Effects of calcium-binding sites in the S2-S3 loop on human and Nematostella vectensis TRPM2 channel gating processes.

As a crucial signaling molecule, calcium plays a critical role in many physiological and pathological processes by regulating ion channel activity. Recently, one study resolved the structure of the transient receptor potential melastatin 2 (TRPM2) channel from Nematostella vectensis (nvTRPM2). This identified a calcium-binding site in the S2-S3 loop, while its effect on channel gating remains unclear. Here, we investigated the role of this calcium-binding site in both nvTRPM2 and human TRPM2 (hTRPM2) by mutagenesis and patch-clamp recording. Unlike hTRPM2, nvTRPM2 cannot be activated by calcium alone. Moreover, the inactivation rate of nvTRPM2 was decreased as intracellular calcium concentration was increased. In addition, our results showed that the four key residues in the calcium-binding site of S2-S3 loop have similar effects on the gating processes of nvTRPM2 and hTRPM2. Among them, the mutations at negatively charged residues (glutamate and aspartate) substantially decreased the currents of nvTRPM2 and hTRPM2. This suggests that these sites are essential for calcium-dependent channel gating. For the charge-neutralizing residues (glutamine and asparagine) in the calcium-binding site, our data showed that glutamine mutating to alanine or glutamate did not affect the channel activity, but glutamine mutating to lysine caused loss of function. Asparagine mutating to aspartate still remained functional, while asparagine mutating to alanine or lysine led to little channel activity. These results suggest that the side chain of glutamine has a less contribution to channel gating than does asparagine. However, our data indicated that both glutamine mutating to alanine or glutamate and asparagine mutating to aspartate accelerated the channel inactivation rate, suggesting that the calcium-binding site in the S2-S3 loop is important for calcium-dependent channel inactivation. Taken together, our results uncovered the effect of four key residues in the S2-S3 loop of TRPM2 on the TRPM2 gating process.

Asparagine 79 is an important amino acid for catalytic activity and substrate specificity of bile salt hydrolase (BSH).

Microbial bile salt hydrolases (BSHs), a member of cholylglycine hydrolase (CGH) family, catalyze the hydrolysis of glycine and taurine-linked bile salts in the small intestine of human. BSH is evolutionarily related to penicillin V acylase (PVA) which hydrolyses a penicillin V and is also a member of CGH family. Although, five of the six amino acids, C2, R16, D19, N170, N79 and R223, supposed to be responsible for catalytic activity of BSH enzyme, are strictly conserved in all CGH family members, N79 is partially conserved in this family. In this study, in order to analyze the correlation between N79 and catalytic activity or substrate specificity of BSH, the polar and acidic N79 was substituted for the aliphatic and hydrophobic V79 by PCR-based site directed mutagenesis and mutant recombinant BSH was expressed in E. coli BLR(DE3). While the effects of the mutation on catalytic activity and substrate specificity of BSH were detected by ninhydrin assay. The effect of this mutation on the stability of the BSH was observed by SDS-PAGE analysis. Although V79 mutation resulted in stable BSH, it reduced the catalytic activity and altered substrate specificity of BSH. The results suggested that N79 might be important for substrate binding and catalytic turnover of BSH.

Effects of calcium-binding sites in the S2-S3 loop on human and Nematostella vectensis TRPM2 channel gating processes / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

