Alanine Insertion Research Articles

The ?-helical membrane spanning domain of cytochrome interacts with cytochrome P450 via nonspecific interactions

Cytochrome b5 (cyt b5) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes. It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an alpha-helical membrane-spanning domain. This study investigated whether there are specific side chain helix-helix packing interactions between the COOH-terminal membrane anchor of cyt b5 and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b5. Insertion of alanine into an alpha-helix causes all amino acids at its carboxyl terminus to be rotated by 100 degrees . The ability of the alanine insertion mutants of cyt b5 to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b5 to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic alpha-helix of cyt b5 does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions.

Transport of Meprin Subunits through the Secretory Pathway: ROLE OF THE TRANSMEMBRANE AND CYTOPLASMIC DOMAINS AND OLIGOMERIZATION

The meprin alpha subunit, a multidomain metalloproteinase, is synthesized as a type I membrane protein and proteolytically cleaved during biosynthesis in the endoplasmic reticulum (ER), consequently losing its membrane attachment and COOH-terminal domains. The meprin alpha subunit is secreted as a disulfide-linked dimer that forms higher oligomers. By contrast, the evolutionarily related meprin beta subunit retains the COOH-terminal domains during biosynthesis and travels to the plasma membrane as a disulfide-linked integral membrane dimer. Deletion of a unique 56-amino acid inserted domain (the I domain) of meprin alpha prevents COOH-terminal proteolytic processing and results in the retention of this subunit within the ER. To determine elements responsible for this retention versus transport to the cell surface, mutagenesis experiments were performed. Replacement of the meprin alpha transmembrane (alphaT) and cytoplasmic (alphaC) domains with their beta counterparts allowed rapid movement of the alpha subunit to the cell surface. The meprin alphaT and alphaC domains substituted into meprin beta delayed movement of this chimera through the secretory pathway. Replacement of glycines in the meprin alphaT domain GXXXG motif with leucine residues, alanine insertions in the meprin alphaT domain, and mutagenesis of basic residues within the meprin alphaC domain did not enhance the movement of the alpha subunit through the secretory pathway. By contrast, a mutant of meprin alpha (C320AalphaDeltaI) that did not form disulfide-linked dimers or higher order oligomers was transported through the secretory pathway, although more slowly than meprin beta. Taken together, the data indicate that the meprin alphaT and alphaC domains together contain a weak signal for retention within the ER/cis-Golgi compartments that is strengthened by oligomerization.

RUNX2 alleles associated with BMD in Scottish women; interaction of RUNX2 alleles with menopausal status and body mass index

Bone mineral density (BMD) is influenced by both environmental and genetic factors. We previously reported the association of the RUNX2 A allele with increased bone mineral density (BMD) and protection against a common form of osteoporotic fracture within a Geelong population. We genotyped 991 women from a Scottish cohort to decipher the role of RUNX2 alleles in regulating BMD. The alleles of RUNX2 within the glutamine–alanine repeat were determined by MspA1I restriction digest. Allele frequencies estimated from Scottish cohort were G allele, 0.87 ± 0.01; A allele, 0.08 ± 0.01; and 11Ala alanine deletion allele, 0.05 ± 0.01. Analysis of covariance (ANCOVA) was used to adjust for the covariates weight and age for BMD at the femoral neck (FN). The A allele was associated with higher FN BMD ( P = 0.035) within a postmenopausal subgroup of the population ( n = 312). The effect of RUNX2 A alleles increased with increasing weight; A alleles were associated with FN BMD in those above the median BMI (BMI > 25), while no association was observed in thin/normal (BMI ≤ 25) postmenopausal women. Glutamine variants and an alanine insertion were identified within the group. These data suggest that the RUNX2 alleles are associated with BMD in a menopause- and weight-dependent manner.

Induction of substrate specificity shifts by placement of alanine insertions within the consensus amphipathic region of the Escherichia coli GABA (gamma-aminobutyric acid) transporter encoded by gabP.

