Fluorescence and NMR investigations on the ligand binding properties of adenylate kinases

Reinstein, Jochen; Vetter, Ingrid R.; Schlichting, Ilme; Rösch, Paul; Wittinghofer, Alfred; Goody, Roger S.

doi:10.1021/bi00484a013

Cited by 107 publications

(118 citation statements)

References 45 publications

(66 reference statements)

Supporting

Mentioning

113

Contrasting

Order By: Relevance

“…The ATP ligand and the AK protein are in equilibrium in solution, with a dissociation constant in the micromolar range [34,35,56]. Upon solvent removal to transition into the gas phase, protein conformation could be rapidly converting into "new, non-native structures" [55].…”

Section: Discussionmentioning

confidence: 99%

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

Yin

Loo

2010

J. Am. Soc. Mass Spectrom.

101

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionmentioning

confidence: 99%

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

Yin

Loo

2010

J. Am. Soc. Mass Spectrom.

101

View full text Add to dashboard Cite

show abstract

“…The initial lag period in the rhodanese recovery in ADP pre-hex can be interpreted as the time required for the accumulation of ATP by adenylate kinase. Indeed, in the presence of Ap5A, a potent inhibitor of adenylate kinase (17), rhodanese reactivation disappeared in ADP raw , whereas it was not affected in ATP. The reason for no rhodanese recovery in AMPPNP pre-hex may be explained by the low concentration of contaminated ADP that is not enough for adenyate kinase to produce ATP.…”

Section: Fig 4 Release Of Groes From Thementioning

confidence: 99%

Discrimination of ATP, ADP, and AMPPNP by Chaperonin GroEL

Motojima

Yoshida

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The double ring chaperonin GroEL binds unfolded protein, ATP, and GroES to the same ring, generating the cis ternary complex in which folding occurs within the cavity capped by GroES (cis folding). The functional role of ATP, however, remains unclear since several reports have indicated that ADP and AMPPNP (5 -adenylyl-␤,␥-imidodiphosphate) are also able to support the formation of the cis ternary complex and the cis folding. To minimize the effect of contaminated ATP and adenylate kinase, we have included hexokinase plus glucose in the reaction mixtures and obtained new results. In ADP and AMPPNP, GroES bound quickly to GroEL but bound very slowly to the GroEL loaded with unfolded rhodanese or malate dehydrogenase. ADP was unable to support the formation of cis ternary complex and cis folding. AMPPNP supported cis folding of malate dehydrogenase to some extent but not cis folding of rhodanese. In the absence of hexokinase, apparent cis folding of rhodanese and malate dehydrogenase was observed in ADP and AMPPNP. Thus, the exclusive role of ATP in generation of the cis ternary complex is now evident.The bacterial chaperonin system consisting of GroEL and GroES facilitates folding of other proteins using the energy of ATP hydrolysis. GroEL is composed of 14 identical 57-kDa subunits, each containing a site for binding and hydrolysis of ATP. Seven GroEL subunits are arranged in a heptamer ring forming a central cavity, and two heptamer rings are stacked back to back. GroES is a dome-shaped, single heptamer ring of 10-kDa subunits. GroEL binds a wide range of unfolded proteins at the apical cavity surface and subsequently binds ATP and GroES to the same GroEL ring (the cis ring, a GroEL heptamer ring that binds to GroES), producing the complex consisting of GroEL, unfolded protein, and GroES (the cis ternary complex). Since the residues of the GroEL apical surface involved in GroES binding are mostly overlapped with substrate protein binding (1), GroES binding results in encapsulating unfolded protein into the enlarged cavity of the cis GroEL ring capped by GroES (the cis cavity) (2). The unfolded protein initiates folding in the cis cavity without a risk of aggregation (the cis folding). ATP hydrolysis in the cis ring and subsequent ATP binding to the opposite side of GroEL ring (the trans ring) induce the release of GroES, ADP, and substrate protein (whether folded or not) from the cis ring (3, 4). When unfolded protein is added to the GroEL-GroES complex, it binds to the trans ring, and its folding is arrested (2). Binding and release from the trans ring enable the folding for some proteins (2), especially large ones that are too large to be encapsulated in the cis cavity, by lowering the concentration of aggregation-prone folding intermediates in bulk solution (5). In contrast, the stringent substrate proteins for chaperonin fold efficiently by the cis folding in the presence of ATP (3, 6).However, it should be noted that the functional significance of ATP for the cis ternary complex formation is unclear yet. I...

show abstract

“…Binding between ATP and AK e has previously been quantified with other biophysical techniques 16,29,30 . These independent studies provided K d app values in the range of 35-53 µM in good agreement with our ITC data.…”

mentioning

confidence: 99%

Overlap between folding and functional energy landscapes for adenylate kinase conformational change

2010

View full text Add to dashboard Cite

Enzyme function is often dependent on fluctuations between inactive and active structural ensembles. Adenylate kinase isolated from Escherichia coli (AK e ) is a small phosphotransfer enzyme in which interconversion between inactive (open) and active (closed) conformations is rate limiting for catalysis. AK e has a modular three-dimensional architecture with two flexible substrate-binding domains that interact with the substrates AmP, ADP and ATP. Here, we show by using a combination of biophysical and mutagenic approaches that the interconversion between open and closed states of the ATP-binding subdomain involves partial subdomain unfolding/refolding in an otherwise folded enzyme. These results provide a novel and, possibly general, molecular mechanism for the switch between open and closed conformations in AK e .

show abstract

Fluorescence and NMR investigations on the ligand binding properties of adenylate kinases

Cited by 107 publications

References 45 publications

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

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

Discrimination of ATP, ADP, and AMPPNP by Chaperonin GroEL

Overlap between folding and functional energy landscapes for adenylate kinase conformational change

Contact Info

Product

Resources

About