As a crucial signaling molecule, calcium plays a critical role in many physiological and pathological processes by regulating ion channel activity. Recently, one study resolved the structure of the transient receptor potential melastatin 2 (TRPM2) channel from Nematostella vectensis (nvTRPM2). This identified a calcium-binding site in the S2-S3 loop, while its effect on channel gating remains unclear. Here, we investigated the role of this calcium-binding site in both nvTRPM2 and human TRPM2 (hTRPM2) by mutagenesis and patch-clamp recording. Unlike hTRPM2, nvTRPM2 cannot be activated by calcium alone. Moreover, the inactivation rate of nvTRPM2 was decreased as intracellular calcium concentration was increased. In addition, our results showed that the four key residues in the calcium-binding site of S2-S3 loop have similar effects on the gating processes of nvTRPM2 and hTRPM2. Among them, the mutations at negatively charged residues (glutamate and aspartate) substantially decreased the currents of nvTRPM2 and hTRPM2. This suggests that these sites are essential for calcium-dependent channel gating. For the charge-neutralizing residues (glutamine and asparagine) in the calcium-binding site, our data showed that glutamine mutating to alanine or glutamate did not affect the channel activity, but glutamine mutating to lysine caused loss of function. Asparagine mutating to aspartate still remained functional, while asparagine mutating to alanine or lysine led to little channel activity. These results suggest that the side chain of glutamine has a less contribution to channel gating than does asparagine. However, our data indicated that both glutamine mutating to alanine or glutamate and asparagine mutating to aspartate accelerated the channel inactivation rate, suggesting that the calcium-binding site in the S2-S3 loop is important for calcium-dependent channel inactivation. Taken together, our results uncovered the effect of four key residues in the S2-S3 loop of TRPM2 on the TRPM2 gating process.

Trade-off between soluble protein production and nutritional storage in Bromeliaceae.

BACKGROUND AND AIMS: Bromeliads are able to occupy some of the most nutrient-poor environments especially because they possess absorptive leaf trichomes, leaves organized in rosettes, distinct photosynthetic pathways [C3, Crassulacean acid metabolism (CAM) or facultative C3-CAM], and may present an epiphytic habit. The more derived features related to these traits are described for the Tillandsioideae subfamily. In this context, the aims of this study were to evaluate how terrestrial predators contribute to the nutrition and performance of bromeliad species, subfamilies and ecophysiological types, whether these species differ in their ecophysiological traits and whether the physiological outcomes are consistent among subfamilies and types (e.g. presence/absence of tank, soil/tank/atmosphere source of nutrients, trichomes/roots access to nutrients). METHODS: Isotopic (15N-enriched predator faeces) and physiological methods (analyses of plant protein, amino acids, growth, leaf mass per area and total N incorporated) in greenhouse experiments were used to investigate the ecophysiological contrasts between Tillandsioideae and Bromelioideae, and among ecophysiological types when a predatory anuran contributes to their nutrition. KEY RESULTS: It was observed that Bromelioideae had higher concentrations of soluble protein and only one species grew more (Ananas bracteatus), while Tillandsioideae showed higher concentrations of total amino acids, asparagine and did not grow. The ecophysiological types that showed similar protein contents also had similar growth. Additionally, an ordination analysis showed that the subfamilies and ecophysiological types were discrepant considering the results of the total nitrogen incorporated from predators, soluble protein and asparagine concentrations, relative growth rate and leaf mass per area. CONCLUSIONS: Bromeliad subfamilies showed a trade-off between two strategies: Tillandsioideae stored nitrogen into amino acids possibly for transamination reactions during nutritional stress and did not grow, whereas Bromelioideae used nitrogen for soluble protein production for immediate utilization, possibly for fast growth. These results highlight that Bromeliaceae evolution may be directly associated with the ability to stock nutrients.

Enzyme Architecture: A Startling Role for Asn270 in Glycerol 3-Phosphate Dehydrogenase-Catalyzed Hydride Transfer.

The side chains of R269 and N270 interact with the phosphodianion of dihydroxyacetone phosphate (DHAP) bound to glycerol 3-phosphate dehydrogenase (GPDH). The R269A, N270A, and R269A/N270A mutations of GPDH result in 9.1, 5.6, and 11.5 kcal/mol destabilization, respectively, of the transition state for GPDH-catalyzed reduction of DHAP by the reduced form of nicotinamide adenine dinucleotide. The N270A mutation results in a 7.7 kcal/mol decrease in the intrinsic phosphodianion binding energy, which is larger than the 5.6 kcal/mol effect of the mutation on the stability of the transition state for reduction of DHAP; a 2.2 kcal/mol stabilization of the transition state for unactivated hydride transfer to the truncated substrate glycolaldehyde (GA); and a change in the effect of phosphite dianion on GPDH-catalyzed reduction of GA, from strongly activating to inhibiting. The N270A mutation breaks the network of hydrogen bonding side chains, Asn270, Thr264, Asn205, Lys204, Asp260, and Lys120, which connect the dianion activation and catalytic sites of GPDH. We propose that this disruption dramatically alters the performance of GPDH at these sites.

