biochemical assays, coupled with crystallography. We are applying our approach to several new targets in DDR and will present some early progress in this space. Results: Apurinic/apyrimidinic endonuclease 1 (APE1) is the major repair enzyme for abasic sites in DNA and contributes to DNA strand break processing. Many studies have associated increased APE1 levels with enhanced growth, migration, and drug resistance in human tumor cells, and with decreased patient survival. APE1 has been implicated in over 20 human cancers, including glioblastoma, making the protein an attractive target for the development of anticancer therapeutics. Despite intensive effort, there are no clinical endonuclease inhibitors of APE1. We have identified 25 diverse small molecule fragments that bind to APE1 at two distinct sites, including the endonuclease site. Pol eta (or PolH) is a DNA polymerase implicated, among other things, in the development of cisplatin resistance in a subset of ovarian cancers. In our quest to develop PolH inhibitors, we have identified 5 diverse fragments that bind to two distinct sites in the polymerase including a new potential allosteric site. Our results on APE1 and PolH represent the first known cases of crystal structures of small molecules bound to these proteins. Flap endonuclease (FEN1) is implicated in several cancers including for example ER/tamoxifen-resistant breast cancer. We are developing a targeted protein degradation approach using PROTACs (Proteolysis-Targeting Chimeras) toward the development of novel therapeutics against FEN1. Conclusions: Our results will help us develop small molecule inhibitors and targeted protein degradation against DDR targets that may be effective as single therapies or be used to make existing therapies more effective.

Cancer cells respond to increases in DNA damage by upregulating their DNA damage response (DDR). The base excision repair (BER) pathway corrects damage to single DNA bases through the action of multiple enzymes, including the central protagonist, apurinic/apyrimidinic endonuclease 1 (APE1). Numerous studies have shown association between increased APE1 levels and enhanced growth, migration, and drug resistance in human tumor cells, as well as with decreased patient survival. APE1 has been implicated in over 20 human cancers, making this an attractive target for developing anticancer therapies. Despite intensive effort, there are currently no clinical endonuclease inhibitors of APE1. We have used a newly developed high-throughput protein X-ray crystallography-based fragment screen to obtain starting points for the design of molecules to block APE1 function. Starting with a proprietary fragment library, we obtained high quality fragment-bound crystal structures showing diversity of chemical matter and hit location, representing the first experimental 3D structures of APE1 bound to drug-like molecules, thereby resolving a primary bottleneck in the path to inhibitor development. The implementation of this unique lead discovery campaign has facilitated three independent strategies toward the development of APE1 inhibitors, including (i) fragment growing and elaboration of hits bound at the endonuclease site; (ii) linking of fragments bound to distinct but proximally located sites, and (iii) use of fragments for the design of hooks to use in targeted protein degradation (TPD) strategies. We are using a combination of computational and medicinal chemistry, structural biology, and biochemical and biophysical studies and will discuss our progress towards these goals.

Cancer will directly affect the lives of over one-third of the population. The DNA Damage Response (DDR) is an intricate system involving damage recognition, cell cycle regulation, DNA repair, and ultimately cell fate determination, playing a central role in cancer etiology and therapy. Two primary therapeutic approaches involving DDR targeting include: combinatorial treatments employing anticancer genotoxic agents; and synthetic lethality, exploiting a sporadic DDR defect as a mechanism for cancer-specific therapy. Whereas, many DDR proteins have proven "undruggable", Fragment- and Structure-Based Drug Discovery (FBDD, SBDD) have advanced therapeutic agent identification and development. FBDD has led to 4 (with -50 more drugs under preclinical and clinical development), while SBDD is estimated to have contributed to the development of >200, FDA-approved medicines. Protein X-ray crystallography-based fragment library screening, especially for elusive or "undruggable" targets, allows for simultaneous generation of hits plus details of protein-ligand interactions and binding sites (orthosteric or allosteric) that inform chemical tractability, downstream biology, and intellectual property. Using a novel high-throughput crystallography-based fragment library screening platform, we screened five diverse proteins, yielding hit rates of -2-8% and crystal structures from -1.8 to 3.2 & Aring;. We consider current FBDD/SBDD methods and some exemplary results of efforts to design inhibitors against the DDR nucleases meiotic recombination 11 (MRE11, a.k.a., MRE11A), apurinic/apyrimidinic endonuclease 1 (APE1, a.k.a., APEX1), and flap endonuclease 1 (FEN1). (c) 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND

