Engineering of Ion Sensing by the Cystathionine ��-Synthase Module of the ABC Transporter OpuA

We have previously shown that the C-terminal cystathionine β-synthase (CBS) domains of the nucleotide-binding domains of the ABC transporter OpuA, in conjunction with an anionic membrane surface function, act as sensor of internal ionic strength (I_in). Here, we show that a surface-exposed cationic region in the CBS module domain is critical for ion sensing. The consecutive substitution of up to five cationic residues led to a gradual decrease of the ionic strength dependence of transport. In fact, a 5-fold mutant was essentially independent of salt in the range from 0 to 250 mm KCl (or NaCl), supplemented to medium of 30 mm potassium phosphate. Importantly, the threshold temperature for transport was lowered by 5–7 °C and the temperature coefficient Q₁₀ was lowered from 8 to ∼1.5 in the 5-fold mutant, indicating that large conformational changes are accompanying the CBS-mediated regulation of transport. Furthermore, by replacing the anionic C-terminal tail residues that extend the CBS module with histidines, the transport of OpuA became pH-dependent, presumably by additional charge interactions of the histidine residues with the membrane. The pH dependence was not observed at high ionic strength. Altogether the analyses of the CBS mutants support the notion that the osmotic regulation of OpuA involves a simple biophysical switching mechanism, in which nonspecific electrostatic interactions of a protein module with the membrane are sufficient to lock the transporter in the inactive state.In their natural habitats microorganisms are often exposed to changes in the concentration of solutes in the environment (1). A sudden increase in the medium osmolality results in loss of water from the cell, loss of turgor, a decrease in cell volume, and an increase in intracellular osmolyte concentration. Osmoregulatory transporters such as OpuA in Lactococcus lactis, ProP in Escherichia coli, and BetP in Corynebacterium glutamicum diminish the consequences of the osmotic stress by mediating the uptake of compatible solutes upon an increase in extracellular osmolality (2–4). For the ATP-binding cassette (ABC)⁵ transporter OpuA, it has been shown that the system, reconstituted in proteoliposomes, is activated by increased concentrations of lumenal ions (increased internal ionic strength) (2, 5, 6). This activation is instantaneous both in vivo and in vitro and only requires threshold levels of ionic osmolytes. Moreover, the ionic threshold for activation is highly dependent of the ionic lipid content (charge density) of the membrane and requires the presence of so-called cystathionine β-synthase (CBS) domains, suggesting that the ionic signal is transduced to the transporter via critical interactions of the protein with membrane lipids.The ABC transporter OpuA consists of two identical nucleotide-binding domains (NBD) fused to CBS domains and two identical substrate-binding domains fused to transmembrane domains. The NBD-CBS and substrate-binding domain-transmembrane domain subunits are named OpuAA and OpuABC, respectively. Two tandem CBS domains are linked to the C-terminal end of the NBD; each domain (CBS1 and CBS2) has a β-α-β-β-α secondary structure (5) (Fig. 1A). The CBS domains are widely distributed in most if not all species of life but their function is largely unknown. Most of the CBS domains are found as tandem repeats but data base searches have also revealed tetra-repeat units (5). The crystal structures of several tandem CBS domains have been elucidated (7–9, 32), and in a number of cases it has been shown that two tandem CBS domains form dimeric structures with a total of four CBS domains per structural module (hereafter referred to as CBS module). The crystal structures of the full-length MgtE Mg²⁺ transporter confirm the dimeric configuration and show that the CBS domains undergo large conformational changes upon Mg²⁺ binding or release (10, 11). In general, ABC transporters are functional as dimers, which implies that two tandem CBS domains are present in the OpuA complex. Preliminary experiments with disulfides engineered at the interface of two tandem CBS domains in OpuA suggest that large structural rearrangements (association-dissociation of the interfaces) play a determining role in the ionic strength-regulated transport. Finally, a subset of CBS-containing proteins has a C-terminal extension, which in OpuA is highly anionic (sequence: ADIPDEDEVEEIEKEEENK) and modulates the ion sensing activity (6).Open in a separate windowFIGURE 1.Domain structure of CBS module of OpuA. A, sequence of tandem CBS domains. The predicted secondary structure is indicated above the sequence. The residues modified in this study are underlined. The amino acid sequence end-points of OpuAΔ61 and OpuAΔ119 are indicated by vertical arrows. B, homology model of tandem CBS domain of OpuA. The CBS domains were individually modeled on the crystal structure of the tandem CBS protein Ta0289 from T. acidophilum (PDB entry 1PVM), using Phyre. Ta0289 was used for the initial modeling, because its primary sequence was more similar to the CBS domains of OpuA than those of the other crystallized CBS proteins. The individual domain models were then assembled with reference to the atomic coordinates of the tandem CBS domains of IMPDH from Streptococcus pyogenes (PDB entry 1ZFJ) to form the tandem CBS pair, using PyMOL (DeLano). The positions of the (substituted) cationic residues are indicated.In this study, we have engineered the surface-exposed cationic residues of the CBS module and the C-terminal anionic tail of OpuA (Fig. 1B). The ionic strength and lipid dependence of the OpuA mutants were determined in vivo and in vitro. We show that substitution of five cationic residues for neutral amino acids is sufficient to inactivate the ionic strength sensor and convert OpuA into a constitutively active transporter. Moreover, by substituting six anionic plus four neutral residues of the C-terminal anionic tail for histidines, the transport reaction becomes strongly pH-dependent.     

