Presence Of Acidic Residues Research Articles

Is the E. coli Homolog of the Formate/Nitrite Transporter Family an Anion Channel? A Computational Study

Formate/nitrite transporters (FNTs) selectively transport monovalent anions and are found in prokaryotes and lower eukaryotes. They play a significant role in bacterial growth and act against the defense mechanism of infected hosts. Because FNTs do not occur in higher animals, they are attractive drug targets for many bacterial diseases. Phylogenetic analysis revealed that they can be classified into eight subgroups, two of which belong to the uncharacterized YfdC-α and YfdC-β groups. Experimentally determined structures of FNTs belonging to different phylogenetic groups adopt the unique aquaporin-like hourglass helical fold. We considered the formate channel from Vibrio cholerae, the hydrosulphide channel from Clostridium difficile, and the uncharacterized channel from Escherichia coli (EcYfdC) to investigate the mechanism of transport and selectivity. Using equilibrium molecular dynamics and umbrella sampling studies, we determined temporal channel radius profiles, permeation events, and potential of mean force profiles of different substrates with the conserved central histidine residue in protonated or neutral form. Unlike the formate channel from V. cholerae and the hydrosulphide channel from C. difficile, molecular dynamics studies showed that the formate substrate was unable to enter the vestibule region of EcYfdC. Absence of a conserved basic residue and presence of acidic residues in the vestibule regions, conserved only in YfdC-α, were found to be responsible for high energy barriers for the anions to enter EcYfdC. Potential of mean force profiles generated for ammonia and ammonium ion revealed that EcYfdC can transport neutral solutes and could possibly be involved in the transport of cations analogous to the mechanism proposed for ammonium transporters. Although YfdC members belong to the FNT family, our studies strongly suggest that EcYfdC is not an anion channel. Absence or presence of specific charged residues at particular positions makes EcYfdC selective for neutral or possibly cationic substrates. Further experimental studies are needed to get a definitive answer to the question of the substrate selectivity of EcYfdC. This provides an example of membrane proteins from the same family transporting substrates of different chemical nature.

How Does a Membrane Protein Know What is In and What is Out? Lipids as Topological Determinants

Primary functions of lipids are to define barrier properties of membranes and provide a scaffold within which membrane proteins are organized. Using a genetic approach to alter the phospholipid composition of the Escherichia coli coupled with biochemical approaches to monitor topological organization of membrane proteins, dependence of lactose permease (LacY) on phosphatidylethanolamine (PE) for proper orientation with respect to the plane of the membrane was determined. Assembly of LacY in the absence of PE results in topological inversion of its N-terminal half, which is largely reversed by post-assembly synthesis of PE. Replacement of PE by the foreign lipids phosphatidylcholine, monoglucosyl diacylglycerol, or diglucosyl diacylglycerol, which exhibit similar properties to PE, restores proper topology thereby supporting common functions for lipids with diverse structures. Topology of LacY in membranes lacking PE is dependent on a connecting flexible hinge region in order for the two halves of LacY to independently respond to the lipid environment. Final topology is determined after LacY exits the translocon by long-range and short-range interactions between the net charge of extra-membrane domains and the net charge density of the phospholipid bilayer surface. PE appears to dampen the translocation potential of acidic residues in normally cytoplasmic domains in favor of the cytoplasmic retention potential of basic residues. Thus a primary role for PE is to allow the presence of acidic residues in the cytoplasmic domains of membrane proteins for functional purposes without affecting protein topological. The topologies of two amino acid permeases (PheP and GabP) unrelated to LacY are also topologically sensitive to membrane lipid composition strongly indicating that lipid environment is a significant determinant of final topological organization of multiple membrane proteins. Supported in part by NIGMS R37-GM20478.

Eyes Absent Proteins: Characterization of Substrate Specificity and Phosphatase Activity of Mutants Associated with Branchial, Otic and Renal Anomalies

The eyes absent (Eya) genes encode a family of proteins that combine the functions of transcriptional cofactors, signal transducers and enzymes, namely protein tyrosine phosphatases. The latter activity resides in the highly conserved C-terminal Eya domain (ED). Here, we investigated the substrate specificity of the Arabidopsis thaliana homologue (AtEya) by using low-molecular-weight compounds and synthetic phosphotyrosine (pY)-containing peptides that correspond either to phosphorylation sites in proteins or to peptides that were selected through the screening of a combinatorial peptide library. AtEya displayed modest peptide substrate specificity and was sensitive to charges adjacent to pY. In general, the presence of acidic residues on the N-terminal side of the phosphorylation site was critical for catalysis, whereas basic amino acids seemed to be preferred with respect to high-affinity binding. We also detected significant acyl phosphatase activity of AtEya; this suggests that Eya proteins might have further substrates in vivo. In addition, we analysed the phosphatase activity of a number of variants of the mouse Eya1 protein that harbours single point mutations that were associated with branchio-oto-renal syndrome (BOR), branchio-oto syndrome (BO) and ocular defects, respectively, in humans. While BOR mutations led to a significantly reduced phosphatase activity, BO mutants as well as those that are associated with ocular defects only displayed activity that was similar to wild-type levels.