The Escherichia coli GABA (gamma-aminobutyric acid) permease GabP is a prototypical APC (amine/polyamine/choline) super-family transporter that has a CAR (consensus amphipathic region) containing multiple specificity determinants, ostensibly organized on two helical surfaces, one hydrophobic [SHS (sensitive hydrophobic surface)] and the other hydrophilic [SPS (sensitive polar surface)]. To gauge the functional effects of placing alanine insertions at close intervals across the entire GabP CAR, 64 insertion variants were constructed. Insertions, particularly those in the SHS and the SPS, were highly detrimental to steady-state [(3)H]GABA accumulation. TSR (transport specificity ratio) analysis, employing [(3)H]nipecotic acid and [(14)C]GABA, showed that certain alanine insertions were associated with a specificity shift (i.e. a change in k (cat)/ K (m)). An insertion (INS Ala-269) located N-terminal to the SHS increased specificity for [(3)H]nipecotic acid relative to [(14)C]GABA, whereas an insertion (INS Ala-321) located C-terminal to the SPS had the opposite effect. Overall, the results are consistent with a working hypothesis that the GabP CAR contains extensive functional surfaces that may be manipulated by insertion mutagenesis to alter the specificity ( k (cat)/ K (m)) phenotype. The thermodynamic basis of TSR analysis provides generality, suggesting that amino acid insertions could affect specificity in many other transporters, particularly those such as the E. coli phenylalanine permease PheP [Pi, Chow and Pittard (2002) J. Bacteriol. 184, 5842-5847] that have a functionally significant CAR-like domain.

Role of the transmembrane domains of prM and E proteins in the formation of yellow fever virus envelope.

Flavivirus envelope proteins have been shown to play a major role in virus assembly. These proteins are anchored into cellular and viral membranes by their C-terminal domain. These domains are composed of two hydrophobic stretches separated by a short hydrophilic segment containing at least one charged residue. We investigated the role of the transmembrane domains of prM and E in the envelope formation of the flavivirus yellow fever virus (YFV). Alanine scanning insertion mutagenesis has been used to examine the role of the transmembrane domains of prM and E in YFV subviral particle formation. Most of the insertions had a dramatic effect on the release of YFV subviral particles. Some of these mutations were introduced into the viral genome. The ability of these mutant viruses to produce infectious particles was severely reduced. The alanine insertions did not affect prM-E heterodimerization. In addition, replacement of the charged residues present in the middle of the transmembrane domains had no effect on subviral particle release. Taken together, these data indicate that the transmembrane domains of prM and E play a crucial role in the biogenesis of YFV envelope. In addition, these data indicate some differences between the transmembrane domains of the hepaciviruses and the flaviviruses.

Definition of an Inhibitory Juxtamembrane WW-like Domain in the Platelet-derived Growth Factor β Receptor

A variety of tumors contain activating mutations in the cytoplasmic juxtamembrane domain of the type III family of receptor-tyrosine kinases, and some constructed mutations in this domain induce ligand-independent receptor activation. To explore the role of this domain in regulation of receptor activity, we subjected the juxtamembrane domain of the murine platelet-derived growth factor (PDGF) beta receptor to alanine-scanning mutagenesis. The mutant receptors were expressed in Ba/F3 cells and tested for constitutive tyrosine phosphorylation, association with phosphatidylinositol 3'-kinase, and their ability to induce cell survival and proliferation in the absence of interleukin-3. The mutant receptors accumulated to similar levels and appeared to undergo a normal PDGF-induced increase in tyrosine phosphorylation. Alanine substitutions at numerous positions located throughout the juxtamembrane domain caused constitutive receptor activation, as did an alanine insertion in the membrane-proximal segment of the juxtamembrane domain and a six-amino acid deletion in the center of the domain. It is possible to model the PDGF receptor juxtamembrane domain as a short alpha-helix followed by a three-stranded beta-sheet very similar to the known structures of WW domains. Strikingly, the activating mutations clustered in the central portions of the first and second beta strands and along one face of the beta-sheet, whereas the loops connecting the strands were largely devoid of mutationally sensitive positions. These findings provide strong support for the model that the activating mutations in the juxtamembrane region stimulate receptor activity by disrupting an inhibitory WW-like domain.

