Engin H. Serpersu

Orientation of Heparin-binding Sites in Native Vitronectin ANALYSES OF LIGAND BINDING TO THE PRIMARY GLYCOSAMINOGLYCAN-BINDING SITE INDICATE THAT PUTATIVE SECONDARY SITES ARE NOT FUNCTIONAL

A primary heparin-binding site in vitronectin has been localized to a cluster of cationic residues near the C terminus of the protein. More recently, secondary binding sites have been proposed. In order to investigate whether the binding site originally identified on vitronectin functions as an exclusive and independent heparin-binding domain, solution binding methods have been used in combination with NMR and recombinant approaches to evaluate ligand binding to the primary site. Evaluation of the ionic strength dependence of heparin binding to vitronectin according to classical linkage theory indicates that a single ionic bond is prominent. It had been previously shown that chemical modification of vitronectin using an arginine-reactive probe results in a significant reduction in heparin binding (Gibson, A., Baburaj, K., Day, D. E., Verhamme, I., Shore, J. D., and Peterson, C. B. (1997) J. Biol. Chem. 272, 5112–5121). The label has now been localized to arginine residues within the cyanogen bromide fragment-(341–380) that contains the primary heparin-binding site on vitronectin. One- and two-dimensional NMR on model peptides based on this primary heparin-binding site indicate that an arginine residue participates in the ionic interaction and that other nonionic interactions may be involved in forming a complex with heparin. A recombinant polypeptide corresponding to the C-terminal 129 amino acids of vitronectin exhibits heparin-binding affinity that is comparable to that of full-length vitronectin and is equally effective at neutralizing heparin anticoagulant activity. Results from this broad experimental approach argue that the behavior of the primary site is sufficient to account for the heparin binding activity of vitronectin and support an exposed orientation for the site in the structure of the native protein.

Metallohistins: A New Class of Plant Metal-Binding Proteins

Two small multimeric histidine-rich proteins, AgNt84 and Ag164, encoded by two nodule-specific cDNAs isolated from nodule cDNA libraries of the actinorhizal host plant Alnus glutinosa, represent a new class of plant metal binding proteins. This paper reports the characterization of the purified in vitro-expressed proteins by size exclusion chromatography, circular dichroism, equilibrium dialysis, metal affinity chromatography coupled with mass spectrometry, and nuclear magnetic resonance spectroscopy. These analyses reveal that each polypeptide is capable of binding multiple atoms of Zn2+, Ni2+, Co2+, Cu2+, Cd2+ and Hg2+. A reversible shift in histidine Cε1 and Cδ2 protons in NMR analysis occurred during titration of this protein with ZnCl2 strongly suggesting that histidine residues are responsible for metal binding. AgNt84 and Ag164 are not related to metal binding metallothioneins and phytochelatins and represent a new class of plant metal binding proteins that we propose to call metallohistins. Possible biological roles in symbioses for AgNt84 and Ag164, and their potential for use in bioremediation are discussed.

Assignment of the Four Disulfides in the N-terminal Somatomedin B Domain of Native Vitronectin Isolated from Human Plasma

The primary sequence of the N-terminal somatomedin B (SMB) domain of native vitronectin contains 44 amino acids, including a framework of four disulfide bonds formed by 8 closely spaced cysteines in sequence patterns similar to those found in the cystine knot family of proteins. The SMB domain of vitronectin was isolated by digesting the protein with endoproteinase Glu-C and purifying the N-terminal 1–55 peptide by reverse-phase high performance liquid chromatography. Through a combination of techniques, including stepwise reduction and alkylation at acidic pH, peptide mapping with matrix-assisted laser desorption ionization mass spectrometry and NMR, the disulfide bonds contained in the SMB domain have been determined to be Cys5:Cys9, Cys19:Cys31, Cys21:Cys32, and Cys25:Cys39. This pattern of disulfides differs from two other connectivities that have been reported previously for recombinant forms of the SMB domain expressed in Escherichia coli. This arrangement of disulfide bonds in the SMB domain from native vitronectin forms a rigid core around the Cys19: Cys31 and Cys21:Cys32 disulfides. A small positively charged loop is created at the N terminus by the Cys5: Cys9 cystine. The most prominent feature of this disulfide-bonding pattern is a loop between Cys25 and Cys39 similar to cystine-stabilized α-helical structures commonly observed in cystine knots. This α-helix has been confirmed in the solution structure determined for this domain using NMR (Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359–29366). It confers function on the SMB domain, comprising the site for binding to plasminogen activator inhibitor type-1 and the urokinase receptor.

