Water-mediated Hydrogen Bonds Research Articles

Induced-Fit Recognition of CCG Trinucleotide Repeats by a Nickel-Chromomycin Complex Resulting in Large-Scale DNA Deformation.

Small-molecule compounds targeting trinucleotide repeats in DNA have considerable potential as therapeutic or diagnostic agents against many neurological diseases. NiII (Chro)2 (Chro=chromomycin A3) binds specifically to the minor groove of (CCG)n repeats in duplex DNA, with unique fluorescence features that may serve as a probe for disease detection. Crystallographic studies revealed that the specificity originates from the large-scale spatial rearrangement of the DNA structure, including extrusion of consecutive bases and backbone distortions, with a sharp bending of the duplex accompanied by conformational changes in the NiII chelate itself. The DNA deformation of CCG repeats upon binding forms a GGCC tetranucleotide tract, which is recognized by NiII (Chro)2 . The extruded cytosine and last guanine nucleotides form water-mediated hydrogen bonds, which aid in ligand recognition. The recognition can be accounted for by the classic induced-fit paradigm.

Exploring the RNA-bound and RNA-free human Argonaute-2 by molecular dynamics simulation method.

Argonaute 2 (Ago2) protein is the major vehicle of microRNAs (miRNAs)-guided gene repression and silencing processes. Although the crystal structure of human Ago2 (hAgo2) has recently been disclosed, the information of dynamically structural character of protein-RNA recognition is still lacking. Molecular dynamics simulations were used to systematically explore hAgo2 in the presence and absence of RNA duplex. Stable direct and water-mediated hydrogen bonds were observed between guide RNA backbone atoms and hAgo2, especially for nucleotides 2-7. In addition, water-mediated hydrogen bonds are indicated to be critical in the specific recognition between hAgo2 and the conserved adenine in position 1 of target RNA. The core domains (N, PAZ, MID, and PIWI) possess rigid body movements during the simulations. The motions of N-PAZ and PIWI-MID are negatively correlated with or without RNA binding and PAZ domain is identified as the most mobile domain in both systems. The reorientation of PAZ domain not only influences the binding of helix-7 and RNA duplex, the initial pairing process, but also the shape of N-PAZ cleft, where the supplemental base pairing occurs. It is speculated that PAZ domain could be a key regulator in hAgo2-mediated miRNA-induced gene regulation.

Crystal Structure of a Homogeneous IgG-Fc Glycoform with the N-Glycan Designed to Maximize the Antibody Dependent Cellular Cytotoxicity.

N-glycosylation on IgG modulates Fc conformation and effector functions. An IgG-Fc contains a human sialo-complex type (hSCT) glycan of biantennary structure with two α2,6-sialylations and without core-fucosylation is an optimized glycoform developed to enhance the antibody dependent cellular cytotoxicity (ADCC). hSCT modification not only enhances the binding affinity to Fc receptors in the presence of antigen but also in some cases provides gain-of-function effector activity. We used enzymatic glyco-engineering to prepare an IgG-Fc with homogeneous hSCT attached to each CH2 domain and solved its crystal structure. A compact form and an open form were observed in an asymmetric unit in the crystal. In the compact structure, the double glycan latches from the two hSCT chains stabilize the CH2 domains in a closed conformation. In the open structure, the terminal sialic acid (N-acetylneuraminic acid or NeuNAc) residue interacts through water-mediated hydrogen bonds with the D249-L251 helix, to modulate the pivot region of the CH2-CH3 interface. The double glycan latches and the sialic acid modulation may be mutually exclusive. This is the first crystal structure of glyco-engineered Fc with enhanced effector activities. This work provides insights into the relationship between the structural stability and effector functions affected by hSCT modification and the development of better antibodies for therapeutic applications.