Ethnic variation in tyrosinase and TYRP1 expression in photoexposed and photoprotected human skin

The relative expression of a number of key mediators of human pigmentation including tyrosinase, tyrosinase related protein-1 (TYRP1), endothelin-1 and adrenocorticotrophic hormone (ACTH) proteins were analysed and quantified in immunohistochemically stained skin sections using semiquantitative computer assisted image analysis. Comparisons were made between a range of different ethnic skin types including European, Chinese, Mexican, Indian and African at both chronically photoexposed and photoprotected sites. Melanocyte number varied little with ethnicity except in the European group which had 60-80% more melanocytes than other skin types (P < 0.01, n = 10; Student Neuman-Keuls). However, melanocyte number was increased approximately twofold in chronically photoexposed skin of all ethnic groups (P < 0.001, n = 48; paired t-test). Tyrosinase protein expression in melanocytes did not vary with ethnicity, but TYRP1 protein was significantly elevated (approximately 2.6-fold) in darkly pigmented African and Indian skin types compared with lightly pigmented Mexican, Chinese and European skin types. In melanocytes from chronically photoexposed skin, there was a modest but significant increase in the expression of tyrosinase protein (approximately 1.2-fold, P < 0.001, n = 48; paired t-test), together with a significant and slightly larger increase in the expression of TYRP1 protein (approximately 1.6-fold, P < 0.005, n = 48; paired t-test). In contrast, the expression of endothelin-1 and ACTH showed no significant variation with either ethnicity or photoexposure. These data are consistent with the view that maintenance of a chronically hyperpigmented phenotype in chronically photoexposed human skin is largely the result of a stable increase in the number of tyrosinase positive melanocytes at these sites. Moreover, the observed ethnic variation in TYRP1 protein expression suggests that TYRP1 may play a significant role in mediating ethnic differences in melanogenesis and constitutive skin pigmentation in vivo.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

Ecological characteristics of plant species and associations along two Dinaric karstic rivers (Bosnia and Herzegovina, the Balkans)

This paper describes the plant communities of two oligotrophic karstic rivers with a slight anthropogenic influence — the Trebi?at and the Li?tica — in South Bosnia and Herzegovina, their sinecology, and the relationship between vegetation and plant species and environmental parameters. According to 87 relevés, a total of 26 plant associations, using Braun-Blanquet methods, were found in the rivers and nearby surveyed area. Only nine associations were common to both rivers. Eight associations were recorded for the territory of Bosnia and Herzegovina for the first time. Associations from both rivers differed according to following parameters measured at the sampling site: water temperature, distance from the mouth (river kilometers), river width, water depth, flow, pH and slope. There were no differences in nutrient concentrations among the associations. According to Canonical Correspondence Analysis (CCA), three environmental variables out of the 11 initially considered were retained as being related to plant distribution. The first two axes explained 56.8% and 35.2% of variance of species-environment relationship in the Trebi?at and Li?tica rivers, respectively. Water depth in the Trebi?at River, and pH and river kilometers in the Li?tica River were found to be the most influential, while nutrients and other physico-chemical parameters were not significant in either river. Water depth vector was identified as an underlying environmental factor determining distribution of Potamogeton lucens and Myriophyllum verticillatum (Potamion), and Nymphaea alba (Nymphaeion albae). According to in situ measurements of light intensity, the highest coefficients of light attenuation, radiance and reflection were found at a station with dense populations of Potamogeton lucens and Nuphar luteum (Potametum lucentis). The vegetation and floristic value of the rivers is discussed as a basis for biodiversity conservation programmes and evaluating the state of these ecosystems in the future.