Asparagine deprivation mediated by Salmonella asparaginase causes suppression of activation-induced T cell metabolic reprogramming.

Salmonellae are pathogenic bacteria that induce immunosuppression by mechanisms that remain largely unknown. Previously, we showed that a putative type II l-asparaginase produced by Salmonella Typhimurium inhibits T cell responses and mediates virulence in a murine model of infection. Here, we report that this putative L-asparaginase exhibits L-asparagine hydrolase activity required for Salmonella Typhimurium to inhibit T cells. We show that L-asparagine is a nutrient important for T cell activation and that L-asparagine deprivation, such as that mediated by the Salmonella Typhimurium L-asparaginase, causes suppression of activation-induced mammalian target of rapamycin signaling, autophagy, Myc expression, and L-lactate secretion. We also show that L-asparagine deprivation mediated by the Salmonella Typhimurium L-asparaginase causes suppression of cellular processes and pathways involved in protein synthesis, metabolism, and immune response. Our results advance knowledge of a mechanism used by Salmonella Typhimurium to inhibit T cell responses and mediate virulence, and provide new insights into the prerequisites of T cell activation. We propose a model in which l-asparagine deprivation inhibits T cell exit from quiescence by causing suppression of activation-induced metabolic reprogramming.

An asparagine residue at the N-terminus affects the maturation process of low molecular weight glutenin subunits of wheat endosperm.

BACKGROUND: Wheat glutenin polymers are made up of two main subunit types, the high- (HMW-GS) and low- (LMW-GS) molecular weight subunits. These latter are represented by heterogeneous proteins. The most common, based on the first amino acid of the mature sequence, are known as LMW-m and LMW-s types. The mature sequences differ as a consequence of three extra amino acids (MET-) at the N-terminus of LMW-m types. The nucleotide sequences of their encoding genes are, however, nearly identical, so that the relationship between gene and protein sequences is difficult to ascertain.It has been hypothesized that the presence of an asparagine residue in position 23 of the complete coding sequence for the LMW-s type might account for the observed three-residue shortened sequence, as a consequence of cleavage at the asparagine by an asparaginyl endopeptidase. RESULTS: We performed site-directed mutagenesis of a LMW-s gene to replace asparagine at position 23 with threonine and thus convert it to a candidate LMW-m type gene. Similarly, a candidate LMW-m type gene was mutated at position 23 to replace threonine with asparagine. Next, we produced transgenic durum wheat (cultivar Svevo) lines by introducing the mutated versions of the LMW-m and LMW-s genes, along with the wild type counterpart of the LMW-m gene.Proteomic comparisons between the transgenic and null segregant plants enabled identification of transgenic proteins by mass spectrometry analyses and Edman N-terminal sequencing. CONCLUSIONS: Our results show that the formation of LMW-s type relies on the presence of an asparagine residue close to the N-terminus generated by signal peptide cleavage, and that LMW-GS can be quantitatively processed most likely by vacuolar asparaginyl endoproteases, suggesting that those accumulated in the vacuole are not sequestered into stable aggregates that would hinder the action of proteolytic enzymes. Rather, whatever is the mechanism of glutenin polymer transport to the vacuole, the proteins remain available for proteolytic processing, and can be converted to the mature form by the removal of a short N-terminal sequence.

Exploring the structural basis of the selective inhibition of monoamine oxidase A by dicarbonitrile aminoheterocycles: role of Asn181 and Ile335 validated by spectroscopic and computational studies.

