Two different heparin-binding domains in the triple-helical domain of ColQ,the collagen tail subunit of synaptic acetylcholinesterase

ColQ, the collagen tail subunit of asymmetric acetylcholinesterase, is responsible for anchoring the enzyme at the vertebrate synaptic basal lamina by interacting with heparan sulfate proteoglycans. To get insights about this function, the interaction of ColQ with heparin was analyzed. For this, heparin affinity chromatography of the complete oligomeric enzyme carrying different mutations in ColQ was performed. Results demonstrate that only the two predicted heparin-binding domains present in the collagen domain of ColQ are responsible for heparin interaction. Despite their similarity in basic charge distribution, each heparin-binding domain had different affinity for heparin. This difference is not solely determined by the number or nature of the basic residues conforming each site, but rather depends critically on local structural features of the triple helix, which can be influenced even by distant regions within ColQ. Thus, ColQ possesses two heparin-binding domains with different properties that may have non-redundant functions. We hypothesize that these binding sites coordinate acetylcholinesterase positioning within the organized architecture of the neuromuscular junction basal lamina.     

Primary structure of a collagenic tail peptide of Torpedo acetylcholinesterase: co-expression with catalytic subunit induces the production of collagen-tailed forms in transfected cells.

The asymmetric forms of cholinesterases are synthesized only in differentiated muscular and neural cells of vertebrates. These complex oligomers are characterized by the presence of a collagen-like tail, associated with one, two or three tetramers of catalytic subunits. The collagenic tail is responsible for ionic interactions, explaining the insertion of these molecules in extracellular basal lamina, e.g. at neuromuscular endplates. We report the cloning of a collagenic subunit from Torpedo marmorata acetylcholinesterase (AChE). The predicted primary structure contains a putative signal peptide, a proline-rich domain, a collagenic domain, and a C-terminal domain composed of proline-rich and cysteine-rich regions. Several variants are generated by alternative splicing. Apart from the collagenic domain, the AChE tail subunit does not present any homology with previously known proteins. We show that co-expression of catalytic AChE subunits and collagenic subunits results in the production of asymmetric, collagen-tailed AChE forms in transfected COS cells. Thus, the assembly of these complex forms does not depend on a specific cellular processing, but rather on the expression of the collagenic subunits.     

Sequences within both the N- and C-terminal domains of phytochrome A are required for PFR ubiquitination and degradation

Photoconversion of the plant photoreceptor phytochrome A (phyA) from its inactive Pr form to its biologically active Pfr from initiates its rapid proteolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that multiple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant PHY genes in vitro and analyzed the Pfr-specific degradation of the resulting photoreceptors expressed in transgenic tobacco. One important site is within the C-terminal half of the polypeptide as its removal stabilizes oat phyA as Pfr. Within this half is a set of conserved lysines that are potentially required for ubiquitin attachment. Substitution of these lysines did not prevent ubiquitination or breakdown of Pfr, suggesting either that they are not the attachment sites or that other lysines can be used in their absence. A small domain just proximal to the C-terminus is essential for the form-dependent breakdown of the holoprotein. Removal of just six amino acids in this domain generated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric photoreceptors generated from potato PHYA and PHYB, we found that the N-terminal half of phyA is also required for Pfr-specific breakdown. Only those chimeras containing the N-terminal sequences from phyA were ubiquitinated and rapidly degraded as Pfr. Taken together, our data demonstrate that, whereas an intact C-terminal domain is essential for phyA degradation, the N-terminal domain is responsible for the selective recognition and ubiquitination of Pfr.     

