Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution

Schaaf, Gabriel; Dynowski, Marek; Mousley, Carl J.; Shah, Sweety D.; Yuan, Peihua; Winklbauer, Eva M.; Campos, Marília Kaphan Freitas de; Trettin, Kyle D.; Quinones, Mary-Chely; Smirnova, Tatyana I.; Yanagisawa, Lora L.; Ortlund, Eric A.; Bankaitis, Vytas A.

doi:10.1091/mbc.e10-11-0903

Cited by 32 publications

(41 citation statements)

References 40 publications

(90 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For example, the Sec14-like PITP, Sfh1, is highly homologous to Sec14. Sfh1 has both PtdInsand PtdCho-binding/transfer activities, as it shares with Sec14 the same PtdIns-binding motif and, more strikingly, the same PtdCho-coordinating substructure that is critical for NPPM binding ( 9,13,22 ). Yet, Sfh1 lipid exchange activities are impervious to NPPM challenge ( 14 ).…”

Section: Protein Purifi Cationmentioning

confidence: 99%

“…This was done because Q 204 A endows Sfh1 the desirable properties of stimulated lipid exchange activity in vitro and enhanced Sec14-like properties in vivo ( 14,22 ). Those features made it an ideal experimental scaffold for manipulating, and subsequently monitoring, the NPPM-resistance/sensitivity properties of Sfh1 and its variants.…”

Section: Docking Simulationsmentioning

confidence: 99%

See 1 more Smart Citation

Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors

Khan

McGrath

Dorosheva

et al. 2016

Journal of Lipid Research

Self Cite

View full text Add to dashboard Cite

This article is available online at http://www.jlr.org Phosphoinositides (PIPs) are phosphorylated derivatives of phosphatidylinositol (PtdIns), and the metabolism of these lipids constitutes a major membrane-associated signaling system in eukaryotic cells (1)(2)(3)(4)(5). The chemical heterogeneity that distinguishes individual PIP species forms one basis for functionally compartmentalizing signaling platform identities on membrane surfaces ( 6, 7 ). Yet while the chemical heterogeneity of PIP species is simple, it translates to an enormous diversity of biological outcomes that derive from PIP signaling. In that regard, recent studies demonstrate additional layers of functional specifi cation for PIP signaling that are of such resolution that production of an individual PIP species by a specifi c PtdIns kinase yields multiple biological outcomes in the same cell ( 8 ). We now appreciate that PtdIns transfer proteins (PITPs) play critical roles in functional compartmentalization of PIP signaling reactions by channeling PtdIns to PtdIns kinases and, subsequently, to distinct sets of effector proteins ( 9-11 ). The Sec14-like PITPs are best studied in this regard, and the multiplicity of Sec14-like PITPs expressed in even simple unicellular eukaryotes highlights the high degree of functional specifi cation for these proteins ( 12,13 ).Emerging evidence that PITPs instruct the biological outcomes of PtdIns kinase activities recommends these proteins as novel targets for chemical intervention with PIP signaling pathways in cells ( 11,14 ). The advantages of targeting PITPs for this purpose are that such interventions can be imposed with selectivities superior to those possible by popular strategies that either target individual PtdIns-kinase isoforms or individual PIP species ( 15,16 ). Proof of concept is exemplifi ed by the identifi cation and validation of several classes of small molecule inhibitors (SMIs) that target Sec14, the major PITP of yeast. Abbreviations: MM-GBSA, molecular mechanics with generalized born and surface area; NPPM, nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanone; PDB, Protein Data Bank; PIP, phosphoinositide; PITP, phosphatidylinositol transfer protein; PtdCho, phosphatidylcholine; PtdIns, phosphatidylinositol; SMI, small molecule inhibitor .

show abstract

Section: Protein Purifi Cationmentioning

confidence: 99%

Section: Docking Simulationsmentioning

confidence: 99%

Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors

Khan

McGrath

Dorosheva

et al. 2016

Journal of Lipid Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…Indeed, the closer one examines the Sec14 conformational dynamics associated with lipid exchange, the more interesting these become. Directed evolution experiments indicate that G-module conformational transitions regulate the kinetics of gating of the hydrophobic pocket for lipid exchange, and that activation of the G-module responds to water organization and dynamics within the lipid binding pocket itself (48). …”

