Neuronal PDZ domains: a promising new molecular target for inhaled anesthetics?

Despite widespread use of volatile general anesthetics in millions of patients each year, the mechanisms by which they exert multiple effects on the behavior of central neurons are poorly understood. PDZ [postsynaptic density 95 (PSD-95), discs large (Dlg), and zonula occludens-1 (ZO-1)] domains are ubiquitous protein interaction modules that participate in neuronal signaling. Recent studies have indicated that clinically relevant concentrations of inhaled anesthetics dose-dependently and specifically inhibit the PDZ domain-mediated protein interactions among multiprotein signaling complexes. These inhibitory effects are immediate, potent, reversible, and occur at a hydrophobic peptidebinding groove on the surface of the PDZ domain. Thus, the PDZ domain might be a new molecular target for inhalational anesthetics.     

Beyond the binding site: the role of the β₂-β₃ loop and extra-domain structures in PDZ domains

A general paradigm to understand protein function is to look at properties of isolated well conserved domains, such as SH3 or PDZ domains. While common features of domain families are well understood, the role of subtle differences among members of these families is less clear. Here, molecular dynamics simulations indicate that the binding mechanism in PSD95-PDZ3 is critically regulated via interactions outside the canonical binding site, involving both the poorly conserved β?-β? loop and an extra-domain helix. Using the CRIPT peptide as a prototypical ligand, our simulations suggest that a network of salt-bridges between the ligand and this loop is necessary for binding. These contacts interconvert between each other on a time scale of a few tens of nanoseconds, making them elusive to X-ray crystallography. The loop is stabilized by an extra-domain helix. The latter influences the global dynamics of the domain, considerably increasing binding affinity. We found that two key contacts between the helix and the domain, one involving the β?-β? loop, provide an atomistic interpretation of the increased affinity. Our analysis indicates that both extra-domain segments and loosely conserved regions play critical roles in PDZ binding affinity and specificity.     

PDZ支架蛋白 PDZK1通过PDZ1与PDZ3结构域与磷脂酶Cβ2羧基末端PDZ结合模块相互作用

磷脂酶C β (PLCβ)在G蛋白偶联受体 (GPCR)介导的细胞信号转导中发挥重要作用. 通过水解磷脂酰肌醇4,5二磷酸 (PIP2),磷脂酶C β可以产生3种重要的第二信使分子：二乙酰甘油 (DAG)、三磷酸肌醇 (IP3)和质子. 在果蝇中,磷脂酶C β通过它的羧基末端盘状同源区域结合模块 (PBM)与盘状同源区域 (PDZ)支架蛋白-失活无后电位D蛋白 (INAD)相互作用,从而调节果蝇的光信号传导 . 在哺乳动物中,磷脂酶C β家族有4个亚型,每1个亚型的羧基末端都有1个典型的盘状同源区域结合模块. 这一结构特点提示我们,磷脂酶C β可能通过其羧基末端的盘状同源区域结合模块与盘状同源区域支架蛋白相互作用,进而调节它们自身的细胞定位和功能. 然而,目前仍对哺乳动物磷脂酶C β家族的盘状同源区域结合蛋白知之甚少. 本文运用分析型凝胶过滤和等温滴定量热技术,系统地研究了不同磷脂酶Cβ亚型的羧基末端盘状同源区域结合模块与不同盘状同源区域蛋白质的结合. 结果表明,磷脂酶Cβ2的羧基末端盘状同源区域结合模块,可以特异地与含有4个盘状同源区域的支架蛋白-盘状同源区域蛋白1 (PDZK1）以2∶1的方式相互结合. 进一步的测定显示,磷脂酶C β2羧基末端盘状同源区域结合模块在盘状同源区域蛋白1上的结合位点为第1和第3个盘状同源区域,而它们与磷脂酶Cβ2的解离常数分别为11.8±3.4 μmol/L 和33.3±8.7 μmol/L.     

