Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes.

Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N-terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix-turn-helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination.

The bromo-adjacent homology domains of PBRM1 associate with histone tails and contribute to PBAF-mediated gene regulation.

A critical component of gene regulation is recognition of histones and their post-translational modifications by transcription-associated proteins or complexes. Although many histone-binding reader modules have been characterized, the bromo-adjacent homology (BAH) domain family of readers is still poorly characterized. A pre-eminent member of this family is PBRM1 (BAF180), a component of the PBAF chromatin-remodeling complex. PBRM1 contains two adjacent BAH domains of unknown histone-binding potential. We evaluated the tandem BAH domains for their capacity to associate with histones and to contribute to PBAF-mediated gene regulation. The BAH1 and BAH2 domains of human PBRM1 broadly interacted with histone tails, but they showed a preference for unmodified N-termini of histones H3 and H4. Molecular modeling and comparison of the BAH1 and BAH2 domains with other BAH readers pointed to a conserved binding mode via an extended open pocket and, in general, an aromatic cage for histone lysine binding. Point mutants that were predicted to disrupt the interaction between the BAH domains and histones reduced histone binding in vitro and resulted in dysregulation of genes targeted by PBAF in cellulo. Although the BAH domains in PBRM1 were important for PBAF-mediated gene regulation, we found that overall chromatin targeting of PBRM1 was not dependent on BAH-histone interaction. Our findings identify a function of the PBRM1 BAH domains in PBAF activity that is likely mediated by histone tail interaction.

KSHV Viral Protein Kinase Interacts with USP9X to Modulate the Viral Lifecycle.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy and one of the most common childhood cancers. Immunosuppressed patients, including HIV-infected patients, are more prone to KSHV-associated disease. KSHV encodes a viral protein kinase (vPK) that is expressed from ORF36. KSHV vPK contributes to the optimal production of infectious viral progeny and upregulation of protein synthesis. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used a bottom-up proteomics approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Subsequently, we validated this interaction using a co-immunoprecipitation assay. We report that both the ubiquitin-like and the catalytic domains of USP9X are important for association with vPK. To uncover the biological relevance of the USP9X/vPK interaction, we investigated whether the knockdown of USP9X would modulate viral reactivation. Our data suggest that depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Understanding how USP9X influences the reactivation of KSHV will provide insights into how cellular deubiquitinases regulate viral kinase activity and how viruses co-opt cellular deubiquitinases to propagate infection. Hence, characterizing the roles of USP9X and vPK during KSHV infection constitutes a first step toward identifying a potentially critical interaction that could be targeted by future therapeutics. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy. KSHV encodes a viral protein kinase (vPK) that aids viral replication. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used an affinity purification approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Overall, our data suggest a proviral role for USP9X.

The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2.

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

Identification of a primary antigenic target of epitope spreading in endemic pemphigus foliaceus.

Epitope spreading is an important mechanism for the development of autoantibodies (autoAbs) in autoimmune diseases. The study of epitope spreading in human autoimmune diseases is limited due to the major challenge of identifying the initial/primary target epitopes on autoantigens in autoimmune diseases. We have been studying the development of autoAbs in an endemic human autoimmune disease, Brazilian pemphigus foliaceus (or Fogo Selvagem (FS)). Our previous findings demonstrated that patients before (i.e. preclinical) and at the onset of FS have antibody (Ab) responses against other keratinocyte adhesion molecules in addition to the main target autoantigen of FS, desmoglein 1 (Dsg1), and anti-Dsg1 monoclonal Abs (mAbs) cross-reacted with an environmental antigen LJM11, a sand fly saliva protein. Since sand fly is prevalent in FS endemic regions, individuals in these regions could develop Abs against LJM11. The anti-LJM11 Abs could recognize different epitopes on LJM11, including an epitope that shares the structure similarity with an epitope on Dsg1 autoantigen. Thus, Ab response against this epitope on LJM11 could be the initial autoAb response detected in individuals in FS endemic regions, including those who eventually developed FS. Accordingly, this LJM11 and Dsg1 cross-reactive epitope on Dsg1 could be the primary target of the autoimmune response in FS. This investigation aimed to determine whether the autoAb responses against keratinocyte adhesion molecules are linked and originate from the immune response to LJM11. The anti-Dsg1 mAbs from preclinical FS and FS individuals were employed to determine their specificity or cross-reactivity to LJM11 and keratinocyte adhesion molecules. The cross-reactive epitopes on autoantigens were mapped. Our results indicate that all tested mAbs cross-reacted with LJM11 and keratinocyte adhesion molecules, and we identified an epitope on these keratinocyte adhesion molecules which is mimicked by LJM11. Thus, the cross-reactivity could be the mechanism by which the immune response against an environmental antigen triggers the initial autoAb responses. Epitope spreading leads to the pathogenic autoAb development and ensuing FS among genetically susceptible individuals.