Organization of the membrane domain of the human liver sodium/bile acid cotransporter.

Mammalian sodium/bile acid cotransporters (SBATs) are glycoproteins with an exoplasmic N-terminus, an odd number of transmembrane regions, and a cytoplasmic C-terminus. Various algorithms predict eight or nine membrane-embedded regions derived from nine hydrophobic stretches of the protein (H1-H9). Three methods were used to define which of these were transmembrane or membrane-associated segments in the liver bile acid transporter. The first was in vitro translation/insertion scanning using either single hydrophobic sequences between the N-terminal domain of the alpha-subunit of the gastric H,K-ATPase and the C-terminal domain of the beta-subunit that contains five N-linked glycosylation exoplasmic flags or using constructs beginning with the N-terminus of the transporter of various lengths and again ending in the C-terminus of the H,K-ATPase beta-subunit. Seven of the predicted segments, but not the amphipathic H3 and H8 sequences, insert as both individual signal anchor and stop transfer sequences in the reporter constructs. These sequences, H3 and H8, are contained within two postulated long exoplasmic loops in the classical seven-transmembrane segment model. The H3 segment acts as a partial stop transfer signal when expressed downstream of the endogenous H2. In a similar manner, the other amphipathic segment, H8, inserts as a signal anchor sequence when translated in the context with the upstream transporter sequence in two different glycosylation constructs. Alanine insertion scanning identified regions of the transporter requiring precise alignment of sequence to form competent secondary structures. The transport activity of these mutants was evaluated either in native protein or in a yellow fluorescent protein (YFP) fusion protein construct. All alanine insertions in H3 and H8 abolished taurocholate uptake, suggesting that both these regions have structures with critical intramolecular interactions. Moreover, these insertions also prevented trafficking to the plasma membrane as assessed by confocal microscopy with a polyclonal antibody against either the C-terminus of the transporter or the YFP signal of the YFP-transporter fusion protein. Two glycosylation signals inserted in the first postulated loop region and four of five such signals in the second postulated loop region were not recognized by the oligosaccharide transferase, and the L256N mutation exhibited 10% glycosylation and was inactive. These findings support a topography with nine membrane-spanning or membrane-associated segments.

Congenital alpha(2)-plasmin inhibitor deficiencies: a review.

Congenital alpha(2)-plasmin inhibitor deficiencies: a review.

Inactivation of the serpin α 2-antiplasmin by stromelysin-1

Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) hydrolyzes the Met 374-Ser 375 (P3-P2), Glu 416-Leu 417 and Ser 432-Leu 433 peptide bonds in human α 2-antiplasmin (α 2-AP), the main physiological plasmin inhibitor. Cleavage is completely abolished in the presence of the MMP inhibitors EDTA or 1,10-phenanthroline. At enzyme/substrate ratio of 1:10 at 37°C, α 2-AP protein cleavage occurs with a half-life of 8 min, and is associated with rapid loss of inhibitory activity towards plasmin with a half-life of 5 min. α 2-AP cleaved by MMP-3 does no longer form a stable complex with plasmin, as shown by SDS-PAGE, and does no longer interact with plasminogen, as shown by crossed immunoelectrophoresis with plasminogen added to the gel. These data are compatible with the removal of a COOH-terminal fragment containing the reactive site peptide bond and the plasmin(ogen)-binding site. In addition, MMP-3 cleaves the Pro 19-Leu 20 peptide bond in α 2-AP, thereby removing the fibrin-binding site from the inhibitor. A dysfunctional α 2-AP variant (Ala-α 2-AP or α 2-AP Enschede), with an alanine insertion in the reactive site sequence converting it from a plasmin inhibitor into a substrate, was also efficiently cleaved by MMP-3 (half-life of 13 min at 37°C and enzyme/substrate ratio of 1:10). Cleavage and inactivation of α 2-AP by MMP-3 may constitute a mechanism favoring local plasmin-mediated proteolysis.

