Differential bonding interactions of inverse agonists of angiotensin II type 1 receptor in stabilizing the inactive state

Although the sartan family of angiotensin II type 1 (AT(1)) receptor blockers (ARBs), which includes valsartan, olmesartan, and losartan, have a common pharmacophore structure, their effectiveness in therapy differs. Although their efficacy may be related to their binding strength, this notion has changed with a better understanding of the molecular mechanism. Therefore, we hypothesized that each ARB differs with regard to its molecular interactions with AT(1) receptor in inducing inverse agonism. Interactions between valsartan and residues Ser(105), Ser(109), and Lys(199) were important for binding. Valsartan is a strong inverse agonist of constitutive inositol phosphate production by the wild-type and N111G mutant receptors. Substituted cysteine accessibility mapping studies indicated that valsartan, but not losartan, which has only weak inverse agonism, may stabilize the N111G receptor in an inactive state upon binding. In addition, the inverse agonism by valsatan was mostly abolished with S105A/S109A/K199Q substitutions in the N111G background. Molecular modeling suggested that Ser(109) and Lys(199) bind to phenyl and tetrazole groups of valsartan, respectively. Ser(105) is a candidate for binding to the carboxyl group of valsartan. Thus, the most critical interaction for inducing inverse agonism involves transmembrane (TM) V (Lys(199)) of AT(1) receptor although its inverse agonist potency is comparable to olmesartan, which bonds with TM III (Tyr(113)) and TM VI (His(256)). These results provide new insights into improving ARBs and development of new G protein-coupled receptor antagonists.     

Exploring new scaffolds for angiotensin II receptor antagonism

Nowadays, AT₁ receptor (AT₁R) antagonists (ARBs) constitute the one of the most prevalent classes of antihypertensive drugs that modulate the renin-angiotensin system (RAS). Their main uses include also treatment of diabetic nephropathy (kidney damage due to diabetes) and congestive heart failure. Towards this direction, our study has been focused on the discovery of novel agents bearing different scaffolds which may evolve as a new class of AT₁ receptor antagonists. To fulfill this aim, a combination of computational approaches and biological assays were implemented. Particularly, a pharmacophore model was established and served as a 3D search query to screen the ChEMBL15 database. The reliability and accuracy of virtual screening results were improved by using molecular docking studies. In total, 4 compounds with completely diverse chemical scaffolds from potential ARBs, were picked and tested for their binding affinity to AT₁ receptor. Results revealed high nanomolar to micromolar affinity (IC₅₀) for all the compounds. Especially, compound 4 exhibited a binding affinity of 199 nM. Molecular dynamics simulations were utilized in an effort to provide a molecular basis of their binding to AT₁R in accordance to their biological activities.     

Analysis of the DNA binding site of Escherichia coli RecA protein

To investigate the DNA binding site of RecA protein, we constructed 15 recA mutants having alterations in the regions homologous to the other ssDNA binding proteins. The in vivo analyses showed that the mutational change at Arg²⁴³, Lys²⁴⁸, Tyr²⁶⁴, or simultaneously at Lys⁶ and Lys¹⁹, or Lys⁶ and Lys²³ caused severe defects in the recA functions, while other mutational changes did not. Purified RecA-K6A-K23A (Lys⁶ and Lys²³ changed to Ala and Ala, respectively) protein was indistinguishable from the wild-type RecA protein in its binding to DNA. However, the RecA-R243A (Arg²⁴³ changed to Ala) and RecA-Y264A (Tyr²⁶⁴ changed to Ala) proteins were defective in binding to both ss- and ds-DNA. In self-oligomerization property, RecA-R243A was proficient but RecA-Y264A was deficient, suggesting that the RecA-R243A protein had a defect in DNA binding site and the RecA-Y264A protein was defective in its interaction with the adjacent RecA molecule. The region of residues 243–257 including the Arg²⁴³ is highly homologous to the DNA binding motif in the ssDNA binding proteins, while the eukaryotic RecA homologues have a similar structure at the amino-terminal side proximal to the nucleotide binding core. The region of residues 243–257 would be a part of the DNA binding site. The other parts of this site would be the Tyr¹⁰³ and the region of residues 178–183, which were cross-linked to ssDNA. These three regions lie in a line in the crystal structure.     

