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Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxinactin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-Nnaphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity.

Understanding the deactivation mechanism of 2-deoxy-d-ribose-5-phosphate aldolase by its natural substrate leads to a single mutant showing complete acetaldehyde resistance.

Recoverin is a Ca2؉ -regulated signal transduction modulator expressed in the vertebrate retina that has been implicated in visual adaptation. An intriguing feature of recoverin is a cluster of charged residues at its C terminus, the functional significance of which is largely unclear. To elucidate the impact of this segment on recoverin structure and function, we have investigated a mutant lacking the C-terminal 12 amino acids. Whereas in myristoylated recoverin the truncation causes an overall decrease in Ca 2؉ sensitivity, results for the non-myristoylated mutant indicate that the truncation primarily affects the high affinity EF-hand 3. The three-dimensional structure of the mutant has been determined by x-ray crystallography. In addition to significant changes in average coordinates compared with wild-type recoverin, the structure provides strong indication of increased conformational flexibility, particularly in the C-terminal domain. Based on these observations, we propose a novel role of the C-terminal segment of recoverin as an internal modulator of Ca 2؉ sensitivity.Many biological processes are triggered or regulated by transient intracellular Ca 2ϩ signals. Because these signals elicit specific cellular responses, the precise detection of changes in cytoplasmic Ca 2ϩ concentration is a crucial step in many signaling pathways and requires sensing of Ca 2ϩ within very different concentration ranges. Ca 2ϩ -binding proteins work as intracellular Ca 2ϩ sensors and regulate their targets with high specificity and high spatial and temporal resolution. To achieve these remarkable tasks, Ca 2ϩ is recognized by specific amino acid sequence motifs, for example the C 2 domain and the EF-hand motif (1, 2). These motifs can detect subtle changes in Ca 2ϩ concentration and allow a fine tuning of Ca 2ϩ signaling. However, it remains a challenging problem to understand at a structural level how minimal changes in cytoplasmic Ca 2ϩ are reliably detected.The EF-hand superfamily of Ca 2ϩ -binding proteins includes, among others, the family of neuronal calcium sensor (NCS) 3 proteins (3), which are named because of their predominant expression in neuronal tissue. NCS proteins are grouped into five subfamilies and show a rather heterogeneous localization and function in the nervous system (4). In the photoreceptor cells of the vertebrate retina, for instance, recoverin and several isoforms of guanylate cyclase activating protein (GCAP) detect changes in Ca 2ϩ concentration during or after illumination and regulate their target proteins in Ca 2ϩ -dependent feedback loops (5).Recoverin inhibits rhodopsin kinase at high cytoplasmic Ca 2ϩ concentration (6 -9), a process that is thought to contribute to light adaptation of photoreceptor cells (9, 10). Recoverin harbors a myristoyl group at its N terminus (11), which is buried in a hydrophobic cleft in the Ca 2ϩ -free state (12). Upon Ca 2ϩ binding to the two functional EF-hands (EF-hand 2 and EFhand 3) (13) the acyl chain is exposed to the solvent. This socalled Ca 2ϩ -myrist...

Recoverin is a Ca2؉ -regulated signal transduction modulator found in vertebrate retina that has been shown to undergo dramatic conformational changes upon Ca 2؉ binding to its two functional EF-hand motifs. To elucidate the differential impact of the N-terminal myristoylation as well as occupation of the two Ca 2؉ binding sites on recoverin structure and function, we have investigated a non-myristoylated E85Q mutant exhibiting virtually no Ca 2؉ binding to EF-2. Crystal structures of the mutant protein as well as the non-myristoylated wild-type have been determined. Although the non-myristoylated E85Q mutant does not display any functional activity, its three-dimensional structure in the presence of Ca 2؉ resembles the myristoylated wildtype with two Ca 2؉ but is quite dissimilar from the myristoylated E85Q mutant. We conclude that the N-terminal myristoyl modification significantly stabilizes the conformation of the Ca 2؉ -free protein (i.e. the T conformation) during the stepwise transition toward the fully Ca 2؉ -occupied state. On the basis of these observations, a refined model for the role of the myristoyl group as an intrinsic allosteric modulator is proposed.Recoverin belongs to an ancient family of calcium-binding proteins termed the neuronal calcium sensor family (1, 2) and is mainly expressed in vertebrate photoreceptor cells (3, 4). The 23-kDa protein is composed of two domains, each of them harboring one non-functional and one functional EF-hand helix-loop-helix motif (5). Vertebrate photoreceptor cells respond to illumination by a decrease of the intracellular transmitters of excitation and adaptation, cGMP and Ca 2ϩ , respectively. In the dark, at high Ca 2ϩ concentration, the two functional EFhands of recoverin (EF-2 and -3) are occupied by Ca 2ϩ (6 -8). In this state, recoverin is able to inhibit the G-protein-coupled receptor kinase GRK1 1 (rhodopsin kinase), thereby prolonging the lifetime of photoexcited rhodopsin (9 -12). Upon illumination, decrease of cytoplasmic Ca 2ϩ causes this inhibition to be relieved. This regulatory circuit is thought to be one out of several Ca 2ϩ -dependent mechanisms that control adaptation of phototransduction to changing background light intensities (13,14).At its N terminus recoverin is heterogeneously acylated, the prevailing modification being a myristoyl chain (15). The observation of a Ca 2ϩ -dependent partitioning of recoverin to membranes led to the proposal that it underwent a Ca 2ϩ -myristoyl switch (16). The mechanics of this switch were unraveled by determining the solution structures of Ca 2ϩ -free and Ca 2ϩ -bound myristoylated recoverin via NMR spectroscopy. In the Ca 2ϩ -free state of recoverin (T state) the myristoyl moiety is buried within a hydrophobic pocket, whereas in the Ca 2ϩ -bound form (R state), the acyl group is extruded and thus available for interaction with other proteins or insertion into a lipid bilayer (17-19). Moreover, the myristoyl chain has been proposed to act as an intrinsic allosteric effector modifying the conformational...