The Erythropoietin Receptor Cytosolic Juxtamembrane Domain Contains an Essential, Precisely Oriented, Hydrophobic Motif

We report that the erythropoietin receptor cytosolic juxtamembrane region is conformationally rigid and contains a hydrophobic motif, composed of residues L 253, I 257, and W 258, that is crucial for Janus kinase 2 (JAK2) activation and receptor signaling. Alanine insertion mutagenesis shows that the orientation of this motif and not its distance from the membrane bilayer is critical. Intragenic complementation studies suggest that L 253 is contained within an α helix functionally continuous to the transmembrane α helix. The α-helical orientation of L 253 is required not for JAK2 activation but for activated JAK2 to induce phosphorylation of the erythropoietin receptor. This motif is highly conserved among cytokine receptors and couples ligand-induced conformational changes in the receptor to intracellular activation of JAK2.

The Transmembrane Domains of Hepatitis C Virus Envelope Glycoproteins E1 and E2 Play a Major Role in Heterodimerization

Oligomerization of viral envelope proteins is essential to control virus assembly and fusion. The transmembrane domains (TMDs) of hepatitis C virus envelope glycoproteins E1 and E2 have been shown to play multiple functions during the biogenesis of E1E2 heterodimer. This makes them very unique among known transmembrane sequences. In this report, we used alanine scanning insertion mutagenesis in the TMDs of E1 and E2 to examine their role in the assembly of E1E2 heterodimer. Alanine insertion within the center of the TMDs of E1 or E2 or in the N-terminal part of the TMD of E1 dramatically reduced heterodimerization, demonstrating the essential role played by these domains in the assembly of hepatitis C virus envelope glycoproteins. To better understand the alanine scanning data obtained for the TMD of E1 which contains GXXXG motifs, we analyzed by circular dichroism and nuclear magnetic resonance the three-dimensional structure of the E1-(350-370) peptide encompassing the N-terminal sequence of the TMD of E1 involved in heterodimerization. Alanine scanning results and the three-dimensional molecular model we obtained provide the first framework for a molecular level understanding of the mechanism of hepatitis C virus envelope glycoprotein heterodimerization.

Mutational Analysis of the Subgroup A Avian Sarcoma and Leukosis Virus Putative Fusion Peptide Domain

Short hydrophobic regions referred to as fusion peptide domains (FPDs) at or near the amino terminus of the membrane-anchoring subunit of viral glycoproteins are believed to insert into the host membrane during the initial stage of enveloped viral entry. Avian sarcoma and leukosis viruses (ASLV) are unusual among retroviruses in that the region in the envelope glycoprotein (EnvA) proposed to be the FPD is internal and contains a centrally located proline residue. To begin analyzing the function of this region of EnvA, 20 substitution mutations were introduced into the putative FPD. The mutant envelope glycoproteins were evaluated for effects on virion incorporation, receptor binding, and infection. Interestingly, most of the single-substitution mutations had little effect on any of these processes. In contrast, a bulky hydrophobic substitution for the central proline reduced viral titers 15-fold without affecting virion incorporation or receptor binding, whereas substitution of glycine for the proline had only a nominal effect on EnvA function. Similar to other viral FPDs, the putative ASLV FPD has been modeled as an amphipathic helix where most of the bulky hydrophobic residues form a patch on one face of the helix. A series of alanine insertion mutations designed to interrupt the hydrophobic patch on the helix had differential effects on infectivity, and the results of that analysis together with the results observed with the substitution mutations suggest no correlation between maintenance of the hydrophobic patch and glycoprotein function.

