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.

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.

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.

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.

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.

Functional reconstitution of an atypical G protein heterotrimer and regulator of G protein signaling protein (RGS1) from Arabidopsis thaliana.

It has long been known that animal heterotrimeric Gαßγ proteins are activated by cell-surface receptors that promote GTP binding to the Gα subunit and dissociation of the heterotrimer. In contrast, the Gα protein from Arabidopsis thaliana (AtGPA1) can activate itself without a receptor or other exchange factor. It is unknown how AtGPA1 is regulated by Gßγ and the RGS (regulator of G protein signaling) protein AtRGS1, which is comprised of an RGS domain fused to a receptor-like domain. To better understand the cycle of G protein activation and inactivation in plants, we purified and reconstituted AtGPA1, full-length AtRGS1, and two putative Gßγ dimers. We show that the Arabidopsis Gα protein binds to its cognate Gßγ dimer directly and in a nucleotide-dependent manner. Although animal Gßγ dimers inhibit GTP binding to the Gα subunit, AtGPA1 retains fast activation in the presence of its cognate Gßγ dimer. We show further that the full-length AtRGS1 protein accelerates GTP hydrolysis and thereby counteracts the fast nucleotide exchange rate of AtGPA1. Finally, we show that AtGPA1 is less stable in complex with GDP than in complex with GTP or the Gßγ dimer. Molecular dynamics simulations and biophysical studies reveal that altered stability is likely due to increased dynamic motion in the N-terminal α-helix and Switch II of AtGPA1. Thus, despite profound differences in the mechanisms of activation, the Arabidopsis G protein is readily inactivated by its cognate RGS protein and forms a stable, GDP-bound, heterotrimeric complex similar to that found in animals.

SmgGDS is a guanine nucleotide exchange factor that specifically activates RhoA and RhoC.

SmgGDS is an atypical guanine nucleotide exchange factor (GEF) that promotes both cell proliferation and migration and is up-regulated in several types of cancer. SmgGDS has been previously shown to activate a wide variety of small GTPases, including the Ras family members Rap1a, Rap1b, and K-Ras, as well as the Rho family members Cdc42, Rac1, Rac2, RhoA, and RhoB. In contrast, here we show that SmgGDS exclusively activates RhoA and RhoC among a large panel of purified GTPases. Consistent with the well known properties of GEFs, this activation is catalytic, and SmgGDS preferentially binds to nucleotide-depleted RhoA relative to either GDP- or GTPγS-bound forms. However, mutational analyses indicate that SmgGDS utilizes a distinct exchange mechanism compared with canonical GEFs and in contrast to known GEFs requires RhoA to retain a polybasic region for activation. A homology model of SmgGDS highlights an electronegative surface patch and a highly conserved binding groove. Mutation of either area ablates the ability of SmgGDS to activate RhoA. Finally, the in vitro specificity of SmgGDS for RhoA and RhoC is retained in cells. Together, these results indicate that SmgGDS is a bona fide GEF that specifically activates RhoA and RhoC through a unique mechanism not used by other Rho family exchange factors.

Acireductone dioxygenase 1 (ARD1) is an effector of the heterotrimeric G protein beta subunit in Arabidopsis.

Heterotrimeric G protein complexes are conserved from plants to mammals, but the complexity of each system varies. Arabidopsis thaliana contains one Gα, one Gß (AGB1), and at least three Gγ subunits, allowing it to form three versions of the heterotrimer. This plant model is ideal for genetic studies because mammalian systems contain hundreds of unique heterotrimers. The activation of these complexes promotes interactions between both the Gα subunit and the Gßγ dimer with enzymes and scaffolds to propagate signaling to the cytoplasm. However, although effectors of Gα and Gß are known in mammals, no Gß effectors were previously known in plants. Toward identifying AGB1 effectors, we genetically screened for dominant mutations that suppress Gß-null mutant (agb1-2) phenotypes. We found that overexpression of acireductone dioxygenase 1 (ARD1) suppresses the 2-day-old etiolated phenotype of agb1-2. ARD1 is homologous to prokaryotic and eukaryotic ARD proteins; one function of ARDs is to operate in the methionine salvage pathway. We show here that ARD1 is an active metalloenzyme, and AGB1 and ARD1 both control embryonic hypocotyl length by modulating cell division; they also may contribute to the production of ethylene, a product of the methionine salvage pathway. ARD1 physically interacts with AGB1, and ARD enzymatic activity is stimulated by AGB1 in vitro. The binding interface on AGB1 was deduced using a comparative evolutionary approach and tested using recombinant AGB1 mutants. A possible mechanism for AGB1 activation of ARD1 activity was tested using directed mutations in a loop near the substrate-binding site.

