PDBsum entry 2k3c

Metal transport

PDB id: 2k3c

Contents

Protein chain

33 a.a. *

* Residue conservation analysis

PDB id:

2k3c

Name:

Metal transport

Title:

Structural and functional characterization of tm ix of the nhe1 isoform of the na+/h+ exchanger

Structure:

Tmix peptide. Chain: a. Engineered: yes

Source:

Synthetic: yes. Other_details: solid-phase peptide synthesis

NMR struc:

40 models

Authors:

T.Reddy,J.Ding,X.Li,B.D.Sykes,L.Fliegel,J.K.Rainey

Key ref:

T.Reddy et al. (2008). Structural and Functional Characterization of Transmembrane Segment IX of the NHE1 Isoform of the Na+/H+ Exchanger. J Biol Chem, 283, 22018-22030. PubMed id: 18508767 DOI: 10.1074/jbc.M803447200

Date:

01-May-08 Release date: 03-Jun-08

Protein chain

No UniProt id for this chain

Struc: 32 a.a.

DOI no: 10.1074/jbc.M803447200

J Biol Chem 283:22018-22030 (2008)

PubMed id: 18508767

Structural and Functional Characterization of Transmembrane Segment IX of the NHE1 Isoform of the Na+/H+ Exchanger.

T.Reddy, J.Ding, X.Li, B.D.Sykes, J.K.Rainey, L.Fliegel.

ABSTRACT

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).

Selected figure(s)

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.

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.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 22018-22030) copyright 2008.

Figures were selected by an automated process.