Jean C. Ingram

Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di‐ and tri‐peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepTSo, a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand‐bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide‐binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepTSo represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.

Molecular cloning of the α-subunit of rat endopeptidase-24.18 (endopeptidase-2) and co-localization with endopeptidase-24.11 in rat kidney by in situ hybridization

Endopeptidase‐24.18 (endopeptidase‐2, EC 3.4.24.18, E‐24.18) is a Zn‐ectoenzyme of rat renal and intestinal microvillar membranes exhibiting an oligometric structure, α2‐β2. The primary structure of the α‐subunit of E‐24.18 has been defined by molecular cloning and its expression mapped in rat kidney by in situ hybridization. A 2.9‐kb cDNA coding for the α‐subunit was isolated and sequenced. It had an open reading frame of 2.244 base pairs coding for a type I membrane protein of 748 amino acids. The deduced amino acid sequence showed 87% identity with that of meprin A, a mouse metallo‐endopeptidase, sharing common properties with the rat enzyme, and 85% identity with the human intestinal enzyme, ‘PABA‐peptide hydrolase’. Northern blot analysis revealed the α‐subunit to be encoded by a single mRNA species of 3.2‐kb. In situ hybridization performed on rat kidney showed a co‐localization of E‐24.18 with endopeptidase‐24.11 in proximal tubules of juxtamedullary nephrons, suggesting that the two enzymes have similar or complementary physiological functions in kidney.

A high-throughput assay of membrane protein stability.

The preparation of purified, detergent-solubilized membrane proteins in a monodisperse and stable form is usually a prerequisite for investigation not only of their function but also for structural studies by X-ray crystallography and other approaches. Typically, it is necessary to explore a wide range of conditions, including detergent type, buffer pH, and the presence of additives such as glycerol, in order to identify those optimal for stability. Given the difficulty of expressing and purifying membrane proteins in large amounts, such explorations must ideally be performed on as small a scale as practicable. To achieve this objective in the UK Membrane Protein Structure Initiative, we have developed a rapid, economical, light-scattering assay of membrane protein aggregation that allows the testing of 48 buffer conditions in parallel on 6 protein targets, requiring less than 2 mg protein for each target. Testing of the assay on a number of unrelated membrane transporters has shown that it is of generic applicability. Proteins of sufficient purity for this plate-based assay are first rapidly prepared using simple affinity purification procedures performed in batch mode. Samples are then transferred by microdialysis into each of the conditions to be tested. Finally, attenuance at 340 nm is monitored in a 384-well plate using a plate reader. Optimal conditions for protein stability identified in the assay can then be exploited for the tailored purification of individual targets in as stable a form as possible.

Investigation of the structure and function of a Shewanella oneidensis arsenical-resistance family transporter

The toxic metalloid arsenic is an abundant element and most organisms possess transport systems involved in its detoxification. One such family of arsenite transporters, the ACR3 family, is widespread in fungi and bacteria. To gain a better understanding of the molecular mechanism of arsenic transport, we report here the expression and characterization of a family member, So_ACR3, from the bacterium Shewanella oneidensis MR-1. Surprisingly, expression of this transporter in the arsenic-hypersensitive Escherichia coli strain AW3110 conferred resistance to arsenate, but not to arsenite. Purification of a C-terminally His-tagged form of the protein allowed the binding of putative permeants to be directly tested: arsenate but not arsenite quenched its intrinsic fluorescence in a concentration-dependent fashion. Fourier transform infrared spectroscopy showed that the purified protein was predominantly α-helical. A mutant bearing a single cysteine residue at position 3 retained the ability to confer arsenate resistance, and was accessible to membrane impermeant thiol reagents in intact cells. In conjunction with successful C-terminal tagging with oligohistidine, this finding is consistent with the experimentally-determined topology of the homologous human apical sodium-dependent bile acid transporter, namely 7 transmembrane helices and a periplasmic N-terminus, although the presence of additional transmembrane segments cannot be excluded. Mutation to alanine of the conserved residue proline 190, in the fourth putative transmembrane region, abrogated the ability of the transporter to confer arsenic resistance, but did not prevent arsenate binding. An apparently increased thermal stability is consistent with the mutant being unable to undergo the conformational transitions required for permeant translocation.

Rat endopeptidase-24.18 α subunit is secreted into the culture medium as a zymogen when expressed by COS-1 cells

Endopeptidase‐24.18 (EC 3.4.24.18, E‐24.18) is an oligomeric Zn‐ectoenzyme. The α and β submits have been cloned from both rat and mouse kidneys. The primary structure of these subunits revealed that they both contain the consensus Zn binding site and that they are members of the astacin family. Analysis of the hydropathy plot also suggested that they are anchored by a C‐terminal hydrophobic domain. In order to verify the mode of anchoring of the rat E‐24.18 α subunit and to test the functionality of the astacin‐like domain in the α subunit when expressed alone, COS‐1 cells were transfected with a cloned cDNA for rat α subunit. Despite the presence of its putative transmembrane domain, the α subunit was not anchored in the plasma membrane but rather secreted as a dimer into the culture medium. When the enzymatic activity of the secreted recombinant protein was tested in the azocasein degradation assay, the α subunit was found to be inactive. Activity could, however, be revealed after mild trypsin digestion. This activity was abolished by replacing the Glu‐157 in the active site by Val. Taken together our results suggest that the α subunit of Endopeptidase‐24.18 contains a latent astacin‐like Zn metallopeptidase activity which could be secreted as a soluble enzyme by kidney and intestine.

