ABSTRACT
Biology provides plenty of examples on achieving complicated structures out of minimal numbers of building blocks. In contrast, structural complexity of designed molecular systems is achieved by increasing the numbers of component molecules. In this study, the component DNA strand assembles into a highly complex crystal structure via an unusual path of divergence and convergence. This assembly path suggests a route to minimalists for increasing structural complexity. The original purpose of this study is to engineer DNA crystals with high resolution, which is the primary motivation and a key objective for structural DNA nanotechnology. Despite great efforts in the last 40 years, engineered DNA crystals have not yet consistently reached resolution better than 2.5 Å, limiting their potential uses. Our research has shown that small, symmetrical building blocks generally lead to high resolution crystals. Herein, by following this principle, we report an engineered DNA crystal with unprecedented high resolution (2.17 Å) assembled from one single DNA component: an 8-base-long DNA strand. This system has three unique characteristics: (1) It has a very complex architecture, (2) the same DNA strand forms two different structural motifs, both of which are incorporated into the final crystal, and (3) the component DNA molecule is only an 8-base-long DNA strand, which is, arguably, the smallest DNA motif for DNA nanostructures to date. This high resolution opens the possibility of using these DNA crystals to precisely organize guest molecules at the Å level, which could stimulate a range of new investigations.
Subject(s)
DNA , Nanostructures , DNA/chemistry , Nanostructures/chemistry , Nanotechnology , Nucleotide Motifs , Engineering , Nucleic Acid ConformationABSTRACT
The growing emergence of multidrug resistance in bacterial pathogens is an immediate threat to human health worldwide. Unfortunately, there has not been a matching increase in the discovery of new antibiotics to combat this alarming trend. Novel contemporary approaches aimed at antibiotic discovery against Gram-negative bacterial pathogens have expanded focus to also include essential surface-exposed receptors and protein complexes, which have classically been targeted for vaccine development. One surface-exposed protein complex that has gained recent attention is the ß-barrel assembly machinery (BAM), which is conserved and essential across all Gram-negative bacteria. BAM is responsible for the biogenesis of ß-barrel outer membrane proteins (ß-OMPs) into the outer membrane. These ß-OMPs serve essential roles for the cell including nutrient uptake, signaling, and adhesion, but can also serve as virulence factors mediating pathogenesis. The mechanism for how BAM mediates ß-OMP biogenesis is known to be dynamic and complex, offering multiple modes for inhibition by small molecules and targeting by larger biologics. In this review, we introduce BAM and establish why it is a promising and exciting new therapeutic target and present recent studies reporting novel compounds and vaccines targeting BAM across various bacteria. These reports have fueled ongoing and future research on BAM and have boosted interest in BAM for its therapeutic promise in combatting multidrug resistance in Gram-negative bacterial pathogens.
ABSTRACT
Roughly 95% of the proteins that make up the chloroplast must be imported from the cytoplasm. The machinery responsible for the translocation of these cargo proteins is called the translocon at the outer membrane of chloroplast (TOC). The TOC core consists of three proteins, Toc34, Toc75, and Toc159; no high-resolution structure has been solved of fully assembled TOC from plants. Efforts toward determining the structure of the TOC have been hindered almost entirely by difficulties in producing sufficient yields for structural studies. In this study, we introduce an innovative method that utilizes synthetic antigen binding fragments (sABs) to isolate TOC directly from wild-type plant biomass including A. thaliana and P. sativum. Binding between the sABs and the POTRA domains was characterized by size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS), X-ray crystallography, and isothermal titration calorimetry. We also demonstrate the isolation of the TOC from P. sativum, laying the framework for large-scale isolation and purification of TOC for functional and structural studies.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Chloroplasts/metabolism , Membrane Proteins/chemistry , Plant Proteins/chemistry , Protein Precursors/chemistry , Protein Transport , Scattering, Small Angle , X-Ray Diffraction , Pisum sativum/metabolismABSTRACT
Sequence-selective recognition of DNA duplexes is important for a wide range of applications including regulating gene expression, drug development, and genome editing. Many small molecules can bind DNA duplexes with sequence selectivity. It remains as a challenge how to reliably and conveniently obtain the detailed structural information on DNA-molecule interactions because such information is critically needed for understanding the underlying rules of DNA-molecule interactions. If those rules were understood, we could design molecules to recognize DNA duplexes with a sequence preference and intervene in related biological processes, such as disease treatment. Here, we have demonstrated that DNA crystal engineering is a potential solution. A molecule-binding DNA sequence is engineered to self-assemble into highly ordered DNA crystals. An X-ray crystallographic study of molecule-DNA cocrystals reveals the structural details on how the molecule interacts with the DNA duplex. In this approach, the DNA will serve two functions: (1) being part of the molecule to be studied and (2) forming the crystal lattice. It is conceivable that this method will be a general method for studying drug/peptide-DNA interactions. The resulting DNA crystals may also find use as separation matrices, as hosts for catalysts, and as media for material storage.