Homology modeling of 3D structure of human chitinase-like protein CHI3L2

The human genome encodes six proteins of family 18 glycosyl hydrolases, two active chitinases and four chitinase-like lectins (chi-lectins) lacking catalytic activity. The present article is dedicated to homology modeling of 3D structure of human chitinase 3-like 2 protein (CHI3L2), which is overexpressed in glial brain tumors, and its structural comparison with homologous chi-lectin CHI3L1. Two crystal structures of CHI3L1 in free state (Protein Data Bank codes 1HJX and 1NWR) were used as structural templates for the homology modeling by Modeller 9.7 program, and the best quality model structure was selected from the obtained model ensemble. Analysis of potential oligosaccharide-binding groove structures of CHI3L1 and CHI3L2 revealed significant differences between these two homologous proteins. 8 of 19 amino acid residues important for ligand binding are substituted in CHI3L2: Tyr³⁴/Asp³⁹, Trp⁶⁹/Lys⁷⁴, Trp⁷¹/Lys⁷⁶, Trp⁹⁹/Tyr¹⁰⁴, Asn¹⁰⁰/Leu¹⁰⁵, Met²⁰⁴/Leu²¹⁰, Tyr²⁰⁶/Phe²¹² and Arg²⁶³/His²⁷¹. The differences between these residues could influence the structure of the ligand-binding groove and substantially change the ability of CHI3L2 to bind oligosaccharide ligands.     

A computer modeling postulated mechanism for angiotensin II receptor activation

The angiotensin II receptor of the AT₁-type has been modeled starting from the experimentally determined three-dimensional structure of bacteriorhodopsin as the template. Intermediate 3D structures of rhodopsin and ₂-adrenergic receptors were built because no direct sequence alignment is possible between the AT₁ receptor and bacteriorhodopsin. Docking calculations were carried out on the complex of the modeled receptor with AII, and the results were used to analyze the binding possibilities of DuP753-type antagonistic non-peptide ligands. We confirm that the positively charged Lys¹⁹⁹ on helix 5 is crucial for ligand binding, as in our model; the charged side chain of this amino acid interacts strongly with the C-terminal carboxyl group of peptide agonists or with the acidic group at the 2-position of the biphenyl moiety of DuP753-type antagonists. Several other receptor residues which are implicated in the binding of ligands and the activation of receptor by agonists are identified, and their functional role is discussed. Therefore, a plausible mechanism of receptor activation is proposed. The three-dimensional docking model integrates most of the available experimental observations and helps to plan pertinent site-directed mutagenesis experiments which in turn may validate or modify the present model and the proposed mechanism of receptor activation.     

Understanding the allosteric trigger for the fructose-1,6-bisphosphate regulation of the ADP-glucose pyrophosphorylase from Escherichia coli

ADP-glucose pyrophosphorylase is the enzyme responsible for the regulation of glycogen synthesis in bacteria. The enzyme N-terminal domain has a Rossmann-like fold with three neighbor loops facing the substrate ATP. In the Escherichia coli enzyme, one of those loops also faces the regulatory site containing Lys³⁹, a residue involved in binding of the allosteric activator fructose-1,6-bisphosphate and its analog pyridoxal-phosphate. The other two loops contain Trp¹¹³ and Gln⁷⁴, respectively, which are highly conserved among all the ADP-glucose pyrophosphorylases. Molecular modeling of the E. coli enzyme showed that binding of ATP correlates with conformational changes of the latter two loops, going from an open to a closed (substrate-bound) form. Alanine mutants of Trp¹¹³ or Gln⁷⁴ did not change apparent affinities for the substrates, but they became insensitive to activation by fructose-1,6-bisphosphate. By capillary electrophoresis we found that the mutant enzymes still bind fructose-1,6-bisphosphate, with similar affinity as the wild type enzyme. Since the mutations did not alter binding of the activator, they must have disrupted the communication between the regulatory and the substrate sites. This agrees with a regulatory mechanism where the interaction with the allosteric activator triggers conformational changes at the level of loops containing residues Trp¹¹³ and Gln⁷⁴.     

Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1–2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNα, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNα were titrated into ¹⁵N-labeled SCR1–2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1–2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn¹¹, Arg¹³, Ala²², Arg²⁸, Ser³², Arg³⁶, Lys⁴¹, Lys⁵⁷, Tyr⁶⁴, Lys⁶⁷, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. With regard to IFNα, the binding is similar to the CR2-C3d interaction with specific residues being Arg¹³, Tyr¹⁶, Arg²⁸, Ser⁴², Lys⁴⁸, Lys⁵⁰, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K_d values of 0.13 and 160 μm, whereas the CR2-gp350 and CR2-IFNα interactions were characterized as single site binding events with affinities of 0.014 and 0.035 μm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.     

