Molecular architecture of Gαo and the structural basis for RGS16-mediated deactivation

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an α subunit with intrinsic GTPase activity and a βγ heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the α subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Gα subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Gα subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Å crystal structure of the enigmatic, neuronal G protein Gαo in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Å structure of apo-RGS16, also presented here, reveals plasticity upon Gαo binding, the determinants for GAP activity, and the structurally unique features of Gαo that likely distinguish it physiologically from other members of the larger Gαi family, affording insight to receptor, GAP and effector specificity

Amino-terminal cysteine residues of RGS16 are required for palmitoylation and modulation of G(i)- and G(q)-mediated signaling

RGS proteins (Regulators of G protein Signaling) are a recently discovered family of proteins that accelerate the GTPase activity of heterotrimeric G protein α subunits of the i, q, and 12 classes. The proteins share a homologous core domain but have divergent amino-terminal sequences that are the site of palmitoylation for RGS-GAIP and RGS4. We investigated the function of palmitoylation for RGS16, which shares conserved amino-terminal cysteines with RGS4 and RGS5. Mutation of cysteine residues at residues 2 and 12 blocked the incorporation of [3H]palmitate into RGS16 in metabolic labeling studies of transfected cells or into purified RGS proteins in a cell-free palmitoylation assay. The purified RGS16 proteins with the cysteine mutations were still able to act as GTPase-activating protein for Giα. Inhibition or a decrease in palmitoylation did not significantly change the amount of protein that was membrane-associated. However, palmitoylation-defective RGS16 mutants demonstrated impaired ability to inhibit both Gi- and Gq-linked signaling pathways when expressed in HEK293T cells. These findings suggest that the amino-terminal region of RGS16 may affect the affinity of these proteins for Gα subunits in vivo or that palmitoylation localizes the RGS protein in close proximity to Gα subunits on cellular membranes

Characterization of ROP GTPase-activated Arabidopsis receptor-like cytoplasmic kinases (RLCK class VI_A)