Temporal Regulation of Dendritic Spines Through NrCAM-Semaphorin3F Receptor Signaling in Developing Cortical Pyramidal Neurons.

Neuron-glial related cell adhesion molecule NrCAM is a newly identified negative regulator of spine density that genetically interacts with Semaphorin3F (Sema3F), and is implicated in autism spectrum disorders (ASD). To investigate a role for NrCAM in spine pruning during the critical adolescent period when networks are established, we generated novel conditional, inducible NrCAM mutant mice (Nex1Cre-ERT2: NrCAMflox/flox). We demonstrate that NrCAM functions cell autonomously during adolescence in pyramidal neurons to restrict spine density in the visual (V1) and medial frontal cortex (MFC). Guided by molecular modeling, we found that NrCAM promoted clustering of the Sema3F holoreceptor complex by interfacing with Neuropilin-2 (Npn2) and PDZ scaffold protein SAP102. NrCAM-induced receptor clustering stimulated the Rap-GAP activity of PlexinA3 (PlexA3) within the holoreceptor complex, which in turn, inhibited Rap1-GTPase and inactivated adhesive ß1 integrins, essential for Sema3F-induced spine pruning. These results define a developmental function for NrCAM in Sema3F receptor signaling that limits dendritic spine density on cortical pyramidal neurons during adolescence.

Formation of a TBX20-CASZ1 protein complex is protective against dilated cardiomyopathy and critical for cardiac homeostasis.

By the age of 40, one in five adults without symptoms of cardiovascular disease are at risk for developing congestive heart failure. Within this population, dilated cardiomyopathy (DCM) remains one of the leading causes of disease and death, with nearly half of cases genetically determined. Though genetic and high throughput sequencing-based approaches have identified sporadic and inherited mutations in a multitude of genes implicated in cardiomyopathy, how combinations of asymptomatic mutations lead to cardiac failure remains a mystery. Since a number of studies have implicated mutations of the transcription factor TBX20 in congenital heart diseases, we investigated the underlying mechanisms, using an unbiased systems-based screen to identify novel, cardiac-specific binding partners. We demonstrated that TBX20 physically and genetically interacts with the essential transcription factor CASZ1. This interaction is required for survival, as mice heterozygous for both Tbx20 and Casz1 die post-natally as a result of DCM. A Tbx20 mutation associated with human familial DCM sterically interferes with the TBX20-CASZ1 interaction and provides a physical basis for how this human mutation disrupts normal cardiac function. Finally, we employed quantitative proteomic analyses to define the molecular pathways mis-regulated upon disruption of this novel complex. Collectively, our proteomic, biochemical, genetic, and structural studies suggest that the physical interaction between TBX20 and CASZ1 is required for cardiac homeostasis, and further, that reduction or loss of this critical interaction leads to DCM. This work provides strong evidence that DCM can be inherited through a digenic mechanism.

A viral kinase mimics S6 kinase to enhance cell proliferation.

