Structural Insights into the Activation of the RhoA GTPase by the Lymphoid Blast Crisis (Lbc) Oncoprotein

The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and ¹H,¹³C,¹⁵N-based NMR spectroscopy. The two domains assume a “chair” shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.     

Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors

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Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its Mechanism

O-Linked glycosylation is one of the most abundant post-translational modifications of proteins. Within the secretory pathway of higher eukaryotes, the core of these glycans is frequently an N-acetylgalactosamine residue that is α-linked to serine or threonine residues. Glycoside hydrolases in family 101 are presently the only known enzymes to be able to hydrolyze this glycosidic linkage. Here we determine the high-resolution structures of the catalytic domain comprising a fragment of GH101 from Streptococcus pneumoniae TIGR4, SpGH101, in the absence of carbohydrate, and in complex with reaction products, inhibitor, and substrate analogues. Upon substrate binding, a tryptophan lid (residues 724-WNW-726) closes on the substrate. The closing of this lid fully engages the substrate in the active site with Asp-764 positioned directly beneath C1 of the sugar residue bound within the −1 subsite, consistent with its proposed role as the catalytic nucleophile. In all of the bound forms of the enzyme, however, the proposed catalytic acid/base residue was found to be too distant from the glycosidic oxygen (>4.3 Å) to serve directly as a general catalytic acid/base residue and thereby facilitate cleavage of the glycosidic bond. These same complexes, however, revealed a structurally conserved water molecule positioned between the catalytic acid/base and the glycosidic oxygen. On the basis of these structural observations we propose a new variation of the retaining glycoside hydrolase mechanism wherein the intervening water molecule enables a Grotthuss proton shuttle between Glu-796 and the glycosidic oxygen, permitting this residue to serve as the general acid/base catalytic residue.     

Structural and Functional Insights into the Mode of Action of a Universally Conserved Obg GTPase

Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed molecular roles in two global processes, ribosome assembly and stringent response. Here, using pre-steady state fast kinetics we demonstrate that ObgE is an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50S subunit. ObgE is a ribosome dependent GTPase; however, upon binding to guanosine tetraphosphate (ppGpp), the global regulator of stringent response, ObgE exhibits an enhanced interaction with the 50S subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits. Furthermore, our cryo-electron microscopy (cryo-EM) structure of the 50S·ObgE·GMPPNP complex indicates that the evolutionarily conserved N-terminal domain (NTD) of ObgE is a tRNA structural mimic, with specific interactions with peptidyl-transferase center, displaying a marked resemblance to Class I release factors. These structural data might define ObgE as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between ObgE and ribosome-associated (p)ppGpp regulators. Together with published data, our results suggest that ObgE might act as a checkpoint in final stages of the 50S subunit assembly under normal growth conditions. And more importantly, ObgE, as a (p)ppGpp effector, might also have a regulatory role in the production of the 50S subunit and its participation in translation under certain stressed conditions. Thus, our findings might have uncovered an under-recognized mechanism of translation control by environmental cues.     

Dynamin-like MxA GTPase: Structural Insights into Oligomerization and Implications for Antiviral Activity

The interferon-inducible MxA GTPase is a key mediator of cell-autonomous innate immunity against a broad range of viruses such as influenza and bunyaviruses. MxA shares a similar domain structure with the dynamin superfamily of mechanochemical enzymes, including an N-terminal GTPase domain, a central middle domain, and a C-terminal GTPase effector domain. Recently, crystal structures of a GTPase domain dimer of dynamin 1 and of the oligomerized stalk of MxA (built by the middle and GTPase effector domains) were determined. These data provide exciting insights into the architecture and antiviral function of the MxA oligomer. Moreover, the structural knowledge paves the way for the development of novel antiviral drugs against influenza and other highly pathogenic viruses.     

Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Insights into the role of the membranes in Rab GTPase regulation

Rab GTPases are molecular switches with essential roles in mediating vesicular trafficking and establishing organelle identity. The conversion from the inactive, cytosolic to the membrane-bound, active species and back is tightly controlled by regulatory proteins. Recently, the roles of membrane properties and lipid composition of different target organelles in determining the activity state of Rabs have come to light. The investigation of several Rab guanine nucleotide exchange factors (GEFs) has revealed principles of how the recruitment via lipid interactions and the spatial confinement on the membrane surface contribute to spatiotemporal specificity in the Rab GTPase network. This paints an intricate picture of the control mechanisms in Rab activation and highlights the importance of the membrane lipid code in the organization of the endomembrane system.     