Ligand promiscuity through the eyes of the aminoglycoside N3 acetyltransferase IIa

Aminoglycoside‐modifying enzymes (AGMEs) are expressed in many pathogenic bacteria and cause resistance to aminoglycoside (AG) antibiotics. Remarkably, the substrate promiscuity of AGMEs is quite variable. The molecular basis for such ligand promiscuity is largely unknown as there is not an obvious link between amino acid sequence or structure and the antibiotic profiles of AGMEs. To address this issue, this article presents the first kinetic and thermodynamic characterization of one of the least promiscuous AGMEs, the AG N3 acetyltransferase‐IIa (AAC‐IIa) and its comparison to two highly promiscuous AGMEs, the AG N3‐acetyltransferase‐IIIb (AAC‐IIIb) and the AG phosphotransferase(3′)‐IIIa (APH). Despite having similar antibiotic selectivities, AAC‐IIIb and APH catalyze different reactions and share no homology to one another. AAC‐IIa and AAC‐IIIb catalyze the same reaction and are very similar in both amino acid sequence and structure. However, they demonstrate strong differences in their substrate profiles and kinetic and thermodynamic properties. AAC‐IIa and APH are also polar opposites in terms of ligand promiscuity but share no sequence or apparent structural homology. However, they both are highly dynamic and may even contain disordered segments and both adopt well‐defined conformations when AGs are bound. Contrary to this AAC‐IIIb maintains a well‐defined structure even in apo form. Data presented herein suggest that the antibiotic promiscuity of AGMEs may be determined neither by the flexibility of the protein nor the size of the active site cavity alone but strongly modulated or controlled by the effects of the cosubstrate on the dynamic and thermodynamic properties of the enzyme.

Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The (15)N-(1)H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH-enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

Deciphering interactions of the aminoglycoside phosphotransferase(3′)-IIIa with its ligands

Aminoglycoside phosphotransferase(3′)‐IIIa (APH) is the enzyme with broadest substrate range among the phosphotransferases that cause resistance to aminoglycoside antibiotics. In this study, the thermodynamic characterization of interactions of APH with its ligands are done by determining dissociation constants of enzyme–substrate complexes using electron paramagnetic resonance and fluorescence spectroscopy. Metal binding studies showed that three divalent cations bind to the apo‐enzyme with low affinity. In the presence of AMPPCP, binding of the divalent cations occurs with 7‐to‐37‐fold higher affinity to three additional sites dependent on the presence and absence of different aminoglycosides. Surprisingly, when both ligands, AMPPCP and aminoglycoside, are present, the number of high affinity metal binding sites is reduced to two with a 2‐fold increase in binding affinity. The presence of divalent cations, with or without aminoglycoside present, shows only a small effect (<3‐fold) on binding affinity of the nucleotide to the enzyme. The presence of metal–nucleotide, but not nucleotide alone, increases the binding affinity of aminoglycosides to APH. Replacement of magnesium (II) with manganese (II) lowered the catalytic rates significantly while affecting the substrate selectivity of the enzyme such that the aminoglycosides with 2′‐NH2 become better substrates (higher Vmax) than those with 2′‐OH.

Aminoglycoside antibiotics bound to aminoglycoside-detoxifying enzymes and RNA adopt similar conformations.