AT1 Receptor blockade limits myocardial injury and upregulates AT2 receptors during reperfused myocardial infarction

Persistent left ventricular (LV) dysfunction after reperfused myocardial infarction (RMI) is a significant problem and angiotensin II (AngII) type 1 receptor (AT₁R) blockers (ARBs) may limit reperfusion injury involving upregulation of AngII type 2 receptors (AT₂R). To determine whether ARBs valsartan and irbesartan limit reperfusion injury and upregulate AT₂R protein during RMI, we randomized dogs with anterior RMI (90 min ischemia; 120 min reperfusion) to 4 groups [valsartan (n = 6); irbesartan (n = 9); vehicle controls (n = 8); and sham (n = 6)] and measured serial in vivo hemodynamics, LV systolic and diastolic function, and inhibition of AngII pressor responses to the ARBs, and ex vivo infarct size, and regional AT₁R and AT₂R protein expression at the end of the reperfusion. Compared to the control group, both ARBs significantly limited the increase in left atrial pressure, promptly limited the deterioration of LV dP/dt_max, dP/dt_min, ejection fraction and diastolic function, limited infarct expansion and thinning, and limited infarct size. Importantly, both ARBs increased AT₂R protein in the postischemic reperfused zone, with no change in AT₁R protein. There were no changes in the sham group. The results suggest that limitation of myocardial injury associated with AT₁R blockade combined with upregulation of AT₂R protein expression contributes to the cardioprotective effects of ARBs during RMI. This beneficial effect of ARBs on persistent LV dysfunction after RMI should be evaluated in the clinical setting to determine the relative benefit of ARBs in patients who undergo reperfusion therapy for acute coronary syndromes.     

Molecular mechanism underlying inverse agonist of angiotensin II type 1 receptor

To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT1) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. Binding affinity of olmesartan and its related compounds to wild-type and mutant AT1 receptors demonstrated that interactions between olmesartan and Tyr113, Lys199, His256, and Gln257 in the AT1 receptor were important. The inositol phosphate production assay of olmesartan and related compounds using mutant receptors indicated that the inverse agonist activity required two interactions, that between the hydroxyl group of olmesartan and Tyr113 in the receptor and that between the carboxyl group of olmesartan and Lys199 and His256 in the receptor. Gln257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr113 in TM III and between the carboxyl group and His256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G protein-coupled receptor antagonists with inverse agonist activity.     

Molecular dynamics simulations of GABA binding to the GABAC receptor: the role of Arg104

GABA is the major inhibitory neurotransmitter in the nervous system and acts at a variety of receptors including GABA_C receptors, which are a subclass of GABA_A receptors. Here we have used molecular dynamics simulations of GABA docked into the extracellular domain of the GABA_C receptor to explain the molecular interactions of the neurotransmitter with the residues that contribute to the binding site; in particular, we have explored the interaction of GABA with Arg¹⁰⁴. The simulations suggest that the amine group of GABA forms cation-π interactions with Tyr¹⁰² and Tyr¹⁹⁸, and hydrogen-bonds with Gln⁸³, Glu²²⁰, Ser²⁴³, and Ser¹⁶⁸, and, most prominently, with Arg¹⁰⁴. Substituting Arg¹⁰⁴ with Ala, Glu, or Lys, which experimentally disrupt GABA_C receptor function, and repeating the simulation revealed fewer and different bonding patterns with GABA, or the rapid exit of GABA from the binding pocket. The simulations therefore unveil interactions of GABA within the binding pocket, and explain experimental data, which indicate that Arg¹⁰⁴ is critical for the efficient functioning of the receptor.     

CIDNP study of the aromatic side chain interactions in myotoxina

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxina, aCrotalus venom toxin which acts as blocker of the Ca²⁺ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr₁, His₅, His₁₀, and (possibly) F₁₂. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr, is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr₁, and proton 4 in each of His₅ and His₁₀ are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca²⁺ ATPase.     

CIDNP study of the aromatic side chain interactions in myotoxina

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxina, aCrotalus venom toxin which acts as blocker of the Ca²⁺ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr₁, His₅, His₁₀, and (possibly) F₁₂. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr, is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr₁, and proton 4 in each of His₅ and His₁₀ are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca²⁺ ATPase.     

Interaction between poly(L-lysine48, L-histidine52) and DNA.