Activated radixin is essential for GABAA receptor alpha5 subunit anchoring at the actin cytoskeleton

Neurotransmitter receptor clustering is thought to represent a critical parameter for neuronal transmission. Little is known about the mechanisms that anchor and concentrate inhibitory neurotransmitter receptors in neurons. GABAA receptor (GABAAR) alpha5 subunits mainly locate at extrasynaptic sites and are thought to mediate tonic inhibition. Notably, similar as synaptic GABAARs, these receptor subtypes also appear in cluster formations at neuronal surface membranes and are of particular interest in cognitive processing. GABAAR alpha5 mutation or depletion facilitates trace fear conditioning or improves spatial learning in mice, respectively. Here, we identified the actin-binding protein radixin, a member of the ERM family, as the first directly interacting molecule that anchors GABAARs at cytoskeletal elements. Intramolecular activation of radixin is a functional prerequisite for GABAAR alpha5 subunit binding and both depletion of radixin expression as well as replacement of the radixin F-actin binding motif interferes with GABAAR alpha5 cluster formation. Our data suggest radixin to represent a critical factor in receptor localization and/or downstream signaling.     

MuSK is required for anchoring acetylcholinesterase at the neuromuscular junction

At the neuromuscular junction, acetylcholinesterase (AChE) is mainly present as asymmetric forms in which tetramers of catalytic subunits are associated to a specific collagen, collagen Q (ColQ). The accumulation of the enzyme in the synaptic basal lamina strictly relies on ColQ. This has been shown to be mediated by interaction between ColQ and perlecan, which itself binds dystroglycan. Here, using transfected mutants of ColQ in a ColQ-deficient muscle cell line or COS-7 cells, we report that ColQ clusterizes through a more complex mechanism. This process requires two heparin-binding sites contained in the collagen domain as well as the COOH terminus of ColQ. Cross-linking and immunoprecipitation experiments in Torpedo postsynaptic membranes together with transfection experiments with muscle-specific kinase (MuSK) constructs in MuSK-deficient myotubes or COS-7 cells provide the first evidence that ColQ binds MuSK. Together, our data suggest that a ternary complex containing ColQ, perlecan, and MuSK is required for AChE clustering and support the notion that MuSK dictates AChE synaptic localization at the neuromuscular junction.     

The 20 C-terminal amino acid residues of the chloroplast ATP synthase gamma subunit are not essential for activity.

It has been suggested that the last seven to nine amino acid residues at the C terminus of the gamma subunit of the ATP synthase act as a spindle for rotation of the gamma subunit with respect to the alpha beta subunits during catalysis (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). To test this hypothesis we selectively deleted C-terminal residues from the chloroplast gamma subunit, two at a time starting at the sixth residue from the end and finishing at the 20th residue from the end. The mutant gamma genes were overexpressed in Escherichia coli and assembled with a native alpha3beta3 complex. All the mutant forms of gamma assembled as effectively as the wild-type gamma. Deletion of the terminal 6 residues of gamma resulted in a significant increase (>50%) in the Ca-dependent ATPase activity when compared with the wild-type assembly. The increased activity persisted even after deletion of the C-terminal 14 residues, well beyond the seven residues proposed to form the spindle. Further deletions resulted in a decreased activity to approximately 19% of that of the wild-type enzyme after deleting all 20 C-terminal residues. The results indicate that the tip of the gammaC terminus is not essential for catalysis and raise questions about the role of the C terminus as a spindle for rotation.     

Function and structure of N-terminal and C-terminal domains of calcineurin B subunit

Calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, plays a critical role in T-cell activation by regulating the activity of NF-AT. CN is a heterodimer consisting of a catalytic subunit (CNA) and a Ca2+-binding regulatory subunit (CNB). CNB is composed of two global domains: the C-terminal domain (DC) and the N-terminal domain (DN), each containing two Ca2+ binding sites. In this study, using purified DN and DC derived from constructed expression systems, we revealed that intact CNB and DC can stimulate the phosphatase activity of CNA, about 2.2 and 1.6 times the phosphatase activity of CNA alone, respectively; DN itself has little effect on the phosphatase activity of CNA. Fluorescence spectroscopy of an ANS-hydrophobic fluorescence probe shows that binding of Ca2+ to CNB, DC or DN leads to exposure of the hydrophobic surface of the proteins and that the hydrophobicity of CNB is the greatest, that of DC is less, and that of DN is the least. The hydrophobic surface of CNB may be an important structural basis for stimulating CN phosphatase activity.     