Viruses depend upon the host cell for manufacturing components of progeny virions. To mitigate the inextricable dependence on host cell protein synthesis, viruses can modulate protein synthesis through a variety of mechanisms. We report that the viral protein kinase (vPK) encoded by open reading frame 36 (ORF36) of Kaposi's sarcoma-associated herpesvirus (KSHV) enhances protein synthesis by mimicking the function of the cellular protein S6 kinase (S6KB1). Similar to S6KB1, vPK phosphorylates the ribosomal S6 protein and up-regulates global protein synthesis. vPK also augments cellular proliferation and anchorage-independent growth. Furthermore, we report that both vPK and S6KB1 phosphorylate the enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 2 (PFKFB2) and that both kinases promote endothelial capillary tubule formation.

Pathogenic IgG4 autoantibodies from endemic pemphigus foliaceus recognize a desmoglein-1 conformational epitope.

Fogo Selvagem (FS), the endemic form of pemphigus foliaceus, is mediated by pathogenic IgG4 autoantibodies against the amino-terminal extracellular cadherin domain of the desmosomal cadherin desmoglein 1 (Dsg1). Here we define the detailed epitopes of these pathogenic antibodies. Proteolytic footprinting showed that IgG4 from 95% of FS donor sera (19/20) recognized a 16-residue peptide (A129LNSMGQDLERPLELR144) from the EC1 domain of Dsg1 that overlaps the binding site for an adhesive-partner desmosomal cadherin molecule. Mutation of Dsg1 residues M133 and Q135 reduced the binding of FS IgG4 autoantibodies to Dsg1 by ∼50%. Molecular modeling identified two nearby EC1 domain residues (Q82 and V83) likely to contribute to the epitope. Mutation of these residues completely abolished the binding of FS IgG4 to Dsg1. Bead aggregation assays showed that native binding interactions between Dsg1 and desmocollin 1 (Dsc1), which underlie desmosome structure, were abolished by Fab fragments of FS IgG4. These results further define the molecular mechanism by which FS IgG4 autoantibodies interfere with desmosome structure and lead to cell-cell detachment, the hallmark of this disease.

Structural complexity and functional diversity of plant NADPH oxidases.

Plant NADPH oxidases also known as respiratory burst oxidase homologs (Rbohs) are a family of membrane-bound enzymes that play diverse roles in the defense response and morphogenetic processes via regulated generation of reactive oxygen species. Rbohs are associated with a variety of functions, although the reason for this is not clear. To evaluate using bioinformatics, the possible mechanisms for the observed functional diversity within the plant kingdom, 127 Rboh protein sequences representing 26 plant species were analyzed. Multiple clusters were identified with gene duplications that were both dicot as well as monocot-specific. The N-terminal sequences were observed to be highly variable. The conserved cysteine (equivalent of Cys890) in C-terminal of AtRbohD suggested that the redox-based modification like S-nitrosylation may regulate the activity of other Rbohs. Three-dimensional models corresponding to the N-terminal domain for Rbohs from Arabidopsis thaliana and Oryza sativa were constructed and molecular dynamics studies were carried out to study the role of Ca2+ in the folding of Rboh proteins. Certain mutations indicated possibly affect the structure and function of the plant NADPH oxidases, thereby providing the rationale for further experimental validation.

The Neural Cell Adhesion Molecule (NCAM) Promotes Clustering and Activation of EphA3 Receptors in GABAergic Interneurons to Induce Ras Homolog Gene Family, Member A (RhoA)/Rho-associated protein kinase (ROCK)-mediated Growth Cone Collapse.

