NMR reveals novel mechanisms of protein activity regulation

Kalodimos, Charalampos G.

doi:10.1002/pro.614

Cited by 45 publications

(57 citation statements)

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“…We investigated the conformational properties of cyclophilin A during the proline isomerization process by using NMR spectroscopy, which can provide atomic-resolution descriptions of the motions of macromolecules in solution (24)(25)(26)(27)(28)(29)(30)(31)(32). In our strategy, NMR data are used as replica-averaged structural restraints in molecular dynamics simulations.…”

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confidence: 99%

Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism

Camilloni

Sahakyan

Holliday

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

103

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Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as "peptidyl-prolyl isomerases" (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simulations, and density functional theory calculations to simultaneously determine the cis-bound and trans-bound conformations of cyclophilin A and its substrate as the enzymatic reaction takes place. We anticipate that this approach will be helpful in elucidating whether the electrostatic handle mechanism that we describe here is common to other PPIases and, more generally, in characterizing other enzymatic processes.enzyme catalysis | NMR spectroscopy D ifferent families of enzymes, often referred to as "peptidylprolyl isomerases" (PPIases), catalyze proline isomerization, a process that involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline (1-3). This isomerization process is an intrinsically slow reaction, typically occurring on the time scale of several minutes under physiological conditions. Hence it often represents a ratelimiting step in biochemical reactions and indeed is used ubiquitously as a molecular switch in regulation (1-7).The possible mechanisms by which PPIases speed up this reaction have been the subject of intense scrutiny (8-16), although consensus descriptions of such mechanisms have not yet emerged. A question of particular relevance is the specific manner in which the electrostatic field in the catalytic site may facilitate the isomerization reaction. To investigate this problem, we considered the case of cyclophilin A, a member of the cyclophilin family of . Previous studies have suggested that conformations resembling those typical of the cis-bound and the trans-bound states are populated through conformational fluctuations in the free state of the enzyme and therefore functional insights into its mechanism of action might be obtained from the study of the free state (21-23).The approach that we followed in studying the mechanism of action of cyclophilin A is based on the simultaneous determination of the structures of the cis-bound and trans-bound states of the complex between the enzyme and its substrate as the catalytic process takes place. Our results re...

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confidence: 99%

Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism

Camilloni

Sahakyan

Holliday

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

103

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“…The signal adapter protein c-CrkII (cellular CT10 regulator of kinase) from chicken contains such a regulatory proline switch [12][13][14][15][16][17][18][19][20]. In c-CrkII, an N-terminal SH2 domain is followed by two SH3 domains (SH3N and SH3C), which are connected by a long linker of about 40 residues ( Fig.…”

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confidence: 99%

Long-Range Energetic Changes Triggered by a Proline Switch in the Signal Adapter Protein c-CrkII

Schmidpeter

Ries

Theer

et al. 2015

Journal of Molecular Biology

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“…1 One of the problems is that it has been extremely challenging to make progress in this context because of the difficulties in obtaining atomic-level information about nonnative states. 6 Cases of protein activity in such states are starting to be uncovered at a pace that, perhaps not by coincidence, corresponds to the development of conceptual and technical methods capable of defining protein states present at low populations. Procedures that combine NMR spectroscopy and molecular simulations methods 9,10 are making it possible to achieve this result and to determine the structures of lowpopulated states.…”

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“…Indeed, a view is gaining momentum according to which the binding events required for regulation can take place in conformations present at low populations in the initial state of the proteinthat is, in excited states. 6,8 Although we are accustomed at thinking about protein activity in terms of structural and dynamical properties of native states, increasing evidence suggests that nonnative states, including excited states and intermediates, do also play a significant role in many cellular processes. 1,3,6-8 It is early to establish just how common is for proteins to be regulated by tuning the populations of their excited states, in a way illustrated clearly in the example reported in this issue of Journal of Molecular Biology.…”

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“…For example, in allosteric regulation, the binding of a ligand in an allosteric site changes the thermodynamic state of a protein, enabling or disabling its function. 6,7 In this type of events, the activity of a protein is carried out from its ground state, and a change in such activity upon regulatory events involves a transition to a new ground state. However, excited states can play a role also in these cases.…”

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