Relief of autoinhibition by conformational switch explains enzyme activation by a catalytically dead paralog

Volkov, Oleg A.; Kinch, Lisa N.; Ariagno, Carson; Deng, Xingming; Zhong, Shihua; Grishin, Nick V.; Tomchick, Diana R.; Chen, James Z.; Phillips, Margaret A.

doi:10.7554/elife.20198

Cited by 20 publications

(31 citation statements)

References 59 publications

(86 reference statements)

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“…Solving the structures of enzyme-pseudoenzyme complexes may assist in providing mechanistic insights. However, given the structural near-identity of pseudoenzymes and their enzyme partners, it seems likely that the statistical disorder exhibited by the crystals described here may be a common problem and could explain the dearth of crystal structures of pseudoenzymes: the PDB contains structures for only three different proteins described as pseudoenzymes (PDB entries 1rxh, 1rxj, 1rxk, 2og4, 5tvm and 5tvf;Eisenberg-Domovich et al, 2004;Pena et al, 2007;Volkov et al, 2016). The first three are of an engineered pseudoenzyme alone (streptavidin), the next is a pre-mRNA-splicing factor and the last two are of an enzymepseudoenzyme complex but do not exhibit statistical disorder.…”

Section: Discussionmentioning

confidence: 96%

Crystal structure of the pseudoenzyme PDX1.2 in complex with its cognate enzyme PDX1.3: a total eclipse

Robinson

Kaufmann

Roux

et al. 2019

Acta Cryst Sect D Struct Biol

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Pseudoenzymes have burst into the limelight recently as they provide another dimension to regulation of cellular protein activity. In the eudicot plant lineage, the pseudoenzyme PDX1.2 and its cognate enzyme PDX1.3 interact to regulate vitamin B6 biosynthesis. This partnership is important for plant fitness during environmental stress, in particular heat stress. PDX1.2 increases the catalytic activity of PDX1.3, with an overall increase in vitamin B6 biosynthesis. However, the mechanism by which this is achieved is not known. In this study, the Arabidopsis thaliana PDX1.2–PDX1.3 complex was crystallized in the absence and presence of ligands, and attempts were made to solve the X‐ray structures. Three PDX1.2–PDX1.3 complex structures are presented: the PDX1.2–PDX1.3 complex as isolated, PDX1.2–PDX1.3‐intermediate (in the presence of substrates) and a catalytically inactive complex, PDX1.2–PDX1.3‐K97A. Data were also collected from a crystal of a selenomethionine‐substituted complex, PDX1.2–PDX1.3‐SeMet. In all cases the protein complexes assemble as dodecamers, similar to the recently reported individual PDX1.3 homomer. Intriguingly, the crystals of the protein complex are statistically disordered owing to the high degree of structural similarity of the individual PDX1 proteins, such that the resulting configuration is a composite of both proteins. Despite the differential methionine content, selenomethionine substitution of the PDX1.2–PDX1.3 complex did not resolve the problem. Furthermore, a comparison of the catalytically competent complex with a noncatalytic complex did not facilitate the resolution of the individual proteins. Interestingly, another catalytic lysine in PDX1.3 (Lys165) that pivots between the two active sites in PDX1 (P1 and P2), and the corresponding glutamine (Gln169) in PDX1.2, point towards P1, which is distinctive to the initial priming for catalytic action. This state was previously only observed upon trapping PDX1.3 in a catalytically operational state, as Lys165 points towards P2 in the resting state. Overall, the study shows that the integration of PDX1.2 into a heteromeric dodecamer assembly with PDX1.3 does not cause a major structural deviation from the overall architecture of the homomeric complex. Nonetheless, the structure of the PDX1.2–PDX1.3 complex highlights enhanced flexibility in key catalytic regions for the initial steps of vitamin B6 biosynthesis. This report highlights what may be an intrinsic limitation of X‐ray crystallography in the structural investigation of pseudoenzymes.

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Section: Discussionmentioning

confidence: 96%

Crystal structure of the pseudoenzyme PDX1.2 in complex with its cognate enzyme PDX1.3: a total eclipse

Robinson

Kaufmann

Roux

et al. 2019

Acta Cryst Sect D Struct Biol

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“…Our recently reported crystal structure of Tb AdoMetDC/prozyme heterodimer (PDB ID: 5TVM and 5TVF) 26 will greatly facilitate hit-to-lead expansion. The molecular space around the identified scaffolds can be efficiently explored through in silico ligand docking.…”

Section: Discussionmentioning

confidence: 99%

“…Heterodimerization of T. brucei AdoMetDC with prozyme leads to greater than 1000-fold activation, resulting in catalytic efficiency similar to that of the homodimeric mammalian enzymes. This activation results from a large conformational change that leads to displacement of an autoinhibitory peptide and stabilization of the active conformation by insertion of the N-terminus of AdoMetDC into the Tb AdoMetDC/prozyme dimer interface 26 . This unique regulatory mechanism, as well as amino acid residue differences in the active site between human and T. brucei AdoMetDC suggests that species-selective trypanosomatid AdoMetDC inhibitors can be identified, strengthening the value of the target.…”

Section: Introductionmentioning

confidence: 99%

Identification of Trypanosoma brucei AdoMetDC Inhibitors Using a High-Throughput Mass Spectrometry-Based Assay

Volkov¹,

Cosner²,

Brockway³

et al. 2017

ACS Infect. Dis.