Establishment of a proper balance of excitatory and inhibitory connectivity is achieved during development of cortical networks and adjusted through synaptic plasticity. The neural cell adhesion molecule (NCAM) and the receptor tyrosine kinase EphA3 regulate the perisomatic synapse density of inhibitory GABAergic interneurons in the mouse frontal cortex through ephrin-A5-induced growth cone collapse. In this study, it was demonstrated that binding of NCAM and EphA3 occurred between the NCAM Ig2 domain and EphA3 cysteine-rich domain (CRD). The binding interface was further refined through molecular modeling and mutagenesis and shown to be comprised of complementary charged residues in the NCAM Ig2 domain (Arg-156 and Lys-162) and the EphA3 CRD (Glu-248 and Glu-264). Ephrin-A5 induced co-clustering of surface-bound NCAM and EphA3 in GABAergic cortical interneurons in culture. Receptor clustering was impaired by a charge reversal mutation that disrupted NCAM/EphA3 association, emphasizing the importance of the NCAM/EphA3 binding interface for cluster formation. NCAM enhanced ephrin-A5-induced EphA3 autophosphorylation and activation of RhoA GTPase, indicating a role for NCAM in activating EphA3 signaling through clustering. NCAM-mediated clustering of EphA3 was essential for ephrin-A5-induced growth cone collapse in cortical GABAergic interneurons, and RhoA and a principal effector, Rho-associated protein kinase, mediated the collapse response. This study delineates a mechanism in which NCAM promotes ephrin-A5-dependent clustering of EphA3 through interaction of the NCAM Ig2 domain and the EphA3 CRD, stimulating EphA3 autophosphorylation and RhoA signaling necessary for growth cone repulsion in GABAergic interneurons in vitro, which may extend to remodeling of axonal terminals of interneurons in vivo.

Staphylococcus aureus lactate- and malate-quinone oxidoreductases contribute to nitric oxide resistance and virulence.

Staphylococcus aureus is a Gram-positive pathogen that resists many facets of innate immunity including nitric oxide (NO·). Staphylococcus aureus NO-resistance stems from its ability to evoke a metabolic state that circumvents the negative effects of reactive nitrogen species. The combination of l-lactate and peptides promotes S. aureus growth at moderate NO-levels, however, neither nutrient alone suffices. Here, we investigate the staphylococcal malate-quinone and l-lactate-quinone oxidoreductases (Mqo and Lqo), both of which are critical during NO-stress for the combined utilization of peptides and l-lactate. We address the specific contributions of Lqo-mediated l-lactate utilization and Mqo-dependent amino acid consumption during NO-stress. We show that Lqo conversion of l-lactate to pyruvate is required for the formation of ATP, an essential energy source for peptide utilization. Thus, both Lqo and Mqo are essential for growth under these conditions making them attractive candidates for targeted therapeutics. Accordingly, we exploited a modelled Mqo/Lqo structure to define the catalytic and substrate-binding residues.We also compare the S. aureus Mqo/Lqo enzymes to their close relatives throughout the staphylococci and explore the substrate specificities of each enzyme. This study provides the initial characterization of the mechanism of action and the immunometabolic roles for a newly defined staphylococcal enzyme family.

In vitro reconstitution of Rab GTPase-dependent vesicle clustering by the yeast lethal giant larvae/tomosyn homolog, Sro7.

Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers that tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here, we describe a novel assay that recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 that results in a protein that is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this, we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9, which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur.

Structural analyses to identify selective inhibitors of glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme.