Polymerase eta (or Pol η or POLH) is a specialized DNA polymerase that is able to bypass certain blocking lesions, such as those generated by ultraviolet radiation (UVR) or cisplatin, and is deployed to replication foci for translesion synthesis as part of the DNA damage response (DDR). Inherited defects in the gene encoding POLH (a.k.a., XPV) are associated with the rare, sun-sensitive, cancer-prone disorder, xeroderma pigmentosum, owing to the enzyme’s ability to accurately bypass UVR-induced thymine dimers. In standard-of-care cancer therapies involving platinum-based clinical agents, e.g., cisplatin or oxaliplatin, POLH can bypass platinum-DNA adducts, negating benefits of the treatment and enabling drug resistance. POLH inhibition can sensitize cells to platinum-based chemotherapies, and the polymerase has also been implicated in resistance to nucleoside analogs, such as gemcitabine. POLH overexpression has been linked to the development of chemoresistance in several cancers, including lung, ovarian, and bladder. Co-inhibition of POLH and the ATR serine/threonine kinase, another DDR protein, causes synthetic lethality in a range of cancers, reinforcing that POLH is an emerging target for the development of novel oncology therapeutics. Using a fragment-based drug discovery approach in combination with an optimized crystallization screen, we have solved the first X-ray crystal structures of small novel drug-like compounds, i.e., fragments, bound to POLH, as starting points for the design of POLH inhibitors. The intrinsic molecular resolution afforded by the method can be quickly exploited in fragment growth and elaboration as well as analog scoping and scaffold hopping using medicinal and computational chemistry to advance hits to lead. An initial small round of medicinal chemistry has resulted in inhibitors with a range of functional activity in an in vitro biochemical assay, leading to the rapid identification of an inhibitor to advance to subsequent rounds of chemistry to generate a lead compound. Importantly, our chemical matter is different from the traditional nucleoside analog-based approaches for targeting DNA polymerases.

Free-standing single-layer β-sheets are extremely rare in naturally occurring proteins, even though β-sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single-layer, anti-parallel β-sheet proteins, comprised of three or four twisted β-hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of β-sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent-exposed tyrosine residues, was identified on the concave surface of the β-sheet. These new modular single-layer β-sheet proteins may serve as a new model system for studying folding and design of β-rich proteins.

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein-protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39-U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemical shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35-U2AF65 and RBM39-SF3b155, the RBM39-UHM-U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM-ULM binding interface, providing a rationale for the known specificity of UHM-ULM interactions. This study therefore establishes a structural basis for specific UHM-ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.

The Dlx5 homeodomain is a transcription factor related to the Drosophila distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including split hand and foot malformation 1 in humans. Our characterization of a Dlx5 homeodomain:(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A Q186H mutation linked to split hand and foot malformation 1 likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and they likely destabilize the formation of functional complexes.

Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys147, resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca2+ for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.

Pili are proteinaceous polymers of linked pilins that protrude from the cell surface of many bacteria and often mediate adherence and virulence. We investigated a set of 20 Bacteroidia pilins from the human microbiome whose structures and mechanism of assembly were unknown. Crystal structures and biochemical data revealed a diverse protein superfamily with a common Greek-key β sandwich fold with two transthyretin-like repeats that polymerize into a pilus through a strand-exchange mechanism. The assembly mechanism of the central, structural pilins involves proteinase-assisted removal of their N-terminal β strand, creating an extended hydrophobic groove that binds the C-terminal donor strands of the incoming pilin. Accessory pilins at the tip and base have unique structural features specific to their location, allowing initiation or termination of the assembly. The Bacteroidia pilus, therefore, has a biogenesis mechanism that is distinct from other known pili and likely represents a different type of bacterial pilus.

The nuclear receptor LRH-1 (Liver Receptor Homolog-1, NR5A2) is a transcription factor that regulates gene expression programs critical for many aspects of metabolism and reproduction. Although LRH-1 is able to bind phospholipids, it is still considered an orphan nuclear receptor (NR) with an unknown regulatory hormone. Our prior cellular and structural studies demonstrated that the signaling phosphatidylinositols PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind and regulate SF-1 (Steroidogenic Factor-1, NR5A1), a close homolog of LRH-1. Here, we describe the crystal structure of human LRH-1 ligand binding domain (LBD) bound by PIP3 – the first phospholipid with a head group endogenous to mammals. We show that the phospholipid hormone binds LRH-1 with high affinity, stabilizing the receptor LBD. While the hydrophobic PIP3 tails (C16/C16) are buried inside the LRH-1 ligand binding pocket, the negatively charged PIP3 head group is presented on the receptor surface, similar to the phosphatidylinositol binding mode observed in the PIP3-SF-1 structure. Thus, data presented in this work reinforce our earlier findings demonstrating that signaling phosphatidylinositols regulate the NR5A receptors LRH-1 and SF-1.

