Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution.
Sec14-superfamily proteins integrate the lipid metabolome with phosphoinositide synthesis and signaling via primed presentation of phosphatidylinositol (PtdIns) to PtdIns kinases. Sec14 action as a PtdIns-presentation scaffold requires heterotypic exchange of phosphatidylcholine (PtdCho) for PtdIns, or vice versa, in a poorly understood progression of regulated conformational transitions. We identify mutations that confer Sec14-like activities to a functionally inert pseudo-Sec14 (Sfh1), which seemingly conserves all of the structural requirements for Sec14 function. Unexpectedly, the "activation" phenotype results from alteration of residues conserved between Sfh1 and Sec14. Using biochemical and biophysical, structural, and computational approaches, we find the activation mechanism reconfigures atomic interactions between amino acid side chains and internal water in an unusual hydrophilic microenvironment within the hydrophobic Sfh1 ligand-binding cavity. These altered dynamics reconstitute a functional "gating module" that propagates conformational energy from within the hydrophobic pocket to the helical unit that gates pocket access. The net effect is enhanced rates of phospholipid-cycling into and out of the Sfh1* hydrophobic pocket. Taken together, the directed evolution approach reveals an unexpectedly flexible functional engineering of a Sec14-like PtdIns transfer protein-an engineering invisible to standard bioinformatic, crystallographic, and rational mutagenesis approaches.
Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA. gabriel.schaaf@zmbp.uni-tuebingen.de
Gabriel
G
Schaaf
Marek
M
Dynowski
Carl J
CJ
Mousley
Sweety D
SD
Shah
Peihua
P
Yuan
Eva M
EM
Winklbauer
Marília K F
MK
de Campos
Kyle
K
Trettin
Mary-Chely
MC
Quinones
Tatyana I
TI
Smirnova
Lora L
LL
Yanagisawa
Eric A
EA
Ortlund
Vytas A
VA
Bankaitis
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D011992:Endosomes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010641:Phenotype; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction; D021601:trans-Golgi Network / Q000378:metabolism
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; C483438:SEC14 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C106163:Sfh1 protein, S cerevisiae
J Magn Reson. 2006 Oct;182(2):229-38
16859937
J Biol Chem. 2006 Nov 17;281(46):34897-908
16997918
Mol Biol Cell. 2007 May;18(5):1928-42
17344474
EMBO J. 1996 Dec 2;15(23):6447-59
8978672
J Cell Biol. 1989 Dec;109(6 Pt 1):2939-50
2687291
J Biol Chem. 1999 May 7;274(19):12990-5
10224048
Mol Cell. 1999 Aug;4(2):187-97
10488334
Trends Biochem Sci. 2010 Mar;35(3):150-60
19926291
J Mol Biol. 2003 Sep 26;332(4):965-77
12972265
Nat Genet. 1995 Feb;9(2):141-5
7719340
Mol Biol Cell. 2002 Jul;13(7):2193-206
12134061
J Biol Chem. 2000 Nov 3;275(44):34293-305
10887188
Nature. 1990 Oct 11;347(6293):561-2
2215682
Nat Rev Mol Cell Biol. 2008 Feb;9(2):99-111
18216767
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Nov 1;62(Pt 11):1156-60
17077504
Nat Genet. 2003 Nov;35(3):264-9
14556008
Annu Rev Biochem. 1998;67:481-507
9759495
EMBO Rep. 2006 Feb;7(2):174-9
16397625
Mol Biol Cell. 2000 Jun;11(6):1989-2005
10848624
Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12346-51
9770489
Nat Chem Biol. 2006 Nov;2(11):576-83
17051233
Mol Cell. 2008 Feb 1;29(2):191-206
18243114
Nature. 1998 Jan 29;391(6666):506-10
9461221
J Cell Sci. 2005 May 15;118(Pt 10):2093-104
15890985
Mol Biol Cell. 2001 Aug;12(8):2396-411
11514624
Traffic. 2005 Dec;6(12):1157-72
16262726
Mol Biol Cell. 1999 Jul;10(7):2235-50
10397762
Traffic. 2010 Sep;11(9):1151-67
20545905
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
Nature. 2005 Dec 1;438(7068):605-11
16319880
Cell. 1991 Feb 22;64(4):789-800
1997207
Biophys J. 2007 May 15;92(10):3686-95
17325006
Protein Sci. 2002 Oct;11(10):2382-92
12237460
Biochim Biophys Acta. 2007 Mar;1771(3):353-404
17382260
J Mol Biol. 2003 Aug 15;331(3):725-34
12899840
J Neurol Sci. 2002 Jun 15;198(1-2):25-9
12039660
EMBO J. 1998 Jul 15;17(14):4004-17
9670016