Poly(Lys⁴⁸, His⁵²), a random copolypeptide of L -lysine (48%) and L -histidine (52%), was used as a model protein for investigating the effects of protonation on the imidazole group of histidines on protein binding to DNA. The complexes formed between poly(Lys⁴⁸, His⁵²) and DNA were examined using absorbance, circular dichroism (CD), and thermal denaturation. Although increasing pH reduces the charges on histidine side chains in the model protein, the protein still binds the DNA with approximately one positive charge per negative charge in protein-bound regions. Nevertheless, CD and melting properties of poly(Lys⁴⁸, His⁵²)-DNA complexes still depend upon the solution pH which determines the protonation state of imidazole group of histidine side chains. At pH 7.0, the complexes show two characteristic melting bands with a t_m (46–51°C) for free base pairs and a t′_m (94°C) for protein-bound base pairs. The t′_m of the complexes is reduced to 90°C at pH 9.2, although at this pH there is still one lysine per phosphate in protein-bound regions. Presumably, the presence of deprotonated histidine residues destabilizes the native structure of protein-bound DNA. The binding of this model protein to DNA causes a red shift of the crossover point and both a red shift and a reduction of the positive CD band of DNA near 275 nm. This phenomenon is similar to that caused by polylysine binding. These effects, however, are greatly diminished when histidine side chains in the model protein are deprotonated. The structure of already formed poly(Lys⁴⁸, His⁵²)·DNA complexes can be perturbed by changing the solution pH. However, the results suggest a readjustment of the complex to accommodate charge interactions rather than a full dissociation of the complex followed by reassociation between the model protein and DNA.     

Nociceptin and its natural and specifically‐modified fragments: Structural studies

The vibrational structures of Nociceptin (FQ), its short bioactive fragments, and specifically‐modified [Tyr¹]FQ (1‐6), [His¹]FQ (1‐6), and [His^1,4]FQ (1‐6) fragments were characterized. We showed that in the solid state, all of the aforementioned peptides except FQ adopt mainly turn and disordered secondary structures with a small contribution from an antiparallel β‐sheet conformation. FQ (1‐11), FQ (7‐17) [His¹]FQ (1‐6), and [His^1,4]FQ (1‐6) have an α‐helical backbone arrangement that could also slightly influence their secondary structure. The adsorption behavior of these peptides on a colloidal silver surface in an aqueous solution (pH = ～8.3) was investigated by means of surface‐enhanced Raman scattering (SERS). All of the peptides, excluding FQ (7‐17), chemisorbed on the colloidal silver surfaces through a Phe⁴ residue, which for FQ, FQ (1‐11), FQ (1‐6), [Tyr¹]FQ (1‐6), and [His¹]FQ (1‐6) lies almost flat on this surface, while for FQ (1‐13) and FQ (1‐13)NH₂ adopts a slightly tilted orientation with respect to the surface. The Tyr¹ residue in [Tyr¹]FQ (1‐6) does not interact with the colloidal silver surface, suggesting that the Tyr¹ and Phe⁴ side chains are located on the opposite sides of the peptide backbone, which can be also true for His¹ and Phe⁴ in [His¹]FQ (1‐6). The lone pair of electrons on the oxygen atom of the ionized carbonyl group of FQ (1‐13) and FQ (7‐17) appears to be coordinated to the colloidal silver nanoparticles, whereas in the case of the remaining peptides, it only assists in the adsorption process, similar to the ? NH₂ group. We also showed that upon adsorption, the secondary structure of these peptides is altered. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1039–1054, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site

Like the translational elongation factor EF-Tu, RNase P interacts with a large number of substrates where RNase P with its RNA subunit generates tRNAs with matured 5′ termini by cleaving tRNA precursors immediately 5′ of the residue at +1, i.e. at the position that corresponds to the first residue in tRNA. Most tRNAs carry a G₊₁C₊₇₂ base pair at the end of the aminoacyl acceptor-stem whereas in tRNA^Gln G₊₁C₊₇₂ is replaced with U₊₁A₊₇₂. Here, we investigated RNase P RNA-mediated cleavage as a function of having G₊₁C₊₇₂ versus U₊₁A₊₇₂ in various substrate backgrounds, two full-size tRNA precursors (pre-tRNA^Gln and pre-tRNA^TyrSu3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G₊₁C₊₇₂ with U₊₁A₊₇₂ influenced ground state binding, cleavage efficiency under multiple and single turnover conditions in a substrate-dependent manner. Interestingly, we observed differences both in ground state binding and rate of cleavage comparing two full-size tRNA precursors, pre-tRNA^Gln and pre-tRNA^TyrSu3. These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step. In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA. Finally, we provide evidence that the structural architecture influences Mg²⁺ binding, most likely in its vicinity.     

