Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis

The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3')-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with (15)N NMR-derived pK(a) and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2'-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3')-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3')-IIIa-induced resistance.     

Tracing the detail: how mutations affect binding modes and thermodynamic signatures of closely related aldose reductase inhibitors

Improvements on the computational methods for affinity prediction from the structure of protein-ligand complexes require a better understanding of the nature of molecular interactions and biomolecular recognition principles. In the present contribution, the binding of two chemically closely related human aldose reductase inhibitors had been studied by high-resolution X-ray analysis (0.92-1.35 ?) and isothermal titration calorimetry against a series of single-site mutants of the wild-type protein. A crucial threonine thought to be involved in a short bromine-to-oxygen halogen bond to the inhibitors in the wild type has been mutated to the structurally similar residues alanine, cysteine, serine and valine. Overall, structurally, the binding mode of the inhibitors is conserved; however, small but significant geometrical adaptations are observed as a consequence of the spatial and electronic changes at the mutation site. They involve the opening of a central bond angle and shifts in consequence of the lost or gained halogen bonds. Remarkably, the tiny structural changes are responded by partly strong modulation of the thermodynamic profiles. Even though the free energy of binding is maximally perturbed by only 7 kJ/mol, much stronger modulations and shifts in the enthalpy and entropy signatures are revealed, which indicate a pronounced enthalpy/entropy compensation. However, an explanatory correlation can be detected when facing these perturbances against the small structural changes. This also provides deeper insights into how single-site mutations can alter the selectivity profile of closely related ligands against a target protein.     

Crystal structure of the LG3 domain of endorepellin, an angiogenesis inhibitor

Endorepellin, the C-terminal region of perlecan, inhibits angiogenesis by disrupting actin cytoskeleton and focal adhesions. The C-terminal laminin-like globular domain (LG3) of endorepellin directs most of this antiangiogenic activity. To investigate the angiostatic mechanism and to identify structural determinants, we have solved crystal structures of the LG3 domain in both apo- and calcium-bound forms at resolutions of 1.5 Å and 2.8 Å, respectively. The conserved core has the jellyroll fold characteristic of LG domains. The calcium-induced structural changes seem very restricted, and the calcium binding site appears to be preformed, suggesting that the bound calcium ion, rather than structural rearrangements, contributes to antiangiogenesis. We have identified H4268 on the EF loop as a key residue for the biochemical function of LG3, since its mutation abolishes antiangiogenic activity, and mutant LG3 can no longer form a direct interaction with integrin. Taken together, we propose that these two distinct structural elements contribute to the angiostatic effect of endorepellin.     

The X-ray structure of zebrafish (Danio rerio) ileal bile acid-binding protein reveals the presence of binding sites on the surface of the protein molecule

The ileal bile acid-binding proteins (I-BABPs), also called ileal lipid-binding proteins or gastrotropins, belong to the family of the fatty acid-binding proteins and play an important role in the solubilization and transport of bile acids in the enterocyte. This article describes the expression, purification, crystallization, and three-dimensional structure determination of zebrafish (Danio rerio) I-BABP both in its apo form and bound to cholic acid. This is the first X-ray structure of an I-BABP. The structure of the apoprotein was determined to a resolution of 1.6 Å, and two different monoclinic crystal forms of the holoprotein were solved and refined to 2.2 Å resolution. Three protein molecules are present in the asymmetric unit of one of the co-crystal forms and two in the other, and therefore, the results of this study refer to observations made on five different protein molecules in the crystalline state. In every case, two cholate ligands were found bound in approximately the same position in the internal cavity of the protein molecules, but an unexpected result is the presence of clear and unambiguous electron density for several cholate molecules bound on hydrophobic patches on the surface of all the five independent protein molecules examined. Isothermal titration calorimetry was used for the thermodynamic characterization of the binding mechanism and has yielded results that are consistent with the X-ray data. Ligand binding is described in detail, and the conformational changes undergone by the protein molecule in the apo-to-holo transition are examined by superposition of the apo- and holoprotein models. The structure of the holoprotein is also compared with that of the liver BABP from the same species and those of other I-BABPs determined by NMR.     