The extracellular domain of the human interleukin-h ( 1 1~5 ) receptor, cornprising 339 amino acids following the signal peptide, has been expressed in baculovirus-infected insect cclls (SflS8). When the soluble receptor secreted into the culture medium was purified by affinity chromatography. using 11,-6 irnmobilized on Sspharose, 6 ing soluble receptor was isolated froiii 1 I conditioned medium of S i t 5 8 suspension cultures. A niolitr absorption coefficient of 9.3 X 10' 1 . mol + cin-' was calculalerl from thc ultraviolet spectrum of the soluble 1L-h receptor. After SDS/PAGE and silver staining, an apparent molecular mass of 48 kDa was estimated for the purified protein. Deglycosylation with pcptide N-glycosidase F resulted in an increase in electrophoretic mobility and ii decrease in thc apparent m o l e c t i l~ mass Croin 48 kDa to about 41 -44 kDa. As expected, the soluble human IL-fi receptor bound human "'Ilabeled TL-6 with low affinity (& = 500 pM). Furthermore, the binding of soluble human IL-6 receptor to immobilized IL-6 was studicd using real-time interaction analysis. The recombinant soluble receptor showed biological activity on HepG2 cells stably transfccted wilh ii cDNA coding for II,-6 (HcpG2-Ild-6 cells). Haptoglobin inRNA synthcsis was induced by thc soluble IL-6 receptor at concentralions iis Itow as 1 0 ng/ml. Five monoclonal antibodies were generated. Two gt-uups of antibodies were identificd m a p ping to amino acids 1-67 and 68-143 of the soluble IL-6 receptor, respectively. The plasmit clearance of soluble '2SI-labeled IL-6 1-eccptor in the absence and presencc of 11,-6 was studied in rBts as a ruodcl system. The kinetics was biphasic. Soluble IL-6 rcceptor/IL-O cotriplexcs were cleiircd more rapidly than the soluble receptor alone. Intravenously injected solublc '251-labelcd IL-6 receptor, as well as coniplexcs with IL-6, rapidly iiccumulatcd in liver and to it lesser extcnt i n skeletal musclc, skin and kidneys, Subsequently, the mdioaclivity appeared in the gut content.?&ywords: interleukin-6; soluble interleukin-0 receptor; baculovirus system ; plasriia clc:uance.

Understanding enzyme stability and activity in extremophilic organisms is of great biotechnological interest, but many questions are still unsolved. Using 2-deoxy-D-ribose-5-phosphate aldolase (DERA) as model enzyme, we have evaluated structural and functional characteristics of different orthologs from psychrophilic, mesophilic and hyperthermophilic organisms. We present the first crystal structures of psychrophilic DERAs, revealing a dimeric organization resembling their mesophilic but not their thermophilic counterparts. Conversion into monomeric proteins showed that the native dimer interface contributes to stability only in the hyperthermophilic enzymes. Nevertheless, introduction of a disulfide bridge in the interface of a psychrophilic DERA did confer increased thermostability, suggesting a strategy for rational design of more durable enzyme variants. Constraint network analysis revealed particularly sparse interactions between the substrate pocket and its surrounding α-helices in psychrophilic DERAs, which indicates that a more flexible active center underlies their high turnover numbers.

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