Dataset Information

Opportunistic complexes of E. coli L-asparaginases with citrate anions.

ABSTRACT: Active sites of enzymes are highly optimized for interactions with specific substrates, thus binding of opportunistic ligands is usually observed only in the absence of native substrates or products. However, during growth of crystals required for structure determination enzymes are often exposed to conditions significantly divergent from the native ones, leading to binding of unexpected ligands to active sites even in the presence of substrates. Failing to recognize this possibility may lead to incorrect interpretation of experimental results and to faulty conclusions. Here, we present several examples of binding of a citrate anion to the active sites of E. coli L-asparaginases I and II, even in the presence of the native substrate, L-Asn. A part of this report focuses on a comprehensive re-interpretation of structural results published previously for complexes of type I L-asparaginase (EcAI) from E. coli. In two re-refined structures a citrate anion forms an acyl-enzyme reaction intermediate with the catalytic threonine. These results emphasize the importance of careful and critical analysis during interpretation of crystallographic data.

SUBMITTER: Lubkowski J

PROVIDER: S-EPMC6667453 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Opportunistic complexes of E. coli L-asparaginases with citrate anions.

Lubkowski Jacek J Chan Waikin W Wlodawer Alexander A

Scientific reports 20190730 1

Active sites of enzymes are highly optimized for interactions with specific substrates, thus binding of opportunistic ligands is usually observed only in the absence of native substrates or products. However, during growth of crystals required for structure determination enzymes are often exposed to conditions significantly divergent from the native ones, leading to binding of unexpected ligands to active sites even in the presence of substrates. Failing to recognize this possibility may lead to ...[more]

PMID: 31363102

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Project description:The combination of ion mobility separation with mass spectrometry is an emergent and powerful structural biology tool, capable of simultaneously assessing the structure, topology, dynamics, and composition of large protein assemblies within complex mixtures. An integral part of the ion mobility-mass spectrometry measurement is the ionization of intact multiprotein complexes and their removal from bulk solvent. This process, during which a substantial portion of protein structure and organization is likely to be preserved, imposes a foreign environment on proteins that may cause structural rearrangements to occur. Thus, a general means must be identified to stabilize protein structures in the absence of bulk solvent. Our approach to this problem involves the protection of protein complex structure through the addition of salts in solution prior to desorption/ionization. Anionic components of the added salts bind to the complex either in solution or during the electrospray process, and those that remain bound in the gas phase tend to have high gas phase acidities. The resulting 'shell' of counterions is able to carry away excess energy from the protein complex ion upon activation and can result in significant structural stabilization of the gas-phase protein assembly overall. By using ion mobility-mass spectrometry, we observe both the dissociation and unfolding transitions for four tetrameric protein complexes bound to populations of 12 different anions using collisional activation. The data presented here quantifies, for the first time, the influence of a large range of counterions on gas-phase protein structure and allows us to rank and classify counterions as structure stabilizers in the absence of bulk solvent. Our measurements indicate that tartrate, citrate, chloride, and nitrate anions are among the strongest stabilizers of gas-phase protein structure identified in this screen. The rank order determined by our data is substantially different when compared to the known Hofmeister salt series in solution. While this is an expected outcome of our work, due to the diminished influence of anion and protein solvation by water, our data correlates well to expected anion binding in solution and highlights the fact that both hydration layer and anion-protein binding effects are critical for Hofmeister-type stabilization in solution. Finally, we present a detailed mechanism of action for counterion stabilization of proteins and their complexes in the gas-phase, which indicates that anions must bind with high affinity, but must dissociate readily from the protein in order to be an effective stabilizer. Anion-resolved data acquired for smaller protein systems allows us to classify anions into three categories based on their ability to stabilize protein and protein complex structure in the absence of bulk solvent.

Dataset Information

Opportunistic complexes of E. coli L-asparaginases with citrate anions.

Publications

Opportunistic complexes of E. coli L-asparaginases with citrate anions.

Similar Datasets

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