Flaviviridae viruses use a common molecular mechanism to escape nucleoside analogue inhibitors

The RNA-dependent RNA polymerases of Flaviviridae viruses are crucial for replication. The Flaviviridae polymerase is organized into structural motifs (A-G), with motifs F, A, C and E containing interrogating, priming and catalytic substrate-interacting sites. Modified nucleoside analogues act as antiviral drugs by targeting Flaviviridae polymerases and integrating into the synthesized product causing premature termination. A threonine mutation of a conserved serine residue in motif B of Flaviviridae polymerases renders resistance to 2′-C-methylated nucleoside analogues. The mechanism how this single mutation causes Flaviviridae viruses to escape nucleoside analogues is not yet known. Given the pivotal position of the serine residue in motif B that supports motif F, we hypothesized the threonine mutation causes alterations in nucleoside exploration within the entry tunnel. Implementing a stochastic molecular software showed the all-atom 2′-C-methylated analogue reaction within the active sites of wild type and serine-threonine mutant polymerases from Hepacivirus and Flavivirus. Compared with the wild type, the serine-threonine mutant polymerases caused a significant decrease of analogue contacts with conserved interrogating residues in motif F and a displacement of metal ion cofactors. The simulations significantly showed that during the analogue exploration of the active site the hydrophobic methyl group in the serine-threonine mutant repels water-mediated hydrogen bonds with the 2′-C-methylated analogue, causing a concentration of water-mediated bonds at the substrate-interacting sites. Collectively, the data are an insight into a molecular escape mechanism by Flaviviridae viruses from 2′-C-methylated nucleoside analogue inhibitors.

Water‐Restructuring Mutations Can Reverse the Thermodynamic Signature of Ligand Binding to Human Carbonic Anhydrase

This study uses mutants of human carbonic anhydrase (HCAII) to examine how changes in the organization of water within a binding pocket can alter the thermodynamics of protein-ligand association. Results from calorimetric, crystallographic, and theoretical analyses suggest that most mutations strengthen networks of water-mediated hydrogen bonds and reduce binding affinity by increasing the enthalpic cost and, to a lesser extent, the entropic benefit of rearranging those networks during binding. The organization of water within a binding pocket can thus determine whether the hydrophobic interactions in which it engages are enthalpy-driven or entropy-driven. Our findings highlight a possible asymmetry in protein-ligand association by suggesting that, within the confines of the binding pocket of HCAII, binding events associated with enthalpically favorable rearrangements of water are stronger than those associated with entropically favorable ones.

A water-mediated allosteric network governs activation of Aurora kinase A.

The catalytic activity of many protein kinases is controlled by conformational changes of a conserved Asp-Phe-Gly (DFG) motif. We used an infrared probe to track the DFG motif of the mitotic kinase Aurora A (AurA) and found that allosteric activation by the spindle-associated protein Tpx2 involves an equilibrium shift toward the active DFG-in state. Förster resonance energy transfer experiments show that the activation loop undergoes a nanometer-scale movement that is tightly coupled to the DFG equilibrium. Tpx2 further activates AurA by stabilizing a water-mediated allosteric network that links the C-helix to the active site through an unusual polar residue in the regulatory spine. The polar spine residue and water network of AurA are essential for phosphorylation-driven activation, but an alternative form of the water network found in related kinases can support Tpx2-driven activation, suggesting that variations in the water-mediated hydrogen bond network mediate regulatory diversification in protein kinases.

Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues.

The flavoenzyme l-6-hydroxynicotine oxidase is a member of the monoamine oxidase family that catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during microbial catabolism of nicotine. While the enzyme has long been understood to catalyze oxidation of the carbon-carbon bond, it has recently been shown to catalyze oxidation of a carbon-nitrogen bond [Fitzpatrick, P. F., et al. (2016) Biochemistry 55, 697-703]. The effects of pH and mutagenesis of active site residues have now been utilized to study the mechanism and roles of active site residues. Asn166 and Tyr311 bind the substrate, while Lys287 forms a water-mediated hydrogen bond with flavin N5. The N166A and Y311F mutations result in ∼30- and ∼4-fold decreases in kcat/Km and kred for (S)-6-hydroxynicotine, respectively, with larger effects on the kcat/Km value for (S)-6-hydroxynornicotine. The K287M mutation results in ∼10-fold decreases in these parameters and a 6000-fold decrease in the kcat/Km value for oxygen. The shapes of the pH profiles are not altered by the N166A and Y311F mutations. There is no solvent isotope effect on the kcat/Km value for amines. The results are consistent with a model in which both the charged and neutral forms of the amine can bind, with the former rapidly losing a proton to a hydrogen bond network of water and amino acids in the active site prior to the transfer of hydride to the flavin.

