Brown, Rhoderick E.

Gao, Yongguang

Malinina, Lucy

Molotkovsky, Julian G.

Ochoa-Lizarralde, Borja

Pike, Helen M.

Samygina, Valeria R.

Zhai, Xiuhong

Elsevier - Publisher Connector 

Focusing the Glycolipid Specificity of Human Glycolipid Transfer Protein by Designer Mutation 

Wednesday, February 6, 2013 593a3055-Pos Board B210
Tryptophan Point Mutants of HET-C2, a Fungal Glycolipid Transfer
Protein, Provide Insights into Glycolipid Binding/Transfer andMembrane
Interaction
Roopa Kenoth1, Xianqiong Zou1, Dhirendra K. Simanshu2, Xiuhong Zhai1,
Helen M. Pike1, Julian G. Molotkovsky3, Dinshaw J. Patel2,
Rhoderick E. Brown1.
1University of Minnesota, Austin, MN, USA, 2Memorial Sloan-Kettering
Cancer Ctr, New York, NY, USA, 3Russian Academy of Sciences, Moscow,
Russian Federation.
Fungal glycolipid transfer protein (GLTP), HET-C2, forms a GLTP-fold, but with
higher binding/transfer preference for simple neutral glycosphingolipids than
broad-selectivity mammalian GLTPs (Kenoth et al., 2010, J. Biol. Chem. 285,
13066-13078). In humanGLTP, three Trp residues function in glycolipid binding
(Trp96), membrane interaction (Trp142) and protein fold stabilization (Trp85).
HET-C2 contains two Trp residues. Trp109 is structurally homologous to GLTP
Trp96, consistent with function as a stacking plate that orients the ceramide-
linked sugar during hydrogen bonding with Asp66, Asn70, and Lys73. HET-C2
Trp208 resides on the surface in a different location than either Trp142 or
Trp85 of GLTP. In this study, we generated single-Trp mutants of HET-C2 to as-
sess potential functional roles. Phe149, a structural homologue of GLTP Trp142,
was also investigated for potential function in membrane interaction. W208F-,
W208A- and F149Y-HET-C2 retained >90% activity and 80-90% intrinsic Trp
intensity; whereas F149A-HET-C2 transfer activity decreased to ~60% but dis-
played ~120% intrinsic Trp intensity. W109Y/F149Y-HET-C2 was nearly inac-
tive and displayed ~8% intrinsic Trp intensity. Thus, neither W208 nor F149 is
essential for activity and most Trp emission intensity (~90%) originates from
Trp109. With wtHET-C2, incubation with POPC vesicles containing glycolipid
decreases Trp fluorescence intensity (25-30%) and blue-shifts Dmax (6-7 nm).
For HET-C2 mutants involving Trp208 or F149, the intensity changes and Dmax
blue-shifts induced by vesicles containing glycolipid become elevated and similar
to human GLTP (~30-40% intensity decrease and ~12 nm Dmax blue-shift). Only
W109Y/F149Y-HET-C2,which lacks Trp109, deviated from the pattern, showing
that the Trp Dmax blue-shift is diagnostic for glycolipid binding in the HET-C2
GLTP-fold; whereas, the Trp intensity decrease reflects both glycolipid binding
and nonspecific bilayer interaction [Support: NIH/NIGMS-GM45928, NIH/
NCI-CA121493, Russian Fdn. Basic Research 012-04-00168, Hormel Fdn.]
3056-Pos Board B211
Elucidation of Calcium Ion and Phospholipid Binding Profiles of Multiple
Dysferlin Isoforms
Anne M. Rice1, Anne Hinderliter1, R. Bryan Sutton2.
1University of Minnesota-Duluth, Duluth, MN, USA, 2Texas Tech University
Health Center, Lubbock, TX, USA.
Dysferlin is a large type II peripheral membrane protein associated with the sar-
colemma and T-tubular system of muscle fibers. It contains seven C2 domains
and two DysF domains and has been implicated in membrane repair. This repair
is thought to be mediated through calcium-triggered exocytosis of intracellular
vesicles, which leads to the formation of a membrane patch. Due to dysferlin’s
structural similarity to synaptotagmin1 it is assumed that its role in the overall
scheme of membrane repair is to act as a calcium and membrane sensor. The
C2A domain is the first of the seven C2 domains and is assumed to be the pro-
tein’s main calcium and membrane sensor.
The C2A domain exists in nature as a combination of two different isoforms
derived from alternative readings of the first exon sequence: the canonical
C2A (WT) isoform derived from exon 1, and the C2Av1 (V1) isoform derived
from exon 1’. The two different isoforms of the C2A domain were studied to
probe the calcium and lipid binding affinities of dysferlin. Using isothermal
titration calorimetry (ITC) the calcium and lipid binding profiles of the two
constructs were established. These profiles indicate a difference in the phospho-
lipid dependence of calcium binding between the two constructs as well as
a uniform calcium dependence on the phospholipid binding. Initial approxima-
tions of the constructs’ binding parameters were estimated individually and can
be fit globally using a partition function approach.
By determining dysferlin’s binding profile we can better characterize its func-
tion in the scheme of membrane repair, as well as form a better understanding
of the intramolecular communication that tunes its physiological response.
3057-Pos Board B212
Focusing the Glycolipid Specificity of Human Glycolipid Transfer Protein
by Designer Mutation
Xiuhong Zhai1, Yongguang Gao1, Borja Ochoa-Lizarralde2,
Valeria R. Samygina2, Helen M. Pike1, Julian G. Molotkovsky3,
Lucy Malinina2, Rhoderick E. Brown1.
1University of Minnesota, Austin, MN, USA, 2CIC bioGUNE, Derio, Spain,
3Russian Academy of Sciences, Moscow, Russian Federation.Human glycolipid transfer protein (GLTP) fold represents a novel structural