Structural Insights into of the Allosteric Activation of the LicT Antiterminator by PTS-Mediated Phosphorylation

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Structural Insights into the Anti-HIV Activity of the Oscillatoria agardhii Agglutinin Homolog Lectin Family

Oscillatoria agardhii agglutinin homolog (OAAH) proteins belong to a recently discovered lectin family. All members contain a sequence repeat of ∼66 amino acids, with the number of repeats varying among different family members. Apart from data for the founding member OAA, neither three-dimensional structures, information about carbohydrate binding specificities, nor antiviral activity data have been available up to now for any other members of the OAAH family. To elucidate the structural basis for the antiviral mechanism of OAAHs, we determined the crystal structures of Pseudomonas fluorescens and Myxococcus xanthus lectins. Both proteins exhibit the same fold, resembling the founding family member, OAA, with minor differences in loop conformations. Carbohydrate binding studies by NMR and x-ray structures of glycan-lectin complexes reveal that the number of sugar binding sites corresponds to the number of sequence repeats in each protein. As for OAA, tight and specific binding to α3,α6-mannopentaose was observed. All the OAAH proteins described here exhibit potent anti-HIV activity at comparable levels. Altogether, our results provide structural details of the protein-carbohydrate interaction for this novel lectin family and insights into the molecular basis of their HIV inactivation properties.     

Structural Insights into the Quinolone Resistance Mechanism of Mycobacterium tuberculosis DNA Gyrase

Mycobacterium tuberculosis DNA gyrase, an indispensable nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and is hence the sole target for quinolone action, a crucial drug active against multidrug-resistant tuberculosis. To understand at an atomic level the quinolone resistance mechanism, which emerges in extensively drug resistant tuberculosis, we performed combined functional, biophysical and structural studies of the two individual domains constituting the catalytic DNA gyrase reaction core, namely the Toprim and the breakage-reunion domains. This allowed us to produce a model of the catalytic reaction core in complex with DNA and a quinolone molecule, identifying original mechanistic properties of quinolone binding and clarifying the relationships between amino acid mutations and resistance phenotype of M. tuberculosis DNA gyrase. These results are compatible with our previous studies on quinolone resistance. Interestingly, the structure of the entire breakage-reunion domain revealed a new interaction, in which the Quinolone-Binding Pocket (QBP) is blocked by the N-terminal helix of a symmetry-related molecule. This interaction provides useful starting points for designing peptide based inhibitors that target DNA gyrase to prevent its binding to DNA.     

Structural and Biochemical Insights into the Activation Mechanisms of Germinal Center Kinase OSR1

The oxidative stress-responsive 1 (OSR1) kinase belongs to the mammalian STE20-like kinase family. OSR1 is activated by with no lysine [K] (WNKs) kinases, and then it phosphorylates cation-coupled Cl-cotransporters, regulating ion homeostasis and cell volume in mammalian cells. However, the specific mechanisms of OSR1 activation remains poorly defined, largely due to its extremely low basal activity. Here, we dissect in detail the regulatory mechanisms of OSR1 activation from the aspects of autoinhibition, upstream kinase WNK, and the newly identified master regulator mouse protein-25 (MO25). Based on our structural and biochemical studies, we propose a “double lock” model, accounting for the tight autoinhibition of OSR1, an effect that has to be removed by WNK before MO25 further activates OSR1. Particularly, the conserved C-terminal (CCT) domain and αAL helix act together to strongly suppress OSR1 basal activity. WNKs bind to the CCT and trigger its conformational rearrangement to release the kinase domain of OSR1, allowing for MO25 binding and full activation. Finally, the regulatory mechanisms of OSR1 activation were further corroborated by cellular studies of OSR1-regulated cell volume control through WNK-OSR1 signaling pathway. Collectively, these results provide insights into the OSR1 kinase activation to facilitate further functional study.     