Section: The Anatomy Of the Sec14 Phospholipid Exchange Cyclementioning

confidence: 99%

“…Although Sec14 has a much higher affinity for PtdIns than for PtdCho, the selectivity for PtdIns vs PtdCho is estimated to be sufficiently small in energetic terms so that H 2 O rearrangements within the hydrophobic pocket are sufficient for negotiating the energy barriers that confront the heterotypic phospholipid exchange cycle. Of particular mechanistic relevance, a functional Sec14 molecule must house both PtdCho and PtdIns binding/exchange capability in order to stimulate production of PtdIns4P in cells (29,48). Those data demonstrate that both homotypic PtdCho- and homotypic PtdIns-exchange reactions are biologically futile activities, and that Sec14-mediated stimulation of PtdIns 4-OH kinase activity requires heterotypic exchange reactions (e.g.…”

Section: Lipid Binding By Sec14-like Proteinsmentioning

confidence: 99%

Phosphatidylinositol transfer proteins and instructive regulation of lipid kinase biology

Grabon

Khan

Bankaitis

2015

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

Self Cite

View full text Add to dashboard Cite

Phosphatidylinositol is a metabolic precursor of phosphoinositides and soluble inositol phosphates. Both sets of molecules represent versatile intracellular chemical signals in eukaryotes. While much effort has been invested in understanding the enzymes that produce and consume these molecules, central aspects for how phosphoinositide production is controlled and functionally partitioned remain unresolved and largely unappreciated. It is in these regards that phosphatidylinositol (PtdIns) transfer proteins (PITPs) are emerging as central regulators of the functional channeling of phosphoinositide pools produced on demand for specific signaling purposes. The physiological significance of these proteins is amply demonstrated by the consequences that accompany deficits in individual PITPs. Although the biological problem is fascinating, and of direct relevance to disease, PITPs remain largely uncharacterized. Herein, we discuss our perspectives regarding what is known about how PITPs work as molecules, and highlight progress in our understanding of how PITPs are integrated into cellular physiology.

show abstract

“…The flux of water into and from the hydrophobic pocket during heterotypic exchange reactions is a major factor in overcoming the differences in Sec14p relative binding affinities for Ptdlns and PtdCho so that heterotypic exchange reactions (Ptdlns for PtdCho or PtdCho for Ptdlns) can actually take place. That Sfh1, a protein that shares 64% sequence identity with Sec14, is functionally distinct to Sec14 is proposed to be attributed to specific reconfigurations in atomic interactions between amino acid side chains and ordered water molecules within the lipid binding cavity (Schaaf et al 2011). Such altered dynamics reconstitutes a functional gating module that communicates conformational energy from within the hydrophobic pocket to the helical structure that gates access to the pocket (Schaaf et al 2011).…”

Section: 6 Ligand Binding By Sec14-like Proteinsmentioning

confidence: 99%

Sec14 Like PITPs Couple Lipid Metabolism with Phosphoinositide Synthesis to Regulate Golgi Functionality

Mousley

Davison

Bankaitis

2012

Subcellular Biochemistry

Self Cite

View full text Add to dashboard Cite

An interface coordinating lipid metabolism with proteins that regulate membrane trafficking is necessary to regulate Golgi morphology and dynamics. Such an interface facilitates the membrane deformations required for vesicularization, forms platforms for protein recruitment and assembly on appropriate sites on a membrane surface and provides lipid co-factors for optimal protein activity in the proper spatio-temporally regulated manner. Importantly, Sec14 and Sec14-like proteins are a unique superfamily of proteins that sense specific aspects of lipid metabolism, employing this information to potentiate phosphoinositide production. Therefore, Sec14 and Sec14 like proteins form central conduits to integrate multiple aspects of lipid metabolism with productive phosphoinositide signaling.

show abstract

Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution

Cited by 32 publications

References 40 publications

Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors

Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors

Phosphatidylinositol transfer proteins and instructive regulation of lipid kinase biology

Sec14 Like PITPs Couple Lipid Metabolism with Phosphoinositide Synthesis to Regulate Golgi Functionality

Contact Info

Product

Resources

About