A dominant negative mutant of the KCC1 K-Cl cotransporter: both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity

K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl cotransport has emerged as an important adjunct strategy for the treatment of sickle cell anemia. However, structure-function relationships among the polypeptide products of the four K-Cl cotransporter (KCC) genes are little understood. We have investigated the importance of the N- and C-terminal cytoplasmic domains of mouse KCC1 to its K-Cl cotransport function expressed in Xenopus oocytes. Truncation of as few as eight C-terminal amino acids (aa) abolished function despite continued polypeptide accumulation and surface expression. These C-terminal loss-of-function mutants lacked a dominant negative phenotype. Truncation of the N-terminal 46 aa diminished function. Removal of 89 or 117 aa (Delta(N)117) abolished function despite continued polypeptide accumulation and surface expression and exhibited dominant negative phenotypes that required the presence of the C-terminal cytoplasmic domain. The dominant negative loss-of-function mutant Delta(N)117 was co-immunoprecipitated with wild type KCC1 polypeptide, and its co-expression did not reduce wild type KCC1 at the oocyte surface. Delta(N)117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2.     

Phosphorylation of Akt at the C-terminal tail triggers Akt Activation

Aberrant hyper-activation of the protein kinase Akt plays a critical role in promoting tumorigenesis. Mechanistically, previous studies establish that phosphorylation of Akt at S473 and T308 by mTORC2 and PDK1, respectively, is necessary for its full activation, thereby having been used as Akt activation markers. Recently, we report that phosphorylation of S477 and T479 at the extreme C-terminus of Akt1 promotes Akt1 activation. We further demonstrate that Akt1 pS477 and pT479 events are governed by Cdk2/Cyclin A or mTORC2 under distinct cellular contexts such as cell cycle progression or growth stimulation conditions. Here, we summarize our major findings regarding the biological significance for pS477/pT479-mediated activation of Akt and also provide perspectives for future follow-up studies.     

Inter and intra-subunit interactions at the subunit interface of chaperonin GroEL are essential for its stability and assembly

Chaperonin GroEL helps in the folding of substrate proteins under normal and stress conditions. Although it remains stable and functional during stress conditions, the quantitative estimation of stability parameters and the specific amino-acid residues playing a role in its stability are not known in sufficient detail. The reason for poor understanding is its large size, multimeric nature, and irreversible unfolding process. The X-ray crystal structure reveals that equatorial domain forms almost all intra and inter-subunit interactions for assembly of GroEL. Considering all these facts, we adopted alternate strategies to use monomeric GroEL, native GroEL and equatorial domain mutants (GroEL_K4E/GroEL_D523K/GroEL_D473C) to study the assembly and stability of GroEL. Loss of inter-subunit interaction involving K4 residue of one subunit and E59, I60, E61, I62 residues of adjacent subunit due to K4E mutation affect the oligomerization efficiency of GroEL subunits while the equilibrium unfolding studies on wild-type monomeric GroEL, native GroEL, and the selected mutants together demonstrate that intra-subunit interactions involving K4 and D523 of the same subunit play a critical role in the thermodynamic stability of both native and monomeric GroEL without affecting the oligomerization of subunits. The stability order between the GroEL_wild-type(M) and its variants is GroEL_wild-type(M) ≥ GroEL_D473C(M)˃GroEL_D523K(M)˃GroEL_K4E.     

Membrane anchoring and interaction between transmembrane domains are crucial for K+ channel function

The small viral channel Kcv is a Kir-like K(+) channel of only 94 amino acids. With this simple structure, the tetramer of Kcv represents the pore module of all complex K(+) channels. To examine the structural contribution of the transmembrane domains (TMDs) to channel function, we performed Ala scanning mutagenesis of the two domains and tested the functionality of the mutants in a yeast complementation assay. The data reveal, in combination with computational models, that the upper halves of both TMDs, which face toward the external medium, are rather rigid, whereas the inner parts are more flexible. The rigidity of the outer TMD is conferred by a number of essential aromatic amino acids that face the membrane and probably anchor this domain in the bilayer. The inner TMD is intimately connected with the rigid part of the outer TMD via π···π interactions between a pair of aromatic amino acids. This structural principle is conserved within the viral K(+) channels and also present in Kir2.2, implying a general importance of this architecture for K(+) channel function.     