Structural Insight into the Activation Mechanism of Human Pancreatic Prophospholipase A2

Pancreatic phospholipase A2 (phospholipase A2 group 1B, G1B) belongs to the superfamily of secreted phospholipase A2 (PLA2) enzymes. G1B has been proposed to be a potential target for diseases such as hypertension, obesity, and diabetes. Human pancreatic prophospholipase A2 (pro-hG1B) is activated by cleavage of the first seven-residue propeptide (phospholipase A2 propeptide, PROP). However, questions still remain on the mode of action for pro-hG1B. In this work, we expressed pro-hG1B in Pichia pastoris and determined the crystal structure at 1.55-Å resolution. The x-ray structure demonstrates that pro-hG1B forms a trimer. In addition, PROP occupies the catalytic cavity and can be self-cleaved at 37 °C. A new membrane-bound surface and activation mechanism are proposed based on the trimeric model of pro-hG1B. We also propose a new autoproteolytic mechanism for pro-hG1B by the reaction triad Asp49-Arg0-Ser(-2) that is similar to the serine protease catalytic triad.Phospholipase A2 (PLA2,⁵ EC 3.1.1.4) hydrolyzes glycerophospholipids at the sn-2 acyl bond to produce free fatty acids. The PLA2 family currently comprises five categories: the secreted PLA2s, the cytosolic PLA2s, the Ca²⁺-independent PLA2s, the platelet-activating factor acetyl hydrolases, and the lysosomal PLA2s. To date, based on the catalytic mechanism as well as functional and structural information, 15 different groups of PLA2 have been reported and named (1).G1B is a member of the secreted PLA2 enzymes. This lipolytic enzyme releases glycerophospholipids and arachidonic acid that serve as the precursors of signal molecules that mediate a multitude of biological functions, such as inflammation. The G1B gene has been reported to be linked to hypertension in three sample populations (2). The concentration of G1B protein in serum is a potential marker for pancreatic acinar cell carcinoma (3). Knock-out mice experiments showed that G1B knockdown can prevent diet-induced obesity and obesity-related insulin resistance (4). After being fed with glucose-rich meals, knock-out mice showed lower postprandial glycemia than wild-type mice (5). A recent report also pointed to the linkage between hG1B and ophthalmic diseases (6). Therefore, hG1B is considered to be a potential target for treatment of obesity, diabetes (4,5), or ophthalmic diseases (6).Pro-hG1B is a digestive zymogen secreted from pancreatic acinar cells in its inactive form (7). It is activated by trypsin in the duodenum. The activation of pro-hG1B by cleavage of the PROP, a heptapeptide of the sequence DSGISPR, is linked to diseases like pancreatitis (8) and acute lung injury (9). Circulating hG1B, mostly in the form of pro-hG1B, indicates pancreatic injury in acute pancreatitis (10). PROP can be used in assays to characterize the severity of acute pancreatitis (11). The C-terminal pentapeptide of PROP (GISPR) is essential for the inhibition of enzyme activity (12). Besides trypsin, pro-hG1B can also be activated by thrombin (13) and 29-kDa trypsin-like endogenous type 1-proPLA2 activator (11). PROP may also play an important role in the folding process as suggested by the refolding experiments of pro-hG1B and hG1B (14).Helix 1 at the N-terminal of hG1B is known to play an important role in enzyme function. An engineered hG1B lacking the N-terminal helix 1 bound to membranes with weaker affinity and exhibited ∼100-fold lower enzymatic activity compared with that of the full-length hG1B. It is inferred that this helix 1 facilitates the membrane binding, thus enhances the enzymatic activity based on polarized infrared spectroscopic experiments (15). Experiments using semi-synthetic hG1B demonstrated that helix 1 residues act as a regulatory domain and mediate interfacial activation (16).It has been generally believed that PLA2 possesses an interfacial binding surface (i-face), which orientates PLA2 for binding to membrane so that PLA2 subsequently hydrolyzes the sn-2 acyl bond of glycerophospholipids (17). A model for this i-face was postulated based on the crystal structure of dimeric porcine G1B (18). Questions still remain on the details of this i-face and the mechanism of its binding to membrane.To date, numerous structures of G1B proteins from various species, including bovine (17, 19–21) and porcine (22,23), have been published. However, there is no report of structure for hG1B or pro-hG1B. The crystal structure of pro-hG1B presented here will illustrate the structural difference of G1B between the human and other species. It will provide structural insight into the activation mechanism of pro-hG1B and potentially facilitate the structure-based design of inhibitor for the treatment of a number of diseases including hypertension, obesity, and diabetes.     

Structural Insights into the Unique Activation Mechanisms of a Non-classical Calpain and Its Disease-Causing Variants

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