Plants have to respond and adapt to a variety of continuously changing environmental factors in order to establish an appropriate developmental strategy to ensure survival. There are ample data showing that protein phosphorylation/dephosphorylation plays a central role in cellular signal transduction in all organisms (Herrmann et al. 2006; Stone and Walker 1995). Interestingly, plants have a similar system as mammals to detect and transfer signals across the cell membrane into the nucleus where adaptations could be initiated. For the detection and transfer of an external signal, mammalian systems have receptor protein kinases. The proteins contain a single hydrophobic transmembrane domain, an extracellular domain and protein kinase domain. The majority of animal receptor kinases are phosphorylated on tyrosine residues within the kinase domain (receptor tyrosin kinases or RTKs; Ullrich and Schlessinger 1990), but a few were discovered which are phosphorylated on serine and threonine residues (Lin et al. 1992). In plants, two different types of transmembrane receptor kinases are known, including receptor-like serine/threonine (Ser/Thr) kinases (receptor-like kinases RLKs; Shiu and Bleecker 2001, 2003; Shiu et al. 2004; Walker 1994), structurally similar to mammalian RTKs, and receptor histidine (His) kinases (Grefen and Harter 2004; Mizuno 2005; Urao et al. 2000). Since the first RLK-encoding gene family was found in Zea mays (Walker and Zhang 1990), thousands of RLK genes have been identified from many different plant species. The Arabidopsis genome contains more than 600 members, representing nearly 2.5% of the annotated protein-coding genes; and more than 1000 members were annotated in the rice genome (Shiu et al. 2004). Approximately 25% of the Arabidopsis RLKs contain only a kinase domain with no apparent signal sequence or transmembrane region and thus were collectively named as receptor like cytoplasmic kinases (RLCKs). Arabidopsis RLCKs can be subdivided into 12 classes with 193 protein coding genes all together. Concerning the function of plant RLCKs, at the present only few members have been characterized and it is very likely that they play major role in the perception and 93 transmission of external signals perceived by RLKs (Zhou et al. 1995; Murase et al. 2004). Recently, our group as well as a group in Germany reported a direct interaction of plant ROP GTPases with receptor-like cytoplasmic kinases (RLCK class VI) from Arabidopsis (Molendijk et al. 2008) and alfalfa (Dorjgotov et al. 2009). Moreover, we provide evidences that kinases belonging to the RLCK Class VI family of Medicago truncatula and Arabidopsis thaliana can be specifically activated by GTP-bound ROP GTPases in vitro further supporting the view that plant Rho (ROP) G-proteins may directly regulate downstream kinase signaling. A further kinase designated as cysteine-rich receptor kinase (NCRK) belonging to a distinct kinase family has also been shown to interact with ROPs (Molendijk et al. 2008). None of these plant specific ROP-interacting kinases has any characteristic domain or motif that could be correlated with their ability to bind ROP GTPases. Plant specific ROP GTPases are versatile molecular switches in many processes during plant growth, development and responses to the environment and thus a possible implication of RLCKs in these ROP-dependent signal transduction pathways is in discussion. Our general aim was to characterize the members of the Arabidopsis thaliana RLCK Class VI family of protein kinases. Despite of their potential significance in ROP GTPase mediated signaling, hardly any functional information was available until now about the fourteen Arabidopsis RLCK Class VI members. Sequence comparison and phylogenetic analysis revealed that gene duplication played a significant role in the formation of this kinase family and allowed the separation of the 14 RCLK VI kinases into two groups with seven members each (A1 to A7 and B1 to B7). The proteins are highly homologous to each other, especially in the kinase domain, but are divergent from the related kinase families. It was established that, several members have an N-terminal UspA (“universal stress protein”) domain (group B members) or an N-terminal serine-rich region (group A members). In order to formulate a possible biological role of AtRLCK_VI kinases, real-time quantitative reverse transcription-polymerase reaction (qRT-PCR) was used to determine relative transcript levels in the various organs (root, rosette leaves, cauline leaves, 94 inflorescence stem, flower buds, open flowers, siliques. exponentially dividing cultured cells) of the Arabidopsis plant as well as under a series of abiotic stress/hormone (osmotic, sugar, salt stress, oxidative stress, cold and hormone treatment) treatments in seedlings. AtRLCK VI genes exhibited diverse expression patterns in the various plant organs as well as in response to stress/hormone treatments..

Structure and function of bacterial dynamin-like proteins

Membrane dynamics are essential for numerous cellular processes in eukaryotic and prokaryotic cells. In eukaryotic cells, membrane fusion and fission are often catalyzed by large GTPases of the dynamin protein family. These proteins couple GTP hydrolysis to membrane deformation, which eventually leads to fusion or fission of the lipid bilayer. Mutations in eukaryotic dynamin-like proteins (DLPs) are associated with various diseases underscoring the importance to fully understand the biochemistry of these proteins. In recent years, a wealth of structural and biochemical data have been published that allow a detailed analysis of how dynamins or DLPs modulate biological membranes. However, less is known about the function of bacterial DLPs, although structural data exist. This review summarizes current knowledge about bacterial dynamins and discusses structural and functional properties in comparison to their eukaryotic counterparts

Signal Recognition Particle (SRP) and SRP Receptor: A New Paradigm for Multistate Regulatory GTPases

The GTP-binding proteins or GTPases comprise a superfamily of proteins that provide molecular switches in numerous cellular processes. The “GTPase switch” paradigm, in which a GTPase acts as a bimodal switch that is turned “on” and “off” by external regulatory factors, has been used to interpret the regulatory mechanism of many GTPases for more than two decades. Nevertheless, recent work has unveiled an emerging class of “multistate” regulatory GTPases that do not adhere to this classical paradigm. Instead of relying on external nucleotide exchange factors or GTPase activating proteins to switch between the on and off states, these GTPases have the intrinsic ability to exchange nucleotides and to sense and respond to upstream and downstream factors. In contrast to the bimodal nature of the GTPase switch, these GTPases undergo multiple conformational rearrangements, allowing multiple regulatory points to be built into a complex biological process to ensure the efficiency and fidelity of the pathway. We suggest that these multistate regulatory GTPases are uniquely suited to provide spatial and temporal control of complex cellular pathways that require multiple molecular events to occur in a highly coordinated fashion