Conformations of ribostamycin and isepamicin, aminoglycoside antibiotics, bound to an aminoglycoside antibiotic, 3′-phosphotransferase, were determined by transferred nuclear Overhauser effect spectroscopy and molecular modeling. Two major conformers of enzyme-bound ribostamycin, a neomycin-group aminoglyeoside were observed. The 3′- and 5″-OH groups (reactive hydroxyl groups) in the conformers are placed in approximate locations. One of the conformers is similar to the structure of paromomycin bound to a 27-nucleotide piece of ribosomal RNA that represents the A-site of the small ribosomal subunit, where rings A and C are in an orthogonal arrangement.Isepamicin, a kanamycin-group aminoglycoside antibiotic, also showed two major enzyme-bound conformations. Both conformations were similar to those observed for bound isepamicin in the active site of an aminoglycoside(6′)-acetyl transferase-Ii. Conformations of other RNA-bound kanamycin-group aminoglycosides were also similar to the enzyme-bound conformations of isepamicin. These observations suggest that aminoglycosides may adopt similar conformations when bound to RNA and protein targets. This may have significant implications in the design of enzyme inhibitors and/or antibiotics.

Effect of protein dynamics and solvent in ligand recognition by promiscuous aminoglycoside-modifying enzymes.

Abstract This article reviews dynamic and thermodynamic aspects of the interactions between various aminoglycoside-modifying enzymes (AGMEs) and their antibiotic ligands. Such global thermodynamic properties as enthalpy, entropy, Gibbs energy, and binding protonation are compared and discussed in terms of functional-group dependence on antibiotics and the influence of the presence of a cofactor. The change in heat capacity as a result of antibiotic binding and the role of solvent in AGME–antibiotic complexation are described for two of the most promiscuous AGMEs, the aminoglycoside acetyltransferase(3)-IIIb [AAC(3)-IIIb] and the aminoglycoside phosphotransferase(3′)-IIIa [APH(3′)-IIIa]. A surprising and highly significant, temperature- and aminoglycoside-dependent interplay of changes in low-frequency vibrational modes of the protein, and solvent reorganization, on the thermodynamics of ligand–protein interactions and the dynamic properties of the protein is described. Overall, this article sets out to link antibiotic promiscuity with changes in enzyme dynamics and solvent. Despite a wide range of sequence homology, these properties appear to be common among AGMEs thus far studied and may be extrapolated to other promiscuous protein–ligand systems.

Protein structure determination using protein threading and sparse NMR data (extended abstract)

It is well known that the NMR method for protein structure determination applies to small proteins and that its effectiveness decreases very rapidly as the molecular weight increases beyond about 30 kD. We have recently developed a method for protein structure determination that can fully utilize partial NMR data as calculation constraints. The core of the method is a threading algorithm that guarantees to find a globally optimal alignment between a query sequence and a template structure, under distance constraints specified by NMR/NOE data. Our preliminary tests have demonstrated that a small number of NMR/NOE distance restraints can significantly improve threading performance in both fold recognition and threading-alignment accuracy, and can possibly extend threadings scope of applicability from structural homologs to structural analogs. An accurate backbone structure generated by NMR-constrained threading can then provide a significant amount of structural information, equivalent to that provided by the NMR method with many NMR/NOE restraints; and hence can greatly reduce the amount of NMR data typically required for accurate structure determination. Our prelimenary study suggest that a small number of NOE restraints may suffice to determine adequately the all-atom structure when those restraints are incorporated in a procedure combining threading, modeling of loops and sidechains, and molecular dynamics simulation. Potentially, this new technique can expand NMRs capability to larger proteins.

Studies of Enzymes That Cause Resistance to Aminoglycosides Antibiotics

Aminoglycoside antibiotics are highly potent, wide-spectrum bactericidals (1, 2). Bacterial resistance to aminoglycosides, however, is a major problem in the clinical use of aminoglycosides. Enzymatic modification of aminoglycosides is the most frequent resistance mode among several resistance mechanisms employed by resistant pathogens (1,3). Three families of aminoglycoside modifying enzymes, O-phosphotransferases, N-acetyltransferases, and N-nucleotidyltransferases, are known to have more than 50 enzymes (1,3,4). In this chapter, determination of enzymatic activity of a single enzyme from each family in the presence and absence of an inhibitor is described.