Significance Maintenance and reprogramming of pluripotency are among the most important issues in stem cell biology and regenerative medicine. Pluripotency is governed by several key transcription factors regulating transcription of other factors. Among these, regulation of OCT4 transcription by NANOG (from Irish myth-ology Tír na nÓg) is a critical interaction. We present here the crystal structure of human NANOG homeodomain in complex with the OCT4 promoter DNA and, through a series of ration-ally designed mutations, we identify key functional residues in the protein–DNA interaction, protein stability, and maintenance of mouse ESC self-renewal. Furthermore, we describe a mutation, NANOG L122A, which enhances DNA binding affinity, protein stability, mouse ESC self-renewal, and reprogramming into ground state from primed state pluripotency.

ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (or dl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminal l-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCE Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.

Flavodoxins in combination with the flavin mononucleotide (FMN) cofactor play important roles for electron transport in prokaryotes. Here, novel insights into the FMN-binding mechanism to flavodoxins-4 were obtained from the NMR structures of the apo-protein from Lactobacillus acidophilus (YP_193882.1) and comparison of its complex with FMN. Extensive reversible conformational changes were observed upon FMN binding and release. The NMR structure of the FMN complex is in agreement with the crystal structure (PDB ID: 3EDO) and exhibits the characteristic flavodoxin fold, with a central five-stranded parallel β-sheet and five α-helices forming an α/β-sandwich architecture. The structure differs from other flavoproteins in that helix α2 is oriented perpendicular to the β-sheet and covers the FMN-binding site. This helix reversibly unfolds upon removal of the FMN ligand, which represents a unique structural rearrangement among flavodoxins.

For over a decade, the Joint Center for Structural Genomics (JCSG.org) has been at the forefront of developing tools and methodologies that enable the application of high-throughput structural biology (HTBSB) approaches to a broad range of challenging biological and biomedical problems. In PSI:Biology (2010-2015) to meet the challenges and embrace the opportunities that arise from our Partnerships projects, we have leveraged our gene-to-structure pipeline to explore challenging projects focused on structural characterization of interaction networks involved in stem cell regulation, T-cell activation and nuclear receptor signaling. These highly collaborative efforts have enabled development of systematic and integrative approaches for identifying and investigating networks of key multi-domain eukaryotic proteins and higher order assemblies of multi-component eukaryotic protein/protein and protein/nucleic acid complexes. In parallel, our biomedical theme project has focused on investigating host/microbe interactions of the microbial communities that inhabit specific niches and environments of the human body, e.g. the human gut microbiome. These efforts to date have been centered on secreted proteins from commensal bacteria in the human gut. The symbiotic relationship and influence on human development, physiology, immunity, and nutrition represent an exciting new frontier for HTBSB where we can investigate how these microorganisms contribute to human health as well as to disease. The JCSG also strives to promote widespread use of PSI resources, materials, methodologies and data to the general scientific community, via Community Nominated Target (CNT) projects and development and use of new technologies and methodologies. We also continue to contribute to the original PSI mission of structural coverage of the expanding protein universe. Supported by NIH U54-GM094586.

Crystal structures of three members (BACOVA_00364 from Bacteroides ovatus, BACUNI_03039 from Bacteroides uniformis and BACEGG_00036 from Bacteroides eggerthii) of the Pfam domain of unknown function (DUF4488) were determined to 1.95, 1.66, and 1.81 Å resolutions, respectively. The protein structures adopt an eight-stranded, calycin-like, β-barrel fold and bind an endogenous unknown ligand at one end of the β-barrel. The amino acids interacting with the ligand are not conserved in any other protein of known structure with this particular fold. The size and chemical environment of the bound ligand suggest binding or transport of a small polar molecule(s) as a potential function for these proteins. These are the first structural representatives of a newly defined PF14869 (DUF4488) Pfam family.