Evolution and virulence contributions of the autotransporter proteins YapJ and YapK of Yersinia pestis CO92 and their homologs in Y. pseudotuberculosis IP32953.

Yersinia pestis, the causative agent of plague, evolved from the gastrointestinal pathogen Yersinia pseudotuberculosis. Both species have numerous type Va autotransporters, most of which appear to be highly conserved. In Y. pestis CO92, the autotransporter genes yapK and yapJ share a high level of sequence identity. By comparing yapK and yapJ to three homologous genes in Y. pseudotuberculosis IP32953 (YPTB0365, YPTB3285, and YPTB3286), we show that yapK is conserved in Y. pseudotuberculosis, while yapJ is unique to Y. pestis. All of these autotransporters exhibit >96% identity in the C terminus of the protein and identities ranging from 58 to 72% in their N termini. By extending this analysis to include homologous sequences from numerous Y. pestis and Y. pseudotuberculosis strains, we determined that these autotransporters cluster into a YapK (YPTB3285) class and a YapJ (YPTB3286) class. The YPTB3286-like gene of most Y. pestis strains appears to be inactivated, perhaps in favor of maintaining yapJ. Since autotransporters are important for virulence in many bacterial pathogens, including Y. pestis, any change in autotransporter content should be considered for its impact on virulence. Using established mouse models of Y. pestis infection, we demonstrated that despite the high level of sequence identity, yapK is distinct from yapJ in its contribution to disseminated Y. pestis infection. In addition, a mutant lacking both of these genes exhibits an additive attenuation, suggesting nonredundant roles for yapJ and yapK in systemic Y. pestis infection. However, the deletion of the homologous genes in Y. pseudotuberculosis does not seem to impact the virulence of this organism in orogastric or systemic infection models.

The plant heterotrimeric G-protein complex.

Heterotrimeric G-protein complexes couple extracellular signals via cell surface receptors to downstream enzymes called effectors. Heterotrimeric G-protein complexes, together with their cognate receptors and effectors, operate at the apex of signal transduction. In plants, the number of G-protein complex components is dramatically less than in other multicellular eukaryotes. An understanding of how multiple signals propagate transduction through the G-protein node can be found in the unique structural and kinetic properties of the plant heterotrimeric G-protein complex. This review addresses these unique features and speculates on why the repertoire of G-protein signaling elements is dramatically simpler than that in all other multicellular eukaryotes.

Distinct developmental phenotypes result from mutation of Set8/KMT5A and histone H4 lysine 20 in Drosophila melanogaster.

Mono-methylation of histone H4 lysine 20 (H4K20me1) is catalyzed by Set8/KMT5A and regulates numerous aspects of genome organization and function. Loss-of-function mutations in Drosophila melanogaster Set8 or mammalian KMT5A prevent H4K20me1 and disrupt development. Set8/KMT5A also has non-histone substrates, making it difficult to determine which developmental functions of Set8/KMT5A are attributable to H4K20me1 and which to other substrates or to non-catalytic roles. Here, we show that human KMT5A can functionally substitute for Set8 during Drosophila development and that the catalytic SET domains of the two enzymes are fully interchangeable. We also uncovered a role in eye development for the N-terminal domain of Set8 that cannot be complemented by human KMT5A. Whereas Set820/20 null mutants are inviable, we found that an R634G mutation in Set8 predicted from in vitro experiments to ablate catalytic activity resulted in viable adults. Additionally, Set8(R634G) mutants retain significant, albeit reduced, H4K20me1, indicating that the R634G mutation does not eliminate catalytic activity in vivo and is functionally hypomorphic rather than null. Flies engineered to express only unmodifiable H4 histones (H4K20A) can also complete development, but are phenotypically distinct from H4K20R, Set820/20 null, and Set8R634G mutants. Taken together, our results demonstrate functional conservation of KMT5A and Set8 enzymes, as well as distinct roles for Set8 and H4K20me1 in Drosophila development.