STUDY HYPOTHESIS: Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development. STUDY FINDING: This study identified a small-molecule GAPDHS inhibitor with micromolar potency and >10-fold selectivity that exerts the expected inhibitory effects on sperm glycolysis and motility. WHAT IS KNOWN ALREADY: Glycolytic ATP production is required for sperm motility and male fertility in many mammalian species. Selective inhibition of GAPDHS, one of the glycolytic isozymes with restricted expression during spermatogenesis, is a potential strategy for the development of a non-hormonal contraceptive that directly blocks sperm function. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Homology modeling and x-ray crystallography were used to identify structural features that are conserved in GAPDHS orthologs in mouse and human sperm, but distinct from the GAPDH orthologs present in somatic tissues. We identified three binding pockets surrounding the substrate and cofactor in these isozymes and conducted a virtual screen to identify small-molecule compounds predicted to bind more tightly to GAPDHS than to GAPDH. Following the production of recombinant human and mouse GAPDHS, candidate compounds were tested in dose-response enzyme assays to identify inhibitors that blocked the activity of GAPDHS more effectively than GAPDH. The effects of a selective inhibitor on the motility of mouse and human sperm were monitored by computer-assisted sperm analysis, and sperm lactate production was measured to assess inhibition of glycolysis in the target cell. MAIN RESULTS AND THE ROLE OF CHANCE: Our studies produced the first apoenzyme crystal structures for human and mouse GAPDHS and a 1.73 Å crystal structure for NAD(+)-bound human GAPDHS, facilitating the identification of unique structural features of this sperm isozyme. In dose-response assays T0501_7749 inhibited human GAPDHS with an IC50 of 1.2 µM compared with an IC50 of 38.5 µM for the somatic isozyme. This compound caused significant reductions in mouse sperm lactate production (P= 0.017 for 100 µM T0501_7749 versus control) and in the percentage of motile mouse and human sperm (P values from <0.05 to <0.0001, depending on incubation conditions). LIMITATIONS, REASONS FOR CAUTION: The chemical properties of T0501_7749, including limited solubility and nonspecific protein binding, are not optimal for drug development. WIDER IMPLICATIONS OF THE FINDINGS: This study provides proof-of-principle evidence that GAPDHS can be selectively inhibited, causing significant reductions in sperm glycolysis and motility. These results highlight the utility of structure-based drug design and support further exploration of GAPDHS, and perhaps other sperm-specific isozymes in the glycolytic pathway, as contraceptive targets. LARGE SCALE DATA: None. Coordinates and data files for three GAPDHS crystal structures were deposited in the RCSB Protein Data Bank (http://www.rcsb.org). STUDY FUNDING AND COMPETING INTERESTS: This work was supported by grants from the National Institutes of Health (NIH), USA, including U01 HD060481 and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and TW/HD00627 from the NIH Fogarty International Center. Additional support was provided by subproject CIG-05-109 from CICCR, a program of CONRAD, Eastern Virginia Medical School, USA. There are no conflicts of interest.

PITPs as targets for selectively interfering with phosphoinositide signaling in cells.

Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs nor PITPs in general have been exploited as targets for chemical inhibition for such purposes. Herein, we validate what is to our knowledge the first small-molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs) and are effective inhibitors in vitro and in vivo. We further establish that Sec14 is the sole essential NPPM target in yeast and that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects and demonstrate that NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof of concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies.

Differences in intradomain and interdomain motion confer distinct activation properties to structurally similar Gα proteins.

Proteins with similar crystal structures can have dissimilar rates of substrate binding and catalysis. Here we used molecular dynamics simulations and biochemical analysis to determine the role of intradomain and interdomain motions in conferring distinct activation rates to two Gα proteins, Gα(i1) and GPA1. Despite high structural similarity, GPA1 can activate itself without a receptor, whereas Gα(i1) cannot. We found that motions in these proteins vary greatly in type and frequency. Whereas motion is greatest in the Ras domain of Gα(i1), it is greatest in helices αA and αB from the helical domain of GPA1. Using protein chimeras, we show that helix αA from GPA1 is sufficient to confer rapid activation to Gα(i1). Gα(i1) has less intradomain motion than GPA1 and instead displays interdomain displacement resembling that observed in a receptor-heterotrimer crystal complex. Thus, structurally similar proteins can have distinct atomic motions that confer distinct activation mechanisms.

Gα12 structural determinants of Hsp90 interaction are necessary for serum response element-mediated transcriptional activation.

The G12/13 class of heterotrimeric G proteins, comprising the α-subunits Gα12 and Gα13, regulates multiple aspects of cellular behavior, including proliferation and cytoskeletal rearrangements. Although guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) are well characterized as effectors of this G protein class, a variety of other downstream targets has been reported. To identify Gα12 determinants that mediate specific protein interactions, we used a structural and evolutionary comparison between the G12/13, Gs, Gi, and Gq classes to identify "class-distinctive" residues in Gα12 and Gα13. Mutation of these residues in Gα12 to their deduced ancestral forms revealed a subset necessary for activation of serum response element (SRE)-mediated transcription, a G12/13-stimulated pathway implicated in cell proliferative signaling. Unexpectedly, this subset of Gα12 mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of Gα13 exhibited robust SRE activation, suggesting a Gα12-specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA-mediated lowering of Hsp90 levels resulted in greater downregulation of Gα12 than Gα13 signaling in SRE activation experiments. Furthermore, the Drosophila G12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and Gα12:Concertina chimeras revealed Gα12-specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify Gα12 determinants of SRE activation, implicate Gα12:Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of Gα12 and Gα13 to allow bifurcated mechanisms of signaling to a common cell proliferative pathway.