Ligand-Induced Structural Transitions, Mutational Analysis, and ‘Open’ Quaternary Structure of the M. tuberculosis Feast/Famine Regulatory Protein (Rv3291c)

Rv3291c is a member of the feast/famine regulatory protein family that is known to form stable protein-DNA complexes. We report a specific oligomeric transition between hexadecameric and octameric/lower-order oligomers in the presence of Phe that supports an effector-mediated model for the disassembly of a nucleosome-like particle. We had generated two mutants, Gly102Thr and Glu104Ala, which are part of the essential 100-106 effector-binding loop. The Gly102Thr mutant adopts an unusual ‘open’ quaternary structure and offers interesting functional insights co-related to the binding of an effector. This is similar to the previously reported Escherichia coli Lrp co-crystallized in the presence of DNA where the interactions of the substrate with the N-terminal DNA binding domain presumably lead to symmetry deviations to the oligomeric association. The present structure represents a direct evidence to support that changes made to the effector-binding domain at the C-terminus also result in a functionally relevant quaternary structural change. Conversely, the Glu104Ala mutant retains the closed quaternary association observed in the native protein and reveals nonsymmetrical interaction effects in the two subunits of the dimer. We also report that the native protein unexpectedly binds Lys but does not recognize Arg and offer a structural explanation for it. Error-scaled difference distance matrix analysis suggests that the protein has a relatively flexible core that is presumably needed to mediate the structural changes necessary for the protein's regulatory functions.     

The thermodynamics of protein-ligand interaction and solvation: insights for ligand design

Isothermal titration calorimetry is able to provide accurate information on the thermodynamic contributions of enthalpy and entropy changes to free energies of binding. The Structure/Calorimetry of Reported Protein Interactions Online database of published isothermal titration calorimetry studies and structural information on the interactions between proteins and small-molecule ligands is used here to reveal general thermodynamic properties of protein-ligand interactions and to investigate correlations with changes in solvation. The overwhelming majority of interactions are found to be enthalpically favoured. Synthetic inhibitors and biological ligands form two distinct subpopulations in the data, with the former having greater average affinity due to more favourable entropy changes on binding. The greatest correlation is found between the binding free energy and apolar surface burial upon complex formation. However, the free-energy contribution per unit area buried is only 30-50% of that expected from earlier studies of transfer free energies of small molecules. A simple probability-based estimator for the maximal affinity of a binding site in terms of its apolar surface area is proposed. Polar surface area burial also contributes substantially to affinity but is difficult to express in terms of unit area due to the small variation in the amount of polar surface buried and a tendency for cancellation of its enthalpic and entropic contributions. Conventionally, the contribution of apolar desolvation to affinity is attributed to gain of entropy due to solvent release. Although data presented here are supportive of this notion, because the correlation of entropy change with apolar surface burial is relatively weak, it cannot, on present evidence, be confidently considered to be correct. Further, thermodynamic changes arising from small differences between ligands binding to individual proteins are relatively large and, in general, uncorrelated with changes in solvation, suggesting that trends identified across widely differing proteins are of limited use in explaining or predicting the effects of ligand modifications.     

Crystal Structure of Human RNA Helicase A (DHX9): Structural Basis for Unselective Nucleotide Base Binding in a DEAD-Box Variant Protein

RNA helicases of the DExD/H-box superfamily are critically involved in all RNA-related processes. No crystal structures of human DExH-box domains had been determined previously, and their structures were difficult to predict owing to the low level of homology among DExH-motif-containing proteins from diverse species. Here we present the crystal structures of the conserved domain 1 of the DEIH-motif-containing helicase DHX9 and of the DEAD-box helicase DDX20. Both contain a RecA-like core, but DHX9 differs from DEAD-box proteins in the arrangement of secondary structural elements and is more similar to viral helicases such as NS3. The N-terminus of the DHX9 core contains two long α-helices that reside on the surface of the core without contributing to nucleotide binding. The RNA-polymerase-II-interacting minimal transactivation domain sequence forms an extended loop structure that resides in a hydrophobic groove on the surface of the DEIH domain. DHX9 lacks base-selective contacts and forms an unspecific but important stacking interaction with the base of the bound nucleotide, and our biochemical analysis confirms that the protein can hydrolyze ATP, guanosine 5′-triphosphate, cytidine 5′-triphosphate, and uridine 5′-triphosphate. Together, these findings allow the localization of functional motifs within the three-dimensional structure of a human DEIH helicase and show how these enzymes can bind nucleotide with high affinity in the absence of a Q-motif.     