Subject(s)
DNA , DNA/chemistry , Crystallography, X-Ray , Nucleic Acid ConformationABSTRACT
The protein N-terminal methyltransferase 1 (NTMT1) is implicated in neurogenesis, retinoblastoma, and cervical cancer. However, its pharmacological potentials have not been elucidated due to the lack of drug-like inhibitors. Here, we report the discovery of the first NTMT1 in vivo chemical probe GD433 by structure-guided optimization of our previously reported lead compound venglustat. GD433 (IC50 = 27 ± 1.1 nM) displays improved potency and selectivity than venglustat across biochemical, biophysical, and cellular assays. GD433 also displays good oral bioavailability and can serve as an in vivo chemical probe to dissect the pharmacological roles of Nα methylation. In addition, we also identified a close analogue (YD2160) that is inactive against NTMT1. The active inhibitor and negative control will serve as valuable tools to examine the physiological and pharmacological functions of NTMT1 catalytic activity.
Subject(s)
Enzyme Inhibitors , Methylation , Structure-Activity Relationship , Enzyme Inhibitors/pharmacologyABSTRACT
TonB-dependent transporters (TDTs) are essential proteins for metal acquisition, an important step in the growth and pathogenesis of many pathogens, including Neisseria gonorrhoeae, the causative agent of gonorrhea. There is currently no available vaccine for gonorrhea; TDTs are being investigated as vaccine candidates because they are highly conserved and expressed in vivo. Transferrin binding protein A (TbpA) is an essential virulence factor in the initiation of experimental infection in human males and functions by acquiring iron upon binding to host transferrin (human transferrin [hTf]). The loop 3 helix (L3H) is a helix finger that inserts into the hTf C-lobe and is required for hTf binding and subsequent iron acquisition. This study identified and characterized the first TbpA single-point substitutions resulting in significantly decreased hTf binding and iron acquisition, suggesting that the helix structure is more important than charge for hTf binding and utilization. The tbpA D355P ΔtbpB and tbpA A356P ΔtbpB mutants demonstrated significantly reduced hTf binding and impaired iron uptake from Fe-loaded hTf; however, only the tbpA A356P ΔtbpB mutant was able to grow when hTf was the sole source of iron. The expression of tbpB was able to restore function in all tbpA mutants. These results implicate both D355 and A356 in the key binding, extraction, and uptake functions of gonococcal TbpA.
Subject(s)
Gonorrhea , Neisseria meningitidis , Transferrin-Binding Protein A , Male , Humans , Transferrin-Binding Protein A/genetics , Transferrin-Binding Protein A/chemistry , Transferrin-Binding Protein A/metabolism , Neisseria gonorrhoeae/metabolism , Transferrin/genetics , Transferrin/metabolism , Point Mutation , Receptors, Transferrin/genetics , Iron/metabolism , Neisseria meningitidis/metabolismABSTRACT
Protein tyrosine phosphatases constitute an important class of drug targets whose potential has been limited by the paucity of drug-like small-molecule inhibitors. We recently described a class of active-site-directed, moderately selective, and potent inhibitors of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP). Here, we report our extensive structure-based design and optimization effort that afforded inhibitors with vastly improved potency and specificity. The leading compound inhibits LMW-PTP potently and selectively (Ki = 1.2 nM, >8000-fold selectivity). Many compounds exhibit favorable drug-like properties, such as low molecular weight, weak cytochrome P450 inhibition, high metabolic stability, moderate to high cell permeability (Papp > 0.2 nm/s), and moderate to good oral bioavailability (% F from 23 to 50% in mice), and therefore can be used as in vivo chemical probes to further dissect the complex biological as well as pathophysiological roles of LMW-PTP and for the development of therapeutics targeting LMW-PTP.