An Extracellular Cu2+ Binding Site in the Voltage Sensor of BK and Shaker Potassium Channels

Copper is an essential trace element that may serve as a signaling molecule in the nervous system. Here we show that extracellular Cu2+ is a potent inhibitor of BK and Shaker K+ channels. At low micromolar concentrations, Cu2+ rapidly and reversibly reduces macrosocopic K+ conductance (GK) evoked from mSlo1 BK channels by membrane depolarization. GK is reduced in a dose-dependent manner with an IC50 and Hill coefficient of ∼2 μM and 1.0, respectively. Saturating 100 μM Cu2+ shifts the GK-V relation by +74 mV and reduces GKmax by 27% without affecting single channel conductance. However, 100 μM Cu2+ fails to inhibit GK when applied during membrane depolarization, suggesting that Cu2+ interacts poorly with the activated channel. Of other transition metal ions tested, only Zn2+ and Cd2+ had significant effects at 100 μM with IC50s > 0.5 mM, suggesting the binding site is Cu2+ selective. Mutation of external Cys or His residues did not alter Cu2+ sensitivity. However, four putative Cu2+-coordinating residues were identified (D133, Q151, D153, and R207) in transmembrane segments S1, S2, and S4 of the mSlo1 voltage sensor, based on the ability of substitutions at these positions to alter Cu2+ and/or Cd2+ sensitivity. Consistent with the presence of acidic residues in the binding site, Cu2+ sensitivity was reduced at low extracellular pH. The three charged positions in S1, S2, and S4 are highly conserved among voltage-gated channels and could play a general role in metal sensitivity. We demonstrate that Shaker, like mSlo1, is much more sensitive to Cu2+ than Zn2+ and that sensitivity to these metals is altered by mutating the conserved positions in S1 or S4 or reducing pH. Our results suggest that the voltage sensor forms a state- and pH-dependent, metal-selective binding pocket that may be occupied by Cu2+ at physiologically relevant concentrations to inhibit activation of BK and other channels.

Pseudospecificity of the Acidic Patch of Plastocyanin for the Interaction with Cytochrome f

The ionic strength dependence of the physiological electron-transfer reaction of plastocyanins (PCus) from a range of sources with a eukaryotic cytochrome f (cyt f) has been studied. The presence of an acidic patch on the surface of PCu is key to this interaction in higher plants, but minor modifications in this surface region have a limited effect. Surprisingly, a similarly small influence results from the repositioning of the acidic patch (as in the fern PCu). The only requirement of a plant PCu to ensure efficient interaction with its cyt f is the presence of acidic residues on the periphery of the protein's hydrophobic patch.

In APCs, the autologous peptides selected by the diabetogenic I-Ag7 molecule are unique and determined by the amino acid changes in the P9 pocket.

We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-A(g7) molecule, the P9 pocket of I-A(g7) plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-A(g7) molecules or a mutant I-A(g7) molecule in which the beta57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-A(g7)-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-A(g7) were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-A(g7) molecule. These peptides did not favor acidic residues toward the carboxyl terminus.

Formation of reversible disulfide bonds with the protein matrix of the endoplasmic reticulum correlates with the retention of unassembled Ig light chains.

Exposed thiols act as intracellular retention elements for unassembled secretory molecules. Yet, some free Ig lambda light chains are secreted despite the presence of an unpaired cysteine (Cys214). This is due largely to the presence of a flanking acidic residue: substitution of Asp213 for Gly or Lys increases pre-Golgi retention and degradation of free lambda. Secretion is restored by exogenous reducing agents or by assembly with heavy chains. In the endoplasmic reticulum (ER), lambda chains form covalent complexes with many proteins through Cys214. These complexes are absent from the Golgi. They are more abundant in transfectants expressing the lambdaGly2I3 and lambdaLys213 mutants that are poorly secreted. Radioactive N-ethylmaleimide labels some monomeric lambda chains isolated from the ER, but not from the Golgi or from the medium, indicating that the Cys214 thiol is masked during ER-Golgi transport. Mass spectrometry reveals the presence of a free cysteine residue disulfide-linked to Cys214. We suggest that thiol-mediated retention involves the formation of reversible disulfide bonds with the protein matrix of the ER. The presence of an acidic residue next to the critical cysteine may allow the masking of the thiol and transport to the Golgi.