Disparate degrees of hypervariable loop flexibility control T-cell receptor cross-reactivity, specificity, and binding mechanism

αβ T-cell receptors (TCRs) recognize multiple antigenic peptides bound and presented by major histocompatibility complex molecules. TCR cross-reactivity has been attributed in part to the flexibility of TCR complementarity-determining region (CDR) loops, yet there have been limited direct studies of loop dynamics to determine the extent of its role. Here we studied the flexibility of the binding loops of the αβ TCR A6 using crystallographic, spectroscopic, and computational methods. A significant role for flexibility in binding and cross-reactivity was indicated only for the CDR3α and CDR3β hypervariable loops. Examination of the energy landscapes of these two loops indicated that CDR3β possesses a broad, smooth energy landscape, leading to rapid sampling in the free TCR of a range of conformations compatible with different ligands. The landscape for CDR3α is more rugged, resulting in more limited conformational sampling that leads to specificity for a reduced set of peptides as well as the major histocompatibility complex protein. In addition to informing on the mechanisms of cross-reactivity and specificity, the energy landscapes of the two loops indicate a complex mechanism for TCR binding, incorporating elements of both conformational selection and induced fit in a manner that blends features of popular models for TCR recognition.     

Insights into the Catalytic Mechanism of 16S rRNA Methyltransferase RsmE (mU1498) from Crystal and Solution Structures

RsmE is the founding member of a new RNA methyltransferase (MTase) family responsible for methylation of U1498 in 16S ribosomal RNA in Escherichia coli. It is well conserved across bacteria and plants and may play an important role in ribosomal intersubunit communication. The crystal structure in monomer showed that it consists of two distinct but structurally related domains: the PUA (pseudouridine synthases and archaeosine‐specific transglycosylases)-like RNA recognition and binding domain and the conserved MTase domain with a deep trefoil knot. Analysis of small-angle X-ray scattering data revealed that RsmE forms a flexible dimeric conformation that may be essential for substrate binding. The S‐adenosyl‐l‐methionine (AdoMet)-binding characteristic determined by isothermal titration calorimetry suggested that there is only one AdoMet molecule bound in the subunit of the homodimer. In vitro methylation assay of the mutants based on the RsmE-AdoMet-uridylic acid complex model showed key residues involved in substrate binding and catalysis. Comprehensive comparisons of RsmE with closely related MTases, combined with the biochemical experiments, indicated that the MTase domain of one subunit in dimeric RsmE is responsible for binding of one AdoMet molecule and catalytic process while the PUA-like domain in the other subunit is mainly responsible for recognition of one substrate molecule (the ribosomal RNA fragment and ribosomal protein complex). The methylation process is required by collaboration of both subunits, and dimerization is functionally critical for catalysis. In general, our study provides new information on the structure-function relationship of RsmE and thereby suggests a novel catalytic mechanism.     

Structure-based design of a protein immunogen that displays an HIV-1 gp41 neutralizing epitope

Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K_d of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.     

RNA targeting by DNA binding drugs: Structural,conformational and energetic aspects of the binding of quinacrine and DAPI to A-form and H-form of poly(rC)·poly(rG)

A key step in the rational design of new RNA binding small molecules necessitates a complete elucidation of the molecular aspects of the binding of existing molecules to RNA structures. This work focuses towards the understanding of the interaction of a DNA intercalator, quinacrine and a minor groove binder 4′,6-diamidino-2-phenylindole (DAPI) with the right handed Watson–Crick base paired A-form and the left-handed Hoogsteen base paired H^L-form of poly(rC)·poly(rG) evaluated by multifaceted spectroscopic and viscometric techniques. The energetics of their interaction has also been elucidated by isothermal titration calorimetry. Results of this study converge to suggest that (i) quinacrine intercalates to both A-form and H^L-form of poly(rC)·poly(rG); (ii) DAPI shows both intercalative and groove-binding modes to the A-form of the RNA but binds by intercalative mode to the H^L-form. Isothermal calorimetric patterns of quinacrine binding to both the forms of RNA and of DAPI binding to the H^L-form are indicative of single binding while the binding of DAPI to the A-form reveals two kinds of binding. The binding of both the drugs to both conformations of RNA is exothermic; while the binding of quinacrine to both conformations and DAPI to the A-form (first site) is entropy driven, the binding of DAPI to the second site of A-form and H^L-conformation is enthalpy driven. Temperature dependence of the binding enthalpy revealed that the RNA–ligand interaction reactions are accompanied by small heat capacity changes that are nonetheless significant. We conclude that the binding affinity characteristics and energetics of interaction of these DNA binding molecules to the RNA conformations are significantly different and may serve as data for the development of effective structure selective RNA-based antiviral drugs.     