Structural and functional separation of the N- and C-terminal domains of the yeast V-ATPase subunit H

The H subunit of the yeast V-ATPase is an extended structure with two relatively independent domains, an N-terminal domain consisting of amino acids 1-348 and a C-terminal domain consisting of amino acids 352-478. We have expressed these two domains independently and together in a yeast strain lacking the H subunit (vma13Delta mutant). The N-terminal domain partially complements the growth defects of the mutant and supports approximately 25% of the wild-type Mg(2+)-dependent ATPase activity in isolated vacuolar vesicles, but surprisingly, this activity is both largely concanamycin-insensitive and uncoupled from proton transport. The C-terminal domain does not complement the growth defects, and supports no ATP hydrolysis or proton transport, even though it is recruited to the vacuolar membrane. Expression of both domains in a vma13Delta strain gives better complementation than either fragment alone and results in higher concanamycin-sensitive ATPase activity and ATP-driven proton pumping than the N-terminal domain alone. Thus, the two domains make complementary contributions to structural and functional coupling of the peripheral V(1) and membrane V(o) sectors of the V-ATPase, but this coupling does not require that they be joined covalently. The N-terminal domain alone is sufficient for activation of ATP hydrolysis in V(1), but the C-terminal domain is essential for proper communication between the V(1) and V(o) sectors.     

Dissection of inhibitory Smad proteins: both N- and C-terminal domains are necessary for full activities of Xenopus Smad6 and Smad7

Smad6 and Smad7 comprise a subclass of vertebrate Smads that antagonize, rather than transduce, TGF-β family signaling. These Anti-Smads can block BMP signaling, as evidenced by their ability to induce a secondary dorsal axis when misexpressed ventrally in Xenopus embryos. Smad7 inhibits additional TGF-β related pathways, and causes spina bifida when misexpressed dorsally. We have performed structure-function analyses to identify domains of Anti-Smads that are responsible for their shared and unique activities. We find that the C-terminal domain of Smad7 displays strong axis inducing activity but cannot induce spina bifida. The isolated N-terminal domain of Smad7 is inactive but restores the ability of the C-terminus to cause spina bifida when the two are co-expressed. By contrast, the N- and C-terminal domains of Smad6 have weak axis inducing activity when expressed individually, but show full activity when co-expressed. Chimeric analysis demonstrates that the C-terminal domain of Smad7, but not Smad6, can induce spina bifida when fused to the N-terminal domain of either Smad6 or Smad7. Thus, although the C-terminal domain is the primary determinant of the intrinsic activity of Xenopus Anti-Smads, the N-terminal domain is essential for full activity, is interchangeable between Smad6 and 7, and can function in trans.     

The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate

The phosphorylated carboxyl-terminal "tail" domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking. Because deletion of the tail domain of NF-H did not alter these axonal properties (Rao, M.V., M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.S. Calcutt, Y. Uchiyama, R.A. Nixon, and D.W. Cleveland. 2002. J. Cell Biol. 158:681-693), we investigated possible functions of the NF-M tail domain by constructing NF-M tail-deleted (NF-MtailDelta) mutant mice using an embryonic stem cell-mediated "gene knockin" approach that preserves normal ratios of the three neurofilament subunits. Mutant NF-MtailDelta mice exhibited severely inhibited radial growth of both motor and sensory axons. Caliber reduction was accompanied by reduced spacing between neurofilaments and loss of long cross-bridges with no change in neurofilament protein content. These observations define distinctive functions of the NF-M tail in regulating axon caliber by modulating the organization of the neurofilament network within axons. Surprisingly, the average rate of axonal transport of neurofilaments was unaltered despite these substantial effects on axon morphology. These results demonstrate that NF-M tail-mediated interactions of neurofilaments, independent of NF transport rate, are critical determinants of the size and cytoskeletal architecture of axons, and are mediated, in part, by the highly phosphorylated tail domain of NF-M.     