Computational analysis of BACE1-ligand complex crystal structures and linear discriminant analysis for identification of BACE1 inhibitors with anti P-glycoprotein binding property

More than 100 years of research on Alzheimer’s disease didn’t yield a potential cure for this dreadful disease. Poor Blood Brain Barrier (BBB) permeability and P-glycoprotein binding of BACE1 inhibitors are the major causes for the failure of these molecules during clinical trials. The design of BACE1 inhibitors with a balance of sufficient affinity to the binding site and little or no interaction with P-glycoproteins is indispensable. Identification and understanding of protein–ligand interactions are essential for ligand optimization process. Structure-based drug design (SBDD) efforts led to a steady accumulation of BACE1-ligand crystal complexes in the PDB. This study focuses on analyses of 153 BACE1-ligand complexes for the direct contacts (hydrogen bonds and weak interactions) observed between protein and ligand and indirect contacts (water-mediated hydrogen bonds), observed in BACE1-ligand complex crystal structures. Intraligand hydrogen bonds were analyzed, with focus on ligand P-glycoprotein efflux. The interactions are dissected specific to subsites in the active site and discussed. The observed protein-ligand and intraligand interactions were used to develop the linear discriminant model for the identification of BACE1 inhibitors with less or no P-glycoprotein binding property. Excellent statistical results and model’s ability to correctly predict a new data-set with an accuracy of 92% is achieved. The results are retrospectively analyzed to give input for the design of potential BACE1 inhibitors.

Lysozyme's lectin-like characteristics facilitates its immune defense function.

Interactions between human lysozyme (HL) and the lipopolysaccharide (LPS) of Klebsiella pneumoniae O1, a causative agent of lung infection, were identified by surface plasmon resonance. To characterize the molecular mechanism of this interaction, HL binding to synthetic disaccharides and tetrasaccharides representing one and two repeating units, respectively, of the O-chain of this LPS were studied. pH-dependent structural rearrangements of HL after interaction with the disaccharide were observed through nuclear magnetic resonance. The crystal structure of the HL-tetrasaccharide complex revealed carbohydrate chain packing into the A, B, C, and D binding sites of HL, which primarily occurred through residue-specific, direct or water-mediated hydrogen bonds and hydrophobic contacts. Overall, these results support a crucial role of the Glu35/Asp53/Trp63/Asp102 residues in HL binding to the tetrasaccharide. These observations suggest an unknown glycan-guided mechanism that underlies recognition of the bacterial cell wall by lysozyme and may complement the HL immune defense function.

Computational and Experimental Characterization of Patient Derived Mutations Reveal an Unusual Mode of Regulatory Spine Assembly and Drug Sensitivity in EGFR Kinase.

The catalytic activation of protein kinases requires precise positioning of key conserved catalytic and regulatory motifs in the kinase core. The Regulatory Spine (RS) is one such structural motif that is dynamically assembled upon kinase activation. The RS is also a mutational hotspot in cancers; however, the mechanisms by which cancer mutations impact RS assembly and kinase activity are not fully understood. In this study, through mutational analysis of patient derived mutations in the RS of EGFR kinase, we identify an activating mutation, M766T, at the RS3 position. RS3 is located in the regulatory αC-helix, and a series of mutations at the RS3 position suggest a strong correlation between the amino acid type present at the RS3 position and ligand (EGF) independent EGFR activation. Small polar amino acids increase ligand independent activity, while large aromatic amino acids decrease kinase activity. M766T relies on the canonical asymmetric dimer for full activation. Molecular modeling and molecular dynamics simulations of WT and mutant EGFR suggest a model in which M766T activates the kinase domain by disrupting conserved autoinhibitory interactions between M766 and hydrophobic residues in the activation segment. In addition, a water mediated hydrogen bond network between T766, the conserved K745-E762 salt bridge, and the backbone amide of the DFG motif is identified as a key determinant of M766T-mediated activation. M766T is resistant to FDA approved EGFR inhibitors such as gefitinib and erlotinib, and computational estimation of ligand binding free energy identifies key residues associated with drug sensitivity. In sum, our studies suggest an unusual mode of RS assembly and oncogenic EGFR activation, and provide new clues for the design of allosteric protein kinase inhibitors.