Solution Structure of Histone Chaperone ANP32B: Interaction with Core Histones H3-H4 through Its Acidic Concave Domain

Eukaryotic gene expression is regulated by histone deposition onto and eviction from nucleosomes, which are mediated by several chromatin-modulating factors. Among them, histone chaperones are key factors that facilitate nucleosome assembly. Acidic nuclear phosphoprotein 32B (ANP32B) belongs to the ANP32 family, which shares N-terminal leucine-rich repeats (LRRs) and a C-terminal variable anionic region. The C-terminal region functions as an inhibitor of histone acetylation, but the functional roles of the LRR domain in chromatin regulation have remained elusive. Here, we report that the LRR domain of ANP32B possesses histone chaperone activity and forms a curved structure with a parallel β-sheet on the concave side and mostly helical elements on the convex side. Our analyses revealed that the interaction of ANP32B with the core histones H3-H4 occurs on its concave side, and both the acidic and hydrophobic residues that compose the concave surface are critical for histone binding. These results provide a structural framework for understanding the functional mechanisms of acidic histone chaperones.     

Structure of the Escherichia coli phosphonate binding protein PhnD and rationally optimized phosphonate biosensors

The phnD gene of Escherichia coli encodes the periplasmic binding protein of the phosphonate (Pn) uptake and utilization pathway. We have crystallized and determined structures of E. coli PhnD (EcPhnD) in the absence of ligand and in complex with the environmentally abundant 2-aminoethylphosphonate (2AEP). Similar to other bacterial periplasmic binding proteins, 2AEP binds near the center of mass of EcPhnD in a cleft formed between two lobes. Comparison of the open, unliganded structure with the closed 2AEP-bound structure shows that the two lobes pivot around a hinge by ∼ 70° between the two states. Extensive hydrogen bonding and electrostatic interactions stabilize 2AEP, which binds to EcPhnD with low nanomolar affinity. These structures provide insight into Pn uptake by bacteria and facilitated the rational design of high signal-to-noise Pn biosensors based on both coupled small-molecule dyes and autocatalytic fluorescent proteins.     

Characterisation of the Nucleotide Exchange Factor ITSN1L: Evidence for a Kinetic Discrimination of GEF-Stimulated Nucleotide Release from Cdc42

Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg²⁺ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex.     

Crystal Structure Determination and Functional Characterization of the Metallochaperone SlyD from Thermus thermophilus

SlyD (sensitive to lysis D; product of the slyD gene) is a prolyl isomerase [peptidyl-prolyl cis/trans isomerase (PPIase)] of the FK506 binding protein (FKBP) type with chaperone properties. X-ray structures derived from three different crystal forms reveal that SlyD from Thermus thermophilus consists of two domains representing two functional units. PPIase activity is located in a typical FKBP domain, whereas chaperone function is associated with the autonomously folded insert-in-flap (IF) domain. The two isolated domains are stable and functional in solution, but the presence of the IF domain increases the PPIase catalytic efficiency of the FKBP domain by 2 orders of magnitude, suggesting that the two domains act synergistically to assist the folding of polypeptide chains. The substrate binding surface of SlyD from T. thermophilus was mapped by NMR chemical shift perturbations to hydrophobic residues of the IF domain, which exhibits significantly reduced thermodynamic stability according to NMR hydrogen/deuterium exchange and fluorescence equilibrium transition experiments. Based on structural homologies, we hypothesize that this is due to the absence of a stabilizing β-strand, suggesting in turn a mechanism for chaperone activity by ‘donor-strand complementation.’ Furthermore, we identified a conserved metal (Ni²⁺) binding site at the C-terminal SlyD-specific helical appendix of the FKBP domain, which may play a role in metalloprotein assembly.     

Crystal structure of the GluR0 ligand-binding core from Nostoc punctiforme in complex with L-glutamate: structural dissection of the ligand interaction and subunit interface

GluR0 from Nostoc punctiforme (NpGluR0) is a bacterial homologue of the ionotropic glutamate receptor (iGluR). We have solved the crystal structure of the ligand-binding core of NpGluR0 in complex with l-glutamate at a resolution of 2.1 Å. The structure exhibits a noncanonical ligand interaction and two distinct subunit interfaces. The side-chain guanidium group of Arg80 forms a salt bridge with the γ-carboxyl group of bound l-glutamate: in GluR0 from Synechocystis (SGluR0) and other iGluRs, the equivalent residues are Asn or Thr, which cannot form a similar interaction. We suggest that the local positively charged environment and the steric constraint created by Arg80 mediate the selectivity of l-glutamate binding by preventing the binding of positively charged and hydrophobic amino acids. In addition, the NpGluR0 ligand-binding core forms a new subunit interface in which the two protomers are arranged differently than the known iGluR and SGluR0 dimer interfaces. The significance of there being two different dimer interfaces was investigated using analytical ultracentrifugation analysis.