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PDBsum entry 2k3c
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Metal transport
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PDB id
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2k3c
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References listed in PDB file
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Key reference
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Title
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Structural and functional characterization of transmembrane segment IX of the nhe1 isoform of the na+/h+ exchanger.
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Authors
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T.Reddy,
J.Ding,
X.Li,
B.D.Sykes,
J.K.Rainey,
L.Fliegel.
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Ref.
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J Biol Chem, 2008,
283,
22018-22030.
[DOI no: ]
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PubMed id
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Abstract
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The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that
regulates intracellular pH by removing one intracellular H(+) in exchange for
one extracellular Na(+). It has a large N-terminal membrane domain of 12
transmembrane segments and an intracellular C-terminal regulatory domain. We
characterized the cysteine accessibility of amino acids of the putative
transmembrane segment IX (residues 339-363). Each residue was mutated to
cysteine in a functional cysteineless NHE1 protein. Of 25 amino acids mutated, 5
were inactive or nearly so after mutation to cysteine. Several of these showed
aberrant targeting to the plasma membrane and reduced expression of the intact
protein, whereas others were expressed and targeted correctly but had defective
NHE1 function. Of the active mutants, Glu(346) and Ser(351) were inhibited
>70% by positively charged [2-(trimethylammonium)-ethyl]methanethiosulfonate
but not by anionic [2-sulfonatoethyl]methanethiosulfonate, suggesting that they
are pore lining and make up part of the cation conduction pathway. Both mutants
also had decreased affinity for Na(+) and decreased activation by intracellular
protons. The structure of a peptide representing amino acids 338-365 was
determined by using high resolution NMR in dodecylphosphocholine micelles. The
structure contained two helical regions (amino acids Met(340)-Ser(344) and
Ile(353)-Ser(359)) kinked with a large bend angle around a pivot point at amino
acid Ser(351). The results suggest that transmembrane IX is critical with
pore-lining residues and a kink at the functionally important residue Ser(351).
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Figure 1.
FIGURE 1. A, model of the Na^+/H^+ exchanger (NHE1 isoform)
(11). The orientation of TM segments 1–12 of the NHE1 isoform
of the Na^+/H^+ exchanger is illustrated. EL 1–6 and IL 1–5
refer to extracellular and intracellular loops as numbered. B,
schematic model of amino acids present in TM IX.
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Figure 7.
FIGURE 7. Assessment of contiguous helicity by secondary
chemical shifts and dihedral angle order parameter. A, ^1H
secondary chemical shifts for TM IX in DPC micelles. ^1H
chemical shifts assignments from homonuclear two-dimensional
NOESY (225-ms mixing time) and two-dimensional TOCSY spectra
collected on an 800-MHz spectrometer. Random coil shifts (59)
were subtracted from the empirical values, and the resulting
secondary chemical shifts are shown for H^  . The solid line is the
chemical shift difference cutoff considered significant (for
helicity) for the ^1H chemical shift index (60). B,  and  backbone
dihedral angle order parameter for each residue of the TM IX
peptide. (dashed line) and (solid
line) dihedral angle order parameters were calculated as
described previously (61). Briefly, if the dihedral angle has
the same value in all 40 structures of the retained NMR
ensemble, its order parameter will be 1. An order parameter of 0
represents a completely random dihedral angle across the
ensemble, and values 0 < order parameter < 1 represent variable
dihedral angles. This parameter is used in place of the
alternative measure of angular standard deviation, which does
not have a defined value for truly random angles.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2008,
283,
22018-22030)
copyright 2008.
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