Structure and function of an ancestral-type β-decarboxylating dehydrogenase from Thermococcus kodakarensis

β-Decarboxylating dehydrogenases, which are involved in central metabolism, are considered to have diverged from a common ancestor with broad substrate specificity. In a molecular phylogenetic analysis of 183 β-decarboxylating dehydrogenase homologs from 84 species, TK0280 from Thermococcus kodakarensis was selected as a candidate for an ancestral-type β-decarboxylating dehydrogenase. The biochemical characterization of recombinant TK0280 revealed that the enzyme exhibited dehydrogenase activities toward homoisocitrate, isocitrate, and 3-isopropylmalate, which correspond to key reactions involved in the lysine biosynthetic pathway, tricarboxylic acid cycle, and leucine biosynthetic pathway, respectively. In T. kodakarensis, the growth characteristics of the KUW1 host strain and a TK0280 deletion strain suggested that TK0280 is involved in lysine biosynthesis in this archaeon. On the other hand, gene complementation analyses using Thermus thermophilus as a host revealed that TK0280 functions as both an isocitrate dehydrogenase and homoisocitrate dehydrogenase in this organism, but not as a 3-isopropylmalate dehydrogenase, most probably reflecting its low catalytic efficiency toward 3-isopropylmalate. A crystallographic study on TK0280 binding each substrate indicated that Thr71 and Ser80 played important roles in the recognition of homoisocitrate and isocitrate while the hydrophobic region consisting of Ile82 and Leu83 was responsible for the recognition of 3-isopropylmalate. These analyses also suggested the importance of a water-mediated hydrogen bond network for the stabilization of the β3-α4 loop, including the Thr71 residue, with respect to the promiscuity of the substrate specificity of TK0280.

Derivatives of 3-Amino-2-methylpyridine as BAZ2B Bromodomain Ligands: In Silico Discovery and in Crystallo Validation.

The 3-amino-2-methylpyridine derivative 1 was identified as ligand of the BAZ2B bromodomain by automatic docking of nearly 500 compounds, selected on the basis of previous fragment hits. Hit expansion by two in silico approaches, pharmacophore search followed by docking, and substructure search resulted in five additional ligands. The predicted binding mode of the six 3-amino-2-methylpyridine derivatives was validated by protein crystallography. A small displacement of residues 1894-1899 of the ZA loop is observed for two of the six ligands. In all structures, the pyridine head is involved in a water-mediated hydrogen bond with the side chain of the conserved Tyr1901 while the 3-amino linker acts as hydrogen bond donor for the backbone carbonyl of Pro1888. Heterogeneous orientations are observed for the tail groups (i.e., the 3-amino substituents). The sulfonyl group in the tail of compounds 1 and 2 is involved in a hydrogen bond with the backbone amide of Asn1894.

High-resolution crystal structure of the therapeutic antibody pembrolizumab bound to the human PD-1.

Pembrolizumab is an FDA-approved therapeutic antibody that targets the programmed cell death-1 (PD-1) to block the immune checkpoint pathway for the treatment of various types of cancer. It receives remarkable attention due to the high degree of efficacy. Very recently, the crystal structure of the Fab fragment of pembrolizumab (PemFab) in complex with the extracellular domain of human PD-1 (PD-1ECD) was reported at a resolution of 2.9 Å. However, this relatively low-resolution structural data fails to provide sufficient information on interfacial water molecules at the binding interface that substantially contribute to affinity and specificity between the therapeutic antibody and target. Here, we present the independently determined crystal structure of the Fv fragment of pembrolizumab (PemFv) in complex with the PD-1ECD at a resolution of 2.15 Å. This high-resolution structure allows the accurate mapping of the interaction including water-mediated hydrogen bonds and provides, for the first time, a coherent explanation of PD-1 antagonism by pembrolizumab. Our structural data also provides new insights into the rational design of improved anti-PD-1 therapeutics.

Urea Mimics Nucleobases by Preserving the Helical Integrity of B-DNA Duplexes via Hydrogen Bonding and Stacking Interactions.