Insertion Scanning Mutagenesis of Subunit a of the F1F0 ATP Synthase near His245and Implications on Gating of the Proton Channel

Subunit a of the E. coli F1F0 ATP synthase was probed by insertion scanning mutagenesis in a region between residues Glu219 and His245. A series of single amino acid insertions, of both alanine and aspartic acid, were constructed after the following residues: 225, 229, 233, 238, 243, and 245. The mutants were tested for growth yield, binding of F1 to membranes, dicyclohexylcarbodiimide sensitivity of ATPase activity, ATP-driven proton translocation, and passive proton permeability of membranes stripped of F1. Significant loss of function was seen only with insertions after positions 238 and 243. In contrast, both insertions after residue 225 and the alanine insertion after residue 245 were nearly identical in function to the wild type. The other insertions showed an intermediate loss of function. Missense mutations of His245 to serine and cysteine were nonfunctional, while the W241C mutant showed nearly normal ATPase function. Replacement of Leu162 by histidine failed to suppress the 245 mutants, but chemical rescue of H245S was partially successful using acetate. An interaction between Trp241 and His245 may be involved in gating a "half-channel" from the periplasmic surface of F0 to Asp61 of subunit a.

Alanine Insertion Scanning Mutagenesis of Lactose Permease Transmembrane Helices

A priori, single residue insertions into transmembrane helices are expected to be highly disruptive to protein structure and function. We have carried out a systematic analysis of the phenotypes associated with Ala insertions into transmembrane helices in lactose permease, a multispanning Escherichia coli inner membrane protein. Insertion of alanine into the center of 7 transmembrane helices was found to abolish stable integration of lactose permease into the membrane or uphill lactose transport. A more detailed Ala insertion scan was made of transmembrane helix III. The results pin-point a central region of approximately 2 helical turns that is crucial for lactose permease stability and/or activity. A Trp scan in this region identified 2 residues essential for lactose permease stability. From these results, it appears that transmembrane helices have differential sensitivities to single residue insertions and that such mutations may be useful for identifying structurally and/or functionally important helix segments.

Molecular cloning of cDNA encoding the c-kit receptor of Shiba goats and a novel alanine insertion specific to goats and sheep in the kinase insert region

The complete open reading frame (ORF) of the c-kit cDNA was cloned from a cerebellar cDNA library of the Shiba goat ( Capra hircus var Shiba) with the dominant black-eyed white phenotype. The analysis of the deduced amino acid sequence revealed the presence of a single amino acid insertion (alanine) in the kinase insert (KI) region. While the newly found alanine insertion is not correlated with the coat color phenotype of goats, it appears to be characteristic of the c-kit genes in goats and sheep. Although the biological significance of the insert remains to be investigated, its phylogenetically limited distribution will provide us with a useful and interesting tool to analyze the problems of evolution of sheep and goats in bovidae.

Different Structural Requirements at Specific Proline Residue Positions in the Conserved Proline-rich Region of Cytochrome P450 2C2