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Human African trypanosomiasis (HAT) is a fatal infectious disease caused by the eukaryotic pathogen Trypanosoma brucei. Available treatments are difficult to administer and have significant safety issues. S-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the parasite polyamine biosynthetic pathway. Previous attempts to develop TbAdoMetDC inhibitors into anti-HAT therapies failed due to poor brain exposure. Here, we describe a large screening campaign of two small-molecule libraries (∼400,000 compounds) employing a new high-throughput (∼7 s per sample) mass spectrometry-based assay for AdoMetDC activity. As a result of primary screening, followed by hit confirmation and validation, we identified 13 new classes of reversible TbAdoMetDC inhibitors with low-micromolar potency (IC50) against both TbAdoMetDC and T. brucei parasite cells. The majority of these compounds were >10-fold selective against the human enzyme. Importantly, compounds from four classes demonstrated high propensity to cross the blood-brain barrier in a cell monolayer assay. Biochemical analysis demonstrated that compounds from eight classes inhibited intracellular TbAdoMetDC in the parasite, though evidence for a secondary off-target component was also present. The discovery of several new TbAdoMetDC inhibitor chemotypes provides new hits for lead optimization programs aimed to deliver a novel treatment for HAT.

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“…brucei cell viability [ 9 ]. Monomeric Tb AdoMetDC is inactive due to autoinhibition by its N-terminus [ 24 ]. Upon heterodimerization with prozyme, the N-terminal α-helix repositions to the heterodimer interface, relieving the autoinhibition and generating the active enzyme.…”

Section: Introductionmentioning

confidence: 99%

A dual regulatory circuit consisting of S-adenosylmethionine decarboxylase protein and its reaction product controls expression of the paralogous activator prozyme in Trypanosoma brucei

Patel¹,

Volkov²,

Leija³

et al. 2018

PLoS Pathog

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Polyamines are essential for cell growth of eukaryotes including the etiologic agent of human African trypanosomiasis (HAT), Trypanosoma brucei. In trypanosomatids, a key enzyme in the polyamine biosynthetic pathway, S-adenosylmethionine decarboxylase (TbAdoMetDC) heterodimerizes with a unique catalytically-dead paralog called prozyme to form the active enzyme complex. In higher eukaryotes, polyamine metabolism is subject to tight feedback regulation by spermidine-dependent mechanisms that are absent in trypanosomatids. Instead, in T. brucei an alternative regulatory strategy based on TbAdoMetDC prozyme has evolved. We previously demonstrated that prozyme protein levels increase in response to loss of TbAdoMetDC activity. Herein, we show that prozyme levels are under translational control by monitoring incorporation of deuterated leucine into nascent prozyme protein. We furthermore identify pathway factors that regulate prozyme mRNA translation. We find evidence for a regulatory feedback mechanism in which TbAdoMetDC protein and decarboxylated AdoMet (dcAdoMet) act as suppressors of prozyme translation. In TbAdoMetDC null cells expressing the human AdoMetDC enzyme, prozyme levels are constitutively upregulated. Wild-type prozyme levels are restored by complementation with either TbAdoMetDC or an active site mutant, suggesting that TbAdoMetDC possesses an enzyme activity-independent function that inhibits prozyme translation. Depletion of dcAdoMet pools by three independent strategies: inhibition/knockdown of TbAdoMetDC, knockdown of AdoMet synthase, or methionine starvation, each cause prozyme upregulation, providing independent evidence that dcAdoMet functions as a metabolic signal for regulation of the polyamine pathway in T. brucei. These findings highlight a potential regulatory paradigm employing enzymes and pseudoenzymes that may have broad implications in biology.

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Relief of autoinhibition by conformational switch explains enzyme activation by a catalytically dead paralog

Cited by 20 publications

References 59 publications

Crystal structure of the pseudoenzyme PDX1.2 in complex with its cognate enzyme PDX1.3: a total eclipse

Crystal structure of the pseudoenzyme PDX1.2 in complex with its cognate enzyme PDX1.3: a total eclipse

Identification of Trypanosoma brucei AdoMetDC Inhibitors Using a High-Throughput Mass Spectrometry-Based Assay

A dual regulatory circuit consisting of S-adenosylmethionine decarboxylase protein and its reaction product controls expression of the paralogous activator prozyme in Trypanosoma brucei

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