Radiation inactivation analysis of kidney microvillar peptidases.

Five membrane peptidases were studied by radiation inactivation analysis of pig kidney microvillar membranes. One heterodimeric enzyme, γ‐glutamyl transferase, presented a target size corresponding to the dimeric M r. The other enzymes are known to be homodimers. Three of these, aminopeptidase A, aminopep‐tidase N and dipeptidyl peptidase IV, gave results clearly indicating the monomer to be the target and, hence, in this group the association of the subunits was not essential for activity. The target size for endopep‐tidase‐24.11 was intermediate between those for monomer and dimer and its functional state was not resolved by the experiments.

Large-scale preparation of bacterial cell membranes by tangential flow filtration.

The preparation of cell membranes by ultracentrifugation of bacterial cell lysates, a pre-requisite for the purification of over-expressed membrane proteins, is both time-consuming and difficult to perform on a large scale. To overcome this bottleneck in the structural investigation of such proteins in the UK Membrane Protein Structure Initiative, we have investigated the alternative use of tangential flow filtration for preparation of membranes from Escherichia coli. This method proved to be superior to the conventional use of ultracentrifuges both in speed and in yield of membrane protein. Moreover, it could more readily be scaled up to process larger quantities of bacterial cells. Comparison of the purity and monodispersity of an over-expressed membrane protein purified from conventionally-prepared membranes and from membranes prepared by filtration revealed no substantial differences. The approach described should therefore be of general use for membrane protein preparation for a wide range of applications, including both structural and functional studies.

Characterization of the Escherichia coli Concentrative Nucleoside Transporter NupC Using Computational, Biochemical, and Biophysical Methods

Members of the concentrative nucleoside transporter (CNT) family of proteins mediate uptake of nucleosides into cells driven by a cation gradient, which then enter salvage pathways for nucleic acid synthesis. In humans, they also transport hydrophilic anticancer and antiviral nucleoside analogue drugs into cells and tissues where they exert their pharmacological effects. Escherichia coli CNT NupC (400 residues) is pyrimidine-specific and driven by a proton gradient. We have used computational, biochemical, and biophysical methods to characterize evolutionary relationships, conservation of residues, structural domains, transmembrane helices, and residues involved in nucleoside binding and/or transport activity in NupC compared with those of sodium-driven Vibrio cholerae CNT (vcCNT) and human CNTs (hCNT1–3). As in the crystal structure of vcCNT, NupC appears to contain eight transmembrane-spanning α-helices. Wild-type NupC and single-cysteine-containing mutants were tested for transport activity in energize...

Putative tripeptidyl peptidase in renal brush border is due to sequential action of two other exopeptidases.

To the Editor: We wish to draw readers’ attention to the need for caution in interpreting assays of peptidases using fluorogenic substrates such as Gly-Pro-Met-2 naphthylamide (NNap). The release of the fluorophore, 2naphthylamine, may indicate the action of a tripeptidyl peptidase releasing the tripeptide, Gly-Pro-Met, but it may also result from the sequential attack of other exopeptidases, first releasing Gly-Pro and then free methionine. The essential criterion of a tripeptidyl peptidase attack is the demonstration of the tripeptide among the products of the reaction. Two recent reports by Knut-Jan Andersen and J. Ken McDonald (1, 2) of a tripeptidyl peptidase active at pH 7 in kidney brush-border membranes do not contain any evidence that a tripeptide was released from the naphthylamide substrate. We have now reinvestigated this question and fail to find a tripeptidyl peptidase hydrolyzing either Gly-Pro-Met-NNap or Gly-Pro-Leu-NNap in pig or rat microvillar membranes (3). The hydrolysis of both substrates was readily assayed fluorimetrically, but the products analyzed by high precision liquid chromatography were Gly-Pro, Met-NNap (or Leu-NNap) and free naphthylamine, with no evidence of tripeptidases. Moreover, the activity monitored by the assay was inhibited by either diisopropylfluorophosphate or amastatin (inhibitors of dipeptidyl peptidase IV and aminopeptidase N, respectively) and could be titrated to zero by polyclonal antibodies raised to pig and rat aminopeptidase N. It was our conclusion that these substrates were hydrolyzed by renal microvilli in a two-step process, first the release of Gly-Pro by dipeptidyl peptidase IV and then the hydrolysis of the amino acid naphthylamide by aminopeptidase N. There are twelve well-characterized peptidases so far identified in renal microvilli (4), but a true tripeptidyl peptidase cannot yet be added to the list.