Subject(s)
Enzyme Inhibitors , Protein Tyrosine Phosphatases , Mice , Animals , Molecular Weight , Protein Tyrosine Phosphatases/metabolism , Catalytic Domain , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistryABSTRACT
Anti-CRISPR (Acr) proteins are encoded by phages to inactivate CRISPR-Cas systems of bacteria and archaea and are used to enhance the CRISPR toolbox for genome editing. Here we report the structure and mechanism of AcrIF24, an Acr protein that inhibits the type I-F CRISPR-Cas system from Pseudomonas aeruginosa. AcrIF24 is a homodimer that associates with two copies of the surveillance complex (Csy) and prevents the hybridization between CRISPR RNA and target DNA. Furthermore, AcrIF24 functions as an anti-CRISPR-associated (Aca) protein to repress the transcription of the acrIF23-acrIF24 operon. Alone or in complex with Csy, AcrIF24 is capable of binding to the acrIF23-acrIF24 promoter DNA with nanomolar affinity. The structure of a Csy-AcrIF24-promoter DNA complex at 2.7 Å reveals the mechanism for transcriptional suppression. Our results reveal that AcrIF24 functions as an Acr-Aca fusion protein, and they extend understanding of the diverse mechanisms used by Acr proteins.
Subject(s)
Bacteriophages , CRISPR-Associated Proteins , CRISPR-Associated Proteins/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism , CRISPR-Cas Systems , Bacteriophages/genetics , Bacteriophages/metabolism , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolismABSTRACT
Venglustat is a known allosteric inhibitor for ceramide glycosyltransferase, investigated in diseases caused by lysosomal dysfunction. Here, we identified venglustat as a potent inhibitor (IC50 = 0.42 µM) of protein N-terminal methyltransferase 1 (NTMT1) by screening 58,130 compounds. Furthermore, venglustat exhibited selectivity for NTMT1 over 36 other methyltransferases. The crystal structure of NTMT1-venglustat and inhibition mechanism revealed that venglustat competitively binds at the peptide substrate site. Meanwhile, venglustat potently inhibited protein N-terminal methylation levels in cells (IC50 = 0.5 µM). Preliminary structure-activity relationships indicated that the quinuclidine and fluorophenyl parts of venglustat are important for NTMT1 inhibition. In summary, we confirmed that venglustat is a bona fide NTMT1 inhibitor, which would advance the study on the biological roles of NTMT1. Additionally, this is the first disclosure of NTMT1 as a new molecular target of venglustat, which would cast light on its mechanism of action to guide the clinical investigations.
Subject(s)
Carbamates/pharmacology , Enzyme Inhibitors , Methyltransferases , Quinuclidines/pharmacology , Carbamates/chemistry , Ceramides , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glycosyltransferases/metabolism , Methylation , Quinuclidines/chemistryABSTRACT
Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide and has been associated with various diseases. Herein, we report the first cell-potent NNMT bisubstrate inhibitor II399, demonstrating a Ki of 5.9â nM in a biochemical assay and a cellular IC50 value of 1.9â µM. The inhibition mechanism and cocrystal structure confirmed II399 engages both the substrate and cofactor binding pockets. Computational modeling and binding data reveal a balancing act between enthalpic and entropic components that lead to II399's low nM binding affinity. Notably, II399 is 1 000-fold more selective for NNMT than closely related methyltransferases. We expect that II399 would serve as a valuable probe to elucidate NNMT biology. Furthermore, this strategy provides the first case of introducing unconventional SAM mimics, which can be adopted to develop cell-potent inhibitors for other SAM-dependent methyltransferases.