PDZ支架蛋白PDZK1通过PDZ1与PDZ3结构域与磷脂酶Cβ2羧基末端PDZ结合模块相互作用（英文）

磷脂酶Cβ(PLCβ)在G蛋白偶联受体(GPCR)介导的细胞信号转导中发挥重要作用.通过水解磷脂酰肌醇4,5二磷酸(PIP2),磷脂酶Cβ可以产生3种重要的第二信使分子:二乙酰甘油(DAG)、三磷酸肌醇(IP3)和质子.在果蝇中,磷脂酶Cβ通过它的羧基末端盘状同源区域结合模块(PBM)与盘状同源区域(PDZ)支架蛋白—失活无后电位D蛋白(INAD)相互作用,从而调节果蝇的光信号传导.在哺乳动物中,磷脂酶Cβ家族有4个亚型,每1个亚型的羧基末端都有1个典型的盘状同源区域结合模块.这一结构特点提示我们,磷脂酶Cβ可能通过其羧基末端的盘状同源区域结合模块与盘状同源区域支架蛋白相互作用,进而调节它们自身的细胞定位和功能.然而,目前仍对哺乳动物磷脂酶Cβ家族的盘状同源区域结合蛋白知之甚少.本文运用分析型凝胶过滤和等温滴定量热技术,系统地研究了不同磷脂酶Cβ亚型的羧基末端盘状同源区域结合模块与不同盘状同源区域蛋白质的结合.结果表明,磷脂酶Cβ2的羧基末端盘状同源区域结合模块,可以特异地与含有4个盘状同源区域的支架蛋白—盘状同源区域蛋白1(PDZK1)以2∶1的方式相互结合.进一步的测定显示,磷脂酶Cβ2羧基末端盘状同源区域结合模块在盘状同源区域蛋白1上的结合位点为第1和第3个盘状同源区域,而它们与磷脂酶Cβ2的解离常数分别为11.8±3.4μmol/L和33.3±8.7μmol/L.     

Linking the experiential,affective and cognitive domains in biology education: a case study – microscopy

A greater emphasis in school curricula on the technology of science would encourage teachers to engage their students more in practical work. This in turn might be expected to improve students’ attitudes towards science and enhance cognitive outcomes. The paper presents findings from a study on first-year university students’ school experience of, attitudes towards, and knowledge of, microscopy. The findings reinforce the general expectations alluded to above. They also draw attention to the importance of the lower secondary science experience – often a suboptimal one owing to a poor resource base – to the formation of student attitudes and cognitive development with respect to science.     

Characterization ofκ-casein and keratin domains in fibrinogen

Summary Following the observation of a close sequence homology between the N-terminal moiety of the -chain of fibrinogen with large parts of-casein, the occurrence of a keratin domain in the middle section of the Achain is suggested.     

Mesoscale organization of domains in the plasma membrane – beyond the lipid raft

The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid–protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the “lipid raft” concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms “membrane raft” or “membrane nanodomain” are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae.     

Direct interaction of eag domains and cyclic nucleotide–binding homology domains regulate deactivation gating in hERG channels

Human ether-á-go-go (eag)-related gene (hERG) potassium channels play a critical role in cardiac repolarization and are characterized by unusually slow closing (deactivation) kinetics. The N-terminal “eag” domain and a C-terminal C-linker/cyclic nucleotide–binding homology domain (CNBHD) are required for regulation of slow deactivation. The region between the S4 and S5 transmembrane domains (S4–S5 linker) is also implicated in this process, but the mechanism for regulation of slow deactivation is unclear. Here, using an eag domain–deleted channel (hERG Δeag) fused to Citrine fluorescent protein, we found that most channels bearing individual alanine mutations in the S4–S5 linker were directly regulated by recombinant eag domains fused to a cyan fluorescent protein (N-eag-CFP) and had robust Förster resonance energy transfer (FRET). Additionally, a channel bearing a group of eight alanine residues in the S4–S5 linker was not measurably regulated by N-eag-CFP domains, but robust FRET was measured. These findings demonstrate that the eag domain associated with all of the S4–S5 linker mutant channels. In contrast, channels that also lacked the CNBHD (hERG Δeag ΔCNBHD-Citrine) were not measurably regulated by N-eag-CFP nor was FRET detected, suggesting that the C-linker/CNBHD was required for eag domains to directly associate with the channel. In a FRET hybridization assay, N-eag-CFP had robust FRET with a C-linker/CNBHD-Citrine, suggesting a direct and specific interaction between the eag domain and the C-linker/CNBHD. Lastly, coexpression of a hERG subunit lacking the CNBHD and the distal C-terminal region (hERG ΔpCT-Citrine) with hERG Δeag-CFP subunits had FRET and partial restoration of slow deactivation. Collectively, these findings reveal that the C-linker/CNBHD, but not the S4–S5 linker, was necessary for the eag domain to associate with the channel, that the eag domain and the C-linker/CNBHD were sufficient for a direct interaction, and that an intersubunit interaction between the eag domain and the C-linker/CNBHD regulated slow deactivation in hERG channels at the plasma membrane.     