Atypical Rho GTPases of the RhoBTB Subfamily: Roles in vesicle trafficking and tumorigenesis

RhoBTB proteins constitute a subfamily of atypical Rho GTPases represented in mammals by RhoBTB1, RhoBTB2, and RhoBTB3. Their characteristic feature is a carboxyl terminal extension that harbors two BTB domains capable of assembling cullin 3-dependent ubiquitin ligase complexes. The expression of all three RHOBTB genes has been found reduced or abolished in a variety of tumors. They are considered tumor suppressor genes and recent studies have strengthened their implication in tumorigenesis through regulation of the cell cycle and apoptosis. RhoBTB3 is also involved in retrograde transport from endosomes to the Golgi apparatus. One aspect that makes RhoBTB proteins atypical among the Rho GTPases is their proposed mechanism of activation. No specific guanine nucleotide exchange factors or GTPase activating proteins are known. Instead, RhoBTB might be activated through interaction with other proteins that relieve their auto-inhibited conformation and inactivated through auto-ubiquitination and destruction in the proteasome. In this review we discuss our current knowledge on the molecular mechanisms of action of RhoBTB proteins and the implications for tumorigenesis and other pathologic conditions

The gateway to chloroplast: re-defining the function of chloroplast receptor proteins

Chloroplast biogenesis often requires a tight orchestration between gene expression (both plastidial and nuclear) and translocation of similar to 3000 nuclear-encoded proteins into the organelle. Protein translocation is achieved via two multimeric import machineries at the outer (TOC) and inner (TIC) envelope of chloroplast, respectively. Three components constitute the core element of the TOC complex: a beta-barrel protein translocation channel Toc75 and two receptor constituents, Toc159 and Toc34. A diverse set of distinct TOC complexes have recently been characterized and these diversified TOC complexes have evolved to coordinate the translocation of differentially expressed proteins. This review aims to describe the recent discoveries relating to the typical characteristics of these distinct TOC complexes, particularly the receptor constituents, which are the main contributors for TOC complex diversification

The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane.

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30-kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane

Concerted Complex Assembly and GTPase Activation in the Chloroplast Signal Recognition Particle

The universally conserved signal recognition particle (SRP) and SRP receptor (SR) mediate the cotranslational targeting of proteins to cellular membranes. In contrast, a unique chloroplast SRP in green plants is primarily dedicated to the post-translational targeting of light harvesting chlorophyll a/b binding (LHC) proteins. In both pathways, dimerization and activation between the SRP and SR GTPases mediate the delivery of cargo; whether and how the GTPase cycle in each system adapts to its distinct substrate proteins were unclear. Here, we show that interactions at the active site essential for GTPase activation in the chloroplast SRP and SR play key roles in the assembly of the GTPase complex. In contrast to their cytosolic homologues, GTPase activation in the chloroplast SRP–SR complex contributes marginally to the targeting of LHC proteins. These results demonstrate that complex assembly and GTPase activation are highly coupled in the chloroplast SRP and SR and suggest that the chloroplast GTPases may forego the GTPase activation step as a key regulatory point. These features may reflect adaptations of the chloroplast SRP to the delivery of their unique substrate protein

Open and cut: allosteric motion and membrane fission by dynamin superfamily proteins.

Cells have evolved diverse protein-based machinery to reshape, cut, or fuse their membrane-delimited compartments. Dynamin superfamily proteins are principal components of this machinery and use their ability to hydrolyze GTP and to polymerize into helices and rings to achieve these goals. Nucleotide-binding, hydrolysis, and exchange reactions drive significant conformational changes across the dynamin family, and these changes alter the shape and stability of supramolecular dynamin oligomers, as well as the ability of dynamins to bind receptors and membranes. Mutations that interfere with the conformational repertoire of these enzymes, and hence with membrane fission, exist in several inherited human diseases. Here, we discuss insights from new x-ray crystal structures and cryo-EM reconstructions that have enabled us to infer some of the allosteric dynamics for these proteins. Together, these studies help us to understand how dynamins perform mechanical work, as well as how specific mutants of dynamin family proteins exhibit pathogenic properties