Catalytic properties of thimet oligopeptidase H600A mutant

Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His⁶⁰⁰. In the present work, the role of His⁶⁰⁰ of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S₁ and S₁′ specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His⁶⁰⁰ residue makes important interactions with the substrate, supporting the prediction that His⁶⁰⁰ moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K_m and k_cat, showing the importance of His⁶⁰⁰ for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His⁶⁰⁰ in TOP catalysis, transferring a proton to the newly generated NH₂-terminus or helping Tyr⁶⁰⁵ and/or Tyr⁶¹² in the intermediate oxyanion stabilization.     

The amino acid sequence of human APOA-I, an apolipoprotein isolated from high density lipoproteins.

The complete amino acid sequence of human A-I has been determined by manual and automated Edman degradation of intact and peptide fragments of A-I. A-I is a single chain protein of 243 residues with the following amino acid composition: Asp₁₆, Asn₅, Thr₁₀, Ser₁₅, Glu₂₇, Gln₁₉, Pro₁₀, Gly₁₀, Ala₁₉, Val₁₃, Met₃, Leu₃₇, Tyr₇, Phe₆, Trp₄, Lys₂₁, His₅, and Arg₁₆. The amino acid sequence contains no linear segments of hydrophobic or hydrophilic residues. A detailed correlation of the amino acid sequence, conformation, and self association of A-I will add further insight into the molecular mechanisms involved in protein-protein and protein-lipid interactions.     

Exploring a potential palonosetron allosteric binding site in the 5-HT3 receptor

Palonosetron (Aloxi) is a potent second generation 5-HT₃ receptor antagonist whose mechanism of action is not yet fully understood. Palonosetron acts at the 5-HT₃ receptor binding site but recent computational studies indicated other possible sites of action in the extracellular domain. To test this hypothesis we mutated a series of residues in the 5-HT3A receptor subunit (Tyr⁷³, Phe¹³⁰, Ser¹⁶³, and Asp¹⁶⁵) and in the 5-HT3B receptor subunit (His⁷³, Phe¹³⁰, Glu¹⁷⁰, and Tyr¹⁴³) that were previously predicted by in silico docking studies to interact with palonosetron. Homomeric (5-HT₃A) and heteromeric (5-HT₃AB) receptors were then expressed in HEK293 cells to determine the potency of palonosetron using both fluorimetric and radioligand methods to test function and ligand binding, respectively. The data show that the substitutions have little or no effect on palonosetron inhibition of 5-HT-evoked responses or binding. In contrast, substitutions in the orthosteric binding site abolish palonosetron binding. Overall, the data support a binding site for palonosetron at the classic orthosteric binding pocket between two 5-HT₃A receptor subunits but not at allosteric sites previously identified by in silico modelling and docking.     

Molecular and Conformational Basis of a Specific and High-Affinity Interaction between AlbA and Albicidin Phytotoxin

The albA gene of Klebsiella oxytoca encodes a protein of 221 amino acids that binds the albicidin phytotoxin with a high affinity (dissociation constant = 6.4 × 10⁻⁸ M). For this study, circular dichroism (CD) spectrometry and an alanine scanning mutagenesis approach were used in combination to investigate the molecular and conformational mechanisms of this high-affinity protein-ligand interaction. CD analysis revealed that AlbA contains a high-affinity binding site, and binding of the albicidin ligand to AlbA in a low-ionic-strength environment induced significant conformational changes. The ligand-dependent conformational changes of AlbA were specific and rapid and reached a stable plateau within seconds after the addition of the antibiotic. However, such conformational changes were not detected when AlbA and albicidin were mixed in the high-ionic-strength buffer that is required for maximal binding activity. Based on the conceptual model of protein-ligand interaction, we propose that a threshold ion strength allows AlbA to complete its conformational rearrangement and resume its original stable structure for accommodation of the bound albicidin. Mutagenesis analysis showed that the replacement of Lys¹⁰⁶, Trp¹¹⁰, Tyr¹¹³, Leu¹¹⁴, Tyr¹²⁶, Pro¹³⁴, and Trp¹⁶² with alanine did not change the overall conformational structure of AlbA but decreased the albicidin binding activity about 30 to 60%. We conclude that these residues, together with the previously identified essential residue His¹²⁵, constitute a high-affinity binding pocket for the ligand albicidin. The results also suggest that hydrophobic and electrostatic potentials of these key amino acid residues may play important roles in the AlbA-albicidin interaction.