Subject(s)
Enzyme Inhibitors , Nicotinamide N-Methyltransferase , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Methyltransferases/metabolism , Niacinamide/pharmacology , Nicotinamide N-Methyltransferase/chemistry , Nicotinamide N-Methyltransferase/metabolismABSTRACT
To combat nutritional immunity, N. gonorrhoeae has evolved systems to hijack zinc and other metals directly from host metal-binding proteins such as calprotectin (CP). Here, we report the 6.1 Å cryoEM structure of the gonococcal surface receptor TdfH in complex with a zinc-bound CP tetramer. We further show that TdfH can also interact with CP in the presence of copper and manganese, but not with cobalt.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Leukocyte L1 Antigen Complex/chemistry , Neisseria gonorrhoeae/metabolism , Zinc/metabolism , Biological Transport , Cryoelectron Microscopy , Gene Expression Regulation, Bacterial , Models, Molecular , Protein ConformationABSTRACT
In Gram-negative bacteria, the biogenesis of ß-barrel outer membrane proteins is mediated by the ß-barrel assembly machinery (BAM). The mechanism employed by BAM is complex and so far- incompletely understood. Here, we report the structures of BAM in nanodiscs, prepared using polar lipids and native membranes, where we observe an outward-open state. Mutations in the barrel domain of BamA reveal that plasticity in BAM is essential, particularly along the lateral seam of the barrel domain, which is further supported by molecular dynamics simulations that show conformational dynamics in BAM are modulated by the accessory proteins. We also report the structure of BAM in complex with EspP, which reveals an early folding intermediate where EspP threads from the underside of BAM and incorporates into the barrel domain of BamA, supporting a hybrid-barrel budding mechanism in which the substrate is folded into the membrane sequentially rather than as a single unit.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Bacterial Outer Membrane Proteins/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Lipids , Molecular Dynamics Simulation , Mutation , Protein FoldingABSTRACT
Lactoferrin-binding protein B (LbpB) is a lipoprotein present on the surface of Neisseria that has been postulated to serve dual functions during pathogenesis in both iron acquisition from lactoferrin (Lf), and in providing protection against the cationic antimicrobial peptide lactoferricin (Lfcn). While previous studies support a dual role for LbpB, exactly how these ligands interact with LbpB has remained unknown. Here, we present the structures of LbpB from N. meningitidis and N. gonorrhoeae in complex with human holo-Lf, forming a 1:1 complex and confirmed by size-exclusion chromatography small-angle X-ray scattering. LbpB consists of N- and C-lobes with the N-lobe interacting extensively with the C-lobe of Lf. Our structures provide insight into LbpB's preference towards holo-Lf, and our mutagenesis and binding studies show that Lf and Lfcn bind independently. Our studies provide the molecular details for how LbpB serves to capture and preserve Lf in an iron-bound state for delivery to the membrane transporter LbpA for iron piracy, and as an antimicrobial peptide sink to evade host immune defenses.
Subject(s)
Bacterial Proteins/genetics , Carrier Proteins/genetics , Lactoferrin/metabolism , Neisseria gonorrhoeae/genetics , Neisseria meningitidis/genetics , Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Neisseria gonorrhoeae/metabolism , Neisseria meningitidis/metabolism , Protein BindingABSTRACT
Understanding the selectivity of methyltransferase inhibitors is important to dissecting the functions of each methyltransferase target. From this perspective, we report a chemoproteomic study to profile the selectivity of a potent protein N-terminal methyltransferase 1 (NTMT1) bisubstrate inhibitor NAH-C3-GPKK (Ki, app = 7 ± 1 nM) in endogenous proteomes. First, we describe the rational design, synthesis, and biochemical characterization of a new chemical probe 6, a biotinylated analogue of NAH-C3-GPKK. Next, we systematically analyze protein networks that may selectively interact with the biotinylated probe 6 in concert with the competitor NAH-C3-GPKK. Besides NTMT1, the designated NTMT1 bisubstrate inhibitor NAH-C3-GPKK was found to also potently inhibit a methyltransferase complex HemK2-Trm112 (also known as KMT9-Trm112), highlighting the importance of systematic selectivity profiling. Furthermore, this is the first potent inhibitor for HemK2/KMT9 reported until now. Thus, our studies lay the foundation for future efforts to develop selective inhibitors for either methyltransferase.
