Biochemical characterization of missense mutations in the Arf/Arl-family small GTPase Arl6 causing Bardet-Biedl syndrome

Bardet-Biedl syndrome (BBS) is a pleiotropically genetic disorder, whose etiology is linked to cilia. Mutations in the Arf/Arl-family GTPase Arl6 have been recently shown to be responsible for BBS type 3. Here we show that BBS mutations alter the guanine nucleotide-binding properties of Arl6. Specifically, substitution of 31st Threonine to Arginine selectively abrogates the GTP-binding ability of Arl6 without affecting GDP-binding/dissociating properties. Furthermore, all the BBS mutations in Arl6 result in low expression of the mutant proteins, which can be restored by the inhibition of the proteasome. These findings implicate that Arl6 mutants are destabilized and eliminated by the proteasome in cells, probably due to the altered nucleotide-binding properties.     

NMR structural studies of the myristoylated N-terminus of ADP ribosylation factor 6 (Arf6)

Arf proteins are guanine nucleotide binding proteins that are implicated in endocytotic pathways and vesicle trafficking. The two widely studied isoforms of Arf proteins (Arf1 and Arf6) have different cellular functions and localizations but similar structures. Arf proteins have an N-terminal helix with a covalently bound myristoyl group. Except structural models, there are no three dimensional structures of the myristoylated N-terminal peptide or the intact myristoylated Arf proteins. However, understanding the role of both the myristoyl group and the N-terminal helix based on the details of their molecular structures is of great interest. In the solution structure of myristoylated N-terminal peptide of Arf6 described here, the myristoyl group folds toward the N-terminus to interact with the hydrophobic residues in particular, the phenyl ring. Also, the structure of the dodecylphosphocholine (DPC) micelle-bound of the peptide together with paramagnetic studies showed that the myristoyl group is inserted into the micelle while residues V4-G10 interact with the surface of the micelle. The structural differences between the unbound and micelle-bound myristoylated N-terminal peptide of Arf6 involves the myristoyl group and the side chains of the hydrophobic residues.     

The Switch that Does Not Flip: The Blue-Light Receptor YtvA from Bacillus subtilis Adopts an Elongated Dimer Conformation Independent of the Activation State as Revealed by a Combined AUC and SAXS Study

Photoreceptors play an important role in plants and bacteria by converting extracellular stimuli into intracellular signals. One distinct class are the blue-light-sensitive phototropins harboring a light-oxygen-voltage (LOV) domain coupled to various effector domains. Photon absorption by the chromophore within the LOV domain results in an activation of the output domain via mechanisms that are hitherto not well understood. The photoreceptor YtvA from Bacillus subtilis is a bacterial analog of phototropins, consists of an LOV and a sulfate transporter/anti-sigma factor antagonist domain, and is involved in the response of the bacterium to environmental stress. We present here analytical ultracentrifugation studies and small-angle X-ray scattering experiments, showing that YtvA is a dimer. On the basis of these results, we present a low-resolution model of the dimer in the dark and the lit state of the protein. In addition, we show that YtvA does not change its oligomerization state or its overall shape upon light activation.     

Domain Features of the Peripheral Stalk Subunit H of the Methanogenic A1AO ATP Synthase and the NMR Solution Structure of H1-47

A series of truncated forms of subunit H were generated to establish the domain features of that protein. Circular dichroism analysis demonstrated that H is divided at least into a C-terminal coiled-coil domain within residues 54-104, and an N-terminal domain formed by adjacent α-helices. With a cysteine at the C-terminus of each of the truncated proteins (H_1-47, H_1-54, H_1-59, H_1-61, H_1-67, H_1-69, H_1-71, H_1-78, H_1-80, H_1-91, and H_47-105), the residues involved in formation of the coiled-coil interface were determined. Proteins H_1-54, H_1-61, H_1-69, and H_1-80 showed strong cross-link formation, which was weaker in H_1-47, H_1-59, H_1-71, and H_1-91. A shift in disulfide formation between cysteins at positions 71 and 80 reflected an interruption in the periodicity of hydrophobic residues in the region ₇₁AEKILEETEKE₈₁. To understand how the N-terminal domain of H is formed, we determined for the first time, to our knowledge, the solution NMR structure of H_1-47, which revealed an α-helix between residues 15-42 and a flexible N-terminal stretch. The α-helix includes a kink that would bring the two helices of the C-terminus into the coiled-coil arrangement. H_1-47 revealed a strip of alanines involved in dimerization, which were tested by exchange to single cysteines in subunit H mutants.     

Characterisation of the Nucleotide Exchange Factor ITSN1L: Evidence for a Kinetic Discrimination of GEF-Stimulated Nucleotide Release from Cdc42

Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg²⁺ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex.     

Calmodulin as a protein linker and a regulator of adaptor/scaffold proteins

Calmodulin (CaM) is a universal regulator for a huge number of proteins in all eukaryotic cells. Best known is its function as a calcium-dependent modulator of the activity of enzymes, such as protein kinases and phosphatases, as well as other signaling proteins including membrane receptors, channels and structural proteins. However, less well known is the fact that CaM can also function as a Ca^2 +-dependent adaptor protein, either by bridging between different domains of the same protein or by linking two identical or different target proteins together. These activities are possible due to the fact that CaM contains two independently-folded Ca^2 + binding lobes that are able to interact differentially and to some degree separately with targets proteins. In addition, CaM can interact with and regulates several proteins that function exclusively as adaptors. This review provides an overview over our present knowledge concerning the structural and functional aspects of the role of CaM as an adaptor protein and as a regulator of known adaptor/scaffold proteins.