Dynamics of allosteric regulation of the phospholipase C-γ isozymes upon recruitment to membranes.

Numerous receptor tyrosine kinases and immune receptors activate phospholipase C-γ (PLC-γ) isozymes at membranes to control diverse cellular processes including phagocytosis, migration, proliferation, and differentiation. The molecular details of this process are not well understood. Using hydrogen-deuterium exchange mass spectrometry, we show that PLC-γ1 is relatively inert to lipid vesicles that contain its substrate, phosphatidylinositol 4,5-bisphosphate (PIP2), unless first bound to the kinase domain of the fibroblast growth factor receptor (FGFR1). Exchange occurs throughout PLC-γ1 and is exaggerated in PLC-γ1 containing an oncogenic substitution (D1165H) that allosterically activates the lipase. These data support a model whereby initial complex formation shifts the conformational equilibrium of PLC-γ1 to favor activation. This receptor-induced priming of PLC-γ1 also explains the capacity of a kinase-inactive fragment of FGFR1 to modestly enhance the lipase activity of PLC-γ1 operating on lipid vesicles but not a soluble analog of PIP2 and highlights potential cooperativity between receptor engagement and membrane proximity. Priming is expected to be greatly enhanced for receptors embedded in membranes and nearly universal for the myriad of receptors and co-receptors that bind the PLC-γ isozymes.

Isoform divergence of the filamin family of proteins.

The vertebrate filamin family (A, B, and C) is part of the spectrin family of actin cross-linking proteins. Family members share high sequence similarity (>64%) and have both common and isoform-distinct functionalities. To identify the basis for isoform-specific functionality, we perform an evolutionary trace of chordate filamin at the granularity of single residues. Our trace methodology is constrained to focus on neofunctionality by requiring that one isoform remain the ancestral type, whereas at least one isoform has an accepted mutation. We call divergence meeting these characteristics "class-distinctive." To obtain a temporal and spatial context for class-distinctive residues, we derive an all-atom model of full-length filamin A by homology modeling and joining individual domains. We map onto our model both conserved and class-distinctive residues along with the period (Teleostei, Amphibian, and Mammalian) in which they diverged. Our phylogenetic analysis suggests that filamins diverged from a common ancestral gene between urochordate and vertebrate lineages. Filamins also diverged the most just after gene duplication, in the Teleostei period, with filamin C remaining closest to ancestral filamin. At the residue level, domains with well-characterized interfaces, IgFLN 17 and IgFLN 21 (immunoglobulin, Ig), have diverged in potentially critical residues in their adhesion protein-binding interfaces, signifying that isoforms may bind or regulate ligand binding differentially. Similarly, isoform divergence in a region associated with F actin-binding regulation suggests that isoforms differentially regulate F-actin binding. In addition, we observe some class-distinctive residues in the vicinity of missense mutations that cause filamin A and B-associated skeletal disorders. Our analysis, utilizing both spatial and temporal granularity, has identified potentially important residues responsible for vertebrate filamin isoform-specific divergence-significantly in regions where few binding partners have been discovered to date- and suggests yet to be discovered filamin-binding partners and isoform-specific differential regulation with these binding partners.

Evolution of a signaling nexus constrained by protein interfaces and conformational States.

Heterotrimeric G proteins act as the physical nexus between numerous receptors that respond to extracellular signals and proteins that drive the cytoplasmic response. The Gα subunit of the G protein, in particular, is highly constrained due to its many interactions with proteins that control or react to its conformational state. Various organisms contain differing sets of Gα-interacting proteins, clearly indicating that shifts in sequence and associated Gα functionality were acquired over time. These numerous interactions constrained much of Gα evolution; yet Gα has diversified, through poorly understood processes, into several functionally specialized classes, each with a unique set of interacting proteins. Applying a synthetic sequence-based approach to mammalian Gα subunits, we established a set of seventy-five evolutionarily important class-distinctive residues, sites where a single Gα class is differentiated from the three other classes. We tested the hypothesis that shifts at these sites are important for class-specific functionality. Importantly, we mapped known and well-studied class-specific functionalities from all four mammalian classes to sixteen of our class-distinctive sites, validating the hypothesis. Our results show how unique functionality can evolve through the recruitment of residues that were ancestrally functional. We also studied acquisition of functionalities by following these evolutionarily important sites in non-mammalian organisms. Our results suggest that many class-distinctive sites were established early on in eukaryotic diversification and were critical for the establishment of new Gα classes, whereas others arose in punctuated bursts throughout metazoan evolution. These Gα class-distinctive residues are rational targets for future structural and functional studies.