Subject(s)
Adenosine/analogs & derivatives , Adenosine/pharmacology , Enzyme Inhibitors/pharmacology , Methyltransferases/antagonists & inhibitors , Oligopeptides/pharmacology , Site-Specific DNA-Methyltransferase (Adenine-Specific)/antagonists & inhibitors , Adenosine/metabolism , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , HeLa Cells , Humans , Methyltransferases/metabolism , Oligopeptides/chemical synthesis , Oligopeptides/metabolism , Protein BindingABSTRACT
Membrane proteins serve essential roles in all aspects of life and make up roughly one-third of all genomes from prokaryotes to eukaryotes. Their responsibilities include mediating cell signaling, nutrient import, waste export, cellular communication, trafficking, and immunity. For their critical role in many cellular processes, membrane proteins serve as targets for up to 50% of drugs currently on the market and remain primary targets in new therapeutics being developed. Despite their importance and abundance in nature, only ~1% of structures in the Protein Data Bank are of transmembrane proteins. This discrepancy can be directly attributed to the biochemical properties of membrane proteins and the difficulty in producing sufficient yields for structural studies or the difficulty in growing well-ordered crystals. Here, we present methods from our work that outline our general pipeline from cloning to structure determination of membrane proteins, with a focus on using X-ray crystallography, which still yields ~90% of all structures being deposited into the Protein Data Bank.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Neisseria gonorrhoeae/metabolism , Cloning, Molecular , Crystallography, X-Ray , Databases, Protein , Models, Molecular , Neisseria gonorrhoeae/genetics , Protein Folding , Protein Structure, SecondaryABSTRACT
Protein N-terminal methyltransferases (NTMTs) catalyze the methylation of the α-N-terminal amines of proteins starting with an X-P-K/R motif. NTMT1 has been implicated in various cancers and in aging, implying its role as a potential therapeutic target. Through structural modifications of a lead NTMT1 inhibitor, BM30, we designed and synthesized a diverse set of inhibitors to probe the NTMT1 active site. The incorporation of a naphthyl group at the N-terminal region and an ortho-aminobenzoic amide at the C-terminal region of BM30 generates the top cell-potent inhibitor DC541, demonstrating increased activity on both purified NTMT1 (IC50 of 0.34 ± 0.02 µM) and the cellular α-N-terminal methylation level of regulator of chromosome condensation 1 (RCC1, IC50 value of 30 µM) in human colorectal cancer HT29 cells. Furthermore, DC541 exhibits over 300-fold selectivity to several methyltransferases. This study points out the direction for the development of more cell-potent inhibitors for NTMT1.
ABSTRACT
Gram-negative bacteria, mitochondria, and chloroplasts all possess an outer membrane populated with a host of ß-barrel outer-membrane proteins (ßOMPs). These ßOMPs play crucial roles in maintaining viability of their hosts, and therefore, it is essential to understand the biogenesis of this class of membrane proteins. In recent years, significant structural and functional advancements have been made toward elucidating this process, which is mediated by the ß-barrel assembly machinery (BAM) in Gram-negative bacteria, and by the sorting and assembly machinery (SAM) in mitochondria. Structures of both BAM and SAM have now been reported, allowing a comparison and dissection of the two machineries, with other studies reporting on functional aspects of each. Together, these new insights provide compelling support for the proposed budding mechanism, where each nascent ßOMP forms a hybrid-barrel intermediate with BAM/SAM in route to its biogenesis into the membrane. Here, we will review these recent studies and highlight their contributions toward understanding ßOMP biogenesis in Gram-negative bacteria and in mitochondria. We will also weigh the evidence supporting each of the two leading mechanistic models for how BAM/SAM function, and offer an outlook on future studies within the field.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Chloroplasts/metabolism , Gram-Negative Bacteria/metabolism , Mitochondria/metabolism , Protein Folding , Amino Acid Motifs , Chloroplasts/chemistry , Mitochondria/chemistry , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/metabolism , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein ConformationABSTRACT
The bisubstrate analogue strategy is a promising approach to develop potent and selective inhibitors for protein methyltransferases. Herein, the interactions of a series of bisubstrate analogues with protein N-terminal methyltransferase 1 (NTMT1) were examined to probe the molecular properties of the active site of NTMT1. Our results indicate that a 2-C to 4-C atom linker enables its respective bisubstrate analogue to occupy both substrate- and cofactor-binding sites of NTMT1, but the bisubstrate analogue with a 5-C atom linker only interacts with the substrate-binding site and functions as a substrate. Furthermore, the 4-C atom linker is the optimal and produces the most potent inhibitor (Ki,app = 130 ± 40 pM) for NTMT1 to date, displaying more than 3000-fold selectivity for other methyltransferases and even for its homologue NTMT2. This study reveals the molecular basis for the plasticity of the active site of NTMT1. Additionally, our study outlines general guidance on the development of bisubstrate inhibitors for any methyltransferases.