Trafficking of the NMDAR2B receptor subunit distal cytoplasmic tail from endoplasmic reticulum to the synapse

NMDA receptor NR2A/B subunits have PDZ-binding domains on their extreme C-termini that are known to interact with the PSD-95 family and other PDZ proteins. We explore the interactions between PSD-95 family proteins and the NR2A/B cytoplasmic tails, and the consequences of these interactions, from the endoplasmic reticulum (ER) through delivery to the synapse in primary rat hippocampal and cortical cultured neurons. We find that the NR2A/B cytoplasmic tails cluster very early in the secretory pathway and interact serially with SAP102 beginning at the intermediate compartment, and then PSD-95. We further establish that colocalization of the distal C-terminus of NR2B and PSD-95 begins at the trans-Golgi Network (TGN). Formation of NR2B/PSD-95/SAP102 complexes is dependent on the PDZ binding domain of NR2B subunits, but association with SAP102 and PSD-95 plays no distinguishable role in cluster pre-formation or initial targeting to the vicinity of the synapse. Instead the PDZ binding domain plays a role in restricting cell-surface clusters to postsynaptic targets.     

Calcium-induced conformational changes in C-terminal tail of polycystin-2 are necessary for channel gating

Polycystin-2 (PC2) is a Ca²⁺-permeable transient receptor potential channel activated and regulated by changes in cytoplasmic Ca²⁺. PC2 mutations are responsible for ∼15% of autosomal dominant polycystic kidney disease. Although the C-terminal cytoplasmic tail of PC2 has been shown to contain a Ca²⁺-binding EF-hand domain, the molecular basis of PC2 channel gating by Ca²⁺ remains unknown. We propose that the PC2 EF-hand is a Ca²⁺ sensor required for channel gating. Consistent with this, Ca²⁺ binding causes a dramatic decrease in the radius of gyration (R_g) of the PC2 EF-hand by small angle x-ray scattering and significant conformational changes by NMR. Furthermore, increasing Ca²⁺ concentrations cause the C-terminal cytoplasmic tail to transition from a mixture of extended oligomers to a single compact dimer by analytical ultracentrifugation, coupled with a >30 Å decrease in maximum interatomic distance (D_max) by small angle x-ray scattering. Mutant PC2 channels unable to bind Ca²⁺ via the EF-hand are inactive in single-channel planar lipid bilayers and inhibit Ca²⁺ release from ER stores upon overexpression in cells, suggesting dominant negative properties. Our results support a model where PC2 channels are gated by discrete conformational changes in the C-terminal cytoplasmic tail in response to changes in cytoplasmic Ca²⁺ levels. These properties of PC2 are lost in autosomal dominant polycystic kidney disease, emphasizing the importance of PC2 to kidney cell function. We speculate that PC2 and the Ca²⁺-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore.     

Mutations in the C-terminal hydrophobic domain of pseudorabies virus gIII affect both membrane anchoring and protein export.

The transmembrane and anchor region of pseudorabies virus gIII is postulated to be in the 35 hydrophobic amino acids (residues 436 to 470) found near the carboxy terminus of the 479-amino-acid envelope protein. In this study, we used a genetic approach to localize the functional gIII membrane anchor between amino acids 443 and 466. Mutant gIII proteins lacking the membrane anchor were not associated with virus particles, indicating that membrane retention is a prerequisite for virion localization. Unexpectedly, the specific hydrophobic gIII sequence defined by these deletions was not required for membrane anchor function since the entire region could be replaced with leucine residues without affecting gIII membrane retention, export, or virion localization. The hydrophobic region appears to encode more than the membrane anchor domain since both efficiency of posttranslational processing and localization to virions are affected by mutations in this region. We speculate that the composition of the hydrophobic domain influences the overall conformation of gIII, which in turn effects the efficiency of gIII export and processing. The virion localization phenotype is probably indirect and reflects the efficiency of protein processing. This conclusion provides insight into the mechanism of glycoprotein incorporation into virions.