Cytochrome P450 is anchored to the endoplasmic reticulum membrane by an N-terminal transmembrane sequence with the catalytic domain facing the cytoplasmic side. Within the peptide sequence linking these two domains is a highly conserved proline-rich region. In cytochrome P450 2C2, this region has the sequence 30PPGPTPFP37. To examine the structural requirements at these proline residues, each proline was replaced with alanine, glycine, valine, or an acidic amino acid, and the activities of the mutated proteins were determined in transfected COS-1 cells. Lauric acid 1omega-hydroxylase activities of Pro30 and Pro33 mutants were less than 10% of wild type for each substitution except for alanine, which was 25-30%. In striking contrast, substitutions at Pro31, including an acidic residue, did not substantially alter activity. At positions 35 and 37, acidic amino acid substitutions reduced activity to less than 10% of wild type while substitution of the other three amino acids had little effect. The tolerance of substitutions of charged residues at Pro31 suggests that the side chain at this position is exposed to a polar environment; conversely, the reduced activity with charged substitutions, but not with uncharged substitutions at positions 35 and 37, suggests that these residues are exposed to a hydrophobic environment, presumably within the folded protein. The loss of activity with substitutions at Pro30 and Pro33 implies that the motif PXXP is important for the formation of a functional cytochrome P450 and that this sequence might have a helical structure with a repeat of three, as in the left-handed poly-L-proline II helix. Insertion of alanine between positions 29 and 30 did not substantially affect activity, but insertions between either 33 and 34 or 37 and 38 resulted in activity less than 25% of wild type. These data indicate that the position of PXXP, relative to the sequence flanking it on the C-terminal side, may be important for its function.

Thermodynamic effects of mutations on the denaturation of T4 lysozyme

We investigated the folding of substantially destabilized mutant forms of T4 lysozyme using differential scanning calorimetry and circular dichroism measurements. Three mutations in an alpha-helix in the protein's N-terminal region, the alanine insertion mutations S44[A] and K48[A], and the substitution A42K had previously been observed to result in unexpectedly low apparent enthalpy changes of melting, compared to a pseudo-wild-type reference protein. The pseudo-wild-type reference protein thermally unfolds in an essentially two-state manner. However, we found that the unfolding of the three mutant proteins has reduced cooperativity, which partially explains their lower apparent enthalpy changes. A three-state unfolding model including a discrete intermediate is necessary to describe the melting of the mutant proteins. The reduction in cooperativity must be considered for accurate calculation of the energy changes of folding. Unfolding in two stages reflects the underlying two-subdomain structure of the lysozyme protein family.

Ala-insertion scanning mutagenesis of the glycophorin A transmembrane helix: a rapid way to map helix-helix interactions in integral membrane proteins.

Alanine insertions into the glycophorin A transmembrane helix are found to disrupt helix-helix dimerization in a way that is fully consistent with earlier saturation mutagenesis data, suggesting that Ala-insertion scanning can be used to rapidly map the approximate location of structurally and/or functionally important segments in transmembrane helices.

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli.

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.

Accommodation of Amino Acid Insertions in an α-Helix of T4 Lysozyme: Structural and Thermodynamic Analysis

One to four alanines were inserted by site-directed mutagenesis at three different locations within the α-helix comprising residues 39 to 50 in bacteriophage T4 lysozyme. All insertion mutants were correctly folded and catalytically active although the insertions led to a thermal destabilization by 1·1 to 4·2 kcal/mol when compared to wild-type. Variants that restored part of the loss in stability associated with the initial alanine insertions could be found by randomizing the inserted amino acids. In selected cases, directed mutagenesis of adjacent residues was also used to regain stability. Structural information obtained from X-ray crystallography and/or 2D-NMR for 10 different variants showed distinct ways in which the protein responded to the amino acid insertions: (1) The inserted amino acids were incorporated into the helix by replacing preceding wild-type amino acids and causing a shift in register towards the N terminus. As a consequence, wild-type amino acids were translocated from the helix into the preceding loop. (2) Insertions caused a "looping out" within the α-helix. In this case the perturbation was confined to a minimal region in the immediate vicinity of the insertion. No change in the length of the helix was detected in either case. The structural response appears to be determined by the maintenance of the hydrophobic interface between the helix and the rest of the protein. This interface remains essentially intact in all variant structures. The results exemplify the plasticity and the adaptability of the protein structure which allows the incorporation of additional amino acids into a secondary structure elements without large structural perturbations, as long as vital internal interactions are preserved. They also suggest the loops in proteins related by evolution can vary in length not only because of insertions within the loops themselves but also as a consequence of insertions within neighboring secondary structure elements.