Urea lesions are formed in DNA because of free radical damage of the thymine base, and their occurrence in DNA blocks DNA polymerases, which has deleterious consequences. Recently, it has been shown that urea is capable of forming hydrogen bonding and stacking interactions with nucleobases, which are responsible for the unfolding of RNA in aqueous urea. Base pairing and stacking are inherent properties of nucleobases; because urea is able to form both, this study attempts to investigate if urea can mimic nucleobases in the context of nucleic acid structures by examining the effect of introducing urea lesions complementary to the four different nucleobases on the overall helical integrity of B-DNA duplexes and their thermodynamic stabilities using molecular dynamics (MD) simulations. The MD simulations resulted in stable duplexes without significant changes in the global B-DNA conformation. The urea lesions occupy intrahelical positions by forming hydrogen bonds with nitrogenous nucleobases, in agreement with experimental results. Furthermore, these urea lesions form hydrogen bonding and stacking interactions with other nucleobases of the same and partner strands, analogous to nucleobases in typical B-DNA duplexes. Direct hydrogen bond interactions are observed for the urea-purine pairs within DNA duplexes, whereas two different modes of pairing, namely, direct hydrogen bonds and water-mediated hydrogen bonds, are observed for the urea-pyrimidine pairs. The latter explains the complexities involved in interpreting the experimental nuclear magnetic resonance data reported previously. Binding free energy calculations were further performed to confirm the thermodynamic stability of the urea-incorporated DNA duplexes with respect to pure duplexes. This study suggests that urea mimics nucleobases by pairing opposite all four nucleobases and maintains the overall structure of the B-DNA duplexes.

Effects of a Single Escape Mutation on T Cell and HIV-1 Co-adaptation.

The mechanistic basis for the progressive accumulation of Y(135)F Nef mutant viruses in the HIV-1-infected population remains poorly understood. Y(135)F viruses carry the 2F mutation within RW8 and RF10, which are two HLA-A(∗)24:02-restricted superimposed Nef epitopes recognized by distinct and adaptable CD8(+) Tcell responses. We combined comprehensive analysis of the Tcell receptor repertoire and cross-reactive potential of wild-type or 2F RW8- and RF10-specific CD8(+) Tcells with peptide-MHC complex stability and crystal structure studies. We find that, by affecting direct and water-mediated hydrogen bond networks within the peptide-MHC complex, the 2F mutation reduces both TCR and HLA binding. This suggests an advantage underlying the evolution of the 2F variant with decreased CD8(+) Tcell efficacy. Our study provides a refined understanding of HIV-1 and CD8(+) Tcell co-adaptation at the population level.

Triazole resistance mediated by mutations of a conserved active site tyrosine in fungal lanosterol 14α-demethylase.

Emergence of fungal strains showing resistance to triazole drugs can make treatment of fungal disease problematic. Triazole resistance can arise due to single mutations in the drug target lanosterol 14α-demethylase (Erg11p/CYP51). We have determined how commonly occurring single site mutations in pathogenic fungi affect triazole binding using Saccharomyces cerevisiae Erg11p (ScErg11p) as a target surrogate. The mutations Y140F/H were introduced into full-length hexahistidine-tagged ScErg11p. Phenotypes and high-resolution X-ray crystal structures were determined for the mutant enzymes complexed with short-tailed (fluconazole and voriconazole) or long-tailed (itraconazole and posaconazole) triazoles and wild type enzyme complexed with voriconazole. The mutations disrupted a water-mediated hydrogen bond network involved in binding of short-tailed triazoles, which contain a tertiary hydroxyl not present in long-tailed triazoles. This appears to be the mechanism by which resistance to these short chain azoles occurs. Understanding how these mutations affect drug affinity will aid the design of azoles that overcome resistance.

Analysis of the binding mode of laulimalide to microtubules: Establishing a laulimalide–tubulin pharmacophore

Laulimalide (LA) is a microtubule-stabilizing agent, currently in preclinical studies. However, studying the binding of this species and successfully synthesizing potent analogues have been challenging. The LA binding site is located between tubulin protofilaments, and therefore LA is in contact with two adjacent -tubulin units. Here, an improved model of the binding mode of LA in microtubules is presented, using the newly available crystal structure pose and an extended tubulin heterodimer complex, as well as molecular dynamics simulations. With this model, a series of LA analogues developed by Mooberry and coworkers are also analyzed in order to establish important pharmacophores in LA binding and cytotoxicity. In the side chain, – interactions are important contributors to LA binding, as are water-mediated hydrogen bonds. An intramolecular hydrogen bond is correlated with high cytotoxicity, and is dependent on macrocycle conformation. Therefore, while the epoxide and olefin groups in the macrocycle do not engage in specific interactions with the protein, they are essential contributions to an active macrocycle conformation, and therefore potency. Calculations reveal that a balance in binding affinity is important for LA activity, where the more potent compounds have larger interactions with the adjacent tubulin unit than the less-active analogs. Several modifications are suggested for the rational design of LA analogues that should not disrupt the active macrocycle conformation.

