Elucidating the site of protein-ATP binding by top-down mass spectrometry

Abstract

A Fourier-transform ion cyclotron resonance (FT-ICR) top-down mass spectrometry strategy for determining the adenosine triphosphate (ATP)-binding site on chicken adenylate kinase is described. Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), but the ability to detect protein-ligand complexes depends on their stability in the gas phase. Previously, we showed that collisionally activated dissociation (CAD) of protein-nucleotide triphosphate complexes yield products from the dissociation of a covalent phosphate bond of the nucleotide with subsequent release of the nucleotide monophosphate (Yin, S. et al., J. Am. Soc. Mass Spectrom. 2008, 19, 1199 -1208. The intrinsic stability of electrostatic interactions in the gas phase allows the diphosphate group to remain noncovalently bound to the protein. This feature is exploited to yield positional information on the site of ATP-binding on adenylate kinase. CAD and electron capture dissociation (ECD) of the adenylate kinase-ATP complex generate product ions bearing monoand diphosphate groups from regions previously suggested as the ATP-binding pocket by NMR and crystallographic techniques. Top-down MS may be a viable tool to determine the ATP-binding sites on protein kinases and identify previously unknown protein kinases in a functional proteomics study. (J Am Soc Mass Spectrom 2010, 21, 899 -907) © 2010 American Society for Mass Spectrometry P roteins function in biology through direct interaction with other molecules. A ligand can be a molecule, an atom, or an ion that binds to a specific site on the protein by a variety of means, including hydrogen bonding, Van der Waals interactions, and ionic forces. Determining the presence of protein-ligand interactions and the structures of proteinligand complexes are of critical importance in biology, and this knowledge is often essential for efforts to establish new molecular drug targets and to develop more potent medicines.A number of biophysical tools are available to characterize the interaction between a protein and its ligand(s). However, mass spectrometry (MS) offers potential advantages in sensitivity, specificity, and speed, especially compared with high-resolution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. With electrospray ionization (ESI) to ionize macromolecules without disrupting covalent bonds while maintaining weak noncovalent interactions, the ESI-MS molecular mass measurement provides direct evidence for protein-ligand associations. The proteinligand interactions are often sufficiently retained upon the transition from solution to the gas phase that the complex size and binding stoichiometry can be measured [1][2][3].Although binding stoichiometry and, in many examples, the relative and absolute solution binding affinities can be measured by ESI-MS methodologies, determining the precise ligand binding site on a target protein is not easily tractable using MS directly. Tandem mass spectrometry (MS/MS) is widely applied...

Molecular Basis for Preventing α-Synuclein Aggregation by a Molecular Tweezer

“…ECD-MS/MS of protein: ligand complexes yields fragments or product ions of the protein chain, some of which are still bound to the ligand. The sequence of these ligand-bound fragments can be mapped onto the full-length protein sequence to determine the ligand binding region(s) (15)(16)(17). This strategy was used previously to measure the binding of CLR01 to amyloid ␤-protein (18) and therefore we applied ESI-MS/MS with ECD here to determine the sites of CLR01 binding to ␣-synuclein.…”

Ligand Migration in the Gaseous Insulin-CB7 Complex—A Cautionary Tale About the Use of ECD-MS for Ligand Binding Site Determination

“…Several studies have reported the use of ESI for transferring noncovalent complexes from solution into the gas phase for interrogation by ECD [22][23][24][25][26][27]. However, structural analysis using gas-phase dissociation techniques such as ECD can only provide accurate information about the solution binding sites if the gas-phase structure does not undergo significant structural changes.…”

“…Moreover, ECD has been shown in many cases to preferentially cleave the protein backbone without disruption of noncovalent interactions [20,21]. This technique has been successfully applied in the localization of binding sites for several noncovalent complexes [22][23][24][25][26][27]. In each of these studies, the gas-phase results have matched closely with previous solution-phase studies on the complexes, thus the use of ECD for structural determination of non-covalent complexes has shown great promise.…”

Influence of Sulfolane on ESI-MS Measurements of Protein–Ligand Affinities

“…With this approach, the identification of peptide fragments retaining the ligand serves to localize the binding site(s). To date, binding sites of several protein-ligand complexes [11][12][13][14][15][16][17], including α-synuclein-spermine [15], adenylate kinase-ATP [16], and anterior gradient-2-PTTIYY hexapeptide interactions [17], were identified using this approach.…”