Amanda Nourse

PUMA binding induces partial unfolding within BCL-xL to disrupt p53 binding and promote apoptosis

Following DNA damage, nuclear p53 induces the expression of PUMA, a BH3-only protein that binds and inhibits the anti-apoptotic BCL-2 repertoire, including BCL-xL. PUMA, unique amongst BH3-only proteins, disrupts the interaction between cytosolic p53 and BCL-xL, allowing p53 to promote apoptosis via direct activation of the BCL-2 effector molecules, BAX and BAK. Structural investigations using nuclear magnetic resonance spectroscopy and X-ray crystallography revealed that PUMA binding induced partial unfolding of two α-helices within BCL-xL. Wild-type PUMA or a PUMA mutant incapable of causing binding-induced unfolding of BCL-xL equivalently inhibited the anti-apoptotic BCL-2 repertoire to sensitize for death receptor (DR)-activated apoptosis, but only wild-type PUMA promoted p53-dependent, DNA damage-induced apoptosis. Our data suggest that PUMA-induced partial unfolding of BCL-xL disrupts interactions between cytosolic p53 and BCL-xL, releasing the bound p53 to initiate apoptosis. We propose that regulated unfolding of BCL-xL provides a mechanism to promote PUMA-dependent signaling within the apoptotic pathways.

Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA

The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus. DOI: http://dx.doi.org/10.7554/eLife.13571.001

Structural polymorphism in the N-terminal oligomerization domain of NPM1

Significance Nucleophosmin (NPM1) is a multifunctional protein with critical roles in ribosome biogenesis, centrosome duplication, and tumor suppression. Despite the established importance of NPM1 as a tumor marker and potential drug target, little is currently known about the molecular mechanisms that govern its various functions. Our manuscript describes that the N-terminal domain of NPM1 (Npm-N) exhibits phosphorylation-dependent structural polymorphism along a broad conformational landscape between two extreme states: a stable, folded pentamer and a globally disordered monomer. We propose that phosphorylation-induced “regulated unfolding” of Npm-N provides a means to modulate NPM1 function and subcellular localization. Our findings will drive future structure-based studies on the roles of regulated unfolding in NPM1 biology and will provide a foundation for NPM1-targeted anticancer drug development. Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer–pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.

Mechanism of polyubiquitination by human Anaphase Promoting Complex: RING repurposing for ubiquitin chain assembly

Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APCs RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.

A Comparison of the Self-association Behavior of the Plant Cyclotides Kalata B1 and Kalata B2 via Analytical Ultracentrifugation

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted. We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mm, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe6, and the second involving a charge-charge interaction with the Asp25 residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.

Heterodimerization of the Sialidase NEU1 with the Chaperone Protective Protein/Cathepsin A Prevents Its Premature Oligomerization

Lysosomal neuraminidase-1 (NEU1) forms a multienzyme complex with β-galactosidase and protective protein/cathepsin A (PPCA). Because of its association with PPCA, which acts as a molecular chaperone, NEU1 is transported to the lysosomal compartment, catalytically activated, and stabilized. However, the mode(s) of association between these two proteins both en route to the lysosome and in the multienzyme complex has remained elusive. Here, we have analyzed the hydrodynamic properties of PPCA, NEU1, and a complex of the two proteins and identified multiple binding sites on both proteins. One of these sites on NEU1 that is involved in binding to PPCA can also bind to other NEU1 molecules, albeit with lower affinity. Therefore, in the absence of PPCA, as in the lysosomal storage disease galactosialidosis, NEU1 self-associates into chain-like oligomers. Binding of PPCA can reverse self-association of NEU1 by causing the disassembly of NEU1-oligomers and the formation of a PPCA-NEU1 heterodimeric complex. The identification of binding sites between the two proteins allowed us to create innovative structural models of the NEU1 oligomer and the PPCA-NEU1 heterodimeric complex. The proposed mechanism of interaction between NEU1 and its accessory protein PPCA provides a rationale for the secondary deficiency of NEU1 in galactosialidosis.

How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic

The 39‐kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a ‘Trojan horse’ antibiotic. The approximately 260‐residue C‐terminal region of MccB is homologous to ubiquitin‐like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB‐catalysed C‐terminal MccA‐acyl‐adenylation is reminiscent of the E1‐catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C‐terminal di‐glycine sequence, MccBs substrate, MccA, is a short peptide with an essential C‐terminal Asn. Furthermore, after an intramolecular rearrangement of MccA‐acyl‐adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate‐linked analogue of acyl‐adenylated aspartic acid. We report six‐crystal structures of MccB in apo, substrate‐, intermediate‐, and inhibitor‐bound forms. Structural and kinetic analyses reveal a novel‐peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic‐active site for catalysing dual adenosine triphosphate‐consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two‐step activation for generating the peptidyl‐antibiotic MccC7.

Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.

Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.

Sgt1 Dimerization Is Required for Yeast Kinetochore Assembly

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study, we show that Sgt1 forms homodimers by performing in vitro and in vivo immunoprecipitation and analytical ultracentrifugation analyses. Analyses of the dimerization of Sgt1 deletion proteins showed that the Skp1-binding domain (amino acids 1–211) contains the Sgt1 homodimerization domain. Also, the Sgt1 mutant proteins that were unable to dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is important for Sgt1-Skp1 binding. Restoring dimerization activity of a dimerization-deficient sgt1 mutant (sgt1-L31P) by using the CENP-B (centromere protein-B) dimerization domain suppressed the temperature sensitivity, the benomyl sensitivity, and the chromosome missegregation phenotype of sgt1-L31P. These results strongly suggest that Sgt1 dimerization is required for kinetochore assembly.

Crystal Structure of the 6-Hydroxymethyl-7,8-Dihydropterin Pyrophosphokinase•Dihydropteroate Synthase Bifunctional Enzyme from Francisella tularensis

The 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) enzymes catalyze sequential metabolic reactions in the folate biosynthetic pathway of bacteria and lower eukaryotes. Both enzymes represent validated targets for the development of novel anti-microbial therapies. We report herein that the genes which encode FtHPPK and FtDHPS from the biowarfare agent Francisella tularensis are fused into a single polypeptide. The potential of simultaneously targeting both modules with pterin binding inhibitors prompted us to characterize the molecular details of the multifunctional complex. Our high resolution crystallographic analyses reveal the structural organization between FtHPPK and FtDHPS which are tethered together by a short linker. Additional structural analyses of substrate complexes reveal that the active sites of each module are virtually indistinguishable from those of the monofunctional enzymes. The fused bifunctional enzyme therefore represents an excellent vehicle for finding inhibitors that engage the pterin binding pockets of both modules that have entirely different architectures. To demonstrate that this approach has the potential of producing novel two-hit inhibitors of the folate pathway, we identify and structurally characterize a fragment-like molecule that simultaneously engages both active sites. Our study provides a molecular framework to study the enzyme mechanisms of HPPK and DHPS, and to design novel and much needed therapeutic compounds to treat infectious diseases.