Structure of the Human Discs Large 1 PDZ2– Adenomatous Polyposis Coli Cytoskeletal Polarity Complex: Insight into Peptide Engagement and PDZ Clustering

The membrane associated guanylate kinase (MAGUK) family member, human Discs Large 1 (hDlg1) uses a PDZ domain array to interact with the polarity determinant, the Adenomatous Polyposis Coli (APC) microtubule plus end binding protein. The hDLG1-APC complex mediates a dynamic attachment between microtubule plus ends and polarized cortical determinants in epithelial cells, stem cells, and neuronal synapses. Using its multi-domain architecture, hDlg1 both scaffolds and regulates the polarity factors it engages. Molecular details underlying the hDlg1-APC interaction and insight into how the hDlg1 PDZ array may cluster and regulate its binding factors remain to be determined. Here, I present the crystal structure of the hDlg1 PDZ2-APC complex and the molecular determinants that mediate APC binding. The hDlg1 PDZ2-APC complex also provides insight into potential modes of ligand-dependent PDZ domain clustering that may parallel Dlg scaffold regulatory mechanisms. The hDlg1 PDZ2-APC complex presented here represents a core biological complex that bridges polarized cortical determinants with the dynamic microtubule cytoskeleton.     

Calcium negatively regulates an intramolecular interaction between the N-terminal recoverin homology and EF-hand motif domains and the C-terminal C1 and catalytic domains of diacylglycerol kinase α

The type I diacylglycerol kinase (DGK) isozymes (α, β and γ) contain a shared recoverin homology (RVH) domain, a tandem repeat of Ca2+-binding EF-hand motifs, two cysteine-rich C1 domains, and the catalytic domain. We previously reported that a DGKα mutant lacking the RVH domain and EF-hands was constitutively active, implying that the N-terminal region (NTR) of DGKα, consisting of the RVH domain and EF-hand motifs, intramolecularly interacts with and masks the activity of the C-terminal region (CTR), containing the C1 and catalytic domains. In this study, we demonstrate that a glutathione S-transferase (GST)-fused DGKα-NTR construct physically binds to a green fluorescent protein (GFP)-fused DGKα-CTR construct. Moreover, co-precipitation of GFP-DGKα-CTR with GST-DGKα-NTR was clearly attenuated by the addition of 1 μM Ca2+. This result indicates that Ca2+ induces dissociation of the physical interaction between DGKα-NTR and DGKα-CTR. In addition to previously reported calcium-dependent changes in the hydrophobicity and net surface charge, Ca2+ also appeared to induce a decrease in the α-helical content of DGKα-NTR. These results suggest that Ca2+-induced conformational changes in the NTR release the intramolecular association between the NTR and the CTR of DGKα.     

Editorial – Clonal plants and environmental heterogeneity – space, time and scale

The fifth Clonal Plant Workshop entitled 'Clonal plants and environmental heterogeneity – space, time and scale' was held at the University of Wales, Bangor, from the 9th to the 14th September 1997. The workshops began in 1988, when a small group of plant scientists with different backgrounds met in the Netherlands to discuss plant clonality. This meeting was followed by workshops in 1990, 1992 and 1995 in Sweden, the Czech Republic and Hungary, respectively. The purpose of the Bangor meeting was to continue to integrate knowledge from a wide range of biological disciplines to attempt a better understanding of clonal plants in the context of the heterogeneous environments in which they occur. This special issue is a collection of a number of the presentations at the Bangor workshop. The effects of spatial and temporal environmental heterogeneity on individuals, populations and communities are, of course, diverse, and this collection of papers is not intended to provide a synthesis of the ecological consequences of environmental heterogeneity. Rather, this special issue more accurately reflects progress in various areas of clonal plant research including population dynamics of ramets and genets, mathematical modelling and molecular techniques. We wish to thank all participants of the workshop for their contribution and trust that they found the meeting as stimulating and enjoyable as we did. We are indebted to the authors and the many referees for their friendly cooperation and to Plant Ecology for publishing the proceedings. We also gratefully acknowledge the financial support of the following that made the workshop possible: The British Ecological Society, The Tansley Fund of the New Phytologist Trust, The Countryside Council for Wales, The Institute of Terrestrial Ecology (Bangor) and Skye Instruments Ltd, Wales.     