Since cyanide potentiates the inhibitory activity of several monoamine oxidase (MAO) inhibitors, a series of carbonitrile-containing aminoheterocycles was examined to explore the role of nitriles in determining the inhibitory activity against MAO. Dicarbonitrile aminofurans were found to be potent, selective inhibitors against MAO A. The origin of the MAO A selectivity was identified by combining spectroscopic and computational methods. Spectroscopic changes induced in MAO A by mono- and dicarbonitrile inhibitors were different, providing experimental evidence for distinct binding modes to the enzyme. Similar differences were also found between the binding of dicarbonitrile compounds to MAO A and to MAO B. Stabilization of the flavin anionic semiquinone by monocarbonitrile compounds, but destabilization by dicarbonitriles, provided further support to the distinct binding modes of these compounds and their interaction with the flavin ring. Molecular modeling studies supported the role played by the nitrile and amino groups in anchoring the inhibitor to the binding cavity. In particular, the results highlight the role of Asn181 and Ile335 in assisting the interaction of the nitrile-containing aminofuran ring. The network of interactions afforded by the specific attachment of these functional groups provides useful guidelines for the design of selective, reversible MAO A inhibitors.

Asparagine-linked glycans determine the cytotoxic capacity of eosinophil cationic protein (ECP).

Eosinophil cationic protein (ECP) is a toxic, granule-stored protein of the eosinophil granulocyte. It is a heterogeneous protein; molecular weights can differ from 15 to 22 kDa, due to glycosylations. We purified high molecular weight ECP from blood donors with the ECP434GG (rs2073342) genotype, with the aim of examining whether removal of carbohydrates could enhance the cytotoxic capacity. The cytotoxic activity of the ECP pools was tested against the NCI-H69 cell line, before and after enzymatic deglycosylation. ECP was also analysed by SELDI-TOF MS to monitor the changes in molecular mass after deglycosylation. Five high molecular weight pools of ECP (HMW-ECP I-V) with decreasing degrees of glycosylation were tested at concentrations ranging from 0.02 to 0.6 µM. The activity ranged from EC50 of >0.6 µM to 0.04 µM; HMW-ECP II had the lowest activity and HMW-ECP V the highest. After deglycosylation with N-glycosidase F, pools HMW-ECP I-III were reduced to the same molecular weight of 15.78 kDa and acquired potent cytotoxic activities. HMW-ECP IV and V with molecular species at 16.3 and 16.1 kDa were highly cytotoxic as such and were only partially deglycosylated, with slight enhancement of the toxic properties. The results suggest the presence of several HMW-ECP molecular species with differences in their post-translational modifications and cytotoxic properties. We conclude that a fraction of native ECP is stored in a non-cytotoxic form, which can be converted into a cytotoxic form by N-deglycosylation, whereas another fraction is stored as a highly cytotoxic form carrying different post-translational modifications.

A protein engineering approach differentiates the functional importance of carbohydrate moieties of interleukin-5 receptor α.

Human interleukin-5 receptor α (IL5Rα) is a glycoprotein that contains four N-glycosylation sites in the extracellular region. Previously, we found that enzymatic deglycosylation of IL5Rα resulted in complete loss of IL5 binding. To localize the functionally important carbohydrate moieties, we employed site-directed mutagenesis at the N-glycosylation sites (Asn(15), Asn(111), Asn(196), and Asn(224)). Because Asn-to-Gln mutagenesis caused a significant loss of structural integrity, we used diverse mutations to identify stability-preserving changes. We also rationally designed mutations at and around the N-glycosylation sites based on sequence alignment with mouse IL5Rα and other cytokine receptors. These approaches were most successful at Asn(15), Asn(111), and Asn(224). In contrast, any replacement at Asn(196) severely reduced stability, with the N196T mutant having a reduced binding affinity for IL5 and diminished biological activity because of the lack of cell surface expression. Lectin inhibition analysis suggested that the carbohydrate at Asn(196) is unlikely involved in direct ligand binding. Taking this into account, we constructed a stable variant, with triple mutational deglycosylation (N15D, I109V/V110T/N111D, and L223R/N224Q). The re-engineered protein retained Asn(196) while the other three glycosylation sites were eliminated. This mostly deglycosylated variant had the same ligand binding affinity and biological activity as fully glycosylated IL5Rα, thus demonstrating a unique role for Asn(196) glycosylation in IL5Rα function. The results suggest that unique carbohydrate groups in multiglycosylated receptors can be utilized asymmetrically for function.

