Conformational Dynamics and Allostery in Pyruvate Kinase

Abstract

Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogendeuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an ␣-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the ␤-strands within the (␤/␣) 8 -barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site.

The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase

“…Our data reveals that removing the lid domain does not alter the folding of the protein or the formation of the tetrameric structure. Thermal shift assays demonstrate that the presence of fructose-1,6-bisphospate decreases the melting temperature of both the wild-type and PK TIM+Reg , which is consistent with the binding of fructose-1,6-bisphosphate inducing an increase in dynamic motion, as previously described (Donovan et al 2016b). Even without the lid domain, analytical ultracentrifugation studies show that PK TIM+Reg forms as a tetramer and small-angle X-ray scattering indicates that the structure is highly likely to be an oblate twisted ring, very similar to that of the wild-type enzyme.…”

The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase

“…Our data reveals that removing the lid domain does not alter the folding of the protein or the formation of the tetrameric structure. Thermal shift assays demonstrate that the presence of fructose-1,6-bisphospate decreases the melting temperature of both the wild-type and PK TIM+Reg , which is consistent with the binding of fructose-1,6-bisphosphate inducing an increase in dynamic motion, as previously described (Donovan et al 2016b). Even without the lid domain, analytical ultracentrifugation studies show that PK TIM+Reg forms as a tetramer and small-angle X-ray scattering indicates that the structure is highly likely to be an oblate twisted ring, very similar to that of the wild-type enzyme.…”

Hydrogen/Deuterium Exchange Measurements May Provide an Incomplete View of Protein Dynamics: a Case Study on Cytochrome c

“…An allosteric mechanism could propagate distal protein destabilization into proximal regions. Similar allosteric signal transmission processes have been demonstrated for other proteins. − …”

“…Two different scenarios could explain the observed HDX pattern. An allosteric mechanism could propagate distal protein destabilization into proximal regions. Similar allosteric signal transmission processes have been demonstrated for other proteins. − Alternatively, the Fe­(II) → Fe­(III) transition may cause destabilization of both coordination bonds, allowing transient rupture of the distal M80–Fe­(III) contact and the proximal Fe­(III)–H18 bond. It is known that the Fe–His bond can be severed, for example, in denaturing solutions − and after photoexcitation, , resulting in a 4-coordinate iron center.…”

Evidence of Allosteric Enzyme Regulation via Changes in Conformational Dynamics: A Hydrogen/Deuterium Exchange Investigation of Dihydrodipicolinate Synthase

“…This study reaffirms the role of HDX MS as a valuable tool for deciphering allosteric aspects of protein regulation. ,− In future work, it will be interesting to further explore the role of enzyme dynamics during turnover and inhibition by complementing HDX MS experiments with MD simulations on substrate diffusion at the water–protein interface and in the protein interior. Earlier conformational sampling simulations focused largely on small molecules such as diatomic gases and water .…”

The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase

“…Our data reveals that removing the lid domain does not alter the folding of the protein or the formation of the tetrameric structure. Thermal shift assays demonstrate that the presence of fructose-1,6-bisphospate decreases the melting temperature of both the wild-type and PK TIM+Reg , which is consistent with the binding of fructose-1,6-bisphosphate inducing an increase in dynamic motion, as previously described (Donovan et al 2016b). Even without the lid domain, analytical ultracentrifugation studies show that PK TIM+Reg forms as a tetramer and small-angle X-ray scattering indicates that the structure is highly likely to be an oblate twisted ring, very similar to that of the wild-type enzyme.…”

Hydrogen/Deuterium Exchange Measurements May Provide an Incomplete View of Protein Dynamics: a Case Study on Cytochrome c

“…An allosteric mechanism could propagate distal protein destabilization into proximal regions. Similar allosteric signal transmission processes have been demonstrated for other proteins. − …”

“…Two different scenarios could explain the observed HDX pattern. An allosteric mechanism could propagate distal protein destabilization into proximal regions. Similar allosteric signal transmission processes have been demonstrated for other proteins. − Alternatively, the Fe­(II) → Fe­(III) transition may cause destabilization of both coordination bonds, allowing transient rupture of the distal M80–Fe­(III) contact and the proximal Fe­(III)–H18 bond. It is known that the Fe–His bond can be severed, for example, in denaturing solutions − and after photoexcitation, , resulting in a 4-coordinate iron center.…”

Evidence of Allosteric Enzyme Regulation via Changes in Conformational Dynamics: A Hydrogen/Deuterium Exchange Investigation of Dihydrodipicolinate Synthase

“…This study reaffirms the role of HDX MS as a valuable tool for deciphering allosteric aspects of protein regulation. ,− In future work, it will be interesting to further explore the role of enzyme dynamics during turnover and inhibition by complementing HDX MS experiments with MD simulations on substrate diffusion at the water–protein interface and in the protein interior. Earlier conformational sampling simulations focused largely on small molecules such as diatomic gases and water .…”

The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase

“…Our data reveals that removing the lid domain does not alter the folding of the protein or the formation of the tetrameric structure. Thermal shift assays demonstrate that the presence of fructose-1,6-bisphospate decreases the melting temperature of both the wild-type and PK TIM+Reg , which is consistent with the binding of fructose-1,6-bisphosphate inducing an increase in dynamic motion, as previously described (Donovan et al 2016b). Even without the lid domain, analytical ultracentrifugation studies show that PK TIM+Reg forms as a tetramer and small-angle X-ray scattering indicates that the structure is highly likely to be an oblate twisted ring, very similar to that of the wild-type enzyme.…”

Hydrogen/Deuterium Exchange Measurements May Provide an Incomplete View of Protein Dynamics: a Case Study on Cytochrome c

“…An allosteric mechanism could propagate distal protein destabilization into proximal regions. Similar allosteric signal transmission processes have been demonstrated for other proteins. − …”

“…Two different scenarios could explain the observed HDX pattern. An allosteric mechanism could propagate distal protein destabilization into proximal regions. Similar allosteric signal transmission processes have been demonstrated for other proteins. − Alternatively, the Fe­(II) → Fe­(III) transition may cause destabilization of both coordination bonds, allowing transient rupture of the distal M80–Fe­(III) contact and the proximal Fe­(III)–H18 bond. It is known that the Fe–His bond can be severed, for example, in denaturing solutions − and after photoexcitation, , resulting in a 4-coordinate iron center.…”

Evidence of Allosteric Enzyme Regulation via Changes in Conformational Dynamics: A Hydrogen/Deuterium Exchange Investigation of Dihydrodipicolinate Synthase

“…This study reaffirms the role of HDX MS as a valuable tool for deciphering allosteric aspects of protein regulation. ,− In future work, it will be interesting to further explore the role of enzyme dynamics during turnover and inhibition by complementing HDX MS experiments with MD simulations on substrate diffusion at the water–protein interface and in the protein interior. Earlier conformational sampling simulations focused largely on small molecules such as diatomic gases and water .…”