Self-association of the Lentivirus protein, Nef.

BACKGROUND: The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization. RESULTS: By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIV(MAC239)Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface. CONCLUSIONS: We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.

Conformational dynamics of the major yeast phosphatidylinositol transfer protein sec14p: insight into the mechanisms of phospholipid exchange and diseases of sec14p-like protein deficiencies.

Molecular dynamics simulations coupled with functional analyses of the major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p identify structural elements involved in regulating the ability of Sec14p to execute phospholipid exchange. The molecular dynamics simulations suggest large rigid body motions within the Sec14p molecule accompany closing and opening of an A(10)/T(4)/A(11) helical gate, and that "state-of-closure" of this helical gate determines access to the Sec14p phospholipid binding cavity. The data also project that conformational dynamics of the helical gate are controlled by a hinge unit (residues F(212), Y(213), K(239), I(240), and I(242)) that links to the N- and C-terminal ends of the helical gate, and by a novel gating module (composed of the B(1)LB(2) and A(12)LT(5) substructures) through which conformational information is transduced to the hinge. The (114)TDKDGR(119) motif of B(1)LB(2) plays an important role in that transduction process. These simulations offer new mechanistic possibilities for an important half-reaction of the Sec14p phospholipid exchange cycle that occurs on membrane surfaces after Sec14p has ejected bound ligand, and is reloading with another phospholipid molecule. These conformational transitions further suggest structural rationales for known disease missense mutations that functionally compromise mammalian members of the Sec14-protein superfamily.

Active nuclear receptors exhibit highly correlated AF-2 domain motions.

Nuclear receptor ligand binding domains (LBDs) convert ligand binding events into changes in gene expression by recruiting transcriptional coregulators to a conserved activation function-2 (AF-2) surface. While most nuclear receptor LBDs form homo- or heterodimers, the human nuclear receptor pregnane X receptor (PXR) forms a unique and essential homodimer and is proposed to assemble into a functional heterotetramer with the retinoid X receptor (RXR). How the homodimer interface, which is located 30 A from the AF-2, would affect function at this critical surface has remained unclear. By using 20- to 30-ns molecular dynamics simulations on PXR in various oligomerization states, we observed a remarkably high degree of correlated motion in the PXR-RXR heterotetramer, most notably in the four helices that create the AF-2 domain. The function of such correlation may be to create "active-capable" receptor complexes that are ready to bind to transcriptional coactivators. Indeed, we found in additional simulations that active-capable receptor complexes involving other orphan or steroid nuclear receptors also exhibit highly correlated AF-2 domain motions. We further propose a mechanism for the transmission of long-range motions through the nuclear receptor LBD to the AF-2 surface. Taken together, our findings indicate that long-range motions within the LBD scaffold are critical to nuclear receptor function by promoting a mobile AF-2 state ready to bind coactivators.

Opinion: alternative views of AMP-activated protein kinase.

Genes most closely related to adenosine monophosphate (AMP)-activated protein kinase, including SAD kinases and Par-1 regulate cell polarity, although AMP-activated protein kinase (AMPK) modulates cellular energy status. LKB1 (Par-4) is required for normal activation of AMPK in the liver and also regulates cell polarity. AMPK is proposed to inhibit energy consuming activity while initiating energy producing activity during energy limitation. Demonstration that metformin, a common drug for Type 2 diabetes, requires LKB1 for full therapeutic benefit has increased interest in AMPK signaling. Despite the potential importance of AMPK signaling for diabetes, metabolic syndrome and even cancer, the developmental processes regulated by AMPK in genetically mutant animals require further elucidation. Mouse conditional null mutants for AMPK activity will allow genetic elucidation of AMPK function in vivo. This perspective focuses on sequence and structural moieties of AMPK and genetic analysis of AMPK mutations. Interestingly, the predicted protein structure of the carboxy-terminus of AMPKalpha resembles the carboxy-terminal KA-1 domain of MARK3, a Par-1 orthologue.