Characterization of rare oncogenic mutations in the kinase domain of Epidermal Growth Factor Receptor (EGFR)

Epidermal growth factor receptor (EGFR) is activated by a variety of oncogenic mutations, many of which map to the intracellular protein kinase domain. While several recurrent mutations in the kinase domain are well characterized, mining of the Protein Kinase Ontology (ProKinO), which relates mutations to key functional motifs, reveals recurrent, but poorly characterized mutations in key functional regions. R776H and M766T are two such mutations in the alphaC‐beta4 and regulator spine of EGFR. We recently showed that the R776H functions as a “superacceptor” when paired with WT EGFR and exerts lateral phosphorylation activity (2). Molecular dynamics simulations identified a critical auto‐inhibitory interaction mediated by R776 that is relieved in the R776H mutant (2). M766T, on the other hand, activates EGFR by disrupting the inactive hydrophobic packing between the C‐helix and DFG motif, and stabilizing a water mediated hydrogen bond with the K745‐E762 salt bridge, formation of which is critical for kinase activation. Our experimental and computational studies provide new insights into kinase mutational activation and framework for identifying and characterizing rare drivers in the cancer kinome.Support or Funding InformationFunding for N.K. from the American Cancer Society (RSG‐10‐ 188‐01‐TBE), Georgia Cancer Coalition (GCC) and University of Georgia is acknowledged. This study was supported in part by resources and technical expertise from the Georgia Advanced Computing Resource Center, a partnership between the University of Georgia's Office of the Vice President for Research and Office of the Vice President for Information Technology.

A Multivalent Marine Lectin from Crenomytilus grayanus Possesses Anti-cancer Activity through Recognizing Globotriose Gb3.

In this study, we report the structure and function of a lectin from the sea mollusk Crenomytilus grayanus collected from the sublittoral zone of Peter the Great Bay of the Sea of Japan. The crystal structure of C. grayanus lectin (CGL) was solved to a resolution of 1.08 Å, revealing a β-trefoil fold that dimerizes into a dumbbell-shaped quaternary structure. Analysis of the crystal CGL structures bound to galactose, galactosamine, and globotriose Gb3 indicated that each CGL can bind three ligands through a carbohydrate-binding motif involving an extensive histidine- and water-mediated hydrogen bond network. CGL binding to Gb3 is further enhanced by additional side-chain-mediated hydrogen bonds in each of the three ligand-binding sites. NMR titrations revealed that the three binding sites have distinct microscopic affinities toward galactose and galactosamine. Cell viability assays showed that CGL recognizes Gb3 on the surface of breast cancer cells, leading to cell death. Our findings suggest the use of this lectin in cancer diagnosis and treatment.

Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives

The Escherichia coli AcrAB-TolC efflux pump is the archetype of the resistance nodulation cell division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these pumps important antibacterial drug discovery targets. We have recently developed novel pyranopyridine-based inhibitors of AcrB, which are orders of magnitude more powerful than the previously known inhibitors. However, further development of such inhibitors has been hindered by the lack of structural information for rational drug design. Although only the soluble, periplasmic part of AcrB binds and exports the ligands, the presence of the membrane-embedded domain in AcrB and its polyspecific binding behavior have made cocrystallization with drugs challenging. To overcome this obstacle, we have engineered and produced a soluble version of AcrB [AcrB periplasmic domain (AcrBper)], which is highly congruent in structure with the periplasmic part of the full-length protein, and is capable of binding substrates and potent inhibitors. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB using a combination of cellular, X-ray crystallographic, and molecular dynamics (MD) simulations studies. The pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein- and water-mediated hydrogen bond network. These detailed insights provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gram-negative pathogens.