amphitropic motif for lipid binding/transfer and reversible membrane translo-
cation. GLTP can transfer various glycosphingolipids known to serve as key
regulators of cell growth, division, surface adhesion, and neurodevelopment.
Previously, we reported structure-guided engineering of point-mutated GLTP
with enhanced selectivity for sulfatide (Samygina et al., 2011, Structure 19,
1644-1654). Herein, we extend the approach to characterize novel designer
GLTPs capable of focused glycolipid binding and transfer. Structure-guided
mutations were directed to three residues, Lys55, Lys87, and Leu92. Different
glycolipids (GlcCer, GalCer, LacCer, sulfatide, disulfatide) were analyzed to
determine mutant GLTP glycolipid transfer specificity using an established
fluorescence resonance energy transfer assay involving anthrylvinyl-labeled
glycolipid and 3-perylenoyl-labeled phosphatidylcholine. Assessment of glyco-
lipid binding by the designer mutants relied on intrinsic changes in tryptophan
fluorescence originating from Trp96, i.e. GLTP Trp signature fluorescence re-
sponse. The response involves large decreases (up to 40%) in emission intensity
accompanied by substantial blue-shift (up to 13 nm) of the emission wave-
length maximum. Insights into the molecular basis for enhanced glycolipid se-
lectivity was provided by X-ray diffraction structural determination of certain
designer mutants complexed with glycolipid. The development of designer
GLTPs with enhanced specificity for select GSLs provides a potential new ther-
apeutic approach for targeting GSL-mediated pathologies [Support: NIH/
NIGMS-GM45928 & NIH/NCI-CA121493, Russian Foundation for Basic Re-
search 012-04-00168, Spanish Ministerio de Ciencia e Innovacion (MICINN
BFU2010-17711), and Hormel Foundation]
3058-Pos Board B213
Several Asparagine Residues Flanking a Hydrophobic Helix are
required to Block Interconversion between Transmembrane and Non-
Transmembrane Configurations
James LeBarron, Erwin London.
Stony Brook University, Stony Brook, NY, USA.
Proteins that are embedded in cellular membranes include polar residues at
both ends of their transmembrane (TM) helices or in loops that connect their
TM helices. These polar residues can prevent the interconversion of these he-
lices from the TM state to a non-TM configuration, with the helices located near
the water-lipid interface. The energetic cost of segment movement containing
polar residues through the hydrophobic membrane is thought to be too large to
allow rapid TM to non-TM interconversion. We have investigated how TM/
non-TM interconversion is influenced by an increasing number of asparagine
residues in the juxta-membrane regions flanking both sides of simple hydropho-
bic helical peptides. The central hydrophobic region of these peptides includes
a Trp whose fluorescence reflects the configuration of the peptide in the bilayer,
and an Asp residue whose protonation state can be controlled via pH. The ends
of the helices contained two Lys as well as a variable number of asparagine res-
idues. At low pH the Asp was uncharged and the TM configuration of the helix
was favored, while at high pH the uncharged Asp favored the non-TM config-
uration of the helix. We find six asparagine residues block non-TM to TM in-
terconversion (insertion) for at least 30 minutes, but do not block the reverse
which takes on the order of seconds. However, two asparagines allowed inter-
conversion between TM and non-TM states, in either direction, on the order of
seconds. This shows that a significant number of polar residues is needed to
block interconversion between the TM and non-TM state, and from the differ-
ence in behavior when Asp is charged or uncharged implies that the hydropho-
bic sequence of the peptide is likely to influence this process.
3059-Pos Board B214
A Buttressed Unilamellar Membrane System for Studying Lipid-Protein
Interactions
Sungsoo Lee, Marc Llaguno, Qiu-xing Jiang.
University of Texas Southwestern Medical Center at Dallas, Dallas, TX,
USA.
Recent studies have demonstrated that annular lipids can directly change the
voltage-gating of voltage-gated ion channels. We recognized that it was a chal-
lenge to insert membrane proteins into a bilayer that mimics the eukaryotic cell
membranes in current model systems, especially the membranes that contain
sphingomyelin and cholesterol. A new system is needed to overcome this tech-
nical barrier. In this study we developed a stable unilamellar vesicle system that
is supported by unidirectionally inserted membrane proteins, and offers the
capability of controlling lipid composition with relative ease. A voltage-
gated potassium channel, KvAP, was used as a model system, and was selec-
tively anchored onto the surface of micron-sized beads. Our data suggested
that with a high surface density of channel molecules, it was feasible to intro-
duce various lipids and form continuous membranes around the beads. The uni-
lamellar nature of the bilayers was demonstrated by cryo-electron microscopic


file:///data/remote/core/dit/data/elsevier/pdf/9ab/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMDAwNjM0OTUxMjA0NTQxOQ==.pdf

Focusing the Glycolipid Specificity of Human Glycolipid Transfer Protein by Designer Mutation

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector