Studying aminoglycoside antibiotic binding to HIV-1 TAR RNA by electrospray ionization mass spectrometry

We analyzed the metal-binding properties of human centrin-2 (HsCen-2) and followed the changes in HsCen-2 structure upon metal-binding using micro-electrospray ionization mass spectrometry (ESI-MS). Apo-HsCen-2 is mostly monomeric. The ESI spectra of HsCen-2 show two charge-state distributions, representing two conformations of the protein. HsCen-2 binds four moles calcium/mol protein: one mol of calcium with high affinity, one additional mol of calcium with lower affinity, and two moles of calcium at low affinity sites. HsCen-2 binds four moles of magnesium/mol protein. The conformation giving the lower charge-state HsCen-2 by ESI, binds calcium and magnesium more readily than does the higher charge-state HsCen-2. Both conformations of HsCen-2 bind calcium more readily than magnesium. Calcium was more effective in displacing magnesium bound to HsCen-2 than vice versa. Binding of a peptide from a known binding partner, the xeroderma pigmentosum complementation group protein C (XPC), to apo-HsCen-2, occurs in the presence or the absence of calcium. Near and far-UV CD spectra of HsCen-2 show little difference with addition of calcium or magnesium. Minor changes in secondary structure are noted. Melting curves derived from temperature dependence of molar ellipticity at 222 nm for HsCen-2 show that calcium increases protein stability whereas magnesium does not. ⌬25 HsCen-2 behaves similarly to HsCen-2. We conclude that HsCen-2 binds calcium and magnesium and that calcium modulates HsCen-2 structure and function by increasing its stability without undergoing significant changes in secondary or tertiary

mentioning

confidence: 99%

Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy

Craig

Benson

Bergen

et al. 2006

“…Relative and absolute binding affinities are readily attainable by strategies that rely on the ability of ESI-MS to resolve complexes with very close physicalchemical characteristics, which are not easily discriminated by other established techniques. Competitive binding experiments have been implemented in which multiple ligands are mixed simultaneously with a target substrate to form a distribution of complexes with abundances reflecting the respective binding affinities [33][34][35][36][37]. Rigorous quantitative determinations of dissociation constants (K d s) have been obtained from titration experiments in which the abundance of free versus bound species in solution was monitored by ESI-MS after each addition of ligand [12, 38 -41].…”

mentioning

confidence: 99%

Mapping noncovalent ligand binding to stemloop domains of the HIV-1 packaging signal by tandem mass spectrometry

Turner

Hagan

Kohlway

et al. 2006

The binding modes and structural determinants of the noncovalent complexes formed by aminoglycoside antibiotics with conserved domains of the HIV-1 packaging signal (⌿-RNA) were investigated using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The location of the aminoglycoside binding sites on the different stemloop structures was revealed by characteristic coverage gaps in the ion series obtained by sustained off-resonance irradiation collision induced dissociation (SORI-CID) of the antibiotic-RNA assemblies. The site positions were confirmed using mutants that eliminated salient structural features of the ⌿-RNA domains. The effects of the mutations on the binding properties of the different substrates served to validate the position of the aminoglycoside site on the wild-type structures. Additional information was provided by docking experiments performed on the different aminoglycoside-stemloop complexes. The results have shown that, in the absence of features disrupting the regular A-helix of the double-stranded stem, aminoglycosides tend to bind in an area situated between the upper stem and the loop regions, as demonstrated for stemloop SL3. The presence of a tandem wobbles motif in SL4 modifies the regular geometry of the upper stem, which does not affect the general site location, but greatly increases its solution binding affinity compared with SL3. The platform motif in SL2 locates the binding site in the stem midsection and confers upon this stemloop an intermediate affinity toward aminoglycosides. In SL3 and SL4, the extensive overlap of the antibiotic site with the region used to bind the nucleocapsid (NC) protein provides the basis for a competition mechanism that could explain the aminoglycoside inhibition of the NC·SL3 and NC·SL4 assemblies. In contrast, the minimal overlap between the aminoglycoside and the NC sites in SL2 accounts for the absence of inhibition of the NC·SL2 complex. . The relatively low mutation frequency of this ϳ120 nt stretch of genomic RNA makes the packaging signal a very promising target for the development of new therapeutic strategies to contend with the rapid emergence of drug resistant strains [7,8]. For this reason, steps have been made toward the identification of possible inhibitors that may bind specific ⌿-RNA structures and disrupt their normal functions [9 -11]. In a systematic investigation of classic nucleic acid ligands, we have recently shown that aminoglycosidic antibiotics are not only capable of binding individual domains of ⌿-RNA, but can also inhibit their interactions with NC in a structure-specific fashion [12]. Initial tests to locate the actual binding sites on the different RNA substrates, which is crucial to explain the mechanism of preferential inhibition, were based on the analysis of mutant-ligand complexes performed by electrospray ionization (ESI) [13,14] and Fourier transform mass spectrometry (FTMS) [15,16].The favorable energetics characteristic of ESI enable the analysis of relatively labile noncovalent complex...

“…The conclusions that have been drawn from these discussions are that the bound state is thermodynamically favored in the gas phase for noncovalent complexes dominated by ionic or polar interactions. Conversely, nonpolar or hydrophobic interactions between molecules are favored as dissociated entities in the gas phase [13][14][15]. For this reason, ESI MS is not recommended as the method of choice studying protein/ligand interactions [12,15].…”

mentioning

confidence: 99%

“…Conversely, nonpolar or hydrophobic interactions between molecules are favored as dissociated entities in the gas phase [13][14][15]. For this reason, ESI MS is not recommended as the method of choice studying protein/ligand interactions [12,15]. The specific interactions taking place in this study occur between the negatively charged carboxylic groups of the sialic acids and positive (or partially positive) regions on the toxin's surface.…”

mentioning

confidence: 99%

Electrospray mass spectrometry of neuac oligomers associated with the c fragment of the tetanus toxin

Conway

Whittal

Baldwin

et al. 2006

The Clostridial neurotoxins, botulinum and tetanus, gain entry into motor neurons by binding to the sialic or N-acetylneuraminic acid (NeuAc) residues of gangliosides and specific protein receptors attached to the cell's surface. While the C-fragment of tetanus toxin (TetC) has been identified to be the ganglioside binding domain, remarkably little is known about how this domain discriminates between the structural features of different gangliosides. We have used electrospray ionization mass spectrometry (ESI-MS) to examine the formation of complexes between TetC and carbohydrates containing NeuAc groups to determine how NeuAc residues contribute to ganglioside binding. ESI-MS was used to obtain an estimate of the dissociation constants (K D values) for TetC binding to a number of related NeuAc-containing carbohydrates (sialyllactose and disialyllactose), as well as six (NeuAc) n oligomers (n ϭ 1-6). K D values were found to range between ϳ10 -35 M. The strength of the interactions between the C fragment and (NeuAc) n are consistent with the topography of the targeting domain of tetanus toxin and the nature of its carbohydrate binding sites. These results suggest that the targeting domain of tetanus toxin contains two binding sites that can accommodate NeuAc (or a dimer) and that NeuAc may play an important role in ganglioside binding and molecular recognition, a process critical for normal cell function and one frequently exploited by toxins, bacteria, and viruses to facilitate their entrance into cells. (J Am Soc Mass Spectrom 2006, 17, 967-976)