scholarly journals Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A

Biochemistry ◽  
2017 ◽  
Vol 56 (47) ◽  
pp. 6292-6303 ◽  
Author(s):  
Shelbi L. Christgen ◽  
Weidong Zhu ◽  
Nikhilesh Sanyal ◽  
Bushra Bibi ◽  
John J. Tanner ◽  
...  
Keyword(s):  
Membrane Binding ◽  
Binding Domain ◽  
Proline Utilization ◽  
Proline Utilization A

scholarly journals Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA

2016 ◽  
Vol 36 (6) ◽  
Author(s):  
Benjamin W. Arentson ◽  
Erin L. Hayes ◽  
Weidong Zhu ◽  
Harkewal Singh ◽  
John J. Tanner ◽  
...  
Keyword(s):  
Dna Binding ◽  
Modular Design ◽  
Quaternary Structure ◽  
Lipid Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Oligomeric State ◽  
Proline Utilization ◽  
Functional Switching ◽  
Proline Utilization A

Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) domains that catalyses the two-step oxidation of proline to glutamate. Trifunctional PutAs also have an N-terminal ribbon–helix–helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability. In the present study, we attempt to construct a trifunctional PutA by fusing the RHH domain of Escherichia coli PutA (EcRHH) to the bifunctional Rhodobacter capsulatus PutA (RcPutA) in order to explore the modular design of functional switching in trifunctional PutAs. The EcRHH–RcPutA chimaera retains the catalytic properties of RcPutA while acquiring the oligomeric state, quaternary structure and DNA-binding properties of EcPutA. Furthermore, the EcRHH–RcPutA chimaera exhibits proline-induced lipid association, which is a fundamental characteristic of functional switching. Unexpectedly, RcPutA lipid binding is also activated by proline, which shows for the first time that bifunctional PutAs exhibit a limited form of functional switching. Altogether, these results suggest that the C-terminal domain (CTD), which is conserved by trifunctional PutAs and certain bifunctional PutAs, is essential for functional switching in trifunctional PutAs.

scholarly journals Structure of a Membrane-binding Domain from a Non-enveloped Animal Virus

2006 ◽  
Vol 281 (39) ◽  
pp. 29278-29286 ◽  
Author(s):  
Lenize F. Maia ◽  
Márcia R. Soares ◽  
Ana P. Valente ◽  
Fabio C. L. Almeida ◽  
Andréa C. Oliveira ◽  
...  
Keyword(s):  
Membrane Binding ◽  
Binding Domain ◽  
Animal Virus

scholarly journals A Membrane Binding Domain in the Ste5 Scaffold Synergizes with Gβγ Binding to Control Localization and Signaling in Pheromone Response

2005 ◽  
Vol 20 (1) ◽  
pp. 21-32 ◽  
Author(s):  
Matthew J. Winters ◽  
Rachel E. Lamson ◽  
Hideki Nakanishi ◽  
Aaron M. Neiman ◽  
Peter M. Pryciak
Keyword(s):  
Membrane Binding ◽  
Binding Domain ◽  
Pheromone Response

scholarly journals Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Richard Izrael ◽  
Lívia Marton ◽  
Gergely N. Nagy ◽  
Hajnalka L. Pálinkás ◽  
Nóra Kucsma ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Cell Line ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Membrane Binding ◽  
Binding Domain ◽  
Regulatory Function ◽  
Phosphocholine Cytidylyltransferase ◽  
Phosphatidylcholine Biosynthesis ◽  
Localization Signal

AbstractThe phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the structurally unique pseudo-heterodimer PfCCT protein in a heterologous cellular context using the thermosensitive CCT-mutant CHO-MT58 cell line. We found that a Plasmodium-specific lysine-rich insertion within the catalytic domain of PfCCT acts as a nuclear localization signal and its deletion decreases the nuclear propensity of the protein in the model cell line. We further showed that the putative membrane-binding domain also affected the nuclear localization of the protein. Moreover, activation of phosphatidylcholine biosynthesis by phospholipase C treatment induces the partial nuclear-to-cytoplasmic translocation of PfCCT. We additionally investigated the cellular function of several PfCCT truncated constructs in a CHO-MT58 based rescue assay. In absence of the endogenous CCT activity we observed that truncated constructs lacking the lysine-rich insertion, or the membrane-binding domain provided similar cell survival ratio as the full length PfCCT protein.

scholarly journals Identification of an 11-residue portion of CTP–phosphocholine cytidylyltransferase that is required for enzyme–membrane interactions

1997 ◽  
Vol 325 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Jilin YANG ◽  
Jinxia WANG ◽  
Irene TSEU ◽  
Maciej KULISZEWSKI ◽  
Wensu LEE ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Fusion Proteins ◽  
Membrane Binding ◽  
Lipid Vesicles ◽  
Lipid Binding ◽  
Binding Domain ◽  
Membrane Interactions ◽  
Phosphocholine Cytidylyltransferase ◽  
N Terminus ◽  
L2 Cells

CTP–phosphocholine cytidylyltransferase (CT) is a key regulatory enzyme in the biosynthesis of phosphatidylcholine (PC) in many cells. Enzyme–membrane interactions appear to play an important role in CT activation. A putative membrane-binding domain appears to be located between residues 236 and 293 from the N-terminus. To map the membrane-binding domain more precisely, glutathione S-transferase fusion proteins were prepared that contained deletions of various domains in this putative lipid-binding region. The fusion proteins were assessed for their binding of [3H]PC/oleic acid vesicles. Fusion proteins encompassing residues 267–277 bound to PC/oleic acid vesicles, whereas fragments lacking this region exhibited no specific binding to the lipid vesicles. The membrane-binding characteristics of the CT fusion proteins were also examined using intact lung microsomes. Only fragments encompassing residues 267–277 competed with full-length 125I-labelled CT, expressed in recombinant Sf9 insect cells, for microsomal membrane binding. To investigate the role of this region in PC biosynthesis, A549 and L2 cells were transfected with cDNA for CT mutants under the control of a glucocorticoid-inducible long terminal repeat (LTR) promoter. Induction of CT mutants containing residues 267–277 in transfectants resulted in reduced PC synthesis. The decrease in PC synthesis was accompanied by a shift in endogenous CT activity from the particulate to the soluble fraction. Expression of CT mutants lacking this region in A549 and L2 cells did not affect PC formation and subcellular distribution of CT activity. These results suggest that the CT region located between residues 267 and 277 from the N-terminus is required for the interaction of CT with membranes.

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