A Strategy for Sequencing Peptides from Dilute Mixtures at the Low Femtomole Level Using Membrane Preconcentration-Capillary Electro-Phoresis-Tandem Mass Spectrometry (MPC-CE-MS/MS)

Abstract A unique method using membrane preconcentration capillary electrophoresis (mPC-CE) on-line with tandem mass spectrometry (mPC-CE-MS/MS) for sequencing <100 fmols of a series of MHC class I synthetic peptides is described. We show how this methodology in conjunction with transient isotachophoresis (tlTP) after analyte elution from the membrane improves conventional CE-MS strategies by permitting the analysis of large sample volumes (>100 μL) while maintaining high analyte resolution and separation efficiencies that are typically afforded by CE. Instrumental parameters, including the internal dimensions of the mPC-CE capillary, collision gas pressure, collision gas type, and collision energy, are all shown to have substantial effect on the abundance of product ions produced in tandem MS/MS spectra when attempting to sequence peptides at the sub 100 femtomole level. Furthermore, we demonstrate that the physico-chemical properties of these analytes can also affect the MS/MS product ion abundance. In particular, the presence of acidic residue tends to reduce mPC-tlTP-CE-MS/MS sensitivity and direct fragmentation processes resulting in incomplete sequence information. This can be overcome by a simple esterification of the carboxylic acid functional group. Ultimately, we present an optimized mPC-tlTP-CE-MS/MS approach that permits peptide sequence determination by consumption of ∼40 femtomole of peptide analyte.

The MRC OX‐47 antigen is a member of the immunoglobulin superfamily with an unusual transmembrane sequence

The MRC OX-47 monoclonal antibody recognizes a membrane antigen present at low levels on many lymphocytes but whose expression is markedly increased on activation with mitogens. cDNA clones for the OX-47 antigen were isolated from an expression library and the protein sequence deduced. It contains a leader sequence giving a mature protein of 251 amino acids with a single putative transmembrane region, a cytoplasmic domain of 40 amino acids and an extracellular domain of 187 amino acids that contained two immunoglobulin-like domains. The putative transmembrane sequence includes a glutamic acid residue within the hydrophobic sequence. The presence of acidic residues within the hydrophobic sequence of transmembrane sequences usually indicates association with other polypeptides and this is predicted for the OX-47 antigen. A sequence of 37 amino acids that included all the transmembrane region was identical to that of the chicken HT7 antigen present on endothelium in brain and erythroblasts. The level of protein sequence identity in the Ig-like domains was lower but HT7 is almost certainly the chicken homologue of the rat OX-47 antigen. The ligand and function of the molecule are unknown. In addition to lymphoblasts the OX-47 antigen was localized on a variety of other cell types including various immature cells, endothelia and cells with excitable membranes.

The use of synthetic peptides for defining the specificity of typrosine protein kinases

The tyrosine protein kinases are a large family of enzymes that may be involved in regulating cell growth and differentiation. An important property of these enzymes is their substrate specificity. Defining the specificity of these enzymes will contribute to a greater understanding of their biological functions. Synthetic peptides provide a useful means for studying the specificities of protein kinases. The utility of synthetic peptides for specificity studies is exemplified by the results obtained with the cyclic nucleotide-dependent protein kinases. A large number of synthetic peptides have been tested as substrates for these enzymes. There are three important conclusions from this work. First, in terms of primary sequence, the two cyclic nucleotide-dependent protein kinases both require the presence of a pair of basic residues on the N-terminal side of the phosphorylatable residue. Second, recognition of a specific secondary structure of these enzymes. Third, the specificities of the two cyclic nucleotide-dependent protein kinases are remarkably similar in terms of both primary and secondary structure recognition. Sequences at the sites of tyrosine phosphorylation often show the presence of numerous acidic residues on the N-terminal side of the tyrosine. This result led to the suggestion that these acidic residues might be important for the recognition of these sites by the tyrosine protein kinases. Specificity studies using synthetic peptidese have provided some experimental verification of this concept. In the case of four tyrosine protein kinases, LSTRA cell tyrosine protein kinase, epidermal growth factor receptor kinase, insulin receptor kinase and pp60 gag-yes tyrosine protein kinase, the presence of acidic residues on the N-terminal side of the tyrosine in a synthetic peptide was a favorable determinant. In the case of a fifth member of the tyrosine protein kinase family, namely pp60 src kinase, the data are less clear that the presence of acidic residues is involved in substrate recognition. This enzyme readily phosphorylated several peptides that did not have acidic residues on the N-terminal side of the tyrosine. Although there is some indication that the presence of acidic residues on the N-terminal side of the tyrosine is a factor in substrate recognition by several of the tyrosine protein kinases, the changes in kinetic parameters with the various peptide substrates were rather small. In addition, some sites phosphorylated in proteins do not have any acidic residues on the N-terminal side of the phosphorylated tyrosine residue. The presence of acidic residues on the primary sequence near the tyrosine is not an absolute determinant for the recognition of sites of phosphorylation by the tyrosine protein kinases. As in the case of the cyclic nucleotide-dependent protein kinases, secondary structure is probably also important for the recognition of sites of phosphorylation by the tyrosine protein kinases.