Structure of the USP15 N-terminal domains: a β-hairpin mediates close association between the DUSP and UBL domains

Ubiquitin specific protease 15 (USP15) functions in COP9 signalosome mediated regulation of protein degradation and cellular signaling through catalyzing the ubiquitin deconjugation reaction of a discrete number of substrates. It influences the stability of adenomatous polyposis coli, IκBα, caspase-3, and the human papillomavirus type 16 E6. USP15 forms a subfamily with USP4 and USP11 related through a shared presence of N-terminal "domain present in ubiquitin specific proteases" (DUSP) and "ubiquitin-like" (UBL) domains (DU subfamily). Here we report the 1.5 ? resolution crystal structure of the human USP15 N-terminal domains revealing a 80 ? elongated arrangement with the DU domains aligned in tandem. This architecture is generated through formation of a defined interface that is dominated by an intervening β-hairpin structure (DU finger) that engages in an intricate hydrogen-bonding network between the domains. The UBL domain is closely related to ubiquitin among β-grasp folds but is characterized by the presence of longer loop regions and different surface characteristics, indicating that this domain is unlikely to act as ubiquitin mimic. Comparison with the related murine USP4 DUSP-UBL crystal structure reveals that the main DU interdomain contacts are conserved. Analytical ultracentrifugation, small-angle X-ray scattering, and gel filtration experiments revealed that USP15 DU is monomeric in solution. Our data provide a framework to advance study of the structure and function of the DU subfamily.     

Cdk1 and BRCA1 target γ-tubulin to microtubule domains

DNA damage is a critical event that requires an appropriate cellular response. This is mediated by checkpoint proteins such as Cdk1 that controls S/G2 and G2/M transition. Cdk1 is required for BRCA1 transport to DNA damage sites inside the nucleus where BRCA1 functions as a scaffold to initiate a signaling cascade. BRCA1 is a multifunctional protein that also ubiquitinates γ-tubulin and, consequently, inhibits microtubule nucleation at the centrosome. Here, we report that γ-tubulin also localizes at confined areas in the microtubule network. Nocodazole-mediated microtubule depolymeration results in disappearance of this γ-tubulin fraction, while microtubule stabilization by taxol preserves this structure. Surprisingly, overexpression of Cdk1 or BRCA1 greatly expands the γ-tubulin coating of microtubules, suggesting that the microtubule-bound γ-tubulin is involved in DNA damage response. This is in accordance with numerous reports of microtubule-associated DNA damage proteins, such as p53, that are transported to the nucleus when DNA damage occurs. γ-Tubulin itself has been reported to form complexes with DNA repair proteins in the nucleus.     

Structural Basis for Asymmetric Association of the βPIX Coiled Coil and Shank PDZ

βPIX (p21-activated kinase interacting exchange factor) and Shank/ProSAP protein form a complex acting as a protein scaffold that integrates signaling pathways and regulates postsynaptic structure. Complex formation is mediated by the C-terminal PDZ binding motif of βPIX and the Shank PDZ domain. The coiled-coil (CC) domain upstream of the PDZ binding motif allows multimerization of βPIX, which is important for its physiological functions. We have solved the crystal structure of the βPIX CC-Shank PDZ complex and determined the stoichiometry of complex formation. The βPIX CC forms a 76-Å-long parallel CC trimer. Despite the fact that the βPIX CC exposes three PDZ binding motifs in the C-termini, the βPIX trimer associates with a single Shank PDZ. One of the C-terminal ends of the CC forms an extensive β-sheet interaction with the Shank PDZ, while the other two ends are not involved in ligand binding and form random coils. The two C-terminal ends of βPIX have significantly lower affinity than the first PDZ binding motif due to the steric hindrance in the C-terminal tails, which results in binding of a single PDZ domain to the βPIX trimer. The structure shows canonical class I PDZ binding with a β-sheet interaction extending to position − 6 of βPIX. The βB-βC loop of Shank PDZ undergoes a conformational change upon ligand binding to form the β-sheet interaction and to accommodate the bulky side chain of Trp − 5. This structural study provides a clear picture of the molecular recognition of the PDZ ligand and the asymmetric association of βPIX CC and Shank PDZ.     

Murein and pseudomurein cell wall binding domains of bacteria and archaea—a comparative view

The cell wall, a major barrier protecting cells from their environment, is an essential compartment of both bacteria and archaea. It protects the organism from internal turgor pressure and gives a defined shape to the cell. The cell wall serves also as an anchoring surface for various proteins and acts as an adhesion platform for bacteriophages. The walls of bacteria and archaea are mostly composed of murein and pseudomurein, respectively. Cell wall binding domains play a crucial role in the non-covalent attachment of proteins to cell walls. Here, we give an overview of the similarities and differences in the biochemical and functional properties of the two major murein and pseudomurein cell wall binding domains, i.e., the Lysin Motif (LysM) domain (Pfam PF01476) and the pseudomurein binding (PMB) domain (Pfam PF09373) of bacteria and archaea, respectively.