TY - JOUR
T1 - The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2)
T2 - Structure drives preference for simple neutral glycosphingolipids
AU - Kamlekar, Ravi Kanth
AU - Simanshu, Dhirendra K.
AU - Gao, Yong Guang
AU - Kenoth, Roopa
AU - Pike, Helen M.
AU - Prendergast, Franklyn G.
AU - Malinina, Lucy
AU - Molotkovsky, Julian G.
AU - Venyaminov, Sergei Yu
AU - Patel, Dinshaw J.
AU - Brown, Rhoderick E.
N1 - Funding Information:
We are grateful for the support by NIH/NIGMS GM45928 & GM34847 , NIH/NCI CA121493 , Spanish Ministerio de Ciencia e Innovacion (MICINN BFU2010-17711 ), Russian Foundation for Basic Research 012-04-00168 ; Abby Rockefeller Mauzé Trust , and the Dewitt Wallace, Maloris, Mayo, and Hormel Foundations . The molecular graphic images shown in Figs. 5, 7 , and S1 were produced using UCSF Chimera ( NIH P41 RR-01081 ) provided by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco .
PY - 2013/2
Y1 - 2013/2
N2 - Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL) production using its C-terminal domain to transport newly synthesized glucosylceramide away from the cytosol-facing glucosylceramide synthase in the cis-Golgi for further anabolic processing. Structural homology modeling against human glycolipid transfer protein (GLTP) predicts a GLTP-fold for FAPP2 C-terminal domain, but no experimental support exists to warrant inclusion in the GLTP superfamily. Here, the biophysical properties and glycolipid transfer specificity of FAPP2-C-terminal domain have been characterized and compared with other established GLTP-folds. Experimental evidence for a GLTP-fold includes: i) far-UV circular dichroism (CD) showing secondary structure with high alpha-helix content and a low thermally-induced unfolding transition (∼ 41 C); ii) near-UV-CD indicating only subtle tertiary conformational change before/after interaction with membranes containing/lacking glycolipid; iii) Red-shifted tryptophan (Trp) emission wavelength maximum (λmax ∼ 352 nm) for apo-FAPP2-C-terminal domain consistent with surface exposed intrinsic Trp residues; iv) 'signature' GLTP-fold Trp fluorescence response, i.e., intensity decrease (∼ 30%) accompanied by strongly blue-shifted λmax (∼ 14 nm) upon interaction with membranes containing glycolipid, supporting direct involvement of Trp in glycolipid binding and enabling estimation of partitioning affinities. A structurally-based preference for other simple uncharged GSLs, in addition to glucosylceramide, makes human FAPP2-GLTP more similar to fungal HET-C2 than to plant AtGLTP1 (glucosylceramide-specific) or to broadly GSL-selective human GLTP. These findings along with the distinct mRNA exon/intron organizations originating from single-copy genes on separate human chromosomes suggest adaptive evolutionary divergence by these two GLTP-folds.
AB - Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL) production using its C-terminal domain to transport newly synthesized glucosylceramide away from the cytosol-facing glucosylceramide synthase in the cis-Golgi for further anabolic processing. Structural homology modeling against human glycolipid transfer protein (GLTP) predicts a GLTP-fold for FAPP2 C-terminal domain, but no experimental support exists to warrant inclusion in the GLTP superfamily. Here, the biophysical properties and glycolipid transfer specificity of FAPP2-C-terminal domain have been characterized and compared with other established GLTP-folds. Experimental evidence for a GLTP-fold includes: i) far-UV circular dichroism (CD) showing secondary structure with high alpha-helix content and a low thermally-induced unfolding transition (∼ 41 C); ii) near-UV-CD indicating only subtle tertiary conformational change before/after interaction with membranes containing/lacking glycolipid; iii) Red-shifted tryptophan (Trp) emission wavelength maximum (λmax ∼ 352 nm) for apo-FAPP2-C-terminal domain consistent with surface exposed intrinsic Trp residues; iv) 'signature' GLTP-fold Trp fluorescence response, i.e., intensity decrease (∼ 30%) accompanied by strongly blue-shifted λmax (∼ 14 nm) upon interaction with membranes containing glycolipid, supporting direct involvement of Trp in glycolipid binding and enabling estimation of partitioning affinities. A structurally-based preference for other simple uncharged GSLs, in addition to glucosylceramide, makes human FAPP2-GLTP more similar to fungal HET-C2 than to plant AtGLTP1 (glucosylceramide-specific) or to broadly GSL-selective human GLTP. These findings along with the distinct mRNA exon/intron organizations originating from single-copy genes on separate human chromosomes suggest adaptive evolutionary divergence by these two GLTP-folds.
KW - Divergent evolution
KW - GLTP superfamily
KW - Glycosphingolipid binding and transfer
KW - Keywords
KW - Membrane interaction
KW - Near-UV and far-UV circular dichroism
KW - Tryptophan fluorescence
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U2 - 10.1016/j.bbalip.2012.10.010
DO - 10.1016/j.bbalip.2012.10.010
M3 - Article
C2 - 23159414
AN - SCOPUS:84870654359
SN - 1388-1981
VL - 1831
SP - 417
EP - 427
JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
IS - 2
ER -