Heterotrimeric G-protein signaling is critical to pathogenic processes in Entamoeba histolytica.

Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGßγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.

A highly conserved interaction involving the middle residue of the SXN active-site motif is crucial for function of class B penicillin-binding proteins: mutational and computational analysis of PBP 2 from N. gonorrhoeae.

Insertion of an aspartate residue at position 345a in penicillin-binding protein 2 (PBP 2), which lowers the rate of penicillin acylation by ~6-fold, is commonly observed in penicillin-resistant strains of Neisseria gonorrhoeae. Here, we show that insertions of other amino acids also lower the penicillin acylation rate of PBP 2, but none supported growth of N. gonorrhoeae, indicating loss of essential transpeptidase activity. The Asp345a mutation likely acts by altering the interaction between its adjacent residue, Asp346, in the ß2a-ß2d hairpin loop and Ser363, the middle residue of the SXN active site motif. Because the adjacent aspartate creates ambiguity in the position of the insertion, we also examined if insertions at position 346a could confer decreased susceptibility to penicillin. However, only aspartate insertions were identified, indicating that only an Asp-Asp couple can confer resistance and retain transpeptidase function. The importance of the Asp346-Ser363 interaction was assessed by mutation of each residue to Ala. Although both mutants lowered the acylation rate of penicillin G by 5-fold, neither could support growth of N. gonorrhoeae, again indicating loss of transpeptidase function. Interaction between a residue in the equivalent of the ß2a-ß2d hairpin loop and the middle residue of the SXN motif is observed in crystal structures of other Class B PBPs, and its importance is also supported by multisequence alignments. Overall, these results suggest that this conserved interaction can be manipulated (e.g., by insertion) to lower the acylation rate by ß-lactam antibiotics and increase resistance, but only if essential transpeptidase activity is preserved.

Recognition of platinum-DNA adducts by HMGB1a.

Cisplatin (CP) and oxaliplatin (OX), platinum-based drugs used widely in chemotherapy, form adducts on intrastrand guanines (5'GG) in genomic DNA. DNA damage recognition proteins, transcription factors, mismatch repair proteins, and DNA polymerases discriminate between CP- and OX-GG DNA adducts, which could partly account for differences in the efficacy, toxicity, and mutagenicity of CP and OX. In addition, differential recognition of CP- and OX-GG adducts is highly dependent on the sequence context of the Pt-GG adduct. In particular, DNA binding protein domain HMGB1a binds to CP-GG DNA adducts with up to 53-fold greater affinity than to OX-GG adducts in the TGGA sequence context but shows much smaller differences in binding in the AGGC or TGGT sequence contexts. Here, simulations of the HMGB1a-Pt-DNA complex in the three sequence contexts revealed a higher number of interface contacts for the CP-DNA complex in the TGGA sequence context than in the OX-DNA complex. However, the number of interface contacts was similar in the TGGT and AGGC sequence contexts. The higher number of interface contacts in the CP-TGGA sequence context corresponded to a larger roll of the Pt-GG base pair step. Furthermore, geometric analysis of stacking of phenylalanine 37 in HMGB1a (Phe37) with the platinated guanines revealed more favorable stacking modes correlated with a larger roll of the Pt-GG base pair step in the TGGA sequence context. These data are consistent with our previous molecular dynamics simulations showing that the CP-TGGA complex was able to sample larger roll angles than the OX-TGGA complex or either CP- or OX-DNA complexes in the AGGC or TGGT sequences. We infer that the high binding affinity of HMGB1a for CP-TGGA is due to the greater flexibility of CP-TGGA compared to OX-TGGA and other Pt-DNA adducts. This increased flexibility is reflected in the ability of CP-TGGA to sample larger roll angles, which allows for a higher number of interface contacts between the Pt-DNA adduct and HMGB1a.