Subject(s)
Catalytic Domain , Methyltransferases/chemistry , Binding Sites/drug effects , Catalytic Domain/drug effects , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Methyltransferases/metabolism , Models, Molecular , Substrate Specificity/drug effectsABSTRACT
Protein N-terminal methyltransferases (NTMTs) methylate the α-N-terminal amines of proteins starting with the canonical X-P-K/R motif. Genetic studies imply that NTMT1 regulates cell mitosis and DNA damage repair. Herein, we report the rational design and development of the first potent peptidomimetic inhibitor for NTMT1/2. Biochemical and cocrystallization studies manifest that BM30 (with a half-maximal inhibitory concentration of 0.89 ± 0.10 µM) is a competitive inhibitor to the peptide substrate and noncompetitive to the cofactor S-adenosylmethionine. BM30 exhibits over 100-fold selectivity to NTMT1/2 among a panel of 41 MTs, indicating its potential to achieve high selectivity when targeting the peptide substrate binding site of NTMT1/2. Its cell-permeable analogue DC432 (IC50 of 54 ± 4 nM) decreases the N-terminal methylation level of the regulator of chromosome condensation 1 and SET proteins in HCT116 cells. This proof-of principle study provides valuable probes for NTMT1/2 and highlights the opportunity to develop more cell-potent inhibitors to elucidate the function of NTMTs in the future.
Subject(s)
Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Methyltransferases/antagonists & inhibitors , Peptidomimetics/chemical synthesis , Peptidomimetics/pharmacology , Chemistry Techniques, Synthetic , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , HCT116 Cells , Humans , Methylation , Methyltransferases/chemistry , Models, Molecular , Peptidomimetics/chemistry , Protein ConformationABSTRACT
Neisseria gonorrhoeae, responsible for the sexually transmitted infection gonorrhea, is an obligate human pathogen exquisitely adapted for survival on mucosal surfaces of humans. This host-pathogen relationship has resulted in evolution by N. gonorrhoeae of pathways that enable the use of host metalloproteins as required nutrients through the deployment of outer membrane-bound TonB-dependent transporters (TdTs). Recently, a TdT called TdfH was implicated in binding to calprotectin (CP) and in removal of the bound zinc (Zn), enabling gonococcal growth. TdfH is highly conserved among the pathogenic Neisseria species, making it a potentially promising candidate for inclusion into a gonococcal vaccine. Currently, the nature and specificity of the TdfH-CP interaction have not been determined. In this study, we found that TdfH specifically interacted with human calprotectin (hCP) and that growth of the gonococcus was supported in a TdfH-dependent manner only when hCP was available as a sole zinc source and not when mouse CP was provided. The binding interactions between TdfH and hCP were assessed using isothermal titration calorimetry where we observed a multistate model having both high-affinity and low-affinity sites of interaction. hCP has two Zn binding sites, and gonococcal growth assays using hCP mutants deficient in one or both of the Zn binding sites revealed that TdfH exhibited a site preference during Zn piracy and utilization. This report provides the first insights into the molecular mechanism of Zn piracy by neisserial TdfH and further highlights the obligate human nature of N. gonorrhoeae and the high-affinity interactions occurring between TdTs and their human ligands during pathogenesis.IMPORTANCE The dramatic rise in antimicrobial resistance among Neisseria gonorrhoeae isolates over the last few decades, paired with dwindling treatment options and the lack of a protective vaccine, has prompted increased interest in identifying new bacterial targets for the treatment and, ideally, prevention of gonococcal disease. TonB-dependent transporters are a conserved set of proteins that serve crucial functions for bacterial survival within the host. In this study, binding between the gonococcal transporter, TdfH, and calprotectin was determined to be of high affinity and host restricted. The current study identified a preferential TdfH interaction at the calprotectin dimer interface. An antigonococcal therapeutic could potentially block this site on calprotectin, interrupting Zn uptake by N. gonorrhoeae and thereby prohibiting continued bacterial growth. We describe protein-protein interactions between TdfH and calprotectin, and our findings provide the building blocks for future therapeutic or prophylactic targets.