Opposing effects of glutamine and asparagine govern prion formation by intrinsically disordered proteins.

Sequences rich in glutamine (Q) and asparagine (N) residues often fail to fold at the monomer level. This, coupled to their unusual hydrogen-bonding abilities, provides the driving force to switch between disordered monomers and amyloids. Such transitions govern processes as diverse as human protein-folding diseases, bacterial biofilm assembly, and the inheritance of yeast prions (protein-based genetic elements). A systematic survey of prion-forming domains suggested that Q and N residues have distinct effects on amyloid formation. Here, we use cell biological, biochemical, and computational techniques to compare Q/N-rich protein variants, replacing Ns with Qs and Qs with Ns. We find that the two residues have strong and opposing effects: N richness promotes assembly of benign self-templating amyloids; Q richness promotes formation of toxic nonamyloid conformers. Molecular simulations focusing on intrinsic folding differences between Qs and Ns suggest that their different behaviors are due to the enhanced turn-forming propensity of Ns over Qs.

Asparaginyl-tRNA synthetase pre-transfer editing assay.

Aminoacyl-tRNA synthetases (AARSs) are a structurally heterogeneous family of enzymes present in prokaryotes, archaea and eukaryotes. They catalyze the attachment of tRNA to its corresponding amino acid via an aminoacyl adenylate intermediate. Errors in protein synthesis will occur if an incorrect amino acid is attached to the tRNA. To prevent such errors, AARSs have evolved editing mechanisms that eliminate incorrect aminoacyl adenylates (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Various AARSs are the targets of natural antibiotics and are considered validated targets for chemotherapy. We have developed a high-throughput screening (HTS) assay measuring the pre-transfer editing activity of pathogen-derived asparaginyl-tRNA synthetase (AsnRS). This was achieved by monitoring the formation of pyrophosphate via cleavage to phosphate, which was quantified by reaction with Malachite Green. L-Aspartate-ß-hydroxamate, an asparagine analogue, was most effective in promoting the editing activity of AsnRS from Brugia malayi (BmAsnRS) and Staphylococcus epidermidis (SeAsnRS) with KM values close to 100 mM. The assay sensitivity was enhanced by the thiol agents, DTT and L-Cysteine, which significantly increased the turn-over of aminoacyl adenylate by BmAsnRS, but not SeAsnRS. The HTS assay was used to screen a library of 37,120 natural-product extracts for inhibitors of BmAsnRS. A small number of extracts that inhibited the pre-transfer editing by BmAsnRS was identified for future isolation of the active component(s). The principle of this assay can be applied to all enzymes having a pre- or post-editing activity.

Ancient atmospheres and the evolution of oxygen sensing via the hypoxia-inducible factor in metazoans.

Metazoan diversification occurred during a time when atmospheric oxygen levels fluctuated between 15 and 30%. The hypoxia-inducible factor (HIF) is a primary regulator of the adaptive transcriptional response to hypoxia. Although the HIF pathway is highly conserved, its complexity increased during periods when atmospheric oxygen concentrations were increasing. Thus atmospheric oxygen levels may have provided a selection force on the development of cellular oxygen-sensing pathways.

A transmembrane amino acid in the GABAA receptor ß2 subunit critical for the actions of alcohols and anesthetics.

Alcohols and inhaled anesthetics enhance the function of GABA(A) receptors containing α, ß, and γ subunits. Molecular analysis has focused on the role of the α subunits; however, there is evidence that the ß subunits may also be important. The goal of our study was to determine whether Asn265, which is homologous to the site implicated in the α subunit (Ser270), contributes to an alcohol and volatile anesthetic binding site in the GABA(A) receptor ß(2) subunit. We substituted cysteine for Asn265 and exposed the mutant to the sulfhydryl-specific reagent octyl methanethiosulfonate (OMTS). We used two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes and found that, after OMTS application, GABA-induced currents were irreversibly potentiated in mutant α(1)ß(2)(N265C)γ(2S) receptors [but not α(1)ß(2)(I264C)γ(2S)], presumably because of the covalent linking of octanethiol to the thiol group in the substituted cysteine. It is noteworthy that this effect was blocked when OMTS was applied in the presence of octanol. We found that potentiation by butanol, octanol, or isoflurane in the N265C mutant was nearly abolished after the application of OMTS, suggesting that an alcohol and volatile anesthetic binding site at position 265 of the ß(2) subunit was irreversibly occupied by octanethiol and consequently prevented butanol or isoflurane from binding and producing their effects. OMTS did not affect modulation or direct activation by pentobarbital, but there was a partial reduction of allosteric modulation by flunitrazepam and alphaxalone in mutant α(1)ß(2)(N265C)γ(2S) receptors after OMTS was applied. Our findings provide evidence that Asn265 may contribute to an alcohol and anesthetic binding site.

Role of the Ser-287-Asn replacement in the hydrolysis spectrum extension of AmpC beta-lactamases in Escherichia coli.

Two AmpC variants harboring the S287N substitution were obtained by mutagenesis from cephalosporinases representative of the phylogenetic groups A and B2 of Escherichia coli. Their biochemical characterization revealed that the S287N replacement led to an important increase in the catalytic efficiency toward extended-spectrum cephalosporins in the AmpC beta-lactamase of group A only.

The carbohydrate at asparagine 386 on HIV-1 gp120 is not essential for protein folding and function but is involved in immune evasion.

BACKGROUND: The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for approximately 50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain. RESULTS: The 386 carbohydrate was not essential for folding of wt gp120. However, its removal improved folding of a gp120 variant lacking the 385-418 disulfide bond, suggesting that it plays an auxiliary role in protein folding in the presence of this disulfide bond. The 386 carbohydrate was not critical for gp120 binding to dendritic cells (DC) and DC-mediated HIV-1 transmission to T cells. In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12. Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent. Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies. CONCLUSION: The carbohydrate at position 386 is not essential for protein folding and function, but is involved in the protection of the CD4BS from antibodies. Removal of this sugar in the context of trimeric Env immunogens may therefore improve the elicitation of neutralizing CD4BS antibodies.

Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS.

BACKGROUND: CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively. RESULTS: Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure. CONCLUSION: Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.

Hb Santa Clara (beta 97His-->Asn), a human haemoglobin variant: functional characterization and structure modelling.

This study examines the functional and structural effects of amino acid substitution at alpha(1)beta(2) interface of Hb Santa Clara (beta 97His-->Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygen-binding experiments. Functional studies outlined an increased oxygen affinity, reduced effect of organic phosphates and a reduced Bohr effect with respect to HbA. In view of the primary role of this interface in the cooperative quaternary transition from the T to R conformational state, a theoretical three-dimensional model of Hb Santa Clara was generated. Structural investigations suggest that replacement of Asn for His beta 97 results in a significant stabilization of the high affinity R-state of the haemoglobin molecule with respect to the low affinity T-state. The role of beta FG4 position has been further examined by computational models of known beta FG4 variants, namely Hb Malmö (beta 97His-->Gln), Hb Wood (beta 97His-->Leu), Hb Nagoya (beta 97His-->Pro) and Hb Moriguchi (beta 97His-->Tyr). These findings demonstrate that, among the various residues at the alpha(1)beta(2) (and alpha(2)beta(1)) intersubunit interface, His beta FG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin.

A novel tripartite motif involved in aquaporin topogenesis, monomer folding and tetramerization.

Aquaporin (AQP) folding in the endoplasmic reticulum is characterized by two distinct pathways of membrane insertion that arise from divergent residues within the second transmembrane segment. We now show that in AQP1 these residues (Asn49 and Lys51) interact with Asp185 at the C terminus of TM5 to form a polar, quaternary structural motif that influences multiple stages of folding. Asn49 and Asp185 form an intramolecular hydrogen bond needed for proper helical packing, monomer formation and function. In contrast, Lys51 interacts with Asp185 on an adjacent monomer to stabilize the AQP1 tetramer. Although these residues are unique to AQP1, they share a highly conserved architecture whose functional properties can be transferred to other family members. These findings suggest a general mechanism by which evolutionary divergence of membrane proteins can confer